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Handbook of Methods In

Environmental Studies

Water and Wastewater Analysis

ABD Publishers
Handbook of Methods in
Environmental Studies
Vol. 1: Water and Wastewater Analysis

B.Sc (Hons), M.Sc (Calcutta University)
M.Tech. (Env. Sc. & Engg.), liT, Mumbai
Ph.D (Indian School of Mines, Dhanbad)

Centre ofMining Environment

Indian School of Mines
Dhanbad-826 004, India

·I: :


ABD Publishers
Jaipur (India)
Handbook of Methods in Environmental Studies
Vol. 1 : Water and Wastewater Analysis
By : S.K. Maiti
ISBN : 978-81-8577-34-07
ISBN 81-8577-34-0

'!~ Author

Second Edition -20U4

No part of this book may be reproduced or transmitted in any form or

by any means electronic mechanical including photocopying, recording or
by any information storage and retrieval system without permission in writing
from the publishers and copyright holder.

Published by :
ABD Publishers
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Distribution by :
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Printed at:
Arora Offset Press
Laxmi Nagar, Delhi 110092.
Dedicated To

My father, Late Shri Mura/idhar Maiti

My mother, Smt. Mondakini Maiti .
Handbook of Methods in Environmental Studies
Vol. 2: Air, Noise, Soil, Overburden, Solid Waste and Ecology
Air quality monitoring: Micrometeorology; Objectives, Siting criteria for AQMS, Ambi-
ent and source emission monitoring, NAAQS, EPA-USA AQS, Monitoring frequency, Re-
porting, AQ monitoring for existing industry and for new industry, Self test
Monitoring of particulate pollutants: Dustfall, Calibration ofHVS, monitoring ofSPM,
RPM, Lead, aeroallergens and aero microbes
Monitoring of gaseous pollutants: Collection of gaseous pollutants, Monitoring of NOx'
S02' Sulphation rate, Oxidants (NBKI ), CO (NDIR), Vehicle exhaust monitoring, Selftest.
Stack monitoring: Objectives, Measurement of emission, Sampling, Steps in stack moni-
toring, Calculation, Self test, Chemicals Apparatus/instruments required for soil analysis


Objectives, dB, SPL, Sources and effects of noise, Noise survey, Noise measurement
instrument- SPL meter - Types and features and Control facilities, Weighting network,
Octave band analyser, Noise dosimeter, Audiometer, Noise rating, Lcq' Ldn, L 10 L~O' L90, NEI,
TNI, NIl, NNJ; Noise mapping; Octave band analysis, Audiometric survey, Standards- CPCB,
OSHA and ISO, C.ommunity noise standards- WHO, CPCB, IS, ILO, air blast, Self test on
noise monitoring


Sampling techniques: Objectives, Steps in sampling, On-site tests and description, Sample
Analysis of physical parameters: Introduction, Coarse fractions, Texture, Bulk density
and pore space, Field moisture, WHC, Wilting point, Infiltration rate, Self test
Analysis of Chemical parameters: pH, Lime requirement, EC, Organic carbon, Organic
matter, Total nitrogen, Av. N, Av. P (Bray's and Olsen's), Total P, P-fixing characteristics
of soil, Exch. K, Exch. Na and SAR, CEC, Av. S, Exch. Ca and Mg, Chloride, Monitoring
ofPb, Fe, Cu, Mn, Zn, Ni and Cr, Cd, DTPA fractions, Hg
Soil microbiology: Collection/processing of sample, Enumeration of bacteria, actinomycetes,
Filamentous fungi, VAM infection in root, Estimation ofVAMF spores, Soil respiration- in
situ and laboratory measurement, Soil enzymes- dehydrogenase, invertase, amylase and
cellulase, Chemicals, apparatus and instruments for soil analysis laboratory


Sampling procedure, Physical characteristics - composition, particle size, moisture content,
density; Chemical composition- proximate analysis, fusing point, energy content, chemical
contents; TCLP test; Self test

Objectives, Assessment of productivity; Diversity, Plant community studies, Litter-fall and
litter decomposition, Study offaunal community; Self test.

ince 1985, when I was a student of M. Tech. (Environmental Science &
Enginering) course at lIT, Mumbai, to this date, when I am a teacher, I
encountered a common problem that the students are not sufficiently drawn
to practical classes because of a near total absence of information required for the
practicals. Notwithstanding the availability of many practical books available in
the market, availability of right information with adequate details still eludes the
students. This has been the prime motive behind my endeavour to write an
environmental analysis handbook that fulfils the needs of practical classes and also
prepares the students well to face viva-voce examinations on water and wastewater
analysis. Keeping this in mind, I have presented all the parameters of water and
wastewater analysis as per drinking water standards (IS: 10500, 1991) and effluent
discharge standards (MOEF, Schedule VI, 1993). I tried to discuss the experimental
details without losing lucidity, the way it should be written in a practical notebook.
For every experiment described, relevant National and International standards have
been mentioned. Also provided are environmental significance of the experiment,
principles of the analysis and chemical reactions involved, glassware and chemicals
required, reagent preparation and storage, etc. Experimental steps have been
elucidated with flow-charts and pictures for ease of visualisation. In order to test
the confidence of the reader, some questions are appended to the experiments
described. A large number of solved examples are also incorporated to facilitate
confidence building.
I have also drawn more than 40 figures, just to explain the experimental
setups. A list of various chemicals and glassware needed in an environmental analysis
laboratory are provided in the Appendix. In addition, a list of general instruments
and sophisticated instruments required for water and wastewater analysis is also
provided in the Appendix along with their costs.
Having been involved in teaching and research in environmental science and
engineering for the past 15 years, I am reasonably confident that, this book will
serve as a handbook to all levels of scholars and professionals involved in water
and wastewater analysis for their day-to-day work.
I have devoted more than 5 years of time to write this handbook and my hard
work will be fruitful if it is of benefit to the students for whom the book was
This being the first edition, there is a lot of scope for qualitative improvement.
I shall be glad to receive any suggestion and constructive criticism in that direction.
No author can take entire credit for publication of a book. In one way or the
other, many persons have helped me in accomplishing the task. My appreciation
goes to my colleagues in the Centre of Mining Environment, Indian School of
Mines, Dhanbad, for their continuous encouragement and for the facilities
provided at the Centre. Prof. Gurdeep Singh and Prof. N.C. Saxena deserve special
mention. Special thanks are due to Dr. I.N. Sinha and Dr. Dinesh Mohan for their
meticulous correction and mental support. I am also thankful to our laboratory staff
for their interest in the work and to Shri Ajoy Bhattacharya for careful drafting of
the figures. Thanks are also due to Dr. P.K. Goel, Reader in Deptt. of Pollution
Studies, Y.C. College of Science, Karad, for his critical suggestions for improve·
ment ofthe contents and getup of the book. It is my pleasure to acknowledge the
help and assistance received from Dr. v.P. Upadhayay (MOEF, Bhubaneswar),
Dr. N.C. Karmakar (ISM, Dhanbad), Dr. Amal Kr. Pal (CU), Dr. Ram Bhagat and
Dr. B. Pati (Vidyasagar University, Midnapore), Dr. P.C. Mishra (Sambalpur
University) Dr. Arvind Kumar (S.K.University, Durnka), Shri P.C. Jha, Rajiv Kr.
and Vinita Arora (CMPDIL, Ranchi), and Shri Suresh Jain (MECON, Ranchi).
I wish to express my sincere appreciation to my wife (Sumita) and children
(Polly and Tukai) for their patience and sacrifice but for which the book would not
have seen the light of the day.
I am also grateful to my publishers for timely publication of the book.

Place: Dhanbad Subodh Kumar Maiti

Date: 1·6·2001

1.1 Water Quality Monitoring

1.2 Quality of Aquatic Environment
1.3 Objective of Water Quality Monitoring
1.4 Description of Monitoring Area 2
1.5 Selection of Sampling Location 2
1.6 Sampling 3
1.7 Water Quality Monitoring in Field 7
1.8 Laboratory Analysis (Priority wise) 8
1.9 Preservation of water Samples 9
1.10 Checking of Analysis 9
1.11 Structuring of Monitoring Report 10
1.12 Setting of Water Analysis Laboratory 13
1.13 'Questions 13


2.1 Standards 15
2.2 Drinking water Standards (India) 15
2.3 International Drinking Water Standards 15
2.4 Coliform Standards 20
2.5 Effiuent Standards 22
2.6 Stream Standards 24
2.7 Wastewater Analysis for Proposed Project 26
2.8 Primary Water Quality Criteria for Bathing Waters 27
2.9 Effiuent Standards for Coal Mines 27
2.10 Standards for Effiuents from Textile Industry 28
2.11 Questions 29

3.1 Acidity 30
3.2 Alkalinity 33
3.3 Biochemical Oxygen Demand (BOD) 37
3.4 Boron 48
3.5 Carbon Dioxide (Free CO 2) 50
3.6 Conductivity or Specific Conductance 52
3.7 Colour 57
3.8 Chemical Oxygen Demand (COD) 60
3.9 Chlorine (Residual) 66
3.10 Chlorine Demand 70
3.11 Cyanide 74
3.11.1 Estimation of Total Cyanide after distillation 74
3.11.2 Estimation of Cyanide by Titration Method 76
3.11.3 Estimation of Cyanide by Colorimetric Method 77
3.11.4 Estimation of Cyanide by Ion Selective Electrode 78
3.12 Dissolved Oxygen 79
3.13 Fluoride 85
3.14 Hardness (Total) 91
3.15 Hardness (Calcium) 94
3.16 Hardness (Magnesium) 96
3.17 Methylene Blue Active Substance (MBAS) 100
3.18 Nitrogen (Total) 104
3.19 Nitrogen (Ammonical) 108
3.19.1 Estimation of Ammonia by Nesslerization 109
3.19.2 Estimation of ammonia by Titration III
3.20 Nitrogen (Nitrate) (N03-N) 113
3.20.1 Estimation of Nitrate by Ultraviolet (UV)
Spectrophotometric Screening Method 114
3.20.2 Estimation of Nitrate by Devarda's Alloy Method 116
3.20.3 Estimation of Nitrate by Phenol Disulphonic Acid
(PDA) Method 117
3.21 Nitrogen (Nitrite) (N02-N) 120
3.22 Nitrogen (Organic) 123
3.23 Odour (TON) 123
3.24 Oil and Grease 125
3.25 pH (Potentia Hydrogenii) 128
3.26 Phenol (Phenolic Compounds) 133
3.27 Phosphorus 138
3.27.1 Estimation of Inorganic Phosphate 139
3.27.2 Estimation of Total Phosphorus 141
3.27.3 Estimation of Particulate Phosphorus 142
3.27.4 Estimation of Organic Phosphorus 142
3.28 Solids 142
3.28.1 Estimation of Total Solids (TS) 143
3.28.2 Estimation of Total Suspended Solids (TSS) 144
3.28.3 Estimation of Total Dissolved Solids (TDS) 146
3.28.4 Estimation of Settleable Solids (Volumetric Method) 148
3.28.5 Estimation of Settleable Solids (Gravimetric Method) 149
3.29 Sodium and Potassium 150
3.29.1 Estimation of sodium 151
3.29.2 Estimation of Potassium 154
3.30 Sulphate 155
3.30.1 Gravimetric Method 156
3.30.2 Turbidimetric Method 158
3.31 Sulphide 161
3.32 Thiocyanate 164
3.33 Transparency (Secchi Disc Method) 167
3.34 Turbidity (Nephelometric Method) 169


4.1 Introduction 174
4.2 Standards of Heavy Metal 174
4.3 Sampling and Preservation of Samples 176
4.4 Digestion of Metals with Nitric Acid 177
4.5 Digestion of Metals with HC1 and HN03 177
4.6 Analysis of Heavy Metals by AAS 178
4.7 Aluminium 181
4.8 Arsenic 182
4.9 Cadmium 183
4.10 Chromium 184
4.11 Copper 185
4.12 Iron 185
4.13 Lead 186
4.14 Manganese 187
4.15 Mercury 188
4.16 Selenium 192
4.17 Silver 193
4.18 Vanadium 193
4.19 Zinc 194
4.20 Check Your Confidence on Heavy Metals 195
5 .1 Coagulation-Flocculation Jar Test of WaterlEffiuents 197
5.2 FoodlMicroorganisms (FIM) Ratio 203
5.3 Mixed Liquor Suspended Solids (MLSS) and Mixed
Liquor Volatile Suspended Solids (MLVSS) 204
5.3.1 Estimation ofMLSS 205
5.3.2 Estimation ofMLVSS 205
5.4 Sludge Volume Index (SVI) 206
5.5 Volatile Fatty Acids (VFAs) 209
6.1 Introduction 213
6.2 Disease Associated with Polluted Water 213
6.3 Coliform Group of Bacteria 214
6.4 Estimation of Coliform Bacteria in Water 215
6.5 Presence-Absence (P-A) coliform Test 223
6.6 Fecal Coliforms (Thermotolerant Coliforms) MPN Test 225
6.7 Fecal Streptococci (FS) Test by MPN Methods 226
6.8 Calculation of Most Probable Number (MPN) 229
6.9 Membrane Filter (MF) Technique 233
6.10 Seven Hour Fecal Coliform Test (Specialised 237
6.11 Check Your Confidence on Coliform Test 240
7.1 Study of Benthic Macroinvertebrates 243
7.1.1 Counting of Benthic Macroinvertebrates 244
7.1.2 Macroinvertebrate Biotic Index (MBI) 247
7.2 Counting of Phytoplankton (Microscopic Algae) 248
7.2.1 Sedgwick-Rafter Cell Method 250
7.2.2 Haemocytometer Method 252
7.2.3 Lackey's Drop Method (Microtransact Method) 253
7.3 Counting of Zooplankton by Sedgwick-Rafter Cell Method 253
7.4 Diversity and other Indices 254
7.4.1 Shannon Index (R) 255
7.4.2 Evenness or Equitability Index 255
7.4.3 Simpson's Index (D) 256
7.4.4 MargalefIndex of Species Richness 256
7.4.5 McIntosh Index (M!) 256
7.4.6 Odum's Index 257
7.4.7 Goodnight and Whitley's Index 257
7.4.8 Kothe's Species Deficit Index 257
7.4.9 Berger-Parker Dominance Index
or Community Dominance Index (DI) 257
7.4.10 Similarity Index 258
7.4.11 Autotrophic Index (AI) 258
7.5 Determination of Phytoplankton Biomass 259
7.5.1 Chlorophyll a Extract Method 259
7.5.2 Gravimetric Method 259
7.6 Phytoplankton Productivity 259
7.7 Estimation of Chlorophyll a 261
7.8 Biological Monitoring for Health of Wastewater
Treatment Plants 264
7.9 Check your Confidence on Biological Monitoring 265



8.1 Toxicity Test

8.1.1 Significance of Toxicity Tests 267
8.1.2 Classification of Toxicity Tests 267
8.2 Measurement of Toxicity in Laboratory 269
8.3 Toxic Unit 271
8.4 Check Your Confidence on Toxicity Measurement 273
9.1 Radioactivity 274
9.2 Measurement of 13 Radioactivity in Water 275
9.3 Measurement of a Radioactivity in Water 277
Appendix I: List of chemicals required for water analysis laboratory 282
Appendix 2: List of glassware required for water analysis laboratory 284
Appendix 3: Instruments for monitoring water/wastewater 286
Appendix 4: Characteristics of three most common acids 287
Appendix 5: Preparation of uniform sodium hydroxide solution 287
Appendix 6: Indicator solutions 288
Appendix 7: Quantities and units 288
Appendix 8: Drinking water standards: IS: 105000 (1991) 296
"This page is Intentionally Left Blank"
Water Sampling and

1.1 Water Quality Monitoring

Monitoring is defmed by the International Organization for Standardization (ISO)
as: "the programme of sampling, measurement and subsequent recording or signal-
ling, or both, of various water characteristics, often with the aim of assessing
conformity to specified objectives". This general definition can be differentiated
into three types of monitoring activities that distinguish between long-term, short-
term and continuous monitoring programmes as follows:
l. Monitoring is the long-term, standardized measurement and observation of the
aquatic environment in order to define status and trends.
2. Surveys are finite duration (short-term) intensive programmes to measure anq
observe the quality of the aquatic environment for a specific purpose.
3. Surveillance is continuous, specific measurement and observation for the pur-.
pose of water quality measurement and operational activities.

1.2 Quality of Aquatic Environment

The quality of aquatic environment is a broader issue which can be described as:
1. water quality,
2. composition and state of the biological life present in the water bodies,
3. nature of the particulate matter present, and
4. physical description of the water body (hydrology, dimension, morphometry,
nature oflake bottom or river bed).

1.3 Objectives of Water Quality Monitoring

l. Identification of baseline conditions in the water-bodies and sources of water
2. Detection of any sign of deterioration in water quality.
3. Identification of water bodies in the water-course system that do not meet the
desired water quality standards.
4. Identification of any contaminated area.
S. Determination of the extent and effect of specific waste discharges.
6. Estimation ofthe pollution load carried by a water-course system or subsystem.
7. Evaluation of the effectiveness ofa water quality management intervention.
8. Development of water quality guidelines and/or standards for specific water
2 Handbook o/Methods in Environmental Studies

9. Development of regulations covering the quantity and quality of waste dis-

10. Development of a water pollution control program.

1.4 Description of the Monitoring Area

The description of a monitoring area should at least consider the following:
1. defmition of the extent ofthe area,
2 a summary of the environmental conditions and processes (including natural and
human activities) that may affect water quality,
3. meteorological and hydrological information,
4. description of the water bodies, and
5. summary of actual and potential uses of water.

1.5 Selecting Sampling Location

Selection of sampling locations requires consideration of the monitoring objectives
and some knowledge of the physical features of the water-course system, as well as
of water and of any discharges of wastes into it (Table 1.1). Sampling locations can
be marked on a map or an aerial photograph, but a final decision on the precise
location of a sampling station can be made only after a field investigation.

Table 1.1
Links between types of monitoring sites and programme objectives.

Type of site Location Objectives

Baseline site Headwater lakes • To establish natural water quality con

or undisturbed ditions.
upstream river • To provide a basis of comparison with
stretches stations having significant direct human
• To test for the influence of long-term
transport of contaminants and the ef-
fects of climatic changes.
Trend site Major river basins, • To test for long-term changes in wa-
large lakes or major ter quality.
aquifers • To provide a basis for statistical identi
fication of the possible causes of mea
sured conditions or identified trends.

Global river Mouth of a major • To determine fluxes of critical pollut-

flux site river. ants from river basin to ocean or re-
gional seas.
• Some trend stations on rivers also
serve as global flux stations.

Source: Based on the GEMS/WATER monitoring program.

Water Sampling and Preservation 3

1.6 Sampling
The objective of sampling is to collect a portion of water which is small enough in
volume to be transported conveniently and handled in the laboratory while still
representative of the characteristics ofthe water available in that water body. Water
is a dynamic system. Its constituents vary with time.


Ask yourself the purpose of study (?), what data are needed (?) and then select
most suitable location that can provide the required information.

Site location for river

Upstream (u/s) of the industrial and domestic discharge points
Immediately downstream (dis) of industrial and domestic discharge points.
• At a place of abstraction for industrial use and public water supply.
Base line stations where water is available in natural state.


Grab or catch samples, also known as spot or snap samples.
Composite samples.
Integrated samples.

a. Grab or catch samples

A sample collected at a particular time and place can represent only the composi-
tion of the source at that time and place. It shows only the prevailing conditions at
the time of sampling and does not represent the average conditions.
Sampling of water from sources such as wells, rivers, streams, lakes, oceans and
reservoir for chemical, physical and bacteriological analysis.
When a source is known to be constant over a considerable time period, in that
case a single grab sample should be considered as representative.
If the sources are known to vary with time, grab sample should be collected at
suitable intervals of time and analyzed separately.
The results can be documented in terms of mean, standard deviation, frequency,
and duration of variations. When the source composition varies in space
(in different locations), collect samples from appropriate locations.

b. Composite samples
The term composite refers to a mixture of grab samples collected at the same sam-
pling point at different times. A composite sample of 24 h period is considered
standard for most of the determination. It provides more meaningful data than the
grab samples.
Sometimes a composite sample representing one shift or a shorter time period or
a complete cycle of a period operation may be preferable.
Take at least 120-150mL of sample in each h, in some cases even at intervals of
4 Handbook ofMethods in Environmental Studies

30 min (if composition varies within an hour) and mix at the end of sampling
period or combine in a single bottle as collected.
• A final volume of 4-5 L is sufficient.
c. Integrated samples
For certain purpose, the information needed is provided best by analyzing mixture
of grab samples collected from different points simultaneously.
Such samples are useful for rivers or streams that vary in composition across
the width and depth. For collection of integrated samples, special sampling
device is needed.
Sample is collected at a known depth without disturbing the surface water.
Water sample should be collected at intervals so that no change in water quality
could pass unnoticed. It depends on the type of data required, purpose of monitor-
ing, availability of funds and personnel.
Number of Samples: Number of samples and how often should samples be col-
lected, are calculated by statistical consideration:


N = Number of samples
t = Student 't' -statistics for a given confidence level
S = overall standard deviation, and
U = acceptable level of uncertainty
For, example, if standard deviation is 0.5 mg/L, U is 0.2 mg/L, and 95% confi-
dence level is desired, then 25 to 30 samples must be taken. (t-test is used for less
than 30 samples).
The frequency is determined by the objectives of monitoring. The following
frequencies may be adopted provisionally:
Once in a year: Generally applicable for long-term ecological evaluation ofbiologi-
cal water quality. The time and place should be the same. Sometimes also used to
know the year-to-year trends, usually as a result of increased human activities in
the watershed.
Four times in a year for studying seasonal variations in water quality. Sometimes
three samples are taken in a year like, pre-monsoon, monsoon and post-monsoon
Weekly samples for one year.
Round the hour sampling for 24 h (i.e. industrial effiuents)
The sampling bottle may be made of either glass or plastic, usually polyethylene. It
must be capable of being tightly sealed either by stopper or cap.
Water Sampling and Preservation 5

The bottles should be soaked with 10% HCI for 24 h and then thoroughly
cleaned and rinsed with distilled water.
If metals are to be analyzed, rinse the container with solution of 1 part concen-
trated HN0 3 to 4 parts water followed by distilled water.
Cleaning solution; acid dichromate: Prepare a saturated water solution of po-
tassium dichromate (K2Crp7). Add 32 mL of this K 2Crp7 solution in 1L of
concentrated H2 S04 (sp. gr. 1.84).


Several different type of·water samplers are available and many of them are
designed for specific purposes. Two most commonly used water samplers are
described here.

a. Dissolved oxygen (DO) sampler

A DO sampler is a metal tube about 10 cm diameter and 30 cm length. One end is

sealed and other end is fixed with a removable cap (usually threaded) as shown in

(ross sKtM)n

Fig 1.1: Dissolved oxygen sampler

SYiptnNort _ _ _ __


1I-4---4-....l/.__ b'''---.:Y--''---+--J!

Fig 1.2: Depth sampler suitable for moderate depth.

6 Handbook ofMethods in Environmental Studies

b. Depth sampler
The depth sampler, sometimes called a grab sampler, is designed in such way that it
can retrieve a sample from any predetermined depth. A simple and relatively inex-
pensive depth sampler suitable for moderate depth « 30m) is illustrated in Fig.l.2.


1. Wherever possible, the container should be rinsed 2 to 3 times with the sample
to be examined.
2. Sample where water is well mixed.
3. Avoid large non-homogeneous matter such as leaves, rags, twigs and other
floating material in the sample.
4. Provide complete information about the source and the conditions under which
the sample was coi!('cted
5. Sample preferably at 20cm depth in a shallow channel (Fig.I.3). Depths> 60cm,
collect 2 samples at 20% and 80% below the surface

Fig.1 .3: Collecting a sample from surface water.

Fig.1.4: Collecting a sample from dug well.

Water Sampling and Preservation 7

6. Sampling from dug wells and similar sources: Prepare the sampling device bottle
with the help of a string and attach a weight at the bottom (Fig. 1.4).


Each sampling bottle must be provided with an identification label on which the
following information is legibly and indelibly written (Table 1.2).


Sample containing bottles should be placed in a box for transportation to the labo-
ratory. Sturdy, insulated wooden or plastic boxes will protect samples from sun-
light, prevent the breakage of bottles and should allow a temperature of 4°C to be
attained and maintained during transport. Fig.I.5 shows a suitable sample transport

1.7 Water Quality Monitoring in Field

Following parameters are recommended for measurement at the sampling site or in
the field immediately after a water sample has been taken.
1. Temperature: must be measured in situ.
2. pH.

Table 1.2:
A typical Label-sheet of container

1. Name of study
2. Sample number/ sample station
identification no.
3. Source/location of sampling point
4. Date and time of collection
5. Volume of sample
6. Nature of collection : Grab/composite sample
7. Given any preservative : Yes/No.
8. Purpose of analysis: : Drinking water/effluent discharge.
9. Field measured parameters : Temperature, pH, DO, or any
other parameters.
10. Purpose of sampling (Please tick
which is applicable).
11. Sample to be refrigerated and used
for general analysis.
1 2. Sample to be refrigerated and used
for bacteriological analysis
1 3. Sample to be used for heavy
metals analysis
14. Brief details of weather any
unusual condition was prevailing
at the time of sampling.

Name of the person and signature

8 Handbook ofMethods in Environmental Studies

~. '.:.' ,,,:'.': .:-:.....: ~.~-: ::. -:.~

:. ~~~~ H ::.
, 2 3 10 S.~ f--Irowtation
I!l 4oR-~'::-tt.----~ bottles
~.e..~.~ . . . .~
•• :

Ju peck or frftziflg rixt'"

Fig.1.5: Sample transport box

3. Electrical conductivity (or specific conductance)

4. Dissolved oxygen (DO) by DO probe. If DO probe is not available, take 300 mL
sample in a BOD bottle and fix the DO by adding 2 mL MnS04 •
5. Transparency (for lakes and reservoirs).
6. Residual chlorine (particularly for chlorinated drinking water) by using colour
1. Collect about 4 L sample in a labelled sample container and refrigerate it at 4°C
(for general analysis).
2. Collect about 100-250 mL ofsample and acidify it with ~SO4 to bring pH < 2 and
label the sample for heavy metals analysis (this is done to minimizes precipita-
tion ~d absorption of heavy metals in container wall).
3. Collect about 250 mL of sample for bacteriological test in a sterilized container
and put a tag for coliform analysis. The sample must be refrigerated at 4°C.

1.8 Laboratory Analysis (Prioritywise)

Step-I: Determine of residual chlorine
Step-2: Determine DO by modified Wrinkler method.
Step-3 :BOD test (take 900 mL sample and incubate 3 replicates)
Step-4: Conduct turbidity test.
Step-5: Conduct TKN test.
Step-6: Conduct acidity test (effluents) (take 100 mL sample).
Step-7: Conduct alkalinity test (take 200 mL sample).
Step-8: Determine colour
Step-9: Determine hardness, metals and others.
Shorter the time between the collection of the sample and its analysis, higher will be the
reliability of results. In general, sample should reach within 48 h to laboratory. Changes
caused by growth of microorganisms are greatly retarded by keeping the sample in
dark and at a low temperature. It is of prime. importance that special preservation
methods are used for the samples that are not to be analyzed immediately.
Water Sampling and Preservation 9

Table 1.3:
Preservation of samples.

Parameters Preservation Method Maximum

holding period

Acidity Refrigerate at 4°C 24 h

Alkalinity Refrigerate at 4°C 24 h
BOD Refrigerate at 4°C 6h
COD 2 mL H2 S0 4 /L 7 day
Residual CI 2 Analyze immediately
Colour Refrigerate at 4°C 24 h
DO Analyse immediately or fix on site 6h
Fluoride Not required 7 day
Metals Acidify with HN0 3 to pH < 2. 6 months
NH3-N Analyse as soon as possible, add 2 mL
40% H2 S0 4 to pH < 2, refrigerate. 24 h
Nitrate Analyse as soon as possible.
N-Kjeldahl Refrigerate, add H2 S0 4 to pH < 2, 7 days
Phosphorus 40 mg HgCI/L, refrigerates at 4°C. 24 h
Sulphides Refrigerate, add 2 mL 2N zinc
acetate/100 mL; add NaOH to pH> 9. 7 days
Sulphate Refrigerate at 4°C 7 days
Turbidity Refrigerate at 4°C
Colifom bacteria Sterilised bottle, no specific preservative,
refrigerates at 4°C 36 h

1.9 Preservation of Water Samples

Suggested chemical preservatives and recommended maximum storage times for
samples for various analyses are summarized in Table 1.3.

1.10 Checking Correctness of Analysis

Basic data checks may be carried out by the laboratory that generates data and/or
by the person or organization that uses or interprets them. Some examples of basic
data checks that may be readily employed are described below.
Total dissolved solids (TDS) = 0.6 (alkalinity as CaCOJ ) + Na+ +K++ Ca2+ + Mg2+
+ CI-+ SO;-+SiO;-+ NO~ + F-

Anion-cation balance: The anions and cations sum, when expressed as milli-
equivalents per liter (meq/L), must balance because all potable waters are electri-
cally neutral. The test is based on the percentage difference defmed as follows:

Lcations - Lanions
% Difference = x 100
Lcations + Lanions
10 Handbook of Methods in Environmental Studies

The criteria for acceptance are as follows:

Anion sum (meq/L) Acceptable differences (meq/L)

0-3 ±0.2
3 - 10 ±2
10 - 800 ±5

An accurate ion balance does not necessarily mean that the analysis is correct.
There may be more than one. error and these may cancel each other. As a result
additional checks are needed. With careful work, the difference will not generally
exceed 2% of the total cations or anions in waters of moderate concentrations (250
to 1000 mg/L). The calculations to see the correctness of results of the actual data
are given in example boxes 1 and 2 later.

Calculate TDS to EC ratio: The numerical value of TDS (mg/L) should not
exceed that of electrical conductivity (~S). The relationship between the two vari-
ables is often described by a constant (commonly between 1.2 and 1.8 for freshwa-
ter) that varies according to chemical composition. For freshwater the normal range
can be calculated from the following relationship:
Conductivity = TDS x F, where F is in the range of 1.2 to 1.8.
Typically, the constant is high for chloride-rich waters and low for sulfate-rich
waters (UNEPIWHO, 1996).
TDS/conductivity = 0.55 to 0.70.
If the ratio ofTDS to EC is outside these limits, measured TDS or measured EC
is suspect; reanalyze the sample (APHA, 1998).

1.11 Structuring of Monitoring Report

The structure described in the following paragraphs may be used as a general
outline for the preparation of monitoring reports, although not all reports will nec-
essarily contain all the elements.
Summary The summary briefly outlines what was done, describes
the significance of key findings, and lists the important
It should be written so that a non-scientist can under-
stand the purpose of the work and the importance of the
Its length should not exceed two or three pages (about
1,000 words).
Often, the summary is the only part of the report, which is
read by senior managers.
Introduction The objectives and the terms of reference (TOR) of the
study should be presented in the introduction.
The problems or issues being addressed should be clearly
stated, previous studies should be described and relevant
scientific literature should be reviewed.
Water Sampling and Preservation 11

Any major restrictions (personnel, access, finance, facili-

ties, etc.) which limit the described scope of the programme
should be identified.
Study area A summary of the geography, hydrology, and other sa-
lient features (which, for example, may include land use,
industry and population distribution) of the area under
study should be provided.
All locations, structures and features mentioned in the
text should be identified on maps.
In certain cases, profiles of river or lake systems may pro-
vide additional useful information.
Methods A separate section should provide details of the methods
and procedures used for all aspects of the study.
A summary of the procedures to be followed for quality
assurance should be presented.
Results Results should be presented in graphical form whenever
possible and graphs should clearly demonstrate the rela-
tionship of the data to the monitoring objectives.
Graph should summarise entire data rather than individual
International units of measurement should be used wher-
ever possible but if local units are used precise descrip-
tion and conversions should be provided.
Analysis of The statistical analysis of results should be described.
results The reliability ofthe statistics and its implication, should
also be given.
Analytical procedures appropriate to the problems should
be used and should focus on the goals of the study or the
Results should be presented in graphical, rather than in
tabular form.
Significance of The report should include a section devoted to interpreta-
results tion of the results in terms of the monitoring objectives.
This will help ensure that the objectives are being met
and that questions are being answered.
This section should emphasise the need to provide infor-
Recommend- Recommendations for proposed future activities should
ations normally be listed in the following two categories;
a. One concerned with scientific matters, the other with man-
agement issues.
b. Ideally, these recommendations should be presented in
order of priority.
Information All sources of information and all literature referred to
sources should be correctly and fully cited so that a permanent
record is available.
12 Handbook o/Methods in Environmental Studies

Example - 1
Problem: An analysis of water from a surface stream yields the following
results. Whether the analysis is acceptable?

.... ,.f:; 8to.m!,,! , Vitt,,+ valef]cy'i,,!, m~'Ol.b.i:::

. "." " '%"'~:::_::
'. "-. >,"'v
=2.0.0 .",
24 •.3/2 ;;;'12.'15
:.r,, 23i1 ~~23 ',',if

.' Concentration
HCOj=317 mg/L ;" 6111 =61 ,
CI- = 89 mg/L ..315.5/1 =3~.,
SO-2=125 mg/L: 96/2=48,'
'Total anions - ~
,': ~ differ~nce ~'i1l:796-JO.3071 + (14.796
". Anions surri Uis . lo:~07 .A~cePt~d}aj~er~ric~ is '5%. I "",,,,,,,,,,,,.,,,'
difference is 17.87%, which is more than ~5% ; :Therefore,
;" >,d hing .wrong ir{tl1is analysis ". ,,;:1,::-";'- .' ", ".J.:"'"
. ~~,::';~;< .". ~:l/<~ $.,<"

Example - 2
Problem: An analysis of water from a surface stream yields the following results.
If an error of 10% is acceptable, should the analysis is acceptable?

" Concentration
+'2'_ 60 . IL ,',,;
.a - mg,i"":0i'
, Mg+2= 10. mg/L .. :
;,Na+ =7mg/L
./<+ =20 mg/!.:
Water Sampling and Preservation 13

Example - 3·
If standard derivation is 0.5 mg/L, test values for 95% confidence level is
1..9 6 and acceptance leve.1 of uncertainty is 0 .2 mg/L (i .e acceptance error), <d
then number of sample needed to meet 95% confidence level is, . '.

X0'J2 .

l ·96
= 24.01 (Say 25)

Also calculate the number of sample needed for 99% confidence level
(t 2 .575) for S =
0.5 mg/L and acceptable level of uncertainty (UI 0.2

N .~

There number . of sample needed as follows:

1. To meet 95% confidence level = 25 nos. or more
-2 . To meet 99% confidence level = 42 nos. or more

1.12 Setting of Water Analysis Laboratory

Appendix Tables I to 6 listed the required chemicals, their amounts and price,
important glasswares, general instruments and sophisticated instruments for
water analysis laboratory.

1.13 Questions
I. What are objectives of water quality monitoring?
2. How to take samples from river, lakes, wells and discharge from a industry?
3. How to clean sample containers?
4. "Samples collected for metal analysis, the polythene containers must be cleaned
with I part HN0 3+ 4 parts of distilled water"- Why? And why it should be
acidify to bring pH < 2?
5. "DO should be measured in the field"; if not, then how to bring sample to
laboratory for DO estimation?
6. What is the general time interval permissible between sample collection and
reaching it to the laboratory? (within 2d or 3d or 4d).
7. List the test to be carried out in the laboratory as per priority.
8. Why residual chlorine should be analysed immediately?
9. "For microbiological analysis sample container must be dechlorinated"-
Why? How to do this? Further, sample should be preserve at 4°C. Why?
10. Name three techniques, those can be used for checking the correctness of
analysis in the laboratory.
11. Is there any relationship between electrical conductivity and total dissolved
solids (TDS).
14 Handbook ofMethods in Environmental Studies

12. Describe the salient points to be considered for the preparation of a standard
monitoring report.
l3. How to prepare 6N, IN, O.lN and O.02NHCI, H 2S04 andHNO] solution. (Hints:
See annexures).
14. How to prepare 15N, 6N, IN and O.lN NaOH solutions. (Hints: Seeannexures).
15. How to prepare phenolphthalein and methyl orange indicator solution. (Hints:
See annexures).
16. An analysis of water from a surface stream yields the following results. If an
error of 10% is acceptable, should the analysis be considered complete. Ca+2 =
32 mgIL; Mg+2 = 42.59 mgIL; Na += 173.57mgIL; K+ = 24.20 mgIL; HC~= 186.46
mg/L; CI- = 275.64 mgIL; SO~2 = 84.25 mg/L; NO; = 9.80 mg/L; F- = 1.24.
(Ans: 3.66%).
17. An analysis of water from a surface stream yields the following results. If an
error of 10% is acceptable, should the analysis be considered complete. Ca+2 =
55 mg/L; Mg+2 =18 mgIL; Na+ = 98 mglL; HCO; = 250 mgIL; CI- = 89 mgIL;
SO~2= 60mgIL. (Ans: 8%).
Water Quality

2.1 Standards
Standards are the concentration to be maintained to achieve the stated objec-
tives. The term standard applies to any define rule, principle or measurement
established by the authority or that has official backing. As per Californian
Federal Act (1967), standard has been defined as " pollution level that can not
legally be exceeded during a specific time period in a specific geographical
area." The primary standards are related to human health and secondary stan-
dards are related to protect human welfare. Three types of standards are of
interest in water quality monitoring study.
• Drinking water standards
• Effluent standards
Stream standards

2.2 Drinking Water Standards (India)

In view of direct consumption of water by human beings, the domestic water
supply is considered to be most critical use of water. In India, agencies like-
Indian Council of Medical Research (ICMR, 1963), Bureau ofIndian Standards
(IS: 10500,1983, 1991) and Ministry of Works and Housing (MWH, 1975) have
formulated certain drinking water standards which are given in Table 2.1. The
most widely used drinking water standard is IS: 10500 (1991).

2.3 International Drinking Water Standards

The World Health Organization (WHO) issued guidelines for drinking water
standards in the years 1971, 1984 and 1993. The different countries also formu-
lated drinking water standards in the line of WHO. The most important ones
are listed below.
• Drinking water standards - United States of America (USA)- as per Safe
Drinking Water Act, 1974, USEPA.
• Canadian guidelines for drinking water quality (1987), and
• European Community (EC) drinking water standards (1980).
16 Handbook of Methods in Environmental Studies

Standards of drinking water by ICMR, IS:10500 and MWH.

Characteristics Indian Council of BIS,IS: 10500 Ministry of Works and

in mg/L Medical Research (1983) Housing (1975)

Desirable Maximum Requirement/ Acceptable Cause of

concen. permissible desirable rejection

1 2 3 4 5 6

1. Colour 5 25 10 5 25
(hazen units)
2. Odour(TON) Nothing disagreeable Unobjectionable Nothing disagreeable
3. Turbidity(NTU) 5 25 10 (Max.) 2.5 10
4. TS - - - 500 1500
5. DS 500 1500 - - -
6. pH 7-8.5 6.5 - 9.2 6.5 - 8.5 7 - 8.5 6.5 - 9.2
7. Total hardness
(as CaCO a) 300 600 300 200 600
8. Calcium 75 200 75 75 200
9. Magnesium 50 150 30 30 150
10. Iron (as Fe) 0.3 1.0 0.3 (max) 0.1 1.0
11. Copper(as CuI 1.0 3.0 0.05 0.05 1.5
12. Manganese 0.1 0.5 0.1 (max) 0.05 0.5
13. Sulphate(SO.) 200 400 150 200 400
14. Chlorides 200 1000 250 200 1000
1 5. Nitrates 20 50 45 45 45
16. Fluoride(as F) 1.0 1.5 0.06-1.2 1.0 1.5
1 7. Phenolics 0.001 0.002 0.001 0.001 0.002
18.Zinc - - 5 5 15
19.Arsenic - 0.05 0.05 0.05 0.05
20. Cyanides - 0.05 0.05 0.05 0.05
21. Cadmium - 0.01 0.01 0.01 0.01
22.Chromium(6+) - - 0.05 0.05 0.05
23.Lead - 0.1 0.1 0.1 0.1
24.Mercury - 0.0001 0.001 0.001 0.001
25.Selenium - 0.01 0.01 0.01 0.01
.26. Anionic detergents
(as MBAS) - - 0.2 0.2 1
27.Residual CI - - 0.2 - -
28.Mineral oils - - 0.01 0.01 0.3
29.PABs - - - 0.2 0.2
30. Pesticides - - - -
31. Radioactive
a. Alpha emitters - 3 pCi/L - 3 3
b. Beta emitters - 30 pCi/L - 30 30
Water Quality Standards 17


The WHO guidelines divide "Drinking water standards- World" under five catego-
ries (Tables 2.2 a - 2.2d):
i. Microbiological and biological quality
n. Inorganic constituents of health significance
iii. Organic constituents of health significance
iv. Aesthetic quality
v. Radioactive constituents

Table 2.2a:
WHO guidelines for drinking water quality: Microbiological and
biological quality.

Organisms Unit Guideline Remarks


A. Piped water supplies
A1. Treated water entering the distribution system
Fecal coliforms No./100 mL 0 Turbidity < 1 NTU, for
disinfection with chlOrine, pH
preferably < 8.0; free chlorine
residual 0.2-0.5 mg/L follow
ing 30 minutes (minimum)
A2. Untreated water entering the distribution system
Fecal coliforms No'/100 mL 0
Coliform No./100 mL 0 In 98% of samples examined
organisms throughout the year - in the
case of large supplies when
sufficient samples are
Coliform No./100 mL 3 In an occasional sample, but
organisms not in consecutive samples.
A3. Water in the distribution system
Fecal coliforms No./100 mL 0
Coliform No./100 mL 0 In 95% of samples examined
throughout the year - in the
case of large supplies when
sufficient samples are
Coliform No./100 mL 3 In an occasional sample, but
organisms not in consecutive samples.

B. Unpiped water supplies

Fecal coliforms, No./100 mL o
Coliform No./100 mL 10 Should not occur repeatedly;
organisms if occurrence is frequent and
if sanitary protection cannot
Table Cont ...
18 Handbook of Methods in Environmental Studies

... Table cant.

be improved, an alternative
source must be found if

C. Bottle drinking water

Fecal coliforms No./100 mL o Source should be free from
fecal contamination.
Coliform No./100 mL o

D. Emergency water supplies

Fecal coliforms No./l00 mL o Advise public to boil water in
case of failure to meet
guideline values.
Coliform No./l00mL o


i) Protozoa (pathogenic) - No guideline value set
ii) Helminths (pathogenic) - No guideline value set
iii) Free-living organisms (algae,others) No guideline value set

Table 2.2b:
WHO guidelines for drinking water quality: Inorganic constituents
of health significance.

Constituents Units Guideline value

1. Arsenic mg/L 0.05

2. Asbestos - No guideline value set
3. Barium - No guideline value set
4. Beryllium - No guideline value set
5. Cadmium mg/L 0.005
6. Chromium mg/L 0.05
7. Cyanide mg/L 0.1
8. Fluoride* mg/L 1.5
9. Hardness - No health related guideline value set
10. Lead mg/L 0.05
11. Mercury mg/L 0.001
12. Nickel - No guideline value set
1 3. N itrate-N mg/L 10
14. Nitrite - No guideline value set
1 5. Selenium mg/L 0.01
16. Silver - No guideline value set
17. Sodium - No guideline value set

* Remarks: Natural or deliberately added; local or climatic conditions may

necessitate adaptation.
Water Quality Standards 19

Table 2.2c:
WHO guidelines for drinking water quality: Radioactive constituents

Constituents Unit Guideline Remarks


Gross alpha activity Bq/L 0.1 a) If the levels are exceeded

more detailed radionuclid
analysis may be necessary.

Gross beta activity Bq/L 1 b) Higher level dos not

necessary imply that the
water is unsuitable for
human consumption.

Table 2.2d:
WHO guidelines for drinking water quality: Aesthetic quality.

Constituents or Unit Guideline Remarks

characteristics values

1. Aluminum mg/L 0.2 -

2. Chloride mg/L 250 -
3. Chlorobenzenes and - No guideline These compounds may
chlorophenols value set affect taste and odour
4. Colour TCU -
15 -
5. Copper mg/L 1 -
6. Detergents - No guideline There should not be any
value set foaming or taste and odor
7. Hardness mg/L 500 -
8. Hydrogen sulfide - Not detectable -
by consumers
9. Iron mg/L 0.3 -
10. Manganese mg/L 0.1 -
11. Dissolved oxygen - No guideline -
value set
12. pH 6.5-8.5 -
13. Sodium mg/L 200 -
14. TDS mg/L 1000 -
15. Sulfate mg/L 400 -
1 6. Taste and odor - Inoffensive to -
most consumers
17. Temperature - No guideline -
value set
1 8. Turbidity NTU 5 Preferably' <: 1 for
disinfection efficiency
19. Zinc mg/L 5 -
Handbook of Methods in Environmental Studies


In United States, there are two types of standards, namely:
Primary drinking water standards and
Secondary drinking water standards.
The primary regulations specify maximum contaminant levels (MCLs), and health
advisory. The MCLs are the maximum permissible level at the tap, are health related
and are legally enforceable. If these concentrations are exceeded or if required
monitoring is not performing the public must be notified.
The secondary drinking water regulations specify the se.condary maximum
contaminats levels (SMCLs). The SMCLs are for contaminants in drinking water
that primarily affect ~sthetic qualities related to public acceptance of drinking
water; they are intended to be guidelines for the States and are not federally en-
forceable. These are outlined in Table 2.3.

Table 2.3:
Primary and secondary drinking water standards in United States.

National Primary Drinking Secondary Drinking

Water Standards Water Standards

Constituents MCl, mg/l Constituents SMCl, (mg/l)

1. Arsenic 0.05 1. Chloride 250
2. Barium 1 2. Colour 1 5 colour unit
3. Cadmium 0.01 3. Copper 1
4. Chromium 0.05 4. Corrosivity Noncorrosive
5. Fluoride 4 5. Fluoride 2
6. Lead 0.05 6. MBAS 0.5
7. Mercury 0.002 7. Iron 0.3
8. Nitrate 10 8. Manganese 0.05
9. Selenium 0.01 9. Odor, TON 3
10.Silver 0.05 10. pH 6.5-8.5
11. Sulfate 250
12.TDS 500
13. Zinc 5.0
14. Sodium 20

Coliforms 1/100 ml

Physical Charl!cteristics
Turbidity, NTU 1-5

2.4 Coliform Standards

As per WHO standards (1973):
In 90% of samples examined throughout the year, coliform bacteria should be
absent or MPN should be < 1/100 mL. No sample should have a coliform MPN
greater than 10/100 mL.
Water Quality Standards 21

• As per Ministry of Works and Housing (1975):

1. Colifonn count in any sample of 100 mL should be zero.
2. Water in the distribution system shall satisfy all the three criteria indicated
a. Colifonn count in 100 mL of any sample should be zero.
b. Colifonn organisms not more than 10 per 100 mL shall be present in any
c. Colifonn organisms should not be detectable in 100 mL of any two consecu-
tive samples or more than 50% of the samples collected.
• As per IS: 10500 (1983):
Water in distribution system
a. Throughout any year, 95% of samples should not contain any coliform
organisms in 100 mL.
b. No sample should contain E. coli in 100 mL.
c. No sample should contain more than 10 colifonns per 100 mL.
d. Colifonn organisms should not be detectable in 100 mL of any two consecu-
tive samples.
Un piped Water supplies
Disinfection is most desirable and considerable; reliance has to be placed on
sanitary inspection and not exclusively on the results on the bacteriological
• As per ICMR standards for drinking water (1963):
Treated water: Throughout any month in 90% of the samples examined, colifonn
bacteria should be absent in 1OOmL of sample. No sample should contain E. coli
in 100 mL. No sample should contain more than 10 colifonn organisms per 100
mL. An MPN of colifonn bacteria of 10 per 100 mL should not occur in consecu-
tive samples.
• Types of Treatment requirements for coliform removal:
Group Limiting monthly average Treatment requirement
coliform density (MPNIIOO mL)

I. Nil No treatment is required

11. Less than 50 Simple chlorination.
Ill. Less than 5000 (20% of samples Complete treatment with rapid
should not exceed 5000) sand filtration and post-chlori-
N. More than 20% of samples may Auxiliary treatments in addition
, exceed 5000 but not more than to complete treatment.
5% of these samples should
exceed 20,000
22 Randbook of Methods in Environmental Studies

2.5 Effluent Standards

The effluent standards pertain to the quality of wastewater originating from com-
munity, agricultural operation and industry. In general, these standards restrict the
quality of pollutants in the effluents that set the desire degree of treatment. The
important discharge standards are given in Table 2.4.
• Tolerance limits of industrial effluent discharge IS:2490 (Part-I), 1981.(includes
35 parameters)
• General standards for discharge of environmental pollutants: Part - A, Effluent
(Schedule-VI, issued in GSR, 422 (E), dated 19th May, 1993, MOEF (w.e.f
19.05.1993). (includes40pararneters)

Table 2.4:
General standards for discharge of environmental pollutants. Part-A, effluent,
(Schedule-VI, 1993). MOEF, GSR 422(E), 19.05.93. (The standards shall
also apply to effluent discharge such as mining, mineral
processing activities and sewage.)

SI. Parameter Inland surface Public sewers Land for Marine

no. water irrigation coastal

1 . * Colour and odour Annex.-1 - Annex.-1 Annex.-1

2. Suspended solids, 100 600 200 *a.
mg/L, Max *b.
3. Particle size of Shall pass - - Floatable
suspended solids. 850 micron solids, max
IS Sieve 3 mm
max 850
4. * Dissolved solids, 2100 2100 2100 -
mg/L. Max.
5. pH value 5.5 to 9.0 5.5-9.0 5.5-9.0 5.5-9.0
6. Temperature, °C, Shall not - - Shall not
Max exceed 5°C exceed
above the 5°C
receiving above the
water temp. receiving
7. Oil & Grease, 10 20 10 20
mg/L, Max
8. Total residual 1.0 - - 1.0
C1 2, mg/L, Max
9. Ammonical N, 50 50 - 50
mg/L, Max.
10. TKN (as N), 100 - - 100
mg/L, Max.
11. Free ammonia 5 5
(as NH3 , mg/L, Max.
78ble Cont...
Water Quality Standards 23

... Table Cant.

12. BOD(5 days.20°C). 30 350 100 100

mg/L. Max.
13. COD. mg/L. Max. 250 250
14. Arsenic (as Asl. 0.2 0.2 0.2 0.2
mg/L. Max.
15. Mercury (as Hgl. 0.01 0.01 0.01
mg/L. Max
16. Lead (as Pbl. 0.1 0.1 2.0
mg/L. Max.
17. Cadmium (as Cdl 2.0 1.0 2.0
mg/L. Max.
1 8. Hexavalent Chromium 0.1 2.0 1.0
(as Cr8 +1. mg/L. Max
1 9. Total Chromium 2.0 2.0 2.0
(as Cr). mg/L. Max
20. Copper (as CuI. 3.0 3.0 3.0
mg/L. Max
21 . Zinc (as Znl. 5.0 15 15
mg/L. Max
22. Selenium (as Sel. 0.05 0.05 0.05
mg/L. Max.
23. Nickel (as Nil. 3.0 3.0 5.0
mg/L. Max
24. ·Boron (as BI. 2 2 2
mg/L. Max
25. ·Percent Na. Max 60
26. ·Residual sodium 5
carbonate. mg/L. Max
27. Cyanida (as CNI. 0.2 2.0 0.2 0.2
mg/L. Max
28. ·Chloride (as CII. 1000 1000 600
mg/L. Max
29. Fluoride (as Fl. 2.0 15 15
mg/L. Max
30. Dissolved phosphate 5.0
(as PI. mg/L. Max
31. ·Sulphate (as S041. 1000 1000 1000
mg/L. Max
32. Sulphide (as SI. 2.0 5.0
mg/L. Max
33. Phenolic compounds 1.0 5.0 5.0
(as C8 H&OHI.
mg/L. Max
34. Radioactive materials
a.Alpha emitters. 10- 7 10-7 10.8 10-7
~Curie/mL. Max
b.Beta emitters. 10-6 10-8 10-7 10-6
~Curie/ml. Max
35. Bioassay test 90% survival 90% survival 90% 90%
of fish after of fish after survival of survival of
96h in 100% 96h in 100% fish after fish after
effluent effluent 96h in 96h in
100% 100%
effluent effluent
Table Cant ...
24 Handbook of Methods in Environmental Studies

... Table Cont.

36. Manganese 2 2 2 2
(as Mn), mg/L, Max.
37. Iron (as Fe). 3 3 3
mg/L, Max.
38. Vanadium (as V), 0.2 0.2 0.2
mg/L, Max
39. Nitrate nitrogen 10 20
mg/L, Max
40. Pesticides (l!g/L), Max.
a. Benzene hexachloride 10 10 10
b. Carboryl 10 10 10
C. DDT 10 10 10
d. Endosulfan 10 10 10
e. Diamethonate 450 450 450
f. Penitrothion 10 10 10
g. Malathion 10 10 10
h. Phorate 10 10 10
i. Methyl parathion 10 10 10
j. Phenthoate 10 10 10
k. Pyrethrums 10 10 10
I. Copper oxychloride 9600 9600 9600
m. Copper sulfate 50 50 50
n. Ziram 1000 1000 1000
O. Sulphur 30 30 30
p. Paraquat 2300 2300 2300
q. Proponil 7300 7300 7300
r. Nitrogen 780 780 780

* a. For process wastewater -100 mg/L

*b. For cooling water effluent 10% of above total suspended matter of ·influent
* All efforts should be made to remove colour and unpleasant odour as far as practi-
cable (Annexure-l)
*4.dissolved soilids, *22 (Boron), *23 (% Na), *24 (Residual sodium carbonate),
*26 (chloride) and *29 (Sulphate) - Omitted by GSR, 80(E), dated 31st December,
1993 (w.e.f 31.12.1993). % Na = (Na)/(Ca + Mg + K + Na) x 100

2.6 Stream Standards

Water resources can be classified or zoned depending upon the designated best
use of water. The CPCB (1979) has adopted a scheme of classification of zoning of
various water bodies (Table 2.5 and Table 2.6).
Table 2.5:
Classification of water bodies.
Designated Class Remarks (parameters)
best use
1 A Drinking water source with conventional treatment
but after disinfection (35 characteristics)
2 B Outdoor bathing (1 3 characteristics)
3 C Drinking water source with conventional treatment
followed by disinfection (25characteristics)
4 D Fish culture and wildlife propagation
(8 characteristics)
5 E Irrigation, industrial cooling or controlled waste
disposal (10 characteristics)
Water Quality Standards 25

Table 2.6:
Quality of water for different uses.

Characteristics A B C D E

1. pH 6.5-8.5 6.5-8.5 6.5-8.5 6.5-8.5 6.0-8.0

2. DO,mg/L,Min 6 5 4 4 -
3. BOD,mg/L,Max. 2 3 3 - -
4. Total Coliform, 50 500 5000 - -
MPN/l00 mL,Max
5. Colour, hazen units,Max. 10 300 300 - -
6. Odour Unobjec - - - -
7. Taste Tasteless - - - -
8. TDS, mg/L, Max. 500 - 1500 - 2100
9. Total hardness 300 - - - -
(as CaC0 3 ),mg/L
10. Ca Hardness 200 - - - -
(as CaC0 3).mg/L
11. Mg hardness 100 - - - -
(as CaC0 3 ).mg/L
12. Copper (as CuI, 1.5 - 1.5 - -
13. Iron (as Fe).mg/L, Max. 0.3 - 50 - -
14. Manganese (an Mn). 0.5 - - - -
mg/L, Max.
15. Chlorides (as CIl, 250 - 600 - 600
mg/L, Max.
1 6. Sulphates (as SO.). 400 - 400 - 1000
17. Nitrates (as N03 ', 20 - 50 - -
18. Fluorides (as F), 1.5 1.5 1.5 - -
mg/L, Max.
19. Phenolic compounds 0.002 0.005 - - -
(as CeHsOH).mg/L,Max.
20. Mercury (as Hg). 0.001 - - - -
mg/L, Max.
21. Cadmium (as Cd), 0.01 - - - -
mg/L, Max.
22. Selenium (as Se), 0.01 - - - -
mg/L, Max.
23. Arsenic (as As), 0.05 0.2 0.2 - -
mg/L, Max.
24. Cyanide (as CN), 0.05 0.05 - - -
mg/L, Max.
25. Lead (as Pb),mg/L,Max. 0.1 - 0.1 - -
26. Zinc (as Zn).mg/L,Max. 15 - 15 - -
27. Chromium (as Cr), 0.05 0.05 - - -
mg/L, Max.
28. Anionic detergents 0.2 1.0 - - -
as MABS, (mg/L,Max.)
29. PAH, mg/L, Max. 0.2 - - - -
30. Mineral oil, mg/L, Max. 0.2 - - - -
31. Barium (as Ba). 0.01 - - - -
mg/L, Max.
... Table Cont .
26 Handbook of Methods in Environmental Studies

... Table Cont.

32. Silver (as Ag),
mg/L, Max.
33. Pesticides 0.05
34. Alpha emitrters, Absent
mC/mL, Max.
35. Beta emitrters, 10.9
mC/mL, Max.
Sodium absorption 26
ratio, Max.
Boron (as B), mgll, Max. 2
Free ammonia (as N), 1.2
Conductivity, pmhos/cm, 1.0 2.25

A, B, C, 0 and E as per Table 2.5

2.7 Wastewater Analysis for Proposed Project

For a proposed project, one should analyze the following characteristics of efflu-
ents (Table 2.7) (As per Handbook ofEnvironmental Procedures and Guidelines,

Table 2.7:
Water quality characteristics to be analyzed for a proposed project.

1. Temperature
2. Colour (Hazen Units)
3. Odour (TON)
4. Turbidity
5. Total suspeneded solids (TSS)' mg/L
6. Total Dissolved solids (TDS), mg/L
7. pH
8. Total alkalinity (as CaC0 3 ), mg/L
9. P value
10. M value
11. Total hardness as CaC0 3 mg/L
1 2. Chloride as CI, mg/L
1 3. Sulfate as SO 4' mg/L
14. Fluorides as F, mg/L
15. Percent sodium (% Na)
16 . Phosphate as PO 4' mg/L
17. Nitrate as N0 3 , mg/L
1 8. Total Kjeldahl nitrogen (TKN), mg/L
19. Total ammonical nitrogen (as N), mg/L
20. BOD, 20°C, 5 days, mg/L
21 . COD, mg/L
22. Disolved oxygen demand (DO), mg/L
23. Total residual chlorine as C1 2 , mg/L
24. Oil and Grease, mg/L
25. Chromium (Cr 6 +), mg/L
Water Quality Standards 27

2.8 Primary Water Quality Criteria for Bathing

(Serial No. 93. as per MOEF notification, 25th September, 2000)
In a water body or its part, water is subjected to several types of uses. Depending
on the types of uses and activities, water quality criteria have been specified to
determine its suitability for a particular purpose. Among the various types of uses
there is one use that demands highest level of water quality or purity and that is
termed as "Designated Best Use" in that stretch of water body. Based on this, water
quality requirements have been specified for different uses in terms of primary
water quality criteria. The primary water quality criteria for bathing water are speci-
fied along with the rationale in Table 2.8.

Table 2.8:
Primary water quality criteria for bathing water.(Water used for organised
outdoor bathing)

Criteria Rationale

1. Fecal Coliform 500 (desirable) To ensure low sewage contamination.

MPN/100 mL 2500 (Maximum Fecal coliform and fecal streptococci
permissible) are considered as they reflect the
bacterial pathogenicity.
2. Fecal Streptococci 100 (desirable) The desirable and permissible limits
MPN/100 mL 500 (Maximum are suggested to allow for fluctuation
Permissible) in environmental conditions such as
seasonal change, changes in flow
conditions etc.
2. pH 6.5 - 8.5 The range provides protection to the
skin and delicate organs like eyes,
nose, ears etc. which are directly
exposed during outdoor bathing.
3. Dissolved Oxygen 5 mg/L or more The minimum dissolved oxygen
concentration of 5 mg/L ensures
reasonable freedom from oxygen
consuming organic pollution
immediately upstream which is
necessary for preventing production of
anaerobic gases (obnoxious gases)
from sediment.
4. Biochemical Oxygen 3 mg/L or less The Biochemical Oxygen Demand of
demand 3 day, 27°C 3 mg/L or less of the water ensures
reasonable freedom from oxygen
demanding pollutants and prevent
production of obnoxious gases".

2.9 Effluent Standards for Coal Mines

Under Clause 3, (Standards for coal mines notification, MOEF, 25111 September, 2000),
the standards for effluent discharge into sewer, stream or land, are given in
Table 2.9.
28 Handbook of Methods in Environmental Studies

Table 2.9:
Standards for effluents from coalmines to be discharged into sewer,
stream or land (MOEF, 2000).

Parameter Standard

pH 5.5 to 9.0
Chemical Oxygen Demand (COD) 250 mg/L
Total Suspended Solids (TSS) 100 mg/L
200 mg/L (Land for irrigation)
Oil & Grease (0 & G) 10 mg/L

Monitoring frequency oj these parameters shall be once in a jortnight.

All other parameters indicated in the general standards for discharge of environ-
mental pollutants under Schedule VI, shall be in addition to the effluent standards
specified under clause 3. Monitoring frequency shall be once in a year for the
optional parameters.

2.10 Standards for Effluents from Textile

(Serial No. 92 as per, MOEF notification, 25th September, 2000)
Standards for the discharge of effluents for textile industry are given in Table 2.10.

Table 2.10:
Standards for the discharge of effluents for textile industry (MOEF, 2000).

Parameter Concentration not to exceed,

mg/L, except pH

pH 5.5-9.0
Total suspended solids 100
Bio-chemical oxygen demand (BOD) 30
Chemical oxygen demand (COD) 250
Total residual chlorine 1
Oil and grease 10
Total chromium as Cr 2
Sulphide as S 2
Phenolic compounds as CsH50H 1

1. Where the treated effluent is discharged into municipal sewer leading to terminal
treatment plant, the BOD may be relaxed to 100 mg/L and COD to 400 mg/L.

2. The quantity of effluent (litre per kilogram of product) shall not exceed 100, 250
and 80 in composite cotton textile industry, composite woollen textile industry
and textile processing industry, respectively.
Water Quality Standards 29.

2.11 Questions
1. Defme the standards ?
2. In WHO {I 984) guidelines drinking water standards are divided into 5 catego-
ries ? Name those categories.
3. What is MCL and SMCL ?
4. List the different drinking water standards available in India.
5. Name the different effluent standards proposed in India.
6. How effluent standards are different from stream standards.
7. How may classes of water has been listed under stream based standard?
8. Name the organisms considered under biological quality of drinking water.
9. How does percent sodium is calculated?
Physical and Chemical
Analysis of Water
and Effluents

3.1 Acidity
Standards of Acidity
Drinking water
IS: 10500 (1991) = No Standards.
Effluents discharge
MOEF (1993)=No Standards.

Acidity of water is its quantitative capacity to react with a strong base to a
designated pH. All waters having a pH lower than 8.5 contain acidity.
There are two types acidity:
Methyl orange acidity also known as mineral acidity (PH < 4.0). (Bromocresol
blue is now recommended as it has a sharper colour change at pH 3.7).
Phenolphthalein acidity or CO 2 acidity, which is due to dissolution of CO 2 in
water and algal photosynthesis.
Both CO 2 and mineral acidity can be measured by means of standard solution of
alkaline reagents. Mineral acids are measured by titration to a pH about 3.7 i.e.
methyl orange acidity. Titration ofa sample to the phenolphthalein end point of pH
8.3 , measured both mineral acidity plus due to week acids, also known as phenol-
phthalein acidity. Two types of acidity are shown in Fig. 3.1.


Phenolphthalein end point

Range of CO 2 acid ity

: } - Methyl orange end pomt

~ Practical range of mineral aCidity

Fig. 3 . 1: Types of acidity.

Analysis of Water and Effluents 31

Measurement of Acidity

The concentration of mineral acids present and contributing the mineral acidity
can be calculated by titrating or neutralising samples to pH 4.3 . The CO 2 and
bicarbonates (carbonic acid) present in the sample can be neutralised completely
by continuing the titration to pH 8.3. Since CaCO] has an equivalent weight of 50,
N/50 NaOH is used as titrating agent so that I mL N/50 NaOH = I mg of acidity.

I . Standard NaOH titrant 4. Potassium biphtalate
2. Phenolpthalin indicatorr 5. Ethyl alcohol
3. Methyl orange indicator 6. Methyl alcohol


1. Carbon dioxide free water: Prepare all stock and standard solutions and
dilution water for the standardisation procedure with distilled or deionised
water that has been freshly boiled for 15 minutes and cooled to room
temperature. The final pH of the water should be > = 6.0 and its conductivity
should be < 2 Il Siemens/cm.
2. Potassium biphathalate solution (approx. O.05N): Crush 15 to 20 g primary
standard potassium biphathalate (KHC sHP4) to about 100 mesh and dry at
120°C for 2 h. Cool in desiccator. Weigh 10.0 ± 0.5 g (to the nearest mg), transfer
to a 1000 mL volumetric flask and make up the volume with CO 2 free water.
3. Standard sodium hydroxide (O.02N) titrant: Dissolve 0.8 g NaOH and dilute to
1000 mL using CO 2 free distilled water. Store in air-tight, mbber stoppered
Pyrex/corning glass bottles to protect from atmospheric CO 2, Standardise
against 0.05 N potassium bipathalate.

NormalityofNaOH = -- - -
204.2 x C

A = g KHC sHP4 weighted into I-L flask.
B = mL ofKHC sHP4solution taken for titration, and
C = mL ofNaOH solution used.

4. Phenolphthalein indicator: Dissolve 0.5 g of phenolphthalein indicator in 500

mL 95% ethyl alcohol. Add 500 mL distilled water. Add drop wise 0.02 N NaOH
till pink colour appears.
5. Methyl orange indicator: Dissolve 0.5 g of methyl orange indicator and dilute
to 1000 mL with CO 2 free distilled water.
32 Handbook of Methods in Environmental Studies

PROCEDURE (Fig. 3.2)

1. Measure suitable volume of sample (50 or 100 mL) in a 250 mL conical flask or
beaker depending upon the method to be followed.
2. Add 2 drops of methyl orange and see the colour. If colour turns yellow, methyl
orange acidity is absent. If the colour turns pink titrate with standard 0.02N
NaOH till colour changes to yellow, characteristic of pH 4.4 - 4.3 . Note down
the volume ofNaOH required (A).
3. Add 2-3 drops phenolphthalein indicator and continue titration with NaOH till
faint pink colour appears indicatiing pH 8.3. Note down the additional volume
ofNaOH required (8).


1. As each mL of 0.02 N NaOH = 1 mg CaC03 , therefore

mL 0.02 N NaOH required x 1000

Acidity as mgIL CaC03
2. In case, ifnormality ofNaOH is other than 0.02 N calculate as follows: Acidity
mineral (A )or due to CO 2 (8)
A or 8 x N x 50 x 1000
as mgIL CaC03 = - - - - - - - -
A = mL NaOH required for sample to raise pH up to 4.4 -4.3 by methyl orange.
B = mL NaOH required for sample to raise pH from 4.4 to 8.3 by phenolphthalein.
N = Normality ofNaOH used .
3. For measuring total acidity use A + B instead of only A or B in the calculation.

O'()2 N
Scheme of acidity determination

100 OIL !lampl'
+2 drop.
. H.thyl' O.anq,

Fig. 3.2: Estimation of acidity.

Analysis a/Water and E.ffluents 33

Check Your Confidence on Acidity

1. "Water having pH <8.5 contain acidity" . Why?
2. Methyl orange acidity (pH <4) also known as ............... ..... .. .acidity.
3. Phenolphthalein acidity (pH < 8.5) is due to (a) ·····and (b) •••••••.
4. How to stored NaOH titrant in the laboratory?
5. A 100·mL sample of water has an initial pH of8.4. 20 mL ofO.02N NaOH is
required to titrate the sample to pH 4.3 . What is the total acidity in mglL as

3.2 Alkalinity

Drinking water
IS: 105000 (1991) = 200 mgIL (maximum), beyond this limit taste becomes
unpleasant. 600 mgIL (permissible in the absence of alternate source).
Effluent discharge
MOEF (1993)= No Standard

The alkalinity of a water is a measure of its capacity to neutralise aCIds. Alkalinity is
a measure of the water ability to absorbed W without significant pH change. That
is, alkalinity is a measure of buffering capacity of water. It is the sum of all the
titrable bases.
The major portion of alkalinity in natural waters is caused by hydroxide, carbon·
ate, and bi·carbonate. For practical purposes, alkalinity due to other materials in
natural water is insignificant and may be ignored. Where bicarbonate concentra·
tion is high Ca and Mg are also high.
In natural water, most of the alkalinity is caused due to CO 2• The free CO2
dissolves in water to form carbonic acid (H2COJ), which fuI1her dissociates into H+
and HCOJ·. The HCO J· thus formed further dissociates into H+ and COJ ~ .

CO 2 + Hp ~ H 2CO J (dissolved CO 2 and carbonic acid)

H2CO J ~ W+ HCO; (bicarbonate)
HCO J ~ W + CO;- (carbonate)

Thus, alkalinity meqlL = [HCO;] + [CO;-] + [OH-] - [W]

Where the quantities in parenthesis are concentration in meqlL or mgIL as

CacoJ •
34 Handbook of Methods in Environmental Studies

Environmental Significance
Alkalinity in itself is not harmful to human beings, still the water supplies with less
than 100 mglL are desirable for domestic use. In large quantities, alkalinity imparts
bitter taste of water. The other applications of alkalinity data are:
1. Chemical coagulation process: alkalinity must be present in excess to neutralise
the acid released by the coagulant.
2. Water softening process.
3. Corrosion control: For calculation of Langelier saturation index.
4. To know the buffer capacity of water.
5. pH changes due to aeration of water: It is common practice to aerate water for
the removal of dissolved CO 2, As CO 2 is acidic gas, its removal tends to decreases
pH (alkaline). Thus high alkaline water tend to have high pH after aeration and
low alkaline water tend to have low pH after aeration .
6. Alkalinity of boiler water is of interest to the environmental engineers.

Estimation of Alkalinity
Alkalinity of a sample can be estimated by titrating with standard sulphuric acid.
Titration to pH 8.3 or de-colourization of phenolphthalein indicator will indicate
complete neutralisation ofOH and Y2 of COl while to pH 4.5 or sharp change from
yellow to pink of methyl orange indicator, that will indicate total alkalinity (complete
neutralisation of OH, COl' HCOJ The alkalinity titration curve is shown in Fig.
3.3. Wherever possible, the titration should be carried out on filtered water at the
point of sampling.


__________ L ____________ _
" :. t co;-. Heoi
OH- + C0 2 - + HCo;
o~-------- __________ ~ ___________
Milliliter. of titront

Fig. 3.3 : Alkalinity titration curve. (note: the point of infection .

One is at pH 88.5 and another infection is at pH 4.5)

Colour, turbidity, iron, aluminium and residual chlorine are prime sources of
interference. Colour and turbidity can be avoided using potentiometric titration .
Residual chlorine can be removed by adding ofa small amount (usually one drop)
of 0.1 mollL sodium thiosulfate solution.
Analysis of Water and Ejjluents 35


I. Sulphuric acid, H 2 S0 4 (sp. gr. 1.84)
2. Phenoplthalein indicator
3. Sodium carbonate, Na2CO]
4. Sodium hydroxide, NaOH
5. Methyl orange indicator
6. Phenolpthalin indicator

l. COz-free distilled water must be used for preparation of all stock and standard
solution. If the distilled water has a pH lower than 6.0, it should be freshly boiled
for 15 min and cooled to room temperature. Deionised water may be substituted
for distilled water provided it has a conductance ofless than 0.2 mS/m and a pH
greater than 6.0.
2. Sodium carbonate solution (0.05 N): Dry 5 to 7 g primary standard Na2 CO] at
250°C for 4 h and cool in a desiccator. Weigh 5.300g, transfer to a 1000-mL
volumetric flask, and fill to the mark with distilled water. Do not keep longer than
I week.
3. Standard H zS04 solution (0.02N): Prepare O.IN H 2S04 by diluting 3.0 mL conc.
H 2S0 4 (sp. gr. = 1.84) to 1000mL. Standardise it against standard Na2C03 (0.05
N). Dilute appropriate volume of H2S0 4 to 1000 mL to obtain standard 0.02 N
4. Phenolphthalein indicator (pH 8.3 indicator): Dissolve 0.5 g in 500 mL 95%
ethyl alcohol. Add 500 mL distilled water. Add drop wise 0.02 N NaOH till pink
colour appears.
5. Methyl orange indicator: Dissolve 0.5 g of methyl orange in 100mL of water
and dilute to 1ObOmL with CO 2 free distilled water.


I. After collection of sample, analyse as soon as possible.

2. Take 100mL sample in a 250 mL conical flask and add 2 to 3 drops of
phenolphthalein indicator. If no colour is produced, the phenolphthalein
alkalinity is zero.
3. Ifpink colour develops, titrate with 0.02 N H2S04 till it disappears or pH is 8.3.
Note the volume of H 2S0 4 required (A).
4. Next, add 2 to 3 drops of methyl orange to the same flask, and continue titration
till pH is down to 4.5 or orange colour changes to pink. Note the volume of
H2S04 added (8).
5. In case pink colour does not appear in step 3 after additionofphenolpthalein,
continue as in (4) above.
6. Calculate Total (T), phenopthalein (P) and methyl orange (MO) alkalinity as
follows and express in mg/L as CaC0J"
36 Handbook of Methods in Environmental Studies

il 0.02 N
H.SO. Scheme of alkalinity determination

100 OIL •• pI.

+2-) drops
[} PMnolpitloltin

Fig. 3.4: Measurement of Alkalinity

• After addition of phenopthalein indicator no colour appears phenopthalein

alkalinity = O.
• If, pink colour develops ~ pink to colurless (end point)~ A
• Next, add 2-3 drops of methyl orange ~ titrate ~ orange to pink (end point) ~
• Phenolphthalein alkalinity (mgIL as CaC03) = (A x 1000) + mL sample
• Methyl orange alkalinity (mgIL as CaC03) = (B x 1000) + mL sample
• Total alkalinity (mgIL as CaC03) = [(A + B) x 1000] + mL sample
In case H2S04 is not 0.02 N, apply the following formula:

A x N x 50 x 1000
Alkalinity (mgIL as CaC03) = - - - - - -
mL of sample

Where, N = Normality of H2 S04 used.

Once the phenolphthalein and total alkalinities are determined, then three types
of alkalinity can be calculated from the Table 3.1.

Check Your Confidence on Alkalinity

1. "Alkalinity is the measure of buffering capacity of water". Explain?
2. What are the most common constituents of alkalinity, and what are the sources
and impacts ofalkalinity?
Analysis of Water and EfJluents 37

Table 3.1:
Relationship between hydroxide (OHI. carbonate (CO]I
an~ bicarbonate (HCO]I alkalinities.

Results of OH alkalinity as CO~- alkalinity as HCOi alkalinity as

titration CaCO] CaCO] CaCO]

P =0 0 0 T
P<Yz T 0 2P T-2P
P=Yz T 0 2P 0
P>Yz T 2P-T 2 (T-PI 0
P=T T 0 0
P = phenolphthalein alkalinity; T = Total alkalinity

3. How alkalinity is measured?

4. How to prepare CO2-free distilled water in the laboratory?
5. 1 mL ofO.02N ~S04 = 1 mgIL alkalinity as CaC03 (trudfalse).
6. List five important environmental significance of alkalinity data?
7. A 100-mL sample of water is titrated with 0.02N H2S04. The initial pH is 9.2 and
6.2mL of acid is required to reach the pH of 8.3 end-point. An additional 9.8mL
is required to reach 4.5 end-point. Detennine the alkalinity present?
8. Determine the total alkalinity. A 200-mL sample of water has an initial pH of 10.
30mL ofO.02N H2S04is required to titrate the sample to pH 4.5 . What is the total
alkalinity of the water as mgIL ofCaC03 • (Ans: 150 mg/L as CaCOJ ).

3.3 Biochemical Oxygen Demand (BOD)

Drinking water
No BOD standard has been prescribed by WHO (1984), IS: 10500 (1983,1991),
MHW (1975) orUSEPA (1974) orICMR(l963)
Emuent discharge
30 mgIL, (MOEF, 1993); IS: 2490 (1981) = In inland surface waters; 350 mgIL
(Public Sewers); 160 mgIL (land for irrigation and marine costal areas).
In India, Central Pollution Control Board (CPCB) recommends BOD measure-
ment at 27°C for 3 days.
For bathing water (MOEF, 2000) = 3 mgIL or less

Application of BOD Data

1. To detennine the strength of domestic and industrial waste in tenns of oxygen
required for stabilization of waste.
38 Handbook of Methods in Environmental Studies

2 To measure the amount of biologically oxidisable organic matter present in

3. BOD is a major criterion parameter in stream pollution control.
4. BOD data are used to assess the self purification capacity of receiving water
5. BOD is one of the regulatory standards for effluent discharge.
6. Used for designing of wastewater treatment plants.

Biochemical oxygen demand (BOD) is defined as the amount of oxygen required by
bacteria in decomposing organic material in a sample under "aerobic condition at
20 "C over a period of 5 days.

Microbes .
C6H.P6 + 602 ) CO2 + ~O + new mIcrobes
Since the test is mainly a bioassay procedure, involving measurement of O2
consumed by bacteria while stabilizing organic matter under aerobic condition, it is
necessary to provide standard conditions of nutrient supply, pH, absence of
microbial growth inhibiting substances and temperature.
Because of low solubility of O2 in water, strong wastes are always diluted to
ensure that the demand does not increase the available oxygen present in the
A mixed group of microorganisms should be present in the sample ifnot, sample
has to be seeded artificially. Temperature is controlled at 20"<:. The test is con-
ducted for 5 days as 70 to 80% of the waste is oxidized during this period. In the
waste, there are two major biodegradable compounds, the BOD could be divided
• Decomposition of carbonaceous compounds (CBOD) and
• Decomposition of nitrogenous compounds.
The corresponding oxygen demand are carbonaceous (CBOD) or first stage
BOD and nitrogenous BOD (NBOD) or second stage BOD. In conventional5-day
BOD test, we measure only the first stage BOD or CBOD (Fig. 3.5)
The typical BOD values of different samples of water are listed in Table 3.2.

Table 3.2:
BOD concentrations of different samples.

Type of sample BOD (mg/L) Remarks

Water 1-3 reasonable

River 5-20 tolerable
Sewage 50-100 very bad
Industrial waste 100-10,000 Extremely poor
Analysis of Water and Effluents 39

Ultimate carbonaceous BOD


co Carbonateous oxygen de~

2 J 4 5 8 9 10 11 12 13 14 15
Time, days

Fig. 3.5: Hypothetical BOD curve showing CBOD and NBOD

Sampling and Storage of Samples

Samples for BOD analysis may be degraded significantly during storage (between
collection and analysis), resulting in low BOD values. To minimize reduction of
BOD, analyze sample promptly or store at 4OC. However, even at low temperature,
keep holding time to a minimum. Warm chilled samples to 200C before analysis.
Samples must be free from all added preservative and stored in glass bottle. Follow-
ing points should be noted before proceeding for BOD test.

1. pH: Should be between 6.5 to 8.5

2 Seeding: Some sample may be sterile - add seeds. The purpose of seeding is to
introduce a biological population capable of oxidizing the organic matter in
waste. For domestic sewage, unchlorinated effluents, surface water etc seed-
ing is not needed.
3. Lack of nutrients: Add nutrients.
4. Presence of residual chlorine: Sample like chlorinated effluents or treated wa-
ter, which may contain residual chlorine, remove by sodium sulfite solution.
5. High DO: Sample may contain high DO (i.e. 9 mgIL) due to algal growth or some
other reason. Reduce the DO content by aerating and agitating the samples.
6. Presence of heavy metals: Cu, Cr, Pb partially Inhibit O2 consumption.
1. Incubation BOD bottles, 300 mL capacity: Clean bottles with a detergent
(or chromic acid), rinse thoroughly, and drain before use so that bottles are free
40 Handbook of Methods in Environmental Studies

from organic matter. As a precaution, against drawing air into the dilution bottle
during incubation, use a water seal. Place a paper or plastic cup over flared
mouth of bottle to reduce evaporation of the water seal during incubation.
2. Incubator: Thermostatically controlled at 20"C. Exclude all light to prevent
possibility of photosynthetic production of DO.


1. Potassium dihydrogen phosphate, KHl04

2 Magnesium sulfate, MgSO4' 7Hp
3. Dipotassium hydrogen phosphate, K 2HP04
4. Disodium hydrogen phosphate, Na2HP04.7Hp
5. Calcium chloride, C:!CI2
6. Ferric chloride, FeCI3"6 Hp
7. Ammonium chloride, NH4CI
8. Sodium thiosulfate, Na2Sp3 - forremoval chlorine.
9. HzS04 (conc.)
10. Sodium hydroxide, NaOH


All reagents prepared for DO determination also required for BOD test. In addition,
prepare following stock solution.
1. Phosphate buCCersolution: Dissolve 8.5gKHl04 + 21.75 g~HP04 + 33.40g
Na2HP04.7H20 + 1.7 gNH4CI in distilled water and dilute lL. Adjust pH to 7.2.
Discard reagent (or any of the following reagents) ifthere is any sign ofbiologi-
cal growth in the stock bottle.
2. Magnesium sulCate: Dissolve 22.5 g MgS04.7Hz0 in IL.
3. Calcium chloride: Dissolve 27.5 g anhydrous CaCl2in IL.
4. Ferric chloride: Dissolve 0.25 g FeClr 6Hp and dilute to IL.
5. Acid and alkali solutions, IN: H2S04and NaOH for neutralisation of sample.
For acid solution, add 28 mL of conc. H 2S04 to lL distilled water. For alkali
solution, add 40 g NaOH in lL distilled water.
6. Sodium thiosulCatesolution (0. IN): Dissolve 25 gNa2Sp3.5Hp and dilute to
lL. This solution is not stable; prepare daily (needed for estimation of residual
chlorine, and dechlorination of sample).
The BOD is determined either by direct method or dilution method depending upon
the expected BOD.

Direct method (for sample with low BOD) (Fig. 3.6)

1. For clean river water, drinking water etc. where BOD is generally less than
7 mglL; dilution is not necessary.
2 To run the test, aerate the sample to bring the DO level near the saturation at the
start of the test.
Analysis of Water and EjJluents 41

'----...,VlFlnal DO
20·C .S da"

300 ml BOO bottle

with tapered $topper
and flared mouth for
water WIll
IHea$Ure mital 00 I
Fig. 3.6: BOD-test (Direct method) (300 mL sample is taken in BOD bottle and DO in
the beginning and after incubation for 5 days at 20°C is measured).

Dilution method (sample having high BOD) (Figs. 3.7 and 3.8)
For polluted water, domestic sewage, industrial effiuents, where BOD values are
generally high, dilution of the sample is needed to provide sufficient dissolved
oxygen. The important pre-requisite steps to be followed are:
a Preparation of dilution water
b. Collection of microbial seedslinoculants
c. Dilution of wastewater sample
d. Determination of DO of the diluted sample and blank. in zero day and after
5 days.

I. Preparation of dilution wat~r

1. Aeration: Aerate the required volume of distilled water in a container by bub-

bling compressed air for 1-2 days to attain DO saturation. Try to maintain the
temperature near 20 OC.
2 pH: Neutral pH (7.2) should be maintained. It is customary to buffer the dilution
water by means of phosphate buffer at about pH 7.2. The buffer is essential to
maintained favorable condition at all times
3. Addition of nutrients: Add -I mL each of phosphate buffer, magnesium sul-
phate, calcium chloride and ferric chloride solution for each litre of dilution
water and mix well. These are added to provide proper osmotic conditions and
essential micronutrients.
4. Seeding: Some samples may not contain a sufficient microbial population,
such as some untreated industrial wastes, disinfected wastes, high temperature
wastes or wastes with extreme pH. In such cases seed the dilution water by
42 Handbook of Methods in Environmental Studies

adding population of microorganisms. In the case of the wastes that are not
expected to have sufficient bacterial populations, add seed to dilution water.
Generally, 2 mL settled sewage or any other given below is considered suffi-
cient for 1L of dilution water.
Seed source
It is necessary to have present populations of microbes capable of oxidising the
biodegradable organic matter present in the sample. Common sources of seeds are:
Domestic wastewater
Unchlorinated or undisinfected effluents from biological wastewater treatment.
Surface water receiving wastewater discharge
Use supernatant from domestic wastewater after settling at room temperature
for at least 1 h but not later than 36 h
When effluent from biological treatment process is used, inhibition ofnitrifica-
tion is recommended.
II. Dilution of sample
1. Neutralize the sample to pH around 7.2 if it is highly alkaline or acidic.
2 The sample should be free from residual chlorine, ifit is present, remove it by
using sodium thiosulphate solution as follows:
Process of the removal offree-residual chlorine from the sample:
a. Take SO mL of the sample and acidify with addition ofl 0 mL I + I acetic acid.
b. Add about I g KI.
c. Titrate with 0.025 N sodium thiosulphate using starch as an indicator.
d. Calculate the volume of sodium thiosulphate required per mL of the sample
and accordingly, add to the sample to be tested for BOD.
S. If the sample is having high DO content. i.e., 9 mgIL DO due to either algal
growth or some other reason, reduce the DO cQntent by aerating and agitating
the samples.
6. Dilution: Unless the approximate BODs of the sample is already known, the
required degree of dilution will not be known. Recommendations of dilution are
given in the Table 3.3. With experience, the analyst will often be able to use the
COD as a guide to the dilution required.
In the absence of prior knowledge, use following dilution: 0.0 to 1% for strong
industrial waste; I to 5% for raw and settled sewage; 5 to 25% for biologically
treated effluents; and 25 to 100% for polluted river waters.
Three types of BOD tests are perform in laboratory:
Direct method
Dilution method (without seeded) i.e when dilution water is not seeded with
bacteria. An outline of the test procedure is given in Fig. 3.7.
Analysis of Water and Effluents 43

Dilution method (with seeded) i.e when dilution water seeded with bacteria. An
outline of the test procedure is given in Fig. 3.8.

Table 3.3:
Recommended dilution for the BOD& test.

Range of BOD values Sample Dilution water Dilution _ Source of

to be determined volume volume (mLI factor Md M sample
(mg/LI (mLI

0-6 Undiluted 0 1 R
4 - 12 500 500 2 R,E
10 - 30 200 800 5 R,E
20 - 60 100 900 10 E,S
40 - 120 50 950 20 S
100 - 300 20 980 50 S,C
200 - 600 10 990 100 S,C
400 - 1200 5 995 200 I,C
1000 - 3000 2 998 500 I
2000 - 6000 1 999 1000 I
R = River water
C = Crude (raw sewage)
E = Biologically purified sewage effluent
S = Settled sewage or weak industrial wastewater
I = Strong industrial wastewater.
1. In preparing dilutions for BOD test, siphon-off or pour careful~ the standard
dilution water into a graduated cylinder of capacity 1000-2000 niL as required.
The sample and dilution water must be mixed thoroughly, but violent agitation
leading to the formation of minute air bubbles must be avoided.
2 Transfer the diluted sample to 4 labelled BOD bottles as demonstrated and
close immediately.
3. Keep one bottle:: for determination of the initial 00 and incubate 3 bottles at
20°C for 5 days. The bottles should have a water seal.
4. Prepare a blank in duplicate by siphoning plain dilution water.

Euenhal Dilution water / ' Test (waste I sample, Vr.

nutrients Air
(Jl()mL-VS /

(20 L)
Air --H:lt-L.o
stone Measure iniAl DO
800 bottle
Dilution water
Fig. 3.7: BOD test- dilution water unseeded IBOD determination in
diluted water sample)
44 Handbook of Methods in Environmental Studies

5. Fix the bottles kept for DO determination and blank by adding 2 mL MnSO.
followed by 2 mL NaOH + KI + NaN3 as described in the estimation of DO.
6. Determine DO in the sample and in the blank on initial (0 day) and after 5 days
(end of test day).

Oilution water SEEDED TnT

( sHdeda $ANPLE

"'UUl"t tiM' DO I
Fig. 3.8: BOD test- dilution water seeded (BOD determination in
diluted water sample)

1. When BOD is determined in an undiluted sample (Direct method):
BODs (mgIL) = DO before incubation (mgIL) - DO after incubation (mgIL)
2 When BOD is determined in diluted water sample
If dilution water is not seeded: BODs (mgIL) =

Ifdilution water is seeded: BODs(mgIL) =

D. DO of the sample bottle on O-day (before incubation).
O2 DO of the sample bottle after 5th day.
B. DO of the blank bottle on O-day.
B2 DO of the blank bottle after 5th day.
f ratio of seed in diluted sample to seed in seed control
Analysis a/Water and EfJIuents 45

(% seed in diluted sample)/(% seed in seed control)

P decimal fraction of the dilution water used.
volume of wastewater
volume of wastewater + dilution water ·


1. For checking, dissolve 150 mg of glucose and 150g glutamic acid (dried at 103"C
for 1h) in distilled water and dilute to 1L. The solution should be freshly pre-
2. Make a 1 in 50 dilution (lmL glucose-glutamic acid solution + 49 mL seeded
dilution water) water and determine the BOD in the usual way. The BOD should
be approximately 220 mg/L. If the result obtained is less than 200 mg/L or more
than 240 mg/L, some error in the seed, dilution water or experimental techniques
should be suspected.

Example-1: (Seeded BOD 5 test)

A 10 mL of sample of sewage mixed with enough water to fill a 300-mL
bottle has an initial DO of 9 mg/L. After 5 days of incubation at 20°C, the final
DO was measured 2 mg/L. The DO in blank, which was 9 mg/L at the start of the
test, was measured to be 8mg/L after 5 days of incubation. Calculate the BODs
at 200C.

Example - 2 (Unseeded BOD 5 test)

For a BOD test, 75 mL of a river water sample is used in the 300 mL of BOD
bottles without seeding with three duplications. The initial DO in three bottles
read 8.86, 8.88 and 8 .83 mg/L respectively. The DO levels after 5 days at
20°C incubation are 5 .49, 5.65 and 5.53 mg/L respectively. Find the 5-days
( for the river water.
46 Handbook of Methods in Environmental Studies

Example-3: (Unseeded BODs test)

A 10-mL sample of sewage was mixed with 290 mL dilution water and filled
in a 300-mL BOD bottle. The initial DO is 5 mg/L. For an accurate test, it is
desirable to have at least a 2 mg/L drop in DO during 5 day run, and the final DO
should be at least 2 mg/L. For what range of BODs would this dilution produce the
desire results.

Check Your Confidence on BODs Test

1. What is CBOD and NBOD?
2 How to reduce high DO before start of BOD test?
3. Discuss BOD estimation for clean river water sample.
4. How to prepare dilution water in the laboratory. Why adjUSbnent of pH, attain-
ment of saturation DO, addition of nutrients, and seeding are given important
5. Why is the BOD bottle stoppered?
6. Why is the test run in the dark (or in a black bottle)?
7. Why is it necessary to dilute the sample?
8. Why is it necessary to seed the sample?
9. Why BODs is not ultimate BOD measured?
10. Why nitrification inhibitor is needed when microbial seeds are collected for
effluent treatment plant?
11. Why sample should be free from residual chlorine for BOD test? How to remove
residual chlorine?
12 A standard 5-day BOD test is run using a mix consisting of 4 parts distilled
water and 1 part wastewater (no seed). The initial 00 ofthe mix is 9 mgIL and the
DO after 5 days is determined to be 2 mgIL. What is BODs?
13. A standard BOD test is run using seeded dilution water. In one bottle, the waste
sample is mixed with seeded dilution water giving a dilution of 1:30. Another
bottle contains just seeded dilution water. Both bottles begin the with DO at the
saturation value of9.2 mgIL. After 5 days, the bottle containing waste has DO
Analysis of Water and Effluents 47

equal to 2 mg/L, while that containing just seeded dilution water has DO equal
to 8 mg/L. Find the BODs of the waste.
14. A mixture consisting of30 mL of waste and 270 mL of seeded dilution water has
an initial DO of8.5 mg/L. After 5 days, it has a final DO of2.4 mg/L. Another
bottle containing just the seeded dilution water has an initial DO
and a fmal DO of8.53 mg/L. Find the BODs of the waste.
15. Determining BODs of an unknown sample: The BODs ofa wastewater is s~s­
peeted to range from 50 to 200 mg/L. Three dilutions are prepared to cover the
range, i.e. 5 mL, 10 mL and 20 mL waste diluted to 300 mL with dilution water. The
initial 00 and final 00 as measured in each bottle are given below. Calculate the
BODs of the wastewater.
Wastewater Dilution . Initial DO after 5 °2consumed P BODs
mL water, mL DO, mg/L daysmg/L mglL mg/L

5 295 92 6.9 23 0.0167 l38*

10 290 9.1 4.4 4.7 0.033 142
~ 280 8.9 1.5 7.4 0.067 llO
* BODs = O2 consumed/dilution factor = 2.3/0.0167 = 138 mg/L.
The third value is to be discarded because the final DO is being less than
2 mg/L. Thus average BODs of the wastewater is 140 mg/L. (Ans).
16. An analysis for BODs is to be run on a sample of wastewater. The BOD is
expected to be in the range of 50 to 350 mg/L and the dilution water is prepared
accordingly. In each case a standard 300-mL BOD is used. The data are
recorded below:
Bottle no. wastewate~mL Initial DO, mg/L DO after 5 days, mg/L
~ 8.9 1.5
2 10 9.1 2.5
3 .5 92 5.8
4 2 92 7.5
a Determine the BODs of the wastewater.
b. If the oxygen utilisation rate (k) is 0.211day at20"C, what will be the BOD) if the
test is run at 27 °C.
17. Seeded BODs test: A 300 mL BOD bottle filled with 15 mL of wastewater and rest
is seeded dilution water (dilution is 1:20) has an initial DO of9 mg/L. After 5
days, DO level drops to 2 mg/L. Another 300-mL BOD bottles containing only
seeded dilution water has initial DO of 9 mg/L and after 5 days, DO is 8 mg/L
(blank test). What would be the BODs of the waste. (Ans. J 14 mg/L).
48 Handbook of Methods in Environmental Studies

3.4 Boron
(Curcumin colorimetric-extraction method: range 0.1 to J mg/L)

Drinking water
IS: 10, 500 (1991) = I mgIL (5 mgIL is pennissible in the absence of alternate
WHO (1993) = 0.3 mgIL (Guide line value).
EC (1980) = I mgIL (Guide level)
Emuent discharge
MOEF, (1993) = 2 mgIL (omitted by GSR, 80 (E), dated 31st December, 1993
(w.e.f. 31.12.93)
Stream standards (CPCB, 1979) = 2 mgIL (E-c1ass water Irrigation purposes).

In most natural waters boron is rarely found in concentrations greater than 1 mgIL,
but even this low concentration can have deleterious effects on agricUlture crops.
Boron (B) is essential to plant growth, but at high concentration it becomes delete-
rious. B concentration in Indian soil ranged from 2 to 8 mgIL.
At high concentration, it effects the central nervous system, while extended
consumption may lead to a condition known as borism. Different methods for
boron estimation are:
I. Colorimetric-curcumin method: O. 10 to I mgIL.
2. Colorimetric-carmine method: 1 to 10 mgIL.

Estimation of Boron
Water sample containing boron is acidified with Hel and evaporated in the pres-
ence ofcurcumin (a dye extracted from tunneric), a red coloured complex rosocyanine
is fonned. The colour product is dissolved in isopropyl alcohol and absorption is
measured at 540 nm in spectrophotometer.
1. Hot-water bath, with temperature control at 55°C.
2 Spectrophotometer.
3. Evaporating dish, 100 to 150 mL capacity.
Nitrate concentration above 20 mgIL begins to interfere. Hardness level about 100
mglL as CaC03 give high results because of the turbidity caused by the insolubility
Analysis of Water and Effluents 49

of the hardness salts in isopropyl alcohol. The turbidity can be eliminated by

filtering the fmal solution through 0.45-1m membrane filter before reading on the
1. Boric acid
2. Oxalic acid
3. Hydrochloric acid, HCI
5. Isopropyl alcohol
6. Sodium hydroxide, NaOH
1. Boron stock solution (ImL = 1 mg B): Dry about 109 of boric acid (H]BO])
powder in a desiccator containing a silica gel for 24h. Dissolve 5. 719g of the dry
boric acid in water and dilute to IL. Store the solution in a plastic bottle.
2. Boron standard solution (ImL = 0.01 mg B): Dilute IOmL of stock boron solu-
tion to IL with water. Store in a plastic bottle.
3. Curcumin solution: Dissolve 40 mg offmely ground curcumin powder and 5 g
of oxalic acid in 80mL of isopropyl alcohol. Add 4 mL of cone. HCI and make up
to I OOmL with isopropyl alcohol.
4. Hydrochloric acid (1+ 19): Add I volume ofHCI (cone.) to 19 volumes of water.
S. Sodium hydroxide solution (20gIL): Dissolve 2 g fNaOH in water and dilute to
Samples should be collected and stored in polythene bottles. Filter the sample
through a 0.45-~m membrane filter as soon as possible after sampling. No preserva-
tion is required.
1. Calibration of standards: Prepare a series of standard boron solution cover-
ing the range from 0 to I mg BIL. Take I, 2, 3, 5 and 10 mL of boron standard
solution (i.e., I mL=O.OI mg B) and diluted to 100 mL, which will give 0.1,0.2,
0.3,0.5 and I mg BIL
2 Sample analysis: Pipette ImL of clear, filter sample (containing 0.1 to I mg BIL)
into an evaporating dish.
3. Run a blank set, containing only distilled water.
4. Add 4 mL of curcumin solution to each sample, standards and blank. Mix the
5. Place the evaporating dish in hot-water bath (55°C) and evaporate to dryness.
After complete drying, allow to cool in room temperature.
6. Add 10 mL isopropyl alcohol in each evaporating dish and stir with a plastic rod
50 Handbook of Methods in Environmental Studies

for complete dissolution of the red-coloured complex.

7. Transfer the content of each evaporating dish into a 25-mL flask. Wash the dish
2-3 times with isopropyl alcohol and make up the mark (up to 25mL).
8. If the solution appears turbid, filter with 0.45-llm membrane filter before taking
reading in spectrophotometer at 540 run. Use reagent blank to set zero absor-

Calculations are not required, as the B concentration can be read directly from the
calibration curve provided that no dilution of the original sample was made. In case
of dilution, mUltiply the reading from the graph by the dilution factor.

Check Your Confidence on Boron Estimation

I. Why presence of boron in drinking water is undesirable?
2. Discuss the principle of boron estimation.
3. List the interfering compounds that effect boron estimation.

3.5 Carbon Dioxide (Free CO 2)

Drinking water
IS: 10500 (1991) = No Standards.
Emuents discharge
MOEF (1993) = No Standards.

Free carbon dioxide in the water accumulates due to microbial activity and respira-
tion of organisms. Surface waters normally contain less than 10 mg free CO2 per litre
while some groundwater may easily exceed that concentration up to 30 to 50 mglL.
This imparts the acidity to the water because of the formation of carbonic acid (C02
+ Hp = H2COJ The CO2 content of a water may contribute significantly to corro-
sion (e.g concentration of CO 2 >20 mgIL is bad). Free CO2 is determined by titrating
the sample using a strong alkali to pH 8.3 .

Estimation of Free CO 2
Free CO2 reacts with sodium carbonate to form sodium bicarbonate, or with so~ium
hydroxide to form sodium carbonate. Completion of reaction is indicated by the
development of the pink colour characteristics of phenolphthalein indicator at the
equivalence pH of8.3.
Analysis o/Water and Effluents 51

CO2 + N~C03 + ~O = 2NaHC03

CO2 + 2 NaOH = N~C03 + ~O


1. Standard sodium hydroxide titrant (O.05N): Dissolve 40g ofNaOH in boiled

CO2 free distilled water and make up the volume to 1 litre. Filter the solution
through a sintered glass filter to remove any Na2CO)" This is the solution of IN
NaOH. Store it in a polythene air tight bottle.
Dilute this solution to 20 times to prepare 0.05N solution at the time of titration.
Standardise the dilute solution with H2S04 ,
2. Phenolphthalein indicator: Dissolve 0.5 g phenolphthalein in 50 mL of95%
ethyl alcohol and add 50 mL distilled water. Add 0.05 N NaOH solution drop-
wise until the solution just turns faintly pink.
PROCEDURE (Fig. 3.9)

After collection, analyse the sample as soon as possible.

1. Take 100 mL of the sample in a conical flask and add few drops of phenolphthalein
2 The colour change to pink indicates the absence offree COl"
3. In case the sample remains colourless, titrate it with 0.05 N NaOH,
4. At the end point pink colour appears.

0.05 N
Standard NaOH

Scheme of Free CO2 determination


( f nO Point I

Fig. 3.9: Estimation of free CO 2 ,

51 Handbook of Methods in Environmental Studies

AxNx44x 1000
Free C02mg/L = --------
Where, A = mL of titrant use
N = normality ofNaOH (0.05)
Precision and bias by the titrimetric method are on the order of 10% of the
known CO2concentration.

Check Your Confidence on F. CO 2 Estimation

1. What are the sources of CO2 in water bodies?
2 How dissolved CO 2imparts acidity?
3. A sample of water collected in the field had a pH of6.8. By the time the water
sample reached the laboratory for analysis, the pH had increased to 7.5. Give a
possible explanation for this change.

3.6 Conductivity or Specific Conductance

1. Drinking water: No standards (IS: 10,500, 1991; ICMR, 1963; MHW, 1975;
WHO, 1984)
2 Effluent discharge: No standards (MOEF, 1993; IS: 2490, 1981).
3. Stream standards, zoning of water bodies (CPCB, 1979):
• Class-D (Fish culture and wild life propagation: 1.0 '.lInhos/cm (maximum).
• Class-E (Irrigation, industrial cooling or controlled waste disposal): 2.25
/lmhoslcm (maximum).

Pure water is a poor conductor of electricity. Acids, bases and salts in water make it
relatively good conductor of electricity and such substances are called electrolytes.
The electrolyte in solution dissociates with positive (cations) and negative (anions)
ions and imparts electrical conductivity. Thus, higher the concentration of electrolyte
in water, the moreis its electrical conductance (i.e'. less the resistance to the flow of
electricity). This gives a rapid method to get an idea about dissolved solids in water.
The important ions that impart conductivity in water are:
i. anions: CI- , S~, CO;, HCO; and NO;
ii. cations: Ca 2+, Mg 2+, Na+ and K+
Conductivity is customarily reported in micromhos per centimeter (/lmhos/cm). In
the International System of units (SI) the reciprocal of ohm is the Siemens (S) and
Analysis of Water and Effluents 53

conductivity is reported as miIlisiemens per meter (mS/m).

I mS/m = 10 J.lS/cm = 10 J.lmhos/cm and I J.lS/cm = I J.lmhos/cm

Freshly distilled water has a conductivity of 0.5 to 2 J.lmhos/cm, and this increases
after a few weeks of storing up to 2 to 4 J.lmhos/cm. This increase is caused by
absorption of atmospheric CO2,

Environmental Significance of Conductivity

1. Used for detection of impurities in water;
2. Used for quantitative measurement of ionic constituents dissolved in water,
which are important for boiler feed water and cooling water etc.;
3. Used for checking correctness of water analysis as there is a distinct relationship
between conductivity and total dissolved solids (TDS).

Estimation of Conductivity

Conductivity is a numerical expression of the ability ofa water sample to carry an

electrical current and varies with the number and types of ions the solution contains.
Most dissolved inorganic substances in water are in the ionised form and hence
contribute to conductance. Conductance G is defined as the reciprocal of resistance

G =- Where, R in ohm and G is ohm-I (sometimes written as mho).
Conductance of a solution is measure between two spatially fixed chemically
inert electrodes. To avoid polarisation at the electrode surface the conductance
measurement is made with an alternating current signal. The conductance (G) ofa
solution is directly proportional to the surface area (A, cm2) and inversely propor-
tional to the distance between the electrode (L,cm). The constant of proportionality
(k) is such that

G=k -
k is called "conductivity" (preferred to "specific conductance"). The unit of k is
1I0hm-cm or mho per centimetre. It can be defmed as the conductance of a conductor
Icm in length and 1 cm 2 in cross-sectional area. The specific conductance depends
on the nature of the conductor (the solution between the electrodes), the ion
concentration, and pressure.
The conductance of a solution measured by conductivity cell depends on cell
parameters, A and L. Although theoretically one could calculate the specific con-
ductance from measurements ofG (or R) and values of A and L, it is easier, and more
accurate to use a solution of known specific conductance, measure G, and calculate
54 Handbook of Methods in Environmental Studies

the ratio AIL. Then specific conductance of a solution is calculated from

Specific conductance, k = G x Kc

Where Kc is equal to LIA and is known as the cell constant. Typical values of
cell constant ranged from 0.10 to 2 cm· l .
Therefore, the cell constant (Kc' cm· l ) is calculated as follows:

conductivity of standard KCl solution, Ilmho/cm

meter conductance

It should be noted that, measured and true conductivity is expressed in same

The temperature of the solution is critical, because electrical conductivity
increases with temperature at a rate of approximately 1.9% per dc.

Equivalent conductance (E)

Although the specific conductance is an important fundamental characteristic of
electrolytic solution, it is not convenient for comparing the relative conductance of
solutions. A property based on a molar amount of electrolyte is the equivalent
conductance, E, the conductance per unit of concentration. As the concentration
is decreased toward zero, E approaches a constant, dec;ignated as EO. With k in
units of micromhos per centimetre, it is necessary to convert concentration to units
of equivalents per cubic centimetre, therefore:

E = 0.001 k/ concentration.

Where the units ofE, k and concentration are mho-cm 2/equivalent, Ilmho/cm,
and equivalentlL, respectively. Some values of equivalent conductance for KCI are
given in Table 3.4.

Table 3.4:
Specific conductance and Equivalent conductance of potassium
chloride solution at 25°C.

KCI concentration, Equivalent conductance, E, Specific conductance,

Molarity or equivalent/L mho-cm 2 /equivalent k,pmho/cm

0 149.9 -
0.0001 148.8 14.9
0.001 146.9 146.9
0.01 141.1 1412
0.1 128.9 12890
1 111.9 111900



Rough estimation of dissolved ionic contents of water sample can be made by

mUltiplying specific conductance (Ilsiemens/cm) by an empirical factor, which may
Analysis a/Water and Effluents 55

vary from 0.19 to 0.55 depending on the soluble components and on the temperature
of measurement.
Conductivity measurement gives rapid and practical estimation of the varia-
tions in the dissolved mineral contents of a water supply. The most commonly
applied relationship is a linear proportionality, as given in the following equation-

Dissolved solids = A x Specific conductance; A = slope of the line

A water quality survey conducted by US Dept of Interior (1997), found that the
value of A varies from 0.60 to 0.74. A higher value of A is associated with high
concentration of sulphate ions. The relationship between dissolved solids, specific
conductance is summarised in Table 3.5.

Relationship between dissolved solids and specific conductance.

Source Dissolved solids Specific conductance Ratio (A)

(DS)(mg/L) (SC) IpS/cm) (DS/SC)

Surface water 107 171 0.64

Ground water 236 392 0.61
Ground water 80 99 0.74
Ground water 173 270 0.65

Conductivity meter.

Standard potassium chloride, (O.OlM): Dissolve 745.60 mganhydrous KCl in double
distilled water or deionised water and make up to 1000 mL at 25°C. This is standard
reference solution, which at 25°C has a specific conductance of 1412 ~mhos/cm. It
is satisfactory for most samples when cell has a constant between 1 and 2 cm·'.

Conductivity measurement is affected by:
nature ofthe various ions, their relative concentration and the ionic strength of
dissolved CO 2; turbidity, and
temperature (the conductivity must be determined at 25°C).

The measurement of conductivity should be made in-situ or in the field immediately
after water sample has been obtained, because conductivity changes with storage
time. Conductivity is also temperature dependant, thus, if the conductivity meter is
not equipped with automatic temperature correction, the temperature of the sample
should be measured and recorded.
56 Handbook of Methods in Environmental Studies

Detennination of cell constant

Conductivity measurement

Determination of cell constant

1. Rinse the conductivity cell at least three portions of standard KCI solutions.
2. Adjust the temperature of a fourth portion to 25°C ± 1°C
3. Immerse the conductivity cell in the standard KCI solution. (Note: the cell should
not be closer than 2 cm to the sides and bottom of the container).
4. Observe and record the temperature of the KCL solution to the nearest O.toC.
Some meters have builtin thennometerl or automatic temperature compensation.
5. Turn the meter on and follow manufacturer's interactions and record the meter
6. Calculate the cell c.:;n.,tant.

Kc = (1412/C K ) x [1 +0.019(t-25)]

CKti Measured conductivity, flmhos/cm.
t = Observed temperature, °C
K c = Cell constant (em-I).

Measurement of sample conductivity

1. Rinse the conductivity cell with at least three portion of the sample.
2. Adjust the temperature of the portion of the sample to 25°C ± 1°C.
3. Immerse the conductivity cell in the solution. There should not be any air
bubble clinging to the electrodes and cell should not be closer than 2 cm to the
sides and bottom of the container. A typical conductivity measurement
procedure is shown in Fig. 3.10.
4. Observe and record the temperature of the KCL solution to the nearest O.I°C.
Some meters have built in thennometerl or automatic temperature compensation.
5. Tum the meter on and follow manufacturer's instructions and record the meter


I'Ilgntl.e Slit >::=::.i-~2...

~."d. to

Fig. 3.10: Conductivity measurement.

Analysis of Water and Ejjluents 57

6. When conductivity of the sample has been measured, the conductivity can be
calculated as follows:

C xK
Conductivity =1 +0.0191 (tt_25)

Where, C m = measured conductivity in units of ~mho/cm at tOe.

Report the reading and the temperature of the sample at the time of reading. Report
the conductivity at 25°e.


When conductivity cell is not in use, the cell must be cleaned with distilled water,
wiped dry and stored in its ca~ing case.

Check Your Confidence on Conductivity

I. What are the important anions and cations that impart electrical conductivity?
2. Distilled water has an electrical conductivity of I ~mhos/cm kept in open bottle.
After 15 days, conductivity was found to be 2 ~mhos/cm. why?
3. How TDS is related to specific conductance?
4. Write at least three importances of conductivity measurement.

3.7 Colour
Standards for Colour
Drinking water
IS: 10500 (1983) = Max. I 0 Hz units;
IS: 10500 (1991) = Max. 5 Hz (above 5 Hz consumer acceptance decreases. (25 Hz
max. permissible limit in the absence of alternate source).
MWH (1975); ICMR (1963) = 5 Hz units is permissible and maximum permissible
is 25 units.
WHO (1984) = Maximum 10Hz units;
WHO (1993)= 15 TCU (time colour unit)
USEPA(1974)= 15 Hz units;
EC (1980) = 1Hz (guide level) and 20 Hz (MAC, maximum admissible concentra-
Note: The standard of 15 Teu is based on the fact that at 15 TeU no colour is detectable
visually. Therefore, consumers' acceptance decreases and colour standards are not based on
health effect (John De Zhane, 1977).
Effluents Discharge
MOEF, 1993 = All efforts should be made to remove colour as far as possible.
58 Handbook of Methods in Environmental Studies

Colour in water may result from the presence of natural metallic ions (iron and
manganese), humus and peat materials, plankton, weeds. For example, Iron oxides
cause reddish water and Manganese oxides cause brown or brackish water. Industrial
wastes from textile and dying operation, pulp and paper production, food processing,
mining and coal processing operations, refinery, slaughter house operation may
add substantial colouration to water in receiving stream.
Colour is removed to make water suitable for general and industrial applica-
tions. Coloured industrial wastewater may require removal of colour discharge into
Colour can be described relative to type and density; the type being related to
whether it is true colour (dissolved) caused by colloidal particles or apparent colour
(filterable), caused by suspended matter. It is express in Hazen units.

Environmental Significance
Coloured water is not aesthetically acceptable to the general public.
True colour caused by organic compounds may exert chlorine demand during
disinfection process and thereby seriously reduce the effectiveness of chlorine
as a disinfectant.
Many industrial wastes are highly coloured, and some contain coloured
substances that are quite resistant to biological destruction.

Estimation of Colour
Waters containing natural colour are yellow-brownish in appearance. It has been
found that potassium chloroplatinate (~PtCI6) tinted with small amounts of cobalt
chloride (CoCI) yield colours that are very much like the natural colours of water.
ImgIL ofPt (KltCI6) gives standard 1 unit of colour. Colour intensity increases
with increase in pH. For this reason recording pH along with colour is advisable.

1mgIL Pt (~PtCI6) = 1 true colour unit (TCU) = 1 Hz

1. Spectrophotometer, pH meter
2. Nessler tube, 50 mL
3. Funnel, graph paper
4. FiIterpaper(Whatrnan No. 41)
5. Pipettes 1 mL, 5 mL, 10 mL

1. Stock standard colour solution: Dissolve 1.246g KltCl 6(equivalent to 500 mg
metallic platinum) and Ig crystallized CoCI 2• 6Hp (equivalentto about 250 mg
Analysis a/Water and Effluents 59

metallic cobalt) in distilled water. Filter it to remove any slight turbidity. Add
100mL conc. HCI and dilute with distilled water to 1000mL. This stock standard
solution, has a colour of 500 Hazen unit.
2 Working standard colour solution: Prepare standards having colour of following
units in 50mL ofNessler's tube. Add 0.5, 1.0, 1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0,6.0
and 7mL of stock coloured solution in 50mL Nessler's tube and filled with
distilled water up to the mark which will give 5, 10, 15,20,25, 30, 35, 40, 45,50, 60
and 70 Hazen units. These standards can serve for several months, provided
that these are protected from dust and evaporation.
1. Filter the sample through a glass fibre filter paper to remove the turbidity.
2 Measure the optical density (OD) with a spectrophotometer at a wavelength

between 385 and 470nm. Put filtered water in the reference cell.
3. Prepare calibration graph (prepared in the range of 10 to 70 units).
4. Record as colour to the nearest whole number.
The colour of the sample is observed by filling a matched Nessler's tube to the 50-
mL mark with sample and comparing it with standards. Look vertically downwards
through tubes towards a white or specyular surface placed at such an angle that
light is reflected upward through the columns ofliquid. If turbidity is present and
has not been removed, reported as "apparent colour". If the colour exceeds 70
units, dilute sample with distilled water in known proportions until the colour is
within the range of the standards. Measure the pH of the each sample.
Calculate colour units by the following equation:

(A x 50)
Colour units (Hazen) = - - -
A = Estimated colour of a diluted sample
B = mL of sample taken for dilution
50= Total volume of sample in Nessler's tube

Check Your Confidence on Colour Estimation

1~ List the environmental significances of colour measurement.
2 Name the sources of colour in drinking water?
3. What is the difference between true colour and apparent colour? How to estimate
4. "1 mg fL ofPt. gives 1 unit of colour" (true/false).
Handbook of Methods in Environmental Studies

3.8 Chemical Oxygen Demand (COD)

cop Standards . . .
Drinking water:
No COD standard has been prescribed by WHO (1984), IS: 10500 (1983,1991),
MHW (1975) or USEPA (1974) or ICMR(l963)
Effluent discharge:
250 mg/L, into inland surface waters and marine coastal areas (MOEF, 1993;
IS: 2490, 1981)

Application of COD Data

1. Extensively used in the analysis of domestic and industrial wastewater.
2. In conjunction with BOD test, COD test is helpful in indicating toxic conditions
and the presence of biologically resistant organic substances.
3. COD test is used widely in the operation of treatment facilities because of the
speed with which results can be obtained.
COD Standards-

Environmental Significance
Chemical Oxygen Demand (COD) test determines the oxygen required for chemical
oxidation of most organic matter and oxidizable inorganic substances with the help
of strong chemical oxidant. The test can be employed for the same purpose as the
BOD test taking into account its limitations.
The intrinsic limitation of the test lies in its inability to differentiate between the
biologically oxidisable and biologically insert material.
COD determination has an advantage over BOD determination in that the result
can be obtained in about 3 h, as compared to 5 days required for BOD test. Further
the test is relatively easy, gives reproducible results and is not affected by
interference's as the BOD test.

Measurement of COD

1. COD determines the amount of oxygen required under specific test conditions
for the oxidation ofwaterbome organic and inorganic matter.
2. The reflux methods are limited by the reagents employed to a maximum COD of
800 mg/L. Simple with higher COD may be processed by appropriate dilution of
the sample.
3. If the sample is diluted, it must consume at least 5 mL of dichromate. Dilute the
sample if more than 20 mL of the potassium dichromate solution is consumed by
the reaction.
Analysis o/Water and Effluents 61

4. COD results are not accurate if the sample contains more than 1000 mg/L chlo-
ride. Consequently, the reflux method should not be applied to samples such as
seawater and brines.

The organic matter and oxidisable inorganic substances present in water or waste-
water get oxidised completely by standard potassium dichromate (K zCrp7) in the
presence ofHzS04 to produce COz + Hp. The excess K ZCrp7 remaining after the
reaction is titrated with ferrous ammonium sulphate [Fe(NH4MS04)Z]. The dichro-
mate consumed gives the 02 required for oxidation of the organic matter. The
contents are refluxed for 2 h.

2~Crp7 + 8HzS04~ 2KzS04+ 2Cr2(S04)3 + 8Hp + 302

C6H1P6+60Z ~ 6COz +6HP

Note: In order for K2Crp7 to oxidize organic matter, the solution is made acidic. An
excess of K2Cr20 7 must be present in COD analysis. Hence excess is added and
actual amount reduced is determined. Ferrous ammonium sulfate is used for back

6Fe2++ Crpi· + 14H+ ~ 6Fe3++2Cr3++7HP

Comments: It is based on the principle that all organic compounds can be oxidized
by strong oxidizing agents in acidic media. Hence:
COD values are always greater than the BOD values. COD values are too large,
if greater amounts of biologically resistant organic matter is present, i.e. lignin.
COD value do not provide any information on the rate at which actual
bio-degradation will take place.
Reflux condensers are used to prevent loss of volatile organic compounds.

1. Fe (II) and hydrogen sulfide exert COD of 0.14 mglmg Fe2+and 0.47 mglmg ~S
2. The chlorides interference is eliminated by addition of mercury salt (mercury
sulfate), which precipitated chlorides as HgCI 2: Hgz+ + 2Cl- = HgCI 2. Although
1g HgS04 is specified for 50 mL sample, a lesser amount may be used where
sample chloride concentration is known to be less than 2000 mg/L or as long as
a 10: I ratio ofHgS04: Cl- is maintained.
3. Addition of A~SO4to conc. H ZS04acts as a catalyst to stimulates the oxidation
of straight chain aliphatic and aromatic compounds.
4. Nitrite (N0z-) exerts a COD of 1.1 mglmg NOz-N. Sulphamic acid is added at 10
mg sulfamic acid for every Img ofNOz-N in the refluxing flask. Add sulfamic
acid to the standard K ZCrp7' since it must be included in the distilled water
62 Handbook of Methods in Environmental Studies

Natural, not very heavily polluted water should be analyzed on the same day or at
least within 24h. If there is to be a delay before analysis, the sample may be pre-
served by adding sulfuric acid, about 2mL H2S04 (d = 1.84) diluted to 1 + 2 to each
100 mL of sample. If samples are to be stored for longer than 24h, deep-freezing is
• Safety precautions:
1. Exercise extreme care when handling conc. H2S04 , especially at the start of
refluxing step.
2. Silver sulfate (Ag2S04 ) is poisonous; avoid contact with the chemical and its
3. Mercuric sulfate (HgS04 ) is very toxic; avoid contact with chemical and its

1. COD Reflux apparatus consisting of a flat bottom 500-mL capacity flask with
2. A heating mantle

1. Potassium dichromate, K2Crp7

2. Conc. sulphuric acid, H2S04 (sp. gr. = 1.84)
3. Sulphamic acid
5. Silver sulfate, Ag2S04
6. Ferrous ammonium sulfate, Fe(NH4MSOA 6Hp
7. Mercuric sulfate, HgS04

1. Standard potassium dichromate, 0.25N or 0.0417 M: Dissolve 12.259 g of
K2Crp7 (dried at 103°C for 24 h) in distilled water and dilute to lL. Add about
120 mg ofsulphamic acid to take care of6 mg/L ofN02-N.
2. Standard ~Cr207solution, 0.025N or 0.00417M: Dilute 100 mL of0.25N
K2Crp7solution to lL. (This solution is required only if COD is determined in
the range of 10 to 50 mg/L).
3. Sulphuric acid - silver sulphate reagent: Add 109 A~SO4 to 1000 mL of conc.
H 2S0 4 and keep overnight for dissolution.
4. Standard ferrous ammonium sulphate (FAS), approx. O.25N: Dissolve 98 g of
[Fe(NH4)iS04)2' 6HPl in about 400 mL distilled water and add 20 mL ofconc.
H2S04, cool and dilute to lL. Standardise the solution daily against the stan-
dard K2Crp7'
5. Standard FAS, O.025N: Dilute 100 mL of standard FAS solution to IL. (or dis-
solve 9.8 g ofFAS in lL distilled water with 20 mL ofconc. sulphuric acid).
Analysis of Water and Effluents 63

Standardisatioll of ferrous ammollium sulphate: Take 10mL of standard

K 2Crp7 and dilute to about 100 mL by adding distilled water. Acidify by 10mL
of H2S04 and cool. Titrate with ferrous ammonium sulphate using 2-3 drops of
ferroin indicator. Calculate normality as follows:

N=(A x B)/C
N = normality of the FAS solution
A = volume ofK2Crp7solution, mL
B = normality ofK2Crp7solution
C = volume ofFAS, mL
6. Ferroin indicator: Dissolve 1.485 g of 1,1 O-phenonthroline monohydrate,
together with 695 mg ofFeS04.7Hp in water and dilute to 100mL. This indicator
solution is also commercially available.
7. Mercuric sulphate (HgS04): Analytical grade crystals

Estimation of COD
1. If the sample contains more than 100 mgIL chloride after dilution, proceed as
2. To 20 mL of sample or aliquot in the flask add 0.5 g mercuric sulfate and shake
thoroughly. This addition is sufficient to complex 40 mg of chloride or 2000
mgIL when 20 mL of sample is used. If more chloride is present, add more HgS04
to maintain a HgS04 : CI- ratio of 10 : 1. It is not important if a slight precipitate
appears as it hardly affects the COD determination.


If a water is expected to have a higher or lower than normal COD, a sample ranging
in size from 10 mL to 50 mL may be used with the volumes, weights and concentra-
tions adjusted accordingly as per Table 3.6.

Table 3.6:
Reagent quantities for different sample sizes.

Sample size Standard H2SO. with HgSO. Cone. of Final vol.

(mL) K2 Cr2 0 7 A9 2SO. (g) FAS(moI/L) Before titration
(mL) (mL) (mL)

10 5.0 15 0.2 0.05 70

20 10.0 30 0.4 0.10 140
30 15.0 45 0.6 0.15 210
40 20.0 60 0.8 0.20 280
50 25.0 75 1.0 0.25 350
64 Handbook of Methods in Environmental Studies


0.4g HgS04

+201llL !>amp"
+10 filL 0.25M 1(2(r20,
Afttr 2 h R.flux
+ 30mL (one. H2!.04
8-'0 drops ferroin

Fig. 3.11: Scheme for COD determination

[Reflux digestion (A) followed titration].

PROCEDURE (Fig. 3.11)

For samples having low COD of less than 50 mgIL the potassium dichromate is
taken with a diluted normality of 0.025 N and the contents are titrated with the
corresponding 0.025 N FAS. On the other hand, for samples containing more than
50 mgIL COD, the higher strength of 0.25 N of these reagents is used.
Steps in COD determination

1. Take a 500 mL capacity flat-bottom conical flask and add 0.4 g ofHgSO4'
2. Add 20 mL of sample or an aliquot of sample diluted to 20 mL with distilled
water and mix well.
3. Add few glass beads followed by 10 mL of 0.025 Nor 0.25 N potassium dichro-
mate depending upon the expected COD.
4. Add slowly 30 mL conc. H2S04 + A~SO 4 reagent and mix thoroughly. The slow
addition along with swirling prevents fatty acids to escape out due to high
5. Mix well. If the colour turns green, take either fresh sample with lesser aliquot or
add more dichromate and H2 S04 , Connect the flask to condenser. Mix the con-
tents before heating. Improper mixing will result in bumping and sample may be
blown out.
6. Reflux for a minimum of 2 h. Cool and then wash down the condenser with
distilled water.
Analysis of Water and Ejjluents 65

7. Dilute for a minimum of 150 mL (about 300 mL), cool to room temperature and
titrate excess K 2Crp7 remaining after retluxing with corresponding standard
ferrous ammonium sulfate using ferroin as an indicator (8-10 drops). Sharp
colour change from blue green to wine red indicates end point or completion of
the titration.
8. Perform blank in the same manner using distilled water instead or sample.

(A-B) x N x 8 x 1000
COD (mgIL) = - - - - - - -
mL of the sample
A = mL offerrous ammonium sulfate used for blank
B = mL offerrous ammonium sulfate used for sample
N = Normality offerrous ammonium sulfate
8 = Milliequivalent weight of oxygen.


The technique and the quality of the reagents may be tested using potassium acid
pthalate as a standard substance. Potassium acid pthalate has a theoretical COD of
1.176 gig.

Stock solution of500 mg/L COD: Dissolve 0.4251 g of potassium acid pthalate in
distilled water and dilute to 1L for a 500 mgIL COD solution. A recovery of 98-100%
of the theoretical oxygen demand can be expected.


Recoveries of known amounts of COD in the series of prepared standards is shown
in Table 3.7.

Table 3.7:
Standard COD sample and COD recovered from the sample.

Prepared COD, Recovered COD, Bias,mg/L Statistically significance

mg/L mg/L

12 .3 12 .34 +0.04 no
40 . 2 37.9 - 2 .3 yes
92.0 88.6 - 3.4 yes
270 257 - 13 yes

Source : ASTM (1995)

Check Your Confidence on COD Test

1. What are the limitations & advantages of COD test over BOD test?
2. Why presence of high level of chloride interferes with COD test? How to
remove chloride interference?
66 Handbook of Methods in Environmental Studies

3. Why AgS04 is added into concentrated H 2S04 during COD test?

4. How to remove interference of nitrite (N02) in COD test?
5. How Fe 2+ and H2S interfere with COD test?
6. How to perform COD test when sample contains high levels of chloride.
7. Which general groups of organic compounds are not oxidized in the COD test?
8. What will be the COD results? (higher, lower or same) if-
a. Mercuric sulfate was not added.
b. Silver sulfate was not added.
c. Ferrous ammonium sulfate was assumed to have the same normality as it did
two weeks prior to the current analysis.
9. Why do COD and BOD analysis usually give different results for same waste.
10. How to check the correctness of COD analysis?
11. List three possible sources of error in COD experiments? How might they be
12. Compare the COD and BOD tests.
Hints: Comparison of COD and BOD test are given below:

BOD test COD test

a. Closely related to natural process. Less relationship to natural process.
b. It requires 5 days analysis time. Rapid analysis time (2h).
c. Difficult to reproduce, both within Good reproducibility.
and between laboratories.
d. Not suitable for toxic waste and Can analyse heavy polluted waste-
difficult to perform for heavy water as well as toxic waste.
polluted water.
5. Best use: Good long term monitoring Best use: Rapid analysis of heavy
of natural water pollution polluted samples e.g. industrial

3.9 Chlorine (Residual)

(lodometric Method)

Drinking water
IS: 10500.(1991) = 0.2 mgIL (residual free chlorine, minimum). To be applicable
only when water is chlorinated. Tested at consumer end. When protection
against viral infection is required, it should be minimum 0.5 mgIL.
Effiuent discharge
MOEF (1993) = 1.0 mgIL (total residual C1 2, maximum), for discharge in inland
surface water and marine coastal areas.
Analysis a/Water and Effluents 67

Chlorine is primarily added to the water (i.e., chlorination) for destroying the
harmful microorganisms in water and wastewater. It is become common practice to
refer to chlorine, hypochlorous acid, and hypochlorite ions are "free chlorine re-
siduals", and the chloramines are called "combined chlorine residuals". An uncon-
trolled excess of chlorine in water, whether free, available or combined, can ad-
versely affect the subsequent use of water. The smallest amount of residual chlo-
rine considered significant is 0.1 mg/L.
Disinfectant residuals are used universally in disinfection practice to control
addition of disinfectant as to ensure effective disinfection without waste of chemicals.

Sampling and Storage of Water Sample

Chlorine in aqueous solution is not stable and the chlorine content of the sample
decreases rapidly. Exposure to sunlight or other strong light or agitation accelerates
the reduction of chlorine. Therefore, chlorine determination is done immediately
after sampling, avoiding excessive light and agitation. Do not store samples to be
analyzed for chlorine.

Estimation of Residual Chlorine

Chlorine is an strong oxidizing agent and liberates free iodine from potassium iodide
(KI) solution at pH 8 or less. The released iodide is titrated with a standard solution
of sodium thiosulphate (Na2Sp3) with starch as an indicator. The end point is
indicated by the disappearance of blue colour. Titration is done at pH 3 to 4 because
the reaction is not stoichiomertic at neutral pH due to partial oxidation ofthiosulphate
to sulphate.

Cl 2+ 2 KI ~ 12 (liberated) + 2 KCl
12 + Starch (indicator) ~ Blue colour solution

12 + 2 Na2Sp3 (titrant) ~ Na2Sp6 + 2 Nal

(blue colour solution) (colourless solutIOn)

Minimum detectable concentration is 40 ~g Cl as CI/L if 0.0 1N Na2Sp3 is used

with a 1000 mL sample. Concentration below 1 mg/L can not be determined accu-
rately by the starch-iodide end point used in this method.

Oxidized form of manganese and other oxidizing agents interfere.
Reducing agents such as organic sulphides also interfere.
Use only acetic acid for acid titration; H 2S04 will only increase interference.
Never use hydrochloric acid.
68 Handbook of Methods in Environmental Studies

Orthotolidin test: The Orthotolidin test is quick and easy method. In 100rnL sample,
ImL of orthotolidin is added. If yellow colour develops, it shows the presence of
chlorine. By comparing the intensity of colour with the standard sample, the residual
chlorine can be find out.


1. Acetic acid (Glacial)
2. Sodium thiosulphate
3. Cone. H2S04
4. Potassium iodide (KI)
5. Potassium dichromate (KFrp7)
6. Starch powder

1. Standard sodium thiosulphate, O.lN: Dissolved 25g Na2Sp3.5 ~O in 1000 rnL
of freshly boiled and cooled distilled water and standardise against potassium
bi-iodate or potassium dichromate after at least 2 weeks storage. This initial
storage is necessary to allow oxidation of lUly bisulphite ion present. Add
about 5 rnL of chloroform as a preservative to minimize bacterial decomposition.
4. Starch indicator (lOg/L): Prepare slurry by adding small quantity of water to
1.0 g starch powder. Add 100 mL boiling water to it and continue boiling for a
few minutes until solution becomes clear. The solution is cooled and preserved
by adding 1.25 g of salicylic acid or a few drops of toluene or chloroform.

PROCEDURE (Fig. 3.12)

I. Standardisation of sodium thiosulphate solution
1. Standardise 0.1 N Na2Sp3 by 0.1 N K2Crp7 sol.ution. 0.1 N potassium dichro-
mate solution is prepared by dissolving 4.904 g anhydrous ~Crp7 of primary
standard quality in distilled water and diluted to 1000 rnL. Store in a glass-
stoppered bottle.
2. To 80 mL of distilled water, add with constant stirring 1 rnL cone. H 2S04, 10 mL
0.1 NK2Crp7and I gKI.
3. Let the reaction mixture stand for 6 min in dark and then add 2 rnL of starch
indicator before titrating with 0.1NNa2Sp3 titrant.
4. The colour changes from blue to colourless end point

Calculation of normality of sodium thiosulphate

N ofK 2Crp7 x Vol. of ~Crp7
N of sodium thiosulphate = ------------
Vol. ofNa2 Sp3

Standard sodium thiosulphate, O.OlN: Improve the stability ofO.01N Na2 Sp3
solution by diluting aged O.1N solution. ImL ofO.OlN Na2Sp3 = 354.5 Ilg CI as
Analysis o/Water and Effluents (f)

0.01 N
5tMd.rd N.2 51 0,

Scheme of residual chlorine determination

100 mL Sallpl'

+Pnch of KI
1'5 ilL aClllc ecid
+StaI'th indicator

SlUE COLDR - Color Loss

lEnd Point)

Fig. 3.12: Estimation of residual chlorine.

II. Estimation of residual chlorine

1. Volume 0/sample: Select a sample volume that will require not more than 20 mL
of 0.01 NNazSPr For a chlorine range ofl to 10mg/L, use a 100-mLsample.
2. Preparation/or titration mixture: In a selected sample volume, add a pinch of
KI and about 5 mL of acetic acid (or enough to reduce pH to between 3.0to 4.0).
Mix with a stirring rod and allow to complete the reaction.
3. Titration: Titrate away from direct sunlight. Titrate free iodine liberated against
0.01 N NaZSZ0 3 with starch as an indicator. The colour changes from blue to
colourless at end point. Note the volume required (A).
4. Blank titration: Prepare a reagent blank using distilled water. Note the volume
oftitrant required (B).
(A - B) x N x 35.45 x 1000
Residual chlorine as Cl/L = ----------
Where, A = mL of titrant required for sample, mL.
B = mL of titrant required for blank, mL
N = normality of titrant (NazSp3)'

For treating 3,000 m3 of water, 12.25 kg of CI-gas used. The chlorine demand
of the water is measured to be 2.6 mg/L. What is the residual CI concentra-
tion in the treated water?

Total Dosage = 12.25 kg/3,OOO m 3 =4.084 mg/L
Residual chlorine = 4.084 mg/L - 2.6 mg/L = 1.484 mg/L (Ans.).
70 Handbook of Methods in Environmental Studies

Check Your Confidence on Residual Chlorine

1. Why it is important to determine disinfectant residual in water treatment
2. The free residual chlorine is sum of ................ and ..................... .
3. What is the minimum residual chlorine that must be present in potable water?
4. Why estimation of residual cholrine should be done immediately in the
5. Why titration is carried out at acidic pH (PH 3 to 4)?
6. Why acetic acid is used to acidify the titration mixture, not HCI or ~S04?
7. Why aging of titrant N~S2 0 3 is necessary for estimation of residual chlorine?
8. What is Orthotolidin test?
9. Compare the relative significance of free chlorine residuals and chloramine
residuals in water treatment practice.

3.10 Chlorine Demand

Significance and Use
Chlorine is added to potable water, wastewater and industrial water for a variety of
purpose. Some of these purposes are as given below:
1. To eliminate or reduce the growth of microorganisms in water.
2. To destroy or modify decomposable organic substances so as to reduce
biochemical oxygen demand of the water.
3. To eliminate or reduce taste, odours, and colour of the water.
4. It is important to avoid over-chlorination in order to minimize chemical
consumption, meet restrictions specified by regulatory agencies, and minimize
equipment degradation.

Chlorine demand is defined as the difference between the amount of chlorine
applied and the amount of free chlorine remaining at the end of the contact period.
The chlorine demand is different for different waters. For effective disinfection,
doses of chlorine, optimum contact time and residual chlorine are required to be
determined. The smallest amount of residual chlorine considered significant is 0.1
mg/L. Temperature, pH and initial chlorine dosages are important variables in
estimating optimum chlorination practice.
Chlorine combines with water to form hypochlorous acid (HOC}) and hydro-
chloric acid (HCI). Hypochlorous acid (HOC I) dissociates to give the OC}- and W
Analysis o/Water and Effluents 71

Quantities of HOC I and OCI- depend on pH of the solution.

HOCI 96% at pH = 6
HOCI 75% at pH = 7
HOCI 22% at pH = 8
HOCI3%atpH = 9
The quantity of HOC1and OCr that is present in water is called the ''free available
chlorine." The killing efficiencies of HOC1in about 40 to 80 times more than OCr

Estimation of Chlorine Demand

Measurement of chlorine demand can be readily made by treating a series of samples
of the water in question with known varying dosages of chlorine or hypochlorite.
The water sample should be at a temperature within the range of interest, and after
the desired contact period, determine the residual chlorine in all samples.
The major interfering substances are oxidisable organic and inorganic matter. In
addition, chlorine and hypochlorous (HOCI) acid also react with wide variety of
substances, including ammonia and naturally occurring humic substances. The
reactions of chlorine with ammonia are as follows:
NH3 + HOCI -+- NH2CI + Hp (monochloramine)
NH2CI + HOCI-+- NHCI 2 + Hp (dichloramine)
NHCI 2 + HOCI -+- NCI3 + ~O (trichloromine)
The chlorine in these compounds is called "Combined available chlorine". These
chloramines are also serve as disinfectants.
1. Bleaching powder
2. Potassium iodide (KI), crystals
3. Sodium thiosulphate
4. Potassium dichromate ~Crp7)
5. Acetic acid (Glacial)
6. Starch powder
7. Bleachin&powder, Ca(OC1)Cl
I. Standard sodium thiosulphate titrant (O.IN): Prepared as given in determination
of residual chlorine,
2. Starch indicator: Same as in residual chlorine
72 Handbook of Methods in Environmental Studies

3. Preparation of standard chlorine solution: Weigh accurately 5.0 g fresh

bleaching powder and transfer to mortar. Add small quantity ofwater to prepare
fme paste. Add some more water, stir and allow to settle for few min. Decant
supernatant. Repeat till a fme suspension is obtained. Dilute to 500 mL.
Alternatively, weigh 350 mg of30% bleaching powder and make its paste and
dilute to 1L. The solution contains approximately 100 mg of chlorinelL).
4. Standardisation ofthe chlorine solution:
a. Transfer 10 mL solution in a conical flask.
b. Add 10 mL of acetic acid (1+1) and 10mLofKlsolution.
c. Titrate with standard 0.1 N N~SP3 solution until the yellow colour of the
liberated iodine is almost discharged.
d. Add 1 mL of starch indicator solution and continue the titration to a
colourless endpoint.
e. Calculate conc~ntration of available chlorine as follows:
A x B x 35.45
Available chlorine, mg/mL = - - - - - -
A = Volume of standard Na2Sp3' mL
B = Normality of standard Na2Sp3
V = Chlorine solution titrated, mL.
1. Take 1000 mL of sample in 10 stoppered bottles.
2 Add standard chlorine solution in ascending order. If chlorine demand of treated
water to be examined, use dose of 0.0 to'300 f,1g CljL. In case ofpoUuted water
sample and treated effluents, use dose of 0.1 mg to 3 mg C~ IL.
3. Allow a contact period of30 minutes for drinking water and suitable higher time
for polluted water, or secondary effluents.
4. Estimate residual chlorine as described earlier.
4. Plot the residual chlorine versus chlorine added. In case of organically polluted
samples, a distinct breakpoint can be obtained. But in case of treated water
sample, it is possible that only a straight line is obtained in absence of any
ammonium compound. A typical break point curve is shown in Fig. 3.13.
5. A residual of 0.2 mg Cl/L after the breakpoint is recommended.

Calculate chlorine dosage in mg/L for each treated sample solution as follows:

Chlorine dosage, mg/L = 2AB

A = chlorinating solution added to each sample aliquot, mL
B = available chlorine/mL of chlorinating solution, mg
Analysis of Water and EfJluents 73

Chlorine demand, mgIL = C - D·

C = chlorine dosage, mgIL
D = free chlorine remaining at the end of contact time, mgIL

Holt ratio, (12 NH3-N

O·S 1 1·S 2

~ 10
:= 9
C>I 8
.,e 7
... 5
;g 10

(hlorlnt dosagt mg/littr

Fig. 3.13: A residual chlorine curve showing a typical break point

(Ammonia nitrogen content of water is 1 mg/L).

What is the daily amount of chlorine needed to treat .30,280 m3 (8MGD) of
water to satisfy 2.8 mg/L residual chlorine demand and provide 0 .5 mg/L
residual chlorine.

Check Your Confidence on Chlorine Demand

1. Define chlorine demand?
2 List 4 applications of chlorine demand test.
3. What is "free available chlorine" and "combined available chlorine"?
4. What are the important variables that effect chlorine demand test?
5. "Quantities of HOC I and OCI are depended on pH of the solution". why?
6. For two samples chlorine demand test is done. One is treated water sample and
other one is organically polluted water sample. In which sample a distinct break
point of chlorination will be obtained and why?
7. A residual of .....,...... CI/L after break point is recommended for drinking water.
74 Handbook ofMethods in Environmental Studies

3.11 Cyanide

Drinking water
IS: 10500 (1983, 1991) = 0.05 mgIL (no relaxation)
ICMR (1963) = 0.05 mgIL
MWH (1975) = 0.05 mgIL
WHO (1984) = 0.1 mgIL
WHO (1993) = 0.07 mgIL
EC (1980) = 0.05 mgIL
Effluent discharge
MOEF (1993) = 0.2 mgIL (Inland surface water, land for irrigation and marine
costal areas); 2 mgIL (Public sewers)

3.11.1 Estimation of Total Cyanide After


After removal of interfering substances, the metal cyanide is converted to HCN
(hydrocyanic acid) gas, which is distilled and absorbed in NaOH solution. The
sodium cyanide in the absorbing solution can be detennined colorimetrically, by
titration, or by selective ion electrode.
Total cyanide is based on the decomposition of nearly all cyanide in the present
of strong acid, magnesium chloride catalyst and heat during a I h reflux distillation.
The minimum conceJ.1tration of cyanide in the absorbing solution that can be accu-
rately detennined:
a. Colorimetrically: 0.005 mgIL to 0.03 mglL.
b. Titration: 0.4 mgIL to I mgIL and
c. Selective ion electrode: 0.05 mgIL to 0.03 mglL.

Distillation Apparatus (Fig. 3.14)
Boiling flask, IL with inlet and provision for water-cooled condenser. The inlet
tube shall be a funnel with 8-mm diameter stem that extended to within 6mm of the
bottom of the flask. The condenser shall be connected to a vacuum-type absorber
which shall be in turn connected to a vacuum line which has provision for fme
control. The flask shall be heated with an electric heater.
Analysis o/Water and Effluents 75


1. Sodium hydroxide, NaOH
2. Sulphuric acid, H2S04
3. Sulfamic acid, NH2S03H
4. Magnesium chloride, MgCl2
5. Lead carbonate, PbC03
1. Sodium hydroxide solution: Dissolve 40g ofNaOH in water and dilute to IL.
2. Magnesium chloride reagent: Dissolve 510g MgClr 6Hp in water and di-
lute to IL.
3. Sulfuric acid (1+1): Slowly add Ivol of H2S04 ( 1.84) to Ivol of water,
stirring and cooling the solution during the addition.
4. Lead carbonate (PbCO): Powder
5. Sulfamic acid (NHzSOJH)

Allihn __ 9mm connlPcting tube

condlPnslPr ' "

Water in

tnt tube

Fig. 3.14: Cyanide distillation apparatus

1. Setup the apparatus as shown in Fig. 3.14.
2. Add 10 mL ofNaOH solution (40gIL) to the absorber and dilute with water, if
necessary, with distilled water to obtain an adequate liquid depth in the
absorber. Do not use more than 225 mL of total volume of absorber solution.
3. Attach the absorber to the vacuum and connect the condenser.
76 Handbook ofMethods in Environmental Studies

4. Place 50 mL of the sample in the flask. If content is suspected to be more than

10 mg/L, use an aliquot so that no more than 5 mg of cyanide is in the distilla-
tion flask and dilute to 500 mL with water.
S. Connect the flask to the condenser.

3.11.2 Estimation of Cyanide by Titration


Cyanide in the alkaline distillate from the preliminary treatment procedure is titrate
with standard AgN0 3 to form the soluble cyanide complex, Ag (CN)". As soon as
all CN- has been complexed, a small excess of Ag+ is added. The excess Ag+ is
detected by the silver sensitive indicator rodanine, which immediately turns from
yellow to a salmon color (pink). The distillation provides a 2: 1 concentration.
The indicator is sensitive to about 0.1 fig Ag/L. If titration shows that CN- is
below 1 mg/L, examine another portion colorimetricaUy.
1. Rhodanine indicator solution: Dissolve 0.02 g of rhodanine (p-dimethyl-
aminobenzal-rhodanine) in 100mL of acetone.
2. Standard silver nitrate, titrant: Dissolve 3.27 g AgN03 in IL of distilled
water. Standardise against standard NaCI solution, using the argentometric
method with potassium chromate as an indicator as directed in chloride estima-
tion. Dilute 500 mL of AgN0 3 solution according to the titer so that 1 mL =
1.00 mg cyanide.
3. Sodium hydroxide solution: Dissolve 1.6g NaOH in 1 L distilled water.
1. Place 100 mL of the absorption solution or an accurately measured aliquot
dilute to 100 mL with NaOH solution (1 .6g/L) in a beaker of conical flask. For
sample with low cyanide concentration « 5 mg/L) do not dilute_
2. Add 0.5 mL indicator solution.
3. Titrate with standard AgN0 3 solution to the first change from yellow to salmon
4. Titrate a blank of 100 mL NaOH solution.
5. Record the results of titration and calculate the cyanide concentration in the
original sample.
(A-B) x 1000 250
Cyanide, rnglL = x -------
mL of original sample mL portion used

A = AgN0 3 solution to titrate sample, mL

B = AgN03 solution to titrate blank, mL
Analysis of Water and EfJluents 77

3.11.3 Estimation of Cyanide by Colorimetric

Cyanide, in the alkaline distillate from preliminary treatment is converted to CNCI
by reaction with chloramine-T at pH <8 without hydrolysing to cyanate (CNO-).
(Caution: CNCI is a toxic gas; avoid inhalation). After reaction is completed, CNCI
forms a red-blue color on addition of a pyridine-barbituric acid reagent. Maximum
color absorbance in aqueous solution is between 575 and 582nrn.

1. Chloramine-T solution: Dissolve Ig of white-coloured, water soluble grade
Chloramine-T powder in 100 mL of water. Prepare fresh weekly and store in
2. Cyanide stock solution (ImL = 1 mg CN-): Dissolve approximately 1.6g
NaOH and 2.51g KCN in IL distilled water (Caution: KCN is highly toxic,
avoid contact or inhalation). Mix thoroughly. Standardise against standard
AgNO) titrant, using 25 mL KCN solution. Check the titer weekly because the
solution gradually loses str-ength.
3. · Cyanide standard solution (lmL = 10 Ilg CN-): Based on the concentration
for the KCN stock solution, calculate the volume required (approx. 10 mL) to
prepare IL ofa 10 I1g cyanide/mL. Dilute with NaOH solution.
4. Cyanide solution (ImL = 1 IlgCN): Dilute 10 mL of 10 I1g CN-/mL solution
to 100 mL with NaOH (1.6g1L) solution. Prepare fresh daily and keep in a
glass-stoppered bottle.
5. Pyridine-barbituric acid reagent: Place 15g of barbituric acid in a 250 mL
volumetric flask and just add water to wash the side of the flask and wet the
barbituric acid. Add 75 mL of pyridine and mix. Add 15 mL of conc. HCr and
cool to room temperature. Dilute to the mark with water and mix until all of the
barbituric acid is dissolved. This solution is stable for approximately 6 months
if stored in an amber bottle under refrigeration. Discard if precipitate develops.
6. Acetate buffer: Dissolve 41 Og of sodium acetate trihydrate (NaC2~02.3HP)
in 500 mL of water. Add glacial acetic acid to yield a solution of pH 4.5,
approximately 500 mL.

Preparation of Standard Curve
Pipette a series of standards containing I to 10 Ilg of cyanide into 50-mL volumet-
ric flasks (0.02 to 0.2 Ilg cyanide/mL). Dilute to 40 mL with NaOH solution (1.6
gIL). Use 40 mL NaOH dilution solution as blank.
Colour development: Add I mL acetate buffer and 2 mL chloramine-T solution,
stopper, and mix by inversion twice. Let stand exactly for 2 min. Add 5 rnL pyri-
dine-barbituric acid reagent, dilute to the mark with distilled water, mix thoroughly
78 Handbook ofMethods in Environmental Studies

and let stand exactly for 8 min. Measure absorbance against distilled water at 578
run. Measure absorbance of blank (0.0 mg CNIL) using 40 mL NaOH dilution
solution and follow the same procedure of colour development.

Estimation of Cyanide In Water Sample

Pipet a portion of absorption solution into- a 50-mL volumetric flask (such that
concentration falls within the standardisation range) and dilute to 40 mL with NaOH
solution. Follow the colour development procedure.,
Slope and intercept ofstandard curve: Calculate the slope on the standard curve,
m, and the intercept b, using following equations.
n:I:ca - :I:c:I:a
n:I:a2- (:I:a)2
:I:a2:I:c - :I:c:I:ac

a = absorbance of standard solution;
c = concentration of cyanide in standard, mgIL
n = number of standard solutions.
m = slope of standard curve, and
b = intercept on "e" axis
The blank concentration, 0.0 mg/L, and the absorbance of the blank must be
included in the calculation of slope and intercept.
Calculate the concentration of cyanide as follows:
50 250
Cyanide, mgIL = (rna + b) x -- x - -
a = Absorbance of sample solution
X = Aliquot of absorbance solution, mL, and
Y = Original sample, mL

3.11.4 Estimation of Cyanide by Ion Selective

1. Place 100-mL aliquot of standard solutions in 250 mL beaker.
2. Place the beakers on a magnetic stirrer, place a TEF-fluorocarbon-coated
Analysis a/Water and Eflluents 79

stirring bar in the solution, stir at a predetermined constant rate, and maintain
constant temperature.
3. Insert the cyanide specific electrode and the reference electrode in the solution
and measure potential or the cyanide concentration following the manufacturer's
4 . Pipet 10 and SO-mL aliquots of standard solution into 2S0-mL beakers and
dilute to 100 mL with NaOH solution (l .6g1L). Follow step-2 and step-3 start-
ing with lowest concentration.
S. Plot concentration values of the standardising solutions on the logarithmic axis
of semi-logarithmic graph paper versus the potentials developed in the
standardising solutions on the linear axis.


1. Place 100 mL of the absorption solution (or an accurately measured aliquot

diluted to 100 mL with NaOH solution (1.6g1L) in a 2S0 mL beaker.
2. Follow step 2 and step3.
3. Use values found from the graph or direct reading ion meter to calculate the
concentration in the original sample.


Calculate the concentration of cyanide in mgIL as follows:

Cyanide,mgIL = Cyanide mglL from graph or meter x (I OO/aliquot) x

(2S0/mL of original sample)

Check Your Confidence on Cyanide Estimation

1. "Cyanide is always absorbed in NaOH solution" , Why?
2. Name three methods of cyanide estimation.
3. Discuss the principle of cyanide estimation by titration method.
4. Name the indicator used in titration method of cyanide estimation.
S. Discuss the principle of cyanide estimation by colorimetric method.

3.12 Dissolved Oxygen (DO)

Drinking water
IS: 10SOO (1991) = No Standards
Effluent discharge
MOEF (1993) = No Standards
Stream Standards (CPCP, 1979)
Class A = 6 mgIL; Class B = S mgIL; Class C =4 mgIL; ClassD = 4 mgIL
MOEF (2000) notification = S mgIL or more for bathing water.
80 Handbook ofMethods in Environmental Studies

1. One of the most important water quality parameters is the amount of dissolved
oxygen (DO) present. The DO levels in. natural and wastewater depend on the
physical, chemical, and biological activities in the water body.
2. Oxygen is considered as poorly soluble in water. Its solubility is related to
pressure and temperature.
3. In fresh water, DO reaches 14.6 mgIL at 0"C and approximately 9.1,8.3 and 7
mgIL at 20"C, 25"C and 35°C, and 1 atmospheric pressure respectively. At tem-
perature of20-30°C, the level of saturated DO is 9 to 7 mgIL.
4. Minimum amount required for health fish population may be as high as 5 to 8
5. The oxygen depleting substances reduces the available DO. During summer
months, the rate of biological oxidation is highly increased, while, unfortu-
nately, the DO concentration is at its minimum due to higher temperature.

Environmental Significance
1. To evaluate the pollution strength of domestic and industrial effluents.
2. For corrosion control: Oxygen is a significant factor in corrosion of iron and
steel pipes in water distribution system and steam boilers. Removal of DO from
boiler-feed water by physical and chemicals means is a common practice in
power industry. So DO test serves as the means of control.
3. DO measures are vital for maintaining aerobic conditions in natural waters that
receive polluted matter.
4. For controlling the rate of aeration during biological treatment of efflu-
5. Rate of biochemical oxidation can be measured by determining residual dis-
solved oxygen in a system at various time interval.
6. For maintenance of desire aquatic life. As DO drops, fish and other aquatic life
are threatened; in the extreme case, killed.
7. As Do levels falls; undesirable odours, tastes and colours reduce the accept-
ability of water.

Estimation of Dissolved Oxygen

Two methods for DO analysis are described in APHA (1995):
1. The azide modification of Winkler method
2. The electrometric method using membrane electrodes
The azide method is a titrimetric procedure based on the oxidising prop-
erty of DO while electrode procedure is based on the rate of diffusion of mo-
lecular oxygen across a membrane. The choice of procedure depends on the
interferences present, the accuracy desired, and, in some cases, convenience
or expedience.
Analysis of Water and EjJ1uents 81


Oxygen present in the sample oxidizes the divalent manganese to its higher va-
lency, which precipitates as brown-hydrated oxides after addition of NaOH and
KI. Upon acidification, manganese reverts to divalent state and liberates iodine
from KI equivalent to DO content in the sample. The liberated iodine is titrated
against Na2Sp) (N/80) using starch as an indicator. The series of reactions, which
take place, can be summarized by the following equations.
MnSO. + 2NaOH -+ Mn(OH)2J.. (white ppt) + Na2S04
If no oxygen is present, a pure white precipitate ofMn(OH)z forms when MnS04
and alkali-iodide reagents (NaOH+ Kl) are added to the sample. If oxygen is present,
the divalent Mn (II) is oxidised to higher valency Mn(lV) (i.e. Mn02) and precipi-
tate as a brown hydrated oxide.
Mnz+ + 20H- + ~OZ -+ MnOzJ.. (brown ppt) + Hp, or
Mn(OH)z + ~OZ -+ MnOz + Hp
The oxidation of Mn(II) to MnOz' sometimes, called fixation of the oxygen.
Under acid condition MnOz reverts to divalent state by oxidising KI to produce Iz,
which is liberated in solution.
MnOz + 21- + 4W -+ Mnz+ + 12 + 2 Hp
The liberated free Iz is titrated against standard solution ofNa2Sp).
Na2Sp) + Iz -+ 2Nal + NaZS.o6

Nitrite (NO~) interference is overcome by the use of sodium azide (NaN). When
HzSO. is added, the following reaction occurs and the NO~ is destroyed.
NaN) + H+ -+ HN) + Na+
HN) + NOz + H+ -+ N z + Np + Hp

Use the azide modification for most wastewater, effluent, and stream samples, es-
pecially if samples contain more than 50 J.lg NOz-NIL and not more than lmg
ferrous ironiL.
Other reducing or oxidizing materials should be absent. If ImL KF solution is
added before the sample is acidified and there is no delay in titration, the method is
applicable in the presence of 100 to 200 mg ferric ironIL.

1. BOD bottle: 250 mL or 300 mL capacity
2. Graduated cylinder (500 mL capacity)
3. Conical flask (500 mL)
4. Burette (50 mL capacity)
5. Pipettes (1 mL,2 mL)
82 Handbook ofMethods in Environmental Studies



1. Monohydrate manganous sulfate (MnS04.HP)

2. Potassium iodide (KI) or sodium iodide (NaI)
3. Conc. H 2S04 (sp. gr. 1.84)
4. Sodium thiosulfate (Na2SPr5HP)
5. Potassium dichromate ~Crp7)
6. Sodium hydroxide (NaOH)
7. Sodium azide (NaN 3)
8. Starch powder


1. Manganous sulfate solution (364g/L): Dissolve 364 g of monohydrate man-

ganous sulfate (MnS04.HP) in distilled water (filter it, if necessary) and dilute
to lL. This solution should not give colour with starch solution when added to
an acidified solution of potassium iodide (KI).
2. Alkaline-iodide-azide solution: Dissolve 500 g ofNaOH and 150 g ofKI (or
135 g NaI) and dilute to 950 mL. Add 109 of sodium azide (NaN 3) dissolved in
40mL of distilled water. Cool the solution and make up the volume to 1000 mL.
This solution should not give colour with starch solution when diluted and acidi-
fied. Store the solution in a dark, rubber stoppered bottle.
3. Concentrated H1SO.: ImL neutralise about 3 mL of alkaline-iodide reagent.
4. Starch indicator: To prepare an aqueous solution take 5g of arrowroot or
soluble starch to approximately 800 mL of boiling water, with stirring. Dilute
to 1L, boil a few minutes, and leave overnight. Use clear supernatant. Preserve
by adding a few drops oftoluene or formalin. Store in a glass-stoppered bottle.
5. Standard sodium thiosulfate solution (O.025N): Dissolve directly 6.025 g
Na2Sp3.5Hp in a previously boiled 1L cooled distilled water, which will give
0.025N. Add 1.5 mL 6NNaOH or 0.4 g solid NaOH per lL. Store in brown
This solution will have to be standardised against standard potassium dichro-
mate ~Crp7) solution for each set of titration.
6. Standard potassium dichromate, O.25N: Dry ~Crp7 at 103°C for 2 h and
take accurately 12.259 g, and dilute to lL.
7. Potassium fluoride solution (400g/L): Dissolve 40 g ofKF.2HP in distilled
water and dilute to 100 mL. This solution is used in the procedure for determin-
ing ferric ion interferences. Store this solution in a plastic bottle.

PROCEDURE (Fig. 3.15)

1. Collect the sample in a BOD bottle (300 mL capacity) taking care to avoid any
bubbling. Fill the sample to the neck of the bottle. Be sure that air bubbles have
not been trapped under the stopper and maintain a water seal around the stop-
per until ready for the next step of analysis.
2. Add I mL of manganous sulphate followed by I mL of alkali-iodide-azide
Analysis of Water and Effluents 83

300mL S . .plt
+ 'lIIl Mn 504 SOlution
+ , IIIl alklli-ildidt-ilZidt

I'hlltd !h! Conttnts

to. SOlllinuh

Iter ~~nat lilt

Allow to prtcip. t.


Add starth indiCator


BLUE COLOR .... Color ltss

lEnd Point)

Fig . 3 .15: Estimation of dissolved oxygen.

84 Handbook ofMethods in Environmental Studies

solution. The tip of the pipette should be below the liquid level while adding
these reagents.
3. Place the stopper carefully to exclude air bubbles and mix by inverting the
bottle repeatedly for at least 15 minutes. An equivalent amount of2 mL of the
contents will come out of the bottle after placing the stopper. Allow the precipi-
tate to settle leaving about 150 mL of clear supernatant.
4. Carefully remove the stopper and immediately add 1 mL of conc. H2S04, close
the bottle and mix with gentle inversion until the precipitate completely
5. Titrate 100 mL or 250 mL contents of the bottle with sodium thiosulphate solu-
tion using starch as an indicator. At the end point the blue colour turns to
colourless. The starch indicator is usually added towards the end of the titration
when a straw pale colour is obtained.

Standardisation of sodium thiosulfate

It can be done either by standard bi-iodate solution or standard potassium dichro-

mate solution.
1. Add 2g KI and 150 mL distilled water in a 500 mL conical flask ..
2. Add few drops of conc. H2S04 and 20 mL ofO.025N K 2Cr20 7•
4. Dilute to 200 mL with distilled water. Keep in dark for 5 min.
5. The liberated iodine is titrated with sodium thiosulfate solution (0.025 N
approx.), adding starch indicator towards the end of titration, when a pale straw
color is reached.

20 x 0.025
Normality of thiosulfate = - - - - - - - - - -
mL of sodium thiosulfate


When only a p~rt of the sample of the content is titrated, i.e. 100 or 250 mL

mL of titrant x Normality x 8 x 1000

DO in mgIL = - - - - - - - - - - - - - -
V 2 (V,-v)N ,

V,=Volume of BOD bottle, mL
V 2= Volume of the contents titrated, mL
v = Volume of MnS04 and iodide azide added, i.e. 1 + 1 = 2 mL

Alum Flocculation Modification

Samples high in suspended solids may consume appreciable quantities of io-
dine in acid solution. The interference due to solids may be removed by alum
Analysis o/Water and EjJluents 85

All the reagents required are as discussed in DO estimation. In addition, the
following two are also required:
1. Alum solution: Dissolve 10 g aluminum potassium sulfate, AlK(S04}2.12 Hp
in distilled water and dilute to 1000 mL. .
2. Ammonium hydroxide: NHpH, concentrated

1. Collect sample in a glass-stoppered bottle of 500 to 1000 mL capacity, using
the same precaution as for regular DO samples.
2. Add 10 mL alum solution and 1 to 2 mL conc. NHpH.
3. Stopper the bottle and invert gently for about 1 min. Let sample settle for about
10 min and siphon clear supernatant into 300-mL DO bottle.

Check Your Confidence on Dissolved Oxygen

1. List five environmental significances of DO measurement.
2. "During summer months DO is minimum"- why?
3. Why DO should be measured in the field or fixed in the field?
4. Why alkali-azide solution is added during DO estimation?
5. Two samples were collected simultaneously at the same spot in a river for DO
analysis. One sample was fixed immediately after collection and other was treated
later in the laboratory. Indicate two possible factors· that could cause lower
results to be obtained in the second sample.
6. How interferences of suspended solids is removed during DO estimation?

3.13 Fluoride
(SPANDS Method)

Drinking water
IS: 10500: (1991); ICMR, (1963) = 1.0 mglL (maximum permissible). Fluoride
may be kept as low as possible. High fluorides may cause fluorosis. 1.5 mgIL
(maximum permissible limit in the absence of alternate source).
WHO (1984) = The guideline value of 1.5 mgIL in drinking water has been
proposed as excessive amounts of fluorides (more than 1.5 mgIL) causes
disfigurement of teeth known as mottled enamel (dental fluorosis).
Effluent discharge
MOEF (1993) = Guideline value of2 mgIL set for discharge into inland surface
water and 15 mgIL for discharge into public sewers and marine coastal areas.
86 Handbook ofMethods in Environmental Studies

Fluorides is more common in ground water than in surface water. The main sources
of fluoride in ground water are different fluoride bearing rocks. In rare instances
the fluoride concentration of naturally occurring water may approach 10 mgIL.
Such water should be defluoridated.
A fluoride concentration of approximately 1 mgIL in drinking water effectively
reduces dental caries or tooth decay without any harmful effect on health.
The actual concentration of fluoride in drinking water depends on the air tem-
perature, because ambient air temperature influences the amount of water that people
drink. The USEPA (1975), published the fluoride primary standards related with
ambient temperature (Table 3.8).

Table 3.8:
USEPA primary drinking water Mel for fluoride (1975) .

Annual average of maximum daily Maximum contaminant levels of

air temperature of community in F (mg/L)
which water system is situated (OC)

12.0 or below 2.4

12. 1 to 14.6 2.2
14.7 to 17.6 2.0
17.7 to 21.4 1.8
21.5 to 26.2 1.6
26.3 to 32.5 1.4

Out of 26 states of India, seven states have considerable amount of fluoride in

potable water (Table 3.9).

Table 3.9:
Status of fluoride in drinking water in some Indian states.

State (name of Average F State (name of Average F

district) conc., mg/L district) conc., mg/l

Tamilnadu Andhra Pradesh

Salem 4.5 Nalangonda 0.8-1.2
Madurai 4.5 Veliore 0.~-1.2
Coimbatore 4.5 Anantpur .0. 8- 1.2
Gujrat Punjab
Surat 4.0 Bhatinda 3.0
Mehsana 4.0 Ferozpur 3.0
Karnataka Haryana
Belgaum 0.8-1.2 Kamal 3.6
Rajasthan (Banswara)
Anandpuri 1.6-2.0
Garhi 1.6-3.3
Ghatola 3.4-4.7
Talwara 1.8-3.1

Choubisa (1997):IJEH,39(4):281-28B.
Analysis of Water and EfJluents 87

Estimation of Fluoride
APHA (1995) recommends the following three methods for fluoride estimation:
a. Ion-selective electrode method: 0.1 to more than 10 mg FIL.
b. SPANDS method (colorimetric): 0 to 1.4 mg FIL.
c. Automated complexone method (colorimetric): 0 to 2.0 mg FIL.


Under acidic condition fluorides (HF) react with zirconium-SPANDS solution and
the colour (colour of SPANDS reagent) gets bleached due to formation of a
colourless complex anion ZrF;-. Since bleaching is a function of fluoride ions, it is
directly proportional to the concentration offluoride (i.e. as the amount of fluoride
increases, the colour produced becomes progressively lighter).


Chlorine, colour and turbidity interfere and must be removed by distillation.

Interference caused by alkalinity, chloride, iron, phosphate and sulphate is not linear
and hence cannot be accounted mathematically.
Samples and standards should be at the same temperature or at most have a 2°C
temperature difference. Temperature is maintained nearly constant throughout colour
development. Different calibration curves are prepared for different temperature


1. Spectrophotometer for use at 570 nm, providing a light path of at least 1 cm or

2. Nessler's tubes with capacity of 100 mL.



I. Sodium fluoride
2. Sodium arsenite
3. ConcHCI
5. Zirconyl chloride octahydrate


1. Stock Fluoride solution (O.221g/L): Dissolve 221 mg anhydrous Sodium

fluoride (NaF) in distilled water and dilute to 1L. ImL = 100 ~g F or 0.1 mg F.
2. Preparation of fluorides standards: Take 10 mL of stock solution and diluted
to 100 mL (lmL=10 ~g F or 0.01 mg F); Prepare a series of standard fluoride
solutions in the range of 0.0 to 5.0 mgIL at intervals of 0.5 mgIL, by dilution of
stock solution with distilled water.
3. SPANDS solution: Dissolve 958 mg SPANOS, [Sodium 2-(para-
88 Handbook ofMethods in Environmental Studies

sulphophenylazo) - 1, 8- dibydroxide - 3, 6 napthalene dilsulphonate] in distilled

water and dilute to 500mL. This solution is stable indefmitely ifprotected from
4. Zirconly-acid reagent: Dissolve 133 mg zirconyl chloride octahydrate (ZtQe~.
8~O) in about 25 mL of distilled water. Add 350 mL conc. Hel and dilute to
500 mL with distilled water.
S. Mix solution (Acid zirconyl+ SPANDS reagent): Mix equal volume of
SPANOS solution and zirconyl acid reagent. The combined reagent is stable
for at least 2 years.
6. Reference solution: Add 10 mL of SPANOS solution to 100 mL distilled water.
Dilute 7 mL conc. Hel to 10 mL and add to diluted SPANOS solution. The
resulting solution, used for setting the reference point (zero) of the
spectrophotometer This solution is very stable and may be reused indefmitely.
7. Sodium arsenite solution: Dissolve 5.0 g NaAs02 and dilute to lL with distilled
water. (caution: Sodium arsenite is toxic avoid ingestion).
1. Preparation ofstandard curve: Prepare fluoride standards in the range of 0 to
lAO mg/L by diluting appropriate quantities of standard fluoride solution to 50
mL with distilled water (Table 3.10).
2. In each 50 mL of Nessler tube, add 10 mL mix solution (or 5 mL of SPANOS
and 5 mL zirconyl acid reagent). Mix well. Take absorbance immediately of the
bleached colour at 570 nm using reference solution for setting zero absorbance.
3. Plot standard graph between concentration and absorbance (optical density).

Table 3.10:
Preparation of Fluoride standard solutions (Fluoride stock solution
1mL = 10 I'g F (Final volume: 50 mL)

F stock solution, mL Fin I'g Distilled water, mL F cone, mgF/L

1 10 49 0.2
2. 20 48 0.4
4 40 46 0.8
6 60 44 1.2
8 80 42 1.6

4. F estimation in sample: Take 50 mL filtered sample and add 10 mL of mix

solution, mix well and read the absorbance immediately of the bleached colour
at 570 nm using reference solution for setting zero absorbance. (Advice: After
adding the mix solution take reading immediately)
5. If sample contain residual chlorine, remove it by adding NaAs02 solution; [1 drop
(0.05mL)=0.lmg el)]. Note: Sodium arsenite concentration of 1300 mg/L
produce an error ofO.lmg/L at ImgFIL).
6. If the transmission fall beyond the range of the standard curve, repeat the
procedure using a smaller sample.
Analysis of Water and Effluents 89

mg F determined photometrically B
Fluoride, mgIL = x 1000 x - -
mL sample used C
The ratio B/C applies only when a sample is diluted to a volume B, and a
portion C taken from it for colour development.

Estimation of Fludtfde After Distillation

In the event of interferences that cannot be controlled in the direct fluoride analysis
procedures, it is possible to remove the fluoride from solution selectively. Distillation
of either fluorosilicic or hydrofluoric acid from an acid solution of higher boiling
point separates the fluoride from other constituents in the original sample.
I. Sulphuric acid, ~SO4 ' concentrated.
2. Silver sulphate, A~S04' crystals.
Distillation assembly: Glassware consisting of a IL round bottom, borosilicate
boiling flask, an adaptor with a thermometer opening, a connecting tube, a condenser
and a thermometer reading up to 200 "C, assemble as shown in Fig. 3.16.

Fig. 3.16: Direct distillation apparatus for fluoride

90 Handbook o/Methods in Environmental Studies

1. Preparation of acid mixture:
a. Place 400 mL distilled water in the boiling flask and carefully add 200 mL
conc H2S04 •
b. Swirl until the flask contents are homogeneous.
c. Add 25-35 glass beads. Make sure that all joints are tight.
d. Start heating slowly at first, then as rapidlyre efficiency of the condenser
permits (the distillate must be cool) until the temperature of the flask contents
reach exactly 180°C.
e. Discard the distillate. The process removes fluoride contamination and adjusts
the acid: water ratio for subsequent distillation.
2. After cooling the acid mixture remaining after step 1 (to 120°C or below), add
300 mL of sample, mix thoroughly, and distil as before until the temperature
reaches 180°C. To prevent sulphate carry-over, do not heat above 180°C. When
high-chloride samples are distilled, add Ag2S04 to the boiling flask at the rate
of 5 mg/mg of chloride.
3. Use H2 S04 solution in the flask repeatedly until the contaminants from samples
accumulate to such as extent that recovery is affected or interferences appear in
the distillate. Check suitability of the acid periodically by distilling standard
fluoride samples, flush the distillation apparatus with 300 mL distilled water.
4. Use the distillate to conduct fluoride analysis. Correct the volumereiationship
if the distillate volume differs from that of the original sample.

Removal of Fluoride from Water

The fluoride present in drinking water is removed by passing through various
types of de fluoridation media, such as, tricalcium phosphate, bone char, bone meal
and activated alumina by a combination of ion-exchange and sorption. Fluoride
can also be removed during lime softening through co-precipitation with magne-
sium hydroxide or by alum coagulation, but at a quite high alum dose.

Check Your Confidence on Fluoride Estimation

1. Fluoride concentration less than 1 mg/L causes ........... and concentration greater
than 1.5 mg/L causes ............... .
2. Why Fluoride concentration is generally more in ground water than in surface
3. In what way does colour formation in the colorimetric procedure for fluoride
differ from that with most other colorimetric analysis?
4. What is the purpose of sample acidification prior to distillation to separate
fluoride from interfering ions.
5. "There will be a diffrence in F standards in tropical and temperate climate"-
justify your answer? Or, As per USA, MLC( 1975) , permissible F concentration
is 4 mg/L, whereas as per IS: 10500 (1991), maximum permissible F level in
drinking water is 1.0 mg/L. Why?
6. How to remove excessive fluorides from drinking water supplies?
Analysis o/Water and Effluents 91

3.14 Hardness (Total)

Drinking water (as mgIL of CaCOJ

WHO (1971) = 100 (Highest desirable) - 500 (Max. pennissible)
WHO (1984) = Max. 500 rnglL (aesthetic quality) - No health related guideline
value set by WHO.
WHO (1993) = No standards. High hardness causes scale deposition and scum
USEPA (1974) = No standards on hardness.
US Dept of Health, Environment and Welfare (1962): Recommended maxi-
mum of 500 mg/L of hardness in drinking water.
MWH (1975) = 200 (Highest desirable) - 600 (Max. penn is sible)
ICMR (1963) = 300 (Highest desirable) - 600 (Max. pennissible)
IS: 10500 (1991) = 300 (Highest value) - 600 (Max. pennissible)
Effluent discharge
MOEF (1993) = No guideline value set

Hardness is defined as the concentration of multivalent metallic cations in solution.
Hard water are generally considered to be those waters that requires considerable
amounts of soap to produce a foam and that also produce scale in hot-water pipes,
heaters, boilers, and other units in which the temperature of the water is increased.
The hardness of waters varies from place to place. In general, surface waters are
softer than groundwater's. The hardness of water reflects the nature of geological
fonnation with which it has been in contact. Water are commonly classified in
tenns of degree of hardness as in the Table 3.11.

Table 3.11 :
Classification of hard water

Hardness range (mg/L as CaC0 3 ) Degree of hardness

0-75 Soft
75-150 moderately hard
150-300 Hard
above 300 Very hard

Causes and sources of hardness: Hardness is caused by the presence of multi va-
lent metallic cations in water. The principle hardness causing cations are divalent
Ca2+, Mg2+, Sr2+ Fe2+ and Mn2+ and anions are HCOl ' S04' CI, NO l and SiOl .
Types of hardness: Carbonate hardness was fonnerly called temporary hardness,
because it can be removed by boiling and is caused by dissolved Ca and Mg bicar-
bonate. Removal process of Ca and Mg bicarbonate is as follows:
92 Handbook ofMethods in Environmental Studies

Ca(HCO J)2 + heat -+ CaCOJ (ppt)J.. + CO2+ Hp

Mg(HCO J )2 + heat -+ MgCO J (ppt)J.. + CO 2+ Hp

Carbonate hardness is especially important since it leads to scaling. Non-
carbonate hardness was formerly called permanent hardness, because it cannot be
removed by boiling. Non-carbonate hardness cations are associated with sulphate,
chloride and nitrate ions of Ca and Mg.

Environmental Significance
Soap consumption by hard-water caus~s economic loss to water consumers.
Precipitated foam by hard water adheres to surfaces of tubs, sinks etc and may
stain clothing, dishes and other items.
Mg hardness, particularly associated with sulphate ion, has a laxative effect in
person unaccustomed to it.
• Carbonate hardness precipitates leads to scaling in boiler, which causes
considerable economic loss.
Determination of hardness serves as a basis for routine control of softening

Estimation of Hardness
In alkaline condition ethylene-diamine-tetra-acetic (EDTA) acid or its sodium salt
(Na2EDTA) reacts with Ca and Mg to form a soluble chelated complex. Ca and Mg
ions develop wine red colour when small amount of dye such as Eriochrome Black
T is added under alkaline condition. When EDTA is added as titrant, the Ca and
Mg will be complexed with EDTA resulting in sharp change from wine red to blue,
which indicates end-point of titration. Hardness is normally expressed as CaCOJ •

M2+ + Eriochrome Black T -+ (M. Eriochrome Black.T)complcx

Wine red

M2+ + EDTA -+ [M.EDTA] compcx


The sharpness of the end point increases with increasing pH. However, pH can
not be increased beyond 10 because of the danger of precipitation ofCaCO J or Mg
(OH)2. The pH for this titration has been recommended at 10.0 ± 0.1. A limit of 5
minutes is set for the duration of the titration to minimise the tendency of CaCO)


1. Ammonium chloride, NH4Cl 2. Ammonium hydroxide, conc.

3. EDTA Mg salt 4. Eriochrome black-T (indicator)
Analysis of Water and Effluents 93

5. Calcium carbonate (AR) 6. HCl (1+1)

7. Na2EDTA salt 8. Hydroxylamine hydrochloride
9. Ethyl alcohol (C 2HPH) 10. Methyl red indicator

1. Buffer solution (pH 10): Dissolve 16.90 gNH4Cl in 143 rnL ofconc NHpH.
Add 1.25 g EDTA Mg salt to obtain sharp change in indicator and dilute to 250
rnL. Alternatively, instead of EDTA Mg salt, 1.179g Na2EDTA plus 0.780g
MgS04 .7Hp can also be used. This has to be titrated with standard calcium
solution to avoid interference produced by EDTA to the buffer. Store in a
plastic or borosilicate glass container for no longer than I month. Stopper tightly
to prevent loss of ammonia (NH3) or pick up of carbon dioxide (C0 2) from air.
2. Eriochrome black T indicator: Mix 0.5 g dye with 100 g NaCI to prepare dry
powder. or dissolve 0.5 g of indicator in 100 rnL of ethyl alcohol.
3. Standard calcium solution (O.OlN) (1 gIL): Weigh accurately 1.0 gAR grade
CaC03 (dried at 180 OC for Ih before weighing) and transfer to 250 mL conical
flask. Place a funnel in the neck of a flask and add 1+1 HCl till CaC0 3dissolves
completely. Add 200 mL distilled water and boil for 20-30 min to remove COl"
Cool and add methyl red indicator. Add NHpH (3N) till intermediate orange
colour develops. Dilute to 1000 rnL to obtain I rnL = I mg CaC03, (IrnL =
0.40 I mg Ca or 0.243 mg Mg).
4. Standard EDTA solution (O.OIM): Dissolve 3.723 g EDTA-disodium salt
and dilute to IL. Standarised against standard calcium solution. I rnL = I mg
CaC03• The reagent is stable for several weeks.
s. Inhibitor: Dissolve 1.5 g hydroxylamine hydrochloride in 100rnL 95% ethyl
alcohol or isopropyl alcohol.
PROCEDURE (Fig. 3.17)

I. Select a sample volume that requires less than 15 mL EDTA titrant and complete
titration within 5 minutes, measured from time of buffer addition.
2. Take 50 mL well mixed sample in a conical flask.
3. Add 1-2mL buffer solution followed by I rnL inhibitor. Usually 1-2 mL buffer
solution is sufficient to give a pH of 10
4. Add a pinch of Eriochrome black T and titrate with standard EDTA (O.OIM)
till wine red colour changes to blue. Note down the volume of EDTA consumed
5. Run a reagent blank with distilled water. Note the volume of EDTA consumed
6. Calculate the volume of EDTA required by sample; C = (A - B).
Low hardness sample: For softened water or natural water oflow hardness (less
than 5 mgIL), take a large sample, say 100 mL to 1000 rnL for titration. Add
proportionately larger amount of buffer, inhibitor and indicator.
• Observation-I: Standardisation of EDTA solution.
• Observation-II: Estimation of Total Hardness against standard EDTA
94 Handbook of Methods in Environmental Studies

0.01 N
Scheme of hardness determination

50 -L $MIpie
.'-1 _L
.'.l lilt.... lOIutioll
+Pinch of £8T "indicator "

WINf 11£0 ..... BlUf(f1ld PaInt)

Fig. 3.17: Estimation of Total Hardness (TItrimetric).

Total hardness (as CaC01 mgIL) = - - - - -
mL of sample
C = mL of EDTA required by sample for titnltion
D = mg CaC01 equivalent to ImL EDTA titrant (lmg for O.OIM EDTA used
here). .

3.15 Hardness (Calcium)

Drinking water
WHO (1963) = 75 mgIL as a maximum acceptable limit for International stan-
dards and 200 mgt!. as an excessive limit.
The USEPA (1991-95) = did not includeCa in the list of Secondary drinlPns.
water standards or express the intention to regulate Ca.
(IS: 10500, 1991) = 75 mgIL - 200 gIL (maximum pemiissible in the absence.
of alternate source)
ICMR (1963), MWH (1975) = 75 mgIL - 200 gIL (maximum permissible in ~e
Analysis of Water and Effluents 95

... cont.
absence of alternate source)
EC (1980) = 100 mgIL (guide level).
Effluent discharge
MOEF (1993) = No guideline value set.

Calcium and magnesium are common constituents of natural water and important
contributor to the hardness of water. The source of Ca and Mg is the rocks from
which it is leached. Being a important contributors of hardness, it reduces the utility
of water for domestic use. Some CaC03 is desirable for domestic water because it
provides a coating in the pipes which protects them against corrosion.

Estimation of Calcium Hardness

When EDTA is added to water containing both Ca and Mg, it combines first with
Ca. Calcium can be determined directly by EDTA, when the pH is made sufficiently
high so that Mg is largely precipitated as the hydroxide and an indicator is used that
combines with Ca only. Several indicators give a colour change when all of the Ca
has been complxed by the EDTA at a pH of 12 to 13. Mg is determined by the
differences between an aliquot titrate at pH 10 and the one titrated at pH 12 to 13.

1. Sodium hydroxide solution, 8% (80 gIL): Dissolve 8 g ofNaOH in distilled
water to prepare 100 mL of solution.
2. Murexide (ammonium purpurate) indicator: The indicator changes from
pink to purple at the end point. Mix 0.2 g of ammonium purpurate and 100 g of
sodium chloride and grind thoroughly to fine powder (i.e. one that passes through
a 40-mesh sieve).
3. EDTA solution (0.01 M): Dissolve 3.723 g ofN a2EDTA in 1L of distilled water.
1 mL = 0.401 mg Ca; ImL = 0.243 mg Mg.

1. Fill the burette with O.OIM EDTA standard solution. (Fig. 3.18)
2. Take 50 mL or a smaller portion diluted to 50 mL so that the Ca content is about
5 to 10 mg. in an Erlenmeyer flask.
3. Add 1 mL ofNaOH to raise pH to 12.0 and a pinch of murexide indicator (0.1
to 0.2 mg) or 1-2 drops of indicator solution. (Note: Titration should be
completed within 5 min of addition ofNaOH solution.)
4. Titrate immediately against EDTA solution till pink colour changes to purple.
Note the volume of EDTA used (A). Confirm the end points by adding 1 to 2
drops oftitrants in excess to make certain that no further colour changes occurs.
96 Handbook of Methods in Environmental Studies

(Note: If 15mL or more titrant is consumed, take a s'maller sample aliquot and
repeat the test).
5. Run a reagent blank with distilled water. Note the mL of EDTA required and
keep it aside to compare endpoints of the sample titration.

0.01 N

Scheme of calcium determination

[j SO .. l 5. ....
+l .. l 11I0Il
-+ Pinch at l1uruid. indicator


Fig.3 .18: Estimation of Ca-hardness (Titrimetric method).

T x 400.5 x 1.05
Calcium (mglL) =
T x 1000 x 1.05
Calcium hardness as CaCO J (mglL) =
T = volume of titrant, mL
V = volume of sample. mL.

3.16 Hardness (Magnesium)

Drinking water
WHO (International Standards of drinking water, 1963) = 50mgIL (max
acceptable level) - 150 mgIL (max allowable level).
Analysis o/Water and Effluents 97

WHO (1984, 1993) = No specific guideline set for Mg.
IS: 10500 (1983, 1991) = 30 mgIL (maximum, beyond this limit, encrustation
to water supply structure and adverse effects on domestic use) - 100 mgIL
(permissible limit in the absence of alternate source).
EC (1980) = 30 mg/L (guide level) and 50 mg/L (MAC).
Taste threshold for magnesium as sulphate has been reported as 100 mgIL and
500 mgIL for the average individuals.
Effluents discharge
MOEF (1993) = No guideline value set for Mg.

Magnesium has been considered as non-toxic to humans at the concentration
expected in water. Magnesium salts have a laxative and diuretic effect particularly
for individuals not accustoms to high dosage.

Estimation of Magnesium Hardness

Magnesium can be determined by calculating the difference between the total hard-
ness and the calcium hardness of the sample. This yields the value of magnesium
hardness as mgIL CaC03 which, when multiplied by 243.1, will be the concentra-
tion of magnesium.
Mg hardness as CaC03 = Total hardness as CaC03 - Ca hardness as CaC03
Magnesium, mgIL = Mg hardness as CaC03 mgIL x 0.2431

Example ~1
Which of the following solutions give 50 mg/L total hardness?
a. 50 mg/L MgC03
b. 21 .1 mg/L MgCO J + 25 mg/L CaC0 3
c. 50 mg/L CaSO.
d. 55 mg/L CaCl z

y;1':~j·, ,. ',,,e,'.,.
The concentratiopl>"gittle above. express~(rin ,mo a
" ,'.p~ f5); ,Q~O:37 ' a'I4id. O.:~q~t.1.ThQ h~@:iel>A~,~n~
'" ' ,withthe relative mo!ecurarweighto.fCIl9;O~ (Fj,1()P);iT
solutions lire ,as follows:'}:""" ,', :",,~,;,,;",.,,;
a. :60mg/L ase'i cc " ' , , '

, b. ' 5,9 mg/Las Ca~O~

c, 37tJ'Qg/L,as CaC03an~ "
,d. ,. 50 mg/L liS CaC0 3
98 Handbook ofMethods in Environmental Studies

A sample of groundwater has 100 mg/L of Ca 2+ and 10 mg/L of Mg2 +. Express
its hardness in units of meq/L and mg/L as CaC0 3 •

Removal of Hardness
In surface water, hardness values generally are 150-200 mg/L and softening is not
usually needed. However, in case of groundwaters, where the hardness level is
more than 1000 mg/L, softening is needed. The softening can be performed by
either lime soda process or ion-exchange process.

Check Your Confidence on Hardness Estimation

1. Are there any health-related guidelines for hardness as per WHO (1984)
2. Name important cations and anions that cause hardness in drinking water?
3. Discuss the principle involves in the EDTA tritimetric estimation of hardness?
4. Why titration should be done at pH 10 and within 5 minutes titration should be
5. How to estimate temporary hardness?
6. Would hardwater be acceptable in most drinking water supplies? Why or Why
7. Would hard water be an acceptable coolant for an industrial plant? Why or why
8. Can carbonate hardness be removed by boiling?
9. Name the important cations associated with non-carbonate hardness?
Analysis of Water and Effiuents 99

Example - 3
Water has the following composition: Calcium = 82mg/L, magnesium = 33mgl
L, sodium = 14 mg/L, bicarbonate = 280 mg/L, sulfate = 36mg/L and chloride
= 82 mg/L Determine carbonate hardness, noncarbonate hardness and total hard-
ness all in terms of mg/L of CaC0 3

SolUtion: ' .. ,' , ,

Step~ 1 : Convert anc()l)c~:mtration (rng/L) to meq/L and mglL as CaC0 3. Construct
'.'~ a 'table asfollows: . f ,,>;!'

Jo'n ,species Mol. Wt. ;';Equivalent ~t. Concentration

' mglL meq/L .• mg/L as CaC0 3

Ca 2 + ., 20.0 82 200
, MgH 12.2 32 , ",. 1,35

";" Na" 23 14 , ":'i;' 30

)'Total cations ·, 365
HCOJ- 61 , 280 230
CI- , 35.5 36 50
SO" 96:1 ," 82 85'
Total anions 365

5tep-2: Construct a bar diagram.

0 200 335 365

Ca 2•
I Mg2+
I Na+ Y·,·:

' HC03- I, SO.. 1
CI- "I
0 230 280

'Step-3: Compute . the hardness distribution: ','

Total hardness (20() + 135) = 335 mg/L as Ca~03.IAns.)
Alkalinity IbicarbonatE)t = 230 mg/L as CaCO J, <
Carbonate hardness ', ,,, alkalinity := 230 mg/L as CaC0 3(Ans.)
Non-carbonate hardness = 365 - 230 = 165 mg/L as CaC0 3.IAns.)

10. A sample of water has the following concentration of ions with the pH near
Cation mg/L Anion mglL
Ca 2+ 95 HC03' 160
Mg2+ 26 S04- 135
Na+ 15 CI- 73
a. What is the total hardness (TH)?
b. What is the carbonate hardness (CH)?
c. What is the non-carbonate hardness?
d. What is the TDS concentration?
e. What is the alkalinity?
• Hints for Q.lO(e): For neutral water, pH around 6-8, the Wand OH- are
insignificant and alkalinity is determined entirely by the bicarbonate. The
bicarbonate concentration is 160 mgIL.
100 Handbook ofMethods in Environmental Studies

Molecular wt bicarbonate = 61.0

Valency =1
meq = 61/1 = 61
meq/L = 160/61= 2.62
Alkalinity as mglL ofCaC03 is = 50 x 2.62 = 131.14 (Aos).
II. How hardness is removed from water?

3.17 Methylene Blue Active Substances


Drinking water
IS: 10500 (1983, 1991) = 0.2 mgIL. (Beyond 0.2 mgIL, it can cause a light
froth in water).
MHW (1975) = 0.2 mgIL (acceptable) and 1.0 mgIL (causes of rejection).
WHO (1984) = No guideline value set.
USEPA (1974) (under secondary drinking water standards) = 0.5 mgIL
Emuents discharge
MOEF (1993) = No guideline value set

Environmental Significance
I. The determination of methylene blue active substances gives an idea of the
presence of detergents in water.
2. The widespread use and discharge of detergents into surface waters can result
in lowering of its aesthetic quality by foam formation and by causing toxicity to
aquatic wildlife. .
3. Biodegradable linear alkyl benzene sulfonates (LAS) have replaced the branched
chain alkyl benzene sulfonates (ABS) in detergent formulations, which were
more resistant to biodegradation. Differentiation between linear and branched
chain alkyl benzene sulfonates, as well as differentiation of the various posi-
tional isomers of either type, is not possible by this test method. While the
methylene blue method may be employed to monitor studies designed to mf?a-
sure biodegradability, it cannot be used to predict this quality.

Estimation of MBAS
This test method covers determination of compounds that react with methylene
blue under the conditions specified in the test procedure. They are referred to as
methylene blue active substances (MBAS), and are calculated and reported in terms
of the reference material, linear alkyl benzene sulfonate (LAS).
Analysis o/Water and Effluents 101

This method is applicable for determining MBAS in water and wastewater. It is

a simple, rapid, control procedure suitable for monitoring the effectiveness ofbio-
degradation or other liner alkyl benzene sulfonate (LAS) removal process. For
greater specificity and interference removal ,the pre-treatment procedure should
be used. Data derived without pre-treatment procedure should be interpreted with
care. This method is applicable in the range from 0.03 to 1.5 mg/L, for a 100-mL

1. This test method is based upon the formation of a blue-coloured chloroform

extractable ion pair by the reaction of cationic methylene blue and an anionic
surfactant (including LAS, other sulfonates, and sulfate esters).
2. The sample is mixed with an acidified, aqueous solution of methylene blue.
Any resulting hydrophobic ion pair which may be formed is extracted success-
fully with chloroform. The combined chloroform extracts are washed with an
acid solution to remove the less hydrophobic ion pairs that can be formed by
potentially interfering substances. The chloroform layer retains the highly hy-
drophobic methylene blue-LAS ion pairs.
3. The intensity ofthe blue colour remaining in the chloroform extract is mea-
sured photometrically at the wavelength of maximum absorption near 650 nm.
This intensity is related to the concentration of LAS by means of a calibration

1. Samples may be preserved against biological oxidation by adding conc ~S04

to adjust the sample to pH 2 or less and storing at 4°C. Analyse the preserve
sample as soon as possible, or within 1 week after collection. Data on decom-
position are not available.
2. Rinse the sample container and cap well to free them of detergent if they have
been used previously and cleaned prior to recycling.


1. Chloroform
2. Methylene blue
3. Phenolphthalein Indicator
4. Sodium dihydrogen phosphate, NaHlO4.Hp
5. Sodium dioctyl sulfo succinate
6. H2S04 solution - 14%, v/v
7. NaOH solution
1. Linear Alkyl Benzene Sulfonate Solution Stock (1 mL= 1 mg LAS): Weigh
the amount of reference material (Le., sodium dioctyl sulfo succinate, Ig in 1L)
necessary to provide the equivalent of 1 g of LAS on a 10% active basis.
102 Handbook o/Methods in Environmental Studies

Dissolve in water and dilute to lL, mixing gently to prevent foam formation.
Record the molecular weight of the LAS reference material as supplied. The
stock solution may be stored at 4°C in the dark for 12 months in a well-stop-
pered flask without deterioration.
2. Linear Alkyl Benzene Sulfonate Standard Solution, (lmL= O.Olmg LAS):
Dilute 10.0 mL of the foam-free stock solution to lL with water that has been
previously adjusted to pH 2 with sulfuric acid and mix. The standard solution
may be stored at 4°C in the dark for at least 12 months in a well-stoppered flask
without deterioration.
3. Methylene Blue Solution (30 mglL): Dissolve O.lg of methylene blue chlo-
ride in 100 mL of water. Transfer 30 mL of this solution to a l-L volumetric
flasks and add 500 mL of water. Add carefully 50 mL of 14% sulfuric acid
stock solution and 50 g of sodium dihydrogen phosphate monohydrate
(NaHl04.HP). Shake until solution is complete and then dilute to lL with
water and mix.
4. Sulfuric Acid Stock Solution (14 % volume per volume): Add carefully 140
mL of cone H 2 S04 (sp gr 1.84) to 700 mL of cold (0 to SoC) water with good
stirring, dilute to I L with water and mix.
5. Sulfuric Acid Solution, Dilute (0.7% volume! volume): Dilute carefully 50mL
of 14% H 2S04 stock solution to IL with water and mix.
6. Phenolphthalein Indicator Solution (5g1L): Dissolve 0.5 g ofphenolphtha-
lein in SO mL of 9S% ethyl alcohol and dilute to 100 mL with water and mix.
7. Sodium Hydroxide Solution (10glL): Dissolve 10 g ofNaOH in water, dilute
to 1L and mix.
8. Phosphate Wash Solution: Dissolve SO g sodium dibydrogen phosphate mono-
hydrate (NaHl04.HP) in 500 mL of water in a I-L volumetric flask. Add
carefully 50 mL cone H 2 S04 stock solution and dilute to volume with water and
mix. The solution has a pH of approximately 1.8.

Preparation of calibration curve
Prepare a series of standards by adding the standard solution (2) from 25-mL
burette to a series of 250-mL separatory funnels and dilute the standards to 100
mL volume with water, yielding solutions as follows:

Standard, mL (lmL=O.OI mg LAS) LAS, mg (per 100 mL of extract)

0.00 (blank) 0.00

1.00 0.01
3.00 0.03
7.00 0.07
9.00 0.09
12.00 0.12
Analysis of Water and Effluents 103

1. Add 3 drops of phenolphthalein indicator solution (6) and just enough NaOH
(7) to produce a pink colour. Add dilute H2 S04 solution, in small increments
until the pink colour is barely discharged.
2. Add 25 mL of methylene blue solution (3) and mix. Add 25 mL of chloroform
and mix thoroughly for 30s with shaking. Vent carefully, permit the phases to
separate and then drain the chloroform layer into a second 250-mL separatory
funnel. Leave ~y emulsion layer in the first separatory funnel. Repeat the ex-
traction, serially, with two additional 25-mL portions of chloroform.
3. Add 50 mL of phosphate wash solution (8) to the combin-:d chloroform extract
in the second separatory funnel and shake vigorously for 30 seconds. Hold the
separatory funnel in a vertical position and swirl the contents. Allow to settle
for I minute. Filter the chloroform layer through a glass wool plug into a con-
ditioned 100-mL volumetric flask.
4. Add 20 mL of chloroform to the second separatory funnel and repeat the shak-
ing, swirling, and settling step. Combine the chloroform layer through the glass
wool into the volumetric flask. Add additional chloroform as needed to bring
the flask to 100-mL volume and mix thoroughly.
5. Using a 50-mm light-path cell, at 650-nm wavelength, set the photometer to
zero absorbance with the extract of the calibration blank.
6. Measure the absorbance of each of the extracts. Because of a tendency to fade
slowly, the absorbance of the extracted methylene blue complex should be mea-
sured within 30 min. Prepare a calibration curve by plotting photometer read-
ing in absorbance against concentration of LAS in mgl100 mL of extract on
rectilinear graph paper, and record the molecular weight of the LAS reference
material, as supplied, on the graph.

Estimation of MBAS from waterl wastewater sample

Select a volume of sample consistent with the anticipated LAS content. If the LAS
concentration is not expected to exceed 1 mgIL, use 100 mL sample. For LAS in
the 10 mgIL range, use a lO-mL sample diluted to 100 mL with water. Follow the
step 3 to 6 as described above.
Calculate and express as MBAS, the apparent concentration of linear alkyl ben-
zene sulfonate (ABS) as follows:

MBAS, mgIL = W x 1000/S

S = sample volume selected.
W = LAS in the sample from the calibration graph, mg.

Include in the report the molecular weight (mw) of the LAS used to prepare the
calibration curve. Report results as:
MBAS (calculated as LAS, mw.......... ) = ..........mgIL.
104 Handbook ofMethods in Environmental Studies

Check your Confidence on MBAS Estimation

I. Discuss the environmental significance ofMBAS estimation.
2. Discuss the principle involved in MBAS estimation.
3. What is LAS?

3.18 Nitrogen (Total)

(Total Kjeldahl Nitrogen, TKN)

Drinking water
IS: 10500 (1991) = No standards have been laid down.
Emuents discbarge
MOEF (1993) = 100 mgIL (maximum) for discharge into inland surface water
and marine coastal areas .

. Four forms of nitrogen namely; nitrate nitrogen (N03-N), nitrite nitrogen (N0z-N),
ammonical nitrogen'(NH3-N) and organic nitrogen may be present in waters and
wastewaters. Analytically, organic nitrogen and ammonical nitrogen can be deter-
mined together and have been referred to as total nitrogen or more correctly total
Kjeldahl nitrogen (TKN).
The presence of total nitrogen is accepted as a chemical evidence of recent
organic pollution particularly of animal origin. The nitrogen forms cause:
• Algal blooms
• Eutrophication
• Toxicity to fish and other organisms
• Health disorders by nitrate and nitrites such as methemoglobinemia.
Total Kjeldahl nitrogen (TKN) is the sum of nitrogen contained in the free
ammonia and other nitrogen compounds which are converted to ammonium sulfate
under the specific digestion conditions (ASTM, 1995). The most reliable results
are obtained with fresh samples. Commonly occurring concentration of TKN in
domestic effluents, according to Metcalf and Eddy (1995) is:
1. For strong sewage: TKN = 85 mgIL (organic, 35 mgIL + NH,-N, SO mgIL)
2. Medium strength: TKN = 40 mgIL (organic, 15 mgIL + NH,-N, 25 mgIL)
3. Low strength: TKN = 20 mgIL (organic, 8 mgIL + NH,-N, 12 mgIL)

Environmental Significance and Use

• Used for measuring organic nitrogen and ammonical nitrogen which are essen-
tial nutrients for growth.
Analysis of Water and EfJluents 105

• Nitrogen is a nutrient for photosynthetic organisms, thus it is important to monitor

and control its discharge into the environment.

Estimation of TKN
Conversion of organic nitrogen to ammonia may occur in samples between collec-
tion and analysis. This effect can be decreased by addition of2 mL H2SO. (sp. gr.
= 1.84) per litre and storage at 4"C. However, it is prudent to analyse all samples as
soon as possible after collection.
In the presence ofconcentrated. sulphuric acid (l\SO.), potassium sulphate ~SO,>
and cupric sulphate (CuSO.> catalyst organically bound nitrogen gets converted
into ammonium bisulphate. Potassium sulphate is added to raise the boiling point
of sulphuric acid from 345"C to 375"C. The digest is diluted, made alkaline with
NaOH and distilled. The liberated ammonia is absorbed in boric acid. The ab-
sorbed ammonia is determined either by titration or by direct nesslerization. Tem-
perature should be kept below 382 "C during digestion to avoid loss of nitrogen.
A Kjeldahl digestion apparatus with 800 mL flask. (Fig. 3.19).

so mL Samplp
+so mL di9.~ton



Fig 3.19: A Kjeldahl digestion and distillation apparatus.

106 Handbook of Methods in Environmental Studies



I. Cupric sulfate, CuS04

2. Conc HZS04
3. Sodium hydroxide
4. Methyl red
5~Boric acid, H)BO)
6. Sodium carbonate, NaZCO)
7. Potassium sulfate, KzS04
8. Phenolphthalein indicator
9. Sodium thiosulfate
10 Methyl blue
II. Ethyl alcohol


Prepare all reagents and dilutions in ammonia-free water.

1. Digestion reagent: Dissolve 134 g ofKzS04 and 7.3 g CuS04in about 800 mL
distilled water. Carefully add 134 mL conc H ZS0 4• When it has cooled to room
temperature, dilute the solution to I L with water. Mix well. Store at a tempera-
ture above 14°C (close to 20°C) to prevent crystallisation.
2. Phenolphthalein indicator (pH 8.3 indicator): Dissolve 5 gin 500 mL 95%
ethyl alcohol. Add 500 mL distilled water. Add dropwise 0.02N NaOH till faint
pink colour appears.
3. Sodium hydroxide-sodium thiosulfate reagent: Dissolve 500 g NaOH and
25 g NaZSp).5Hp in ammonia free distilled water and dilute to IL.
4. Mixed indicator: Dissolve 200 mg of methyl red in 100 mL 95% ethyl alcohol
and 100 mg methyl blue in 50 mL 95% ethyl alcohol. Mix the two solutions.
S. Boric acid + mixed indicator solution: Dissolve 20 g H)BO) in distilled wa-
ter, add 10 mL mixed indicator and dilute to 1000 mL.
6. Standard H 1SO4 solution (O.02N): Prepare a stock solution of approx. 0.1 N
HZS0 4 by diluting 3mL conc. HZS04(sp. gr. 1.84) to 1000mL with water. Dilute
20mL of~S04 (assume O.IN) to 100mL to obtain standard 0.02 N ~S04.
Standardise it against 0.02N standard NaZCO). Dissolve 1.060 g of oven dried
at 1400C anhydrous NaZCO) diluting to IL with water to prepare 0.02N solu-
tion. (For 0.02N, ImL HZS0 4 = 280 ~g N = 0.28 mg N).

The important steps in determination ofTKN are as follows:

• Selection of sample volume
• Digestion of sample
• Distillation of sample
Analysis of Water and Effluents 107

Selection of sample volume

Organic-N in sample, mg/L Sample size, mL

0-1 500
1-10 250
10-20 100
20-50 50
50-100 25
Place a measured volume of sample in an 800-mL Kjeldahl flask. Select the
sample size based on concentration of organic nitrogen present in the sample. Pre-
pare a 500 mL reagent blank.

Digestion of the sample

1. Take 50 mL sample in a 800-mL Kjeldahl flask. (Selection of sample volume

will be based on expected concentration of organic-N present in the sample).
Add few glass beads.
2. Then carefully add 50 mL of digestion reagent and heat.
3. As digestion continues, coloured - turbid samples will become transparent and
pale green. After digestion, allow it to cool, dilute to 300 mL with water and
mix well.
4. Tilt the flask away from the face, and carefully add 50 mL of sodium hydrox-
ide-thiosulfate reagent to form an alkaline layer at flask bottom. Swirl the
Kjeldahl flask to ensure complete mixing. The pH of the solution should
exceed 11.


1. Take 50 mL of boric acid cum indicator solution (pink colour solution) in 500
mL of flask and place it below the condenser of the distillation assembly so that
the lower open end of the condenser is dipped in solution.
2. Collect at least 200 mL of distillate in flask (Pink solution turns to green due to
absorption of ammonia). It is good, to put a mark at the level of250 mL in the
flask, so that one could easily know when 200 mL distillate will be collected.
Remove the flask with distillate.
3. Titrate the distillate with O.2N H 2S04 to its original colour (i.e.,change of green
to pink colour indicates the end point of titration)
4. Run distilled water blank in the same manner.


(A - B) x 280
Total-N (TKN) ,mg/L= - - - - -
mL of Sample

A= volume of H2S04 required for sample, mL
B= volume of H 2S04 required for blank, mL
108 Handbook of Methods in Environmental Studies

If normality of H 2S04 is not 0.02N, then use following formula:

(A-B) x N x 14 x 1000
Total N (TKN) (mgIL) = - - - - - - -
mL of sample.

N= normality of H 2S04 use as titrant.

Check Your Confidence on Total Nitrogen (TKN)

1. What form of nitrogen is generally present in water and wastewater?
2. What is TKN and what it consists of'?
3. List three major impacts of nitrogen pollution.
4. Why TKN should be analysed as soon as possible in the laboratory?
5. What is the purpose of addition ofK2 S04 in the digestion catalysts mixture?
6. "Temperature should be kept below 380°C during digestion of sample". Why?
7. How organic nitrogen is calculated?

3.19 Nitrogen (Ammonical) (NH3-N)

Drinking water
IS: 10500 (1983, 1991) = No standards
Canadian MACL (1987) = 0.5 mgIL.
Effluents discharge
MOEF (1993) = 50 mgIL (max.) for inland surface water, public sewers and
marine coastal areas; Free ammonia (as NH) ) = 5 mgIL for inland surface
water and marine coastal areas.

Ammonia is present naturally in surface waters and wastewater. Its concentra-

tion is generally low in groundwater because it is absorbs by soil particles and
clays and not leached readily from soils. Ammonia concentration encountered in
water varies from less than 10 Ilg ammonia- NIL in some natural surface and ground-
water to more than 30 mgIL in some wastewater.
When nitrogenous organic matter is destroyed by microbial activity; ammonia
is produced and is therefore found many surface and groundwater. The properties
of the two forms of ammonia nitrogen i.e. free ammonia and ammonium ions,
depends on the pH as below.

pH 6 7 8 9 10 11
%NH) 0 I 4 25 78 96
% NH; 100 99 96 75 22
Analysis of Water and Effluents 109

If it is not possible to carry out the detennination soon after sampling, the sample
should be refrigerated at 4°C. Chemical preservation can be achieved by adding
either 20-40 mg HgCI2 or I mL HzSO4 to I L of sample.
Four different methods for detennining ammonia-N are included in APHA
(1993). The selection of methods depends on the concentration of ammonia and
presence of interferences. These methods are:
I. Two colorimetric methods: The Nesslerisation and phenate method.
• The Nesslerization method has been dropped as a standard method (APHA,
1995), although it has been considered as a classic water quality measurement
for more than a century. The use of mercury in this test and its problems of
disposal were the reasons of deletion.
• The phenate method is used to analyse both freshwater and seawater for low
NH)-N concentration.
2. One volumetric method: The distillation followed by titration is used for NH)-
N concentration greater than 5 mgIL.
3. The instrumental method using an ammonia-selective electrode is applicable
over range from 0.03 to 1400 mg N~-NIL.

3.19.1 E_stimation of Ammonia by Nesslerization

(Color;met;c method)
Use direct Nesslerisation for purified drinking waters, natural water, and highly
purified wastewater effluents, all of which should be low in colour and have NH)-
N concentration not exceeding 20 mg NH)-NIL.

Ammonia produces a yellowish-brown coloured compound when reacted with
alkaline Nessler reagent (KzHgI4 or 2KI + HgI z)' provided the sample is clarified
properly. Pretreatment with ZnSO4 and NaOH precipitates Ca, Fe, Mg, and sulfide,
which fonn turbidity and apparent colour. Addition of EDTA (before Nessler's
reagent) or Rochelle salt solution prevents precipitation of residual Ca and Mg in
presence of the alkaline Nessler's reagent.
2KzHgI4 + NH) + 3KOH -+ I-Hg-O-Hg-NHz + 7KI + 2H zO
(yellow-brown colour)

Colour, turbidity, Ca, Mg, salts and Fe in the sample constitute the prime sources
of interference.
I. Spectrophotometer, use at 400 to 500 nm.
2. pH meter.
110 Handbook ofMethods in Environmental Studies

1. Zinc sulfate
2. Sodium hydroxide
3. Potassium iodide, KI
4. Rochelle salt (potassium sodium tartrate tetrahydrate)
6. Mercuric iodide
7. Ammonium chloride

1. Zinc sulphate solution: Dissolve 100 g ofZnS04 .7Hp in distilled water and
dilute to 100 mL.
2. Sodium hydroxide, 6N: Dissolve 24 g ofNaOH and dilute to 100 mL.
3. EDTA reagent: Dissolve 50 g of EDTA in 60 mL of water containing 109
NaOH. If necessary, apply gentle heat to complete dissolution. Cool to room
temperature and dilute to 100 mL.
4. Rochelle salt solution: Dissolve 50 g of potassium sodium tartrate tetrahydrate
(KNaC4Hp6.4HP) in 100 mL of water. Remove NH3 by boiling off 30 mL
solution. After cooling dilute to 100 mL.
5. Nessler reagent: Dissolve 1OOg HgCl2 and 70 g KI in a small quantity ofwater.
Add this mixture to a cooled solution of 160 g NaOH in 500 mL of water and
dilute to 1L. Keep overnight. Filter through a glass fibre filter before using.
Store supernatant in a coloured bottle.
6. Stock ammonia solution: Dissolve 3.819 g anhydrous NH4Cl, dried at 100°C,
dilute to IL. 1 mL = 1 mg N= 1.22 mg NH 3•
7. Standard ammonia solution: Dilute 10 mL of stock ammonia solution to IL
with distilled water. ImL=lO ~g N = 12.2 ~g NH3 •


1. Take 100 mL of sample in a flask

2. Add 1 mL of ZnS04 solution and or 0.50 mL NaOH to obtain a pH of 10.5.
Allow to settle and filter the supernatant through Whatman filter paper No. 42.
3. Take a suitable aliquot of sample and dilute to 50 mL.
4. Add 3 drop of Rochelle salt solution or 1 drop of EDTA. Mix well.
5. Add 3 mL Nessler's reagent, ifEDTA is used or add 1 mL Nessler's reagent if
Rochells salt solution is used. Make up to 100 mL.
6. Mix well and read absorbance (optical density) after 10 minutes at 410 nm
against a blank prepared in the same way using distilled water instead of sample.
7. Prepare a calibration curve using suitable aliquots of standard solution in the
range of 5 to 120 ~g/I 00 mL (0.5 to 1.2 mgIL). For reference follow the same
procedure as per steps I to 5 above, but use the standard solution in place of
8. The value of ammonia is obtained from the standard graph and multiplied by
the dilution factor.
Analysis of Water and Effluents 111

3.19.2 Estimation of Ammonia by Titration


When sample contains more than 2 mgIL of ammonia-N, as in the case with
domestic sewage and many industrial wastes, the concentration can be determined
by titration with a standard solution of H2S04 after distillation and absorption of
ammonia in boric acid solution.


The Table 3.12 is useful in selecting sample volume for the distillation followed
by titration method.

Table 3.12:
Selection of sample volume for ammonia estimation.

Ammonia-N in sample. mg/L Sample volume. mL

5-10 250
10-20 100
20-50 50
50-100 25


Kjeldahl distillation assembly (Fig. 3.20).


1. Sodium tetraborate
2. Sodium thiosulfate, Na2Sp3.5Hp
3. Ethyl alcohol
4. Boric acid, H3B03
5. Sodium carbonate, Na2C03
6. Sodium hydroxide, NaOH
7. Methyl red indicator
9. H2 S04


1. Use ammonia-free water in making all reagents and dilution: This should be
prepared for each batch of samples.
Distillation to prepare ammoniafree water: To each lL of tap water add 2mL
ofa solution of ferrous sulfate (100 gIL FeS04 .7HP) and sufficient H2S04 to a
slight acid reaction to methyl orange. Distil with care. Reject the fIrst 50 mL of
distillate and then proceed until three-quarters of the volume of water has dis-
tilled over. Test the absence of ammonia in the distillate with Nessler's reagent.
2. Borate buffer solution: Add 88 mL 0.1 N NaOH (4g1L) solution to 500 mL
112 Handbook ofMethods in Environmental Studies

approximately 0.025M di-sodium tetraborate (NalBp,) solution (9.5g

NalBp,.lO Hp per litre) and dilute to 1000 mL.
3. Sodium hydroxide, 6N : Dissolve 24g ofNaOH and dilute to 100 mL.
4. Mixed indicator solution: Dissolve 200 mg of methyl red indicator in 100 mL
95% ethyl alcohol. Dissolve 100 mg methyle blue in 90 mL 95% ethyl alcohol.
Combine these two solutions. Prepare monthly.
S. Indicator boric acid solution: Dissolve 20 g boric acid in ammonia free dis-
tilled water, add 10 mL mixed indicator solution and dilute to IL. Prepare
6. Standard sulfuric acid titrant, O.02N: Prepare O.IN HlS04 by diluting 3mL
conc. HlS04 to 1L. Dilute appropriate volume ofHlS04 to 1L to obtained 0.02N
Hl S04 • 1 mL of 0.02N Hl S04 = 0.28 mg N. Standardise it against standard
0.02 N NalCO) solution
7. Dechlorinating agent: For chlorinated drinking water and effluent samples.
Use 1 mL of either (a or b) of the following reagents to remove 1 mgIL of
residual chlorine in 500 mL sample
a. Sodium thiosulfate: Dissolve 3.5 g NalSPr5HP in water and dilute to 1 L.
Prepare weekly.
b. Sodium sulfite: Dissolve 0.9 g NalSOJ in water and dilute to 1L. Prepare weekly.
PROCEDURE (Fig. 3.20)
Distillation of NH3
1. Take 100 mL of sample in a 500 mL ofKjeldahl flask and add 20 mL of borate
buffer and adjust pH to 9.0 with 6N NaOH solution. Add a few glass-beads of
boiling chips.
2. The contents is heated on low heat for first 10-30 minutes and raise the heat
thereafter for about an 1 h to released all the residual ammonia.
3. Take 50 mL of boric acid cum indicator solution (pink colour solution) in 500
mL of flask and place it below the condenser of the distillation assembly so that
the lower open end of the condenser is dipped in solution.
4. Collect at least 200 mL of distillate in a flask (Pink solution turns to green due
to absorption of ammonia). It is good, to put a mark at the level of 250 mL in
the flask, so that one could easily know when 200 mL distillate is collected.
5. Remove the flask with distillate. Titrate the distillate with 0.02N Hl S04, to its
original colour (i.e. green to pink colour indicates the end point of titration).
6. Run a distilled water blank in the same manner.
(T J - T l ) x 280
NH)-N,mgIL= - - - - -
Ifnormality of HlS04 is other than 0.02N, use following formula:
(T - Tl ) x N x 14 x 1000
NH)-N, mgIL = --=---=-------

mL of Sample
Analysis of Water and Eflluents 113

T. = Volume of H2S04 titrant used against sample (mL)
T2 = Volume of H S0 titrant used against distilled water Blank (mL)
2 4
N = Nonnality of H2S04 titrant.

100llll ~ ..
• 20 fill borat.

,Ollll boric acid- Gr..n _ Pink

mi.,d .ndiUltor
(tnd point)

Fig. 3.20: Ammonia estimation by distillation followed by titration.

Check Your Confidence on Ammonical Nitrogen

1. What are the sources ofNH)-N in water and domestic sewage?
2. List different methods of estimation ofNH)-N as per Standard Methods.
3. Why Nesslerization method has been discontinued as standard method in APHA
4. Why estimation ofNH)-N is done under alkaline conditions?
5. What is Nessler's reagent?
6. What is the purpose of adding EDTA or Rochelle salt?
7. How to prepare ammonia-free distilled water in the laboratory?

3.20 Nitrogen (Nitrate) (N03-N)

Drinking water
IS: 10500 (1983) = 45 mgIL (maximum)
IS: 10500 (1991) = 45 mgIL (maximum, beyond this limit methemoglobinemia
takes place); 100 mgIL(maximum pennissible in the absence ofaltemate source).
WHO (1984) = 10 mgIL
U.S.EPA (1974) = 10 mgIL
114 Handbook of Methods in Environmental Studies

... cont.
Effluents discharge:
MOEF (1993) = 10 mgIL (inland surface waters) and 20 mgIL (marine coastal
Stream standards:
CPCB (1979) = 20 mgIL (Class A); and 50 mgIL (Class C)

Environmental Significance
1. Nitrate nitrogen (NO]-N) is the highest oxidisable form of nitrogen and occurs
in trace quantities in surface waters but may attain high levels in some ground-
2. Nitrate is an important plant nutrient and causes eutrophicatiori in receiving
water bodies.
3. High concentration in drinking water (>40 mgIL) may cause blue-baby disease.
Certain bacteria commonly found in the intestinal tract of infants can convert
nitrates (NO;) to highly toxic nitrites (NO;). Nitrites have a greater affmity for
haemoglobin in the blood stream than does oxygen, they replace that needed
oxygen, a condition is known as methemoglobinemia. The resulting oxygen
starvation causes a bluish discoloration of the infant and known as blue-baby
syndrome. In extreme. cases, the victims may die from suffocation. Usually
after the age of about 6 months, the digestive system of a child is sufficiently
developed that this syndrome does not occur.

Estimation of Nitrate
Following methods are available for determination of nitrate:
a. Ultraviolet spectrophotometric method ,
b. Ion chromatographic method
c. Nitrate electrode method
d. Cadmium reduction method (NO; is reduced to NO;).
e. Devarda's alloy method (reduction to ammonia) (UNEP/WHO, 1996).
f. Nitrate can also be determined colorimetrically by phenol disulphonic acid
method and Brucine method.


Start nitrate determination promptly after sampling. If storage is necessary, store

for up to 24 hr at 4°C; for longer storage preserve with 2mL conc. H2SO/L and
store at 4OC. When sample is preseryed with acid, NO; and NO; cannot be deter-
mined individually.

3.20.1 Estimation of Nitrate by Ultraviolet (UV)

Spectrophotometric Screening Method
A{l, ultraviolet (UV) technique measures the absorbance of nitrate at 220 om, which
Analysis o/Water and Effluents 115

is suitable for screening uncontaminated water (low in organic matter). A second

measurement made at 275 nm, may be used to correct the nitrate value (because
275 nm is not absorbed by nitrate, but absorbed by other matter). The nitrate cali-
bration curve follows Beer's law up to II mg N0 3-N/L.

Spectrophotometer, for use at 220 nm and 275 nm with matched silica cell of Icm
or longer path.

1. Stock nitrate solution (ImL= 100 Ilg N03-N): Dry potassium nitrate (KNO])
in an oven at 105°C for 24 h. Dissolve 721.8 mg in water and dilute to 1000 mL.
Preserve with 2 mL chloroform per L. This solution is stable for at least
6 months.
2. Intermediate nitrate solution (1 mL = 10 Ilg N03-N): Dilute 100 mL of
stock nitrate solution to 1000 mL with water. Preserve with 2 mL chloroform
per L. This solution is stable for at least 6 months.
3. Hydrochloric acid solution, IN: Dilute 83 mL of cone. HCI to IL.

1. Treatment of sample
Take 50 mL of clear sample and (filter it, if necessary) add I mL HCl solution
and mix thoroughly.
2. Preparation of standard curve
Prepare nitrate calibration standards in the range of 0 to 7 mg NOJ-NIL. Dilute
the following volumes of intermediate nitrate solution i.e., 0, 1,2,4,7, 10, 15,
20,25,30 and 35 mL to 50 mL with distilled water. Treat the standards in same
manner as sample.
3. Spectrophotometric reading
Read absorbance against distilled water set at zero absorbance. Use a wave-
lengt~ of 220 nm to obtain N0 3-N reading and a wavelength of 275 nm to
determine interference due to dissolved organic matter.

I. For samples and standards, subtract two times the absorbance reading at 275
nm from the reading at 220 nm to obtain absorbance due to NOJ-N only.
2. Construct a standard curve by plotting absorbance due to nitrate against
NOJ-N concentration.
3. Obtain sample concentration directly from the standard c' .rve.
Note: If correction value is more than 10% of the reading at 220 nm, do not use this
116 Handbook of Methods in Environmental Studies

3.20.2 Estimation of Nitrate by Devarda's Alloy

This method involves oxidation, distillation and titration. One of its attractive fea-
tures is that it can be perfonned on the residues remaining in the blank flask after
the distillation process required in the detennination of ammonia nitrogen.

This method is suitable for nitrate concentration exceeding I mgIL especially for
wastewater and polluted surface waters. This analysis may be carried out on the
original sample or on the residue from the detennination of ammonia.
Nitrate is reduced to ammonia by nascent hydrogen by the use of Devarda's
alloy (59% AI, 39% Cu, 2% Zn). The resulting ammonia is distilled and its con-
centration detennined by titration.

Ammonia must be removed from the sample before the main procedure is started.
This is achieved either by pre-treatment or by using the distillation residue from
the detennination of ammonia The air in the laboratory and the distilled water
used for solutions and during the procedure should be free of ammonia.

The same glassware and distilling apparatus as for the ammonia nitrogen detenni-

The following reagents are required in addition to those used for the ammonia
nitrogen detennination.
1. Mixed indicator solution
2. Boric acid, 2% (20 gIL)
3. Devarda's alloy, powdered: If fme powder is not available, the material
should be ground to pass through a 0.07-0.1 mm sieve (200-140 mesh). It should
be as free as possible from nitrogen.
4. Hel, 0.00714 N: Dilute 6 mL conc. HCI (12N) to 1000 mL and standardise
against sodium carbonate solution.
5. Sodium hydroxide, ION: 400 gIL.
For determination in an original sample
I. Before assembling the apparatus, thoroughly clean the distillation flask and
condenser. In order to free the apparatus from possible contamination by am-
monia, add to the flask about 350 mL water, preferably ammonia-free, and
distil until the distillate is shown to be free from ammonia by testing with
Nessler's reagent. Empty the distillation flask and allow it to cool.
Analysis of Water and Ejjluents 117

2. Measure 200 mL of the sample in the flask.

3. Add 10 mL of ION NaO H. Evaporate in the distillation flask to about 100 mL.
Allow the residue to cool.
4. Continue as indicated in step 6 onward, below.
For determination in the residue from the analysis for ammonia
5. At the end of the distillation procedure for the analysis of ammonia, allow the
residue to cool.
6. To the cool residue add sufficient ammonia-free water to bring the volume in
the distillation flask to about 350 mL. Add 1 g ofDevarda's alloy and immedi-
ately connect the flask to the condenser.
7. After few minutes start the distillation, keeping the lower end of the delivery
tube from the condenser below the surface of the liquid in the receiver through-
out the distillation.
8. Place 50mL of the indicating boric acid solution in the receiver and distil at a
rate of about 10mL per minute.
9. As the absorbent solution changes colour, titrate with 0.00714 N HCI, continu-
ing the distillation until the addition of one drop of the standard acid produces
a permanent pink colour in the solution.
10. After the completion of titration, remove the receiver from the apparatus before
the source of heat is withdrawn.
11. Carry out the blank determination as appropriate. For each sample, correct the
fmal titration figure for any ammonia in the reagent used.
(A - B) x N x 14 x 1000
Nitrate nitrogen (as N), mgIL = -n
A = volume of 0.0714 N acid used for titration of the distillate of the sample,
B = volume of 0.0714 N acid used for titration of the distillate of the blank, mL
V = volume of the undiluted sample, mL.
n = concentration ofnitrite-N, in mgIL determined separately.
Note: The same calculation without subtracting nitrite gives the result for total
oxidised nitrogen, i.e. nitrate + nitrite.
The result is reported as nitrate nitrogen (as N) mgIL and should be rounded to
two significant figures.

3.20.3 Estimation of Nitrate by Phenol Disulph-

onic Acid (PDA) Method
Nitrate reacts with phenol disulphonic acid and produces a nitro-derivative which
in alkaline solution develops yellow colour due to rearrangement of its structure.
118 Handbook ofMethods in Environmental Studies

The yellow colour follows Beer's law and is proportional to the concentration of
nitrate present in the sample.

Chloride and nitrite are the two main courses of interferences. Pretreatment of
sample is necessary when the interfering radicals are present.

I. Spectrocolorimeter or spectrophotometer having a range of 300-700nm.

2. Nessler's tubes: capacity, 100 mL
3. Beakers: capacity, 100 mL

1. Ag2S04
2. Conc. H2S04 (sp. gr., 1.84)
3. KOH
5. Phenol
6. NHpH, Conc.
7. KN0 3
8. Potash alum, Aluminium hydroxide
1. Standard silver sulfate: Dissolve 4.40 g A~S04 in distilled water and dilute
to 1000 mL; 1 mL = 1 mg Cl.
2. Phenol disulfonic acid (PDA): Dissolve 25 g white phenol in 150 mL conc.
H2 S04 , Add 75 mL fuming H2 S04(15 % free S03)' stir well and heat for 2 h on
water-bath. If fuming sulfuric acid is not available, add additional 85 mL conc.
H2 S04 to the 150 mL H2 S04 , stir well and heat for 2 h.
3. Ammonium hydroxide: Concentrated
4. Potassium hydroxide (12 N): Dissolve 673 g KOH in distilled water and
make up to 1000 mL with distilled water.
5. Stock nitrate solution: Dissolve 721.8 mg anhydrous potassium nitrate and
dilute to 1000 mL with distilled water; 1mL = 100 Ilg N03-N
6. Standard nitrate solution: Evaporate 50 mL stock nitrate solution to dryness
on water-bath. Dissolve residue in 2 mL PDA reagent and dilute to 500 mL.
1 mL = 10 Ilg N.
7. EDTA: Weigh 50 g EDTA and make a paste with 20 mL distilled water. Add 60
mL NHpH and mix well.
8. Aluminium hydroxide: Dissolve 125 g potash alum in 1000 mL. Heat to 60°C
and add 55-60 mL NHpH and allow to stand for 1 h. Decant the supernatant
and wash the precipitate a number of times till it is free from CI or nitrite.
Analysis o/Water and Effluents 119

I. Pre-treatment of sample
1. Colour removal: If the sample has a colour in excess of 10units, add 3 mL
aluminium hydroxide to 150 mL of sample. Stir well and allow to settle for a
few minutes. Filter and use the filtrate discarding the first portion of the
2. Nitrite removal: (a) Generally nitrite occurs along with ammonia and gets eliPli-
nated in the routine test due to decomposition of nitrite and NH; to N2 (b)
Oxidise NO~ to NO; under acidic condition using KMn04 (c) Add sulfamic
acid to the sample to suppress nitrite interference.
3. Chloride removal: Determine the chloride content of the sample and precipi-
tate out it as AgCI by using silver sulphate solution at the rate of ImL to re-
move 1mg of chloride. One should be very careful while adding A~SO 4 be-
cause excess Ag will precipitates as silver oxide when alkali is added to the
II. Colour development
4. Neutralize the clarified sample to pH 7.0.
5. Take a suitable aliquot of the sample in a beaker and evaporate to dryness on
6. Dissolve the residue using glass rod with 2mL phenol disulfonic acid (PDA)
reagent. Dilute to the original volume and transfer to a Nessler tube.
7. Add 8-lOmL 12 N KOH. If turbidity is developed add the EDTA reagent drop-
wise till it dissolves. Filter and make up to 100mL.
8. Prepare blank in the same way using distilled water instead of sample.
9. Read the colour developed at 410 nm with a light path of 1 cm. Record nitrate
nirogen in mgIL from the standard graph.
10. Prepare calibration curve using suitable aliquots of standard nitrate in the range
of 0 to Img NIL following the above procedure.
I. Construct a standard curve by plotting the nitrate-nitrogen in mglL against
2. Obtain sample concentration directly from the standard curve.

Check Your Confidence on Nitrate Estimation

I. Why nitrate is found in lesser quantities in surface water and higher quantities
in ground water?
2. List two important environmental significance of nitrate.
3. What are the different methods are used for nitrate estimation?
4. What are the advantages of Devarda's alloy method for nitrate estimation?
5. What is the principle ofPDA method of nitrate estimation?
6. How nitrate is measured by UV -spectrophotometric screening method?
120 Handbook of Methods in Environmental Studies

3.21 Nitrogen (Nitrite) (N02-N)

Drinking water: No Standards (IS: 10500, 1991; WHO, 1989, 1993; USEPA,
1974; ICMR, 1963).
Effluents discharge: No Standards (MOEF, 1993)

Nitrite (NO~) is fonned in waters by oxidation of ammonia compounds (by aerobic
nitrifying bacteria, e.g. l'!itrosomonas) or by reduction of nitrate (by facultative
anaerobic denitrifyiub bacteria, e.g. Pseudomonas). Such oxidation and reduction
may occur in wastewater treatment plants, water distribution system, and natural
waters. As an intennediate stage in nitrogen cycle, it is unstable. Very high nitrite
levels are usually associated with waters having unsatisfactory microbiological
APHA (1995) described two methods of nitrite-nitrogen estimation:
1. Colorimetric method: Suitable for concentration of 5 to 1000 Ilg NOz-NIL (ap-
plicable in the range of 0.05 - Img/L of N0 2-N). Sample containing higher
concentration must be diluted. (UNEPIWHO, 1996 also recommended this
2. Ion Chromatography method: Not discussed here.

PRINCIPLE (Colorimetric method)

Under acidic condition (PH 2-2.5) nitrite ion (NO~) as nitrous acid (HNO z) reacts
with sulfanilamide to fonn a diazonium salt, which combines with N-(l-naphthyl)-
ethylenediamine dihydrochloride (NED dihydrochloride) to fonn a bright coloured
pinkish red azo-dye. The colour produced is directly proportional to the amount of
nitrite present in the sample. The colour obeys Beer's law up to 180 Ilg IL with 1
cm light path at 543 nm. Higher nitrite concentration can be detennined by diluting
a sample.

Sulfanilamide + HNO z + HCI ~ Diazonium salt + Hp.

Diazonium salt + NED-dihydrochloride ~ Red coloured azo dye.

Chlorine in the samples or nitrogen trichloride which nonnally coexist with nitrite
produce a false colour. This can be minimised by addition of naphthyl amine hydro-
chloride. Nitrite detennination should be carried out in filtered and turbidity free
samples. Presences of strong oxidants or reductants in the sample readily affect the
nitrite concentration. High alkalinity (> 600 mglL as CaC03) gives low results
owing to a shift in pH.
Analysis o/Water and Effluents 121


Never use acid preservation for samples to be analysed for nitrite. Make determi-
nation promptly on fresh samples to prevent bacterial conversion of nitrite to ni-
trate or ammonia. For short-term preservation of 1 to 2 days, store at 4°C.

1. Spectrophotometer for use at 543 nm, providing a light path of l-cm or longer.
2. Nessler's tubes or 100 mL capacity volumetric flasks.


1. Sulfanilamide
2. Sodium nitrite, KN02
3. Chloroform, CHCl3
4. N-( I-naphthyl)-ethylenediamine dihydrochloride (NED dihydrochloride)
5. Phosphoric acid, Hl0 4, 85%
6. Conc H2S04

1. Colour reagent: To 800 mL water add 100 mL of 85% phosphoric acid and 10
g sulfanilamide. After dissolving sulfanilamide completely, add 1 g NED-
dihydrochloride. Mix to dissolve and dilute to 1000 mL with water. Solution is
stable for about a month when stored in a dark bottle in refrigerator.
2. Preparation o/standard nitrite solution: The standard nitrite solution could be
prepared by anyone of the following two methods .
• ASTM (1995) and
• Methods suggested in UNEP/WHO (1996)
A. Preparation of standard nitrite solution as per ASTM (1995)
a. Stock nitrite solution (lmL= 100 pg nitrite N or 100 mg NIL) : Place potas-
sium nitrite (KN0 2) in a tarred l25-mL beaker and dry for about 24h to a
constant weight in a dissector containing conc. H2S04 • Take 0.6072 g dry
potassium nitrite and add 50 mL of water to the beaker, stir until dissolved,
and transfer quantitatively to alL volumetric flask. Dilute to the mark with
water and preserve with 2 mL of chloroform. Store in a sterilised bottle under
refrigeration. This solution is slable for about a month.
b.Nitrite solution, standards (lmL = lpg nitrite-N or lmg N IL): Dilute 10 mL
of stock nitrite solution to 1 L with water. This solution is unstable. Prepare
freshly when needed.
B. Preparation of standard nitrite solution as per UNEPIWHO (1996)

a. Stock nitrite solution (lmL= 100 pg nitrite N or 100 mg N IL): Distilled

water free of nitrate and nitrite must be used in the preparation of this solu-
122 Handbook ofMethods in Environmental Studies

tion. Dissolve 0.4926 g of anhydrous sodium nitrite (24 h in desiccator) in

distilled water and dilute to 1L. Preserve with 2 mL chlorofonnl L.
b. Working nitrite solution (ImL = Ipg nitrite-N or Img NIL): Distilled water
free of nitrite and nitrate must be used in the preparation of this solution.
Dilute 10 mL of the stock solution to lL.
3. Preparation of calibration curve: Prepare following series of standards in 50
mL Nessler's tube. Working nitrite solution: 1 mL = 1 ~g nitrite N.

Volume ofworking NO]-N Concentration when diluted

solution, mL to 50 mL (mglL ofNO] -N)
0.0 (Blank) 0.0
1.0 0.02
2.0 0.04
4.0 0.08
5.0 0.10
10.0 0.20
15.0 0.30


1. Take 50 mL of sample in a 100 mL volumetric flask.

2. Removal of suspended solids: If sample contains suspended solids, filter it
through Whatman no. 42 filter paper.
3. Check the pH of the solution. If pH is not between 5 to 9, neutralise pH by
adding 1 N HCI or NHpH as required.
4. Add 2 mL of colour reagent, mix well and makeup the volume to 100 mL. At
this stage pH should be 2-2.5.
5. Measure the colour after 10 min at 543 nm. Prepare calibration curve in the
range of 0 to 1 mg N02-NIL at the interval ot(f.1 mg N02-NIL.
6. Prepare a blank in the same way by using distilled water instead of the sample.
7. Calculate and record as N0 2-N in mgIL.


Read the concentration ofN02-N in samples directly from the calibration curve.
If less than 50 mL of sample is taken, calculate the concentration as follows: .

mgIL from standard curve x 50

Nitrite (as N), mgIL = - - - - - - - - - - -

Check Your Confidence on Nitrite Estimation

1. How nitrite is formed in wastewater or in a water body?
2. High level of nitrite in effluents of sewage treatment plant indicates what?
3. What is the principle of nitrite determination?
4. Why high alkalinity (> 600 mgIL) gives low results of nitrite.
Analysis of Water and Effluents 123

3.22 Nitrogen (Organic)

Organic nitrogen can be estimated by subtracting the concentration of ammonical
nitrogen from TKN.

Organic nitrogen = Total Kjeldahl nitrogen (TKN) - Ammonical nitrogen

3.23 Odour (TON)

Drinking water
ICMR (1963); MWH (1975) = Nothing disagreeable.
IS: 10,500 (1983, 1991) = Should be unobjectionable.
WHO (1984) = Should be inoffensive to most consumers.
WHO (1993) = Taste and odour should be acceptable to consumers.
USEPA (1989) = issued a secondary maximum contaminant level (SMCL) of
3 TON for odour. Secondary standard are not related to health.
EC = (1980) 0 (guide level) and 2 TON at 12°C and 3 TON at 25°C.
All efforts should be made to remove unpleasant odour as far as possible
(Ministry of Environment and Forests, 1993).

Many substances with which water comes into contact in nature of human use may
impart perceptible taste and odour. These includes minerals, metals and salts from
soil, inorganic substances are more likely to produce taste unaccompanied by odour.
Organic substance on the other hand, is likely to produce both taste and odour.
Principles among are the reduced product of sulphur as H 2S, which imparts rotten
egg odours. Certain species of algae secret substances may results both taste and
odour problems. The combination of two more substances, neither of which would
produce taste or odour by itself, but may produce odour ifmixes. This synergistic
effect may noted either in case of organic and chlorine.


In domestic wastewater, gases produced by the decomposition of organic matter
cause odour. The most characteristics odour of stale or septic wastewater is that of
H2 S.
124 Handbook ofMethods in Environmental Studies

Environmental Significance
• It causes psychological stress rather than to harm to the human body.
• Offensive odour can cause poor appetite for food, lowered water consumption,
impaired respiration, nausea and vomiting and mental perturbation.

Estimation of Odour (TON)

It is described by type and threshold odour number (TON) which is related to
odour-free water required for dilution an odorous sample to a non-odorous level.
The ultimate odour-testing device is the human nose .

1. Sample bottle, glass-stoppered

2. Constant-temperature bath
3. Odour flask: Glass -stoppered, 500 mL capacity, to hold sample dilutions testing.
4. Pipettes, measuring cylinder, thermometer
5. Odour-free water: prepare odour-free water by passing distilled water through
activated carbon.

1. Erlenmeyer flask: 500mL capacity, wide mouth, glass stoppered or covered by

watch glass. Rinse with tap water and clean with chromic acid solution.
2. Determine approximate range of threshold odour number (TON) by adding
200 mL, 50 mL, 12 mL and 2.8 mL sample to separate 500 mL flasks and
odour-free to make total volume of200 mL. Use a spare flask containing only
odour-free water as reference for comparison. The test should run at 40°C.
3. Stopper and warm flasks to 40°C in a water bath. Prolonged or direct heating is
avoided. Mix vigorously by swirling the flask 3 to 4 times. Remove the stopper
and place the nose at the tip of the flask. Test for odour, using normal inhalation.
4. The test is made by two or more testers. One makes dilution and other determines
odour. The person making the test select the odours sample from among three
flasks, two of which contain odour-free water. The test is carried out at 40 OC.
5. It odour tester fails to detect an odour, the dilution is decreased (increased
concentration of sample) until a dilution is found at which odour is perceptible.
The various dilution of the sample and corresponding TON is given in
Table 3.13.

Analysis o/Water and EjJIuents 125

A = mL of sample
B = mL of odour free wat

Table 3.13 :
Threshold odour number (TON) corresponding to various dilution.

Sample volume diluted to TON Sample volume diluted to TON

200mL 200 mL

200 1 140 1.4

100 2 70 3
50 4 35 6
25 8 17 12
8 .3 24 4 50
2 .8 70 2 100
1.4 140 1 200

A sample of water requires 150 mL of odour free water to render the odour
barely detectable. What is the volume of sample and what is the TON .

. Volume of sampl~ (Art Odour free water (8) = 200 mL

,' A+i50 F 200 "'::;..v
' Tbertlfor~, A-=
TQN= (At B)!
(50+ 1
. =

Check Your Confidence on Odour Estimation

1. What substance causes odour problems in drinking water and domestic
2. List the impacts of odour of drinking water to the consumers.
3. How to get odour free water?

3.24 Oil and Grease

Drinking water
IS: 10500 (I983, 1991) = 0.01 mgIL (beyond this limit, undesirable taste and
126 Handbook ofMethods in Environmental Studies

... cont.
odour after chlorination is produced). 0.03 mgIL (permissible in absence of
alternate source).
MHW (1975) = 0.01 mgIL (acceptable); 0.03 mgIL (cause of rejection)
WHO (1984) = No guideline value set.
Effluent discharge
MOEF (1993) = 10 mgIL (inland surface discharge and land for irrigation) and
20 mgIL (public sewer and marine coastal areas).
Effluent discharge from coal mine (MOEF 2000) = 10 mgIL

In the determination of oil and grease, an absolute quantity of a specific substance
is not measured. Rather a group of substances with similar physical characteristics
are determined quantitatively on the basis of their solubility in an organic extracting
solvent. Thus, oil and grease are a class distinguished on the basis of its solubility
characteristics, not is chemical composition. Oil and grease is defmed as any material
recovered as a substance soluble in the solvent. This test is applicable to natural
water and domestic wastewater. This method is also suitable for most industrial
The 12th edition of APHA (1971) standard methods prescribes the use of
petroleum ether as the solvent for natural and treated waters and n-hexane for pol-
luted water.
In the 14th, 17th, and 19th editions of APHA (1981, 1989, 1995) standard
methods only trichlorofluroethane has been recommended.
Two test methods are referred in APHA (1995):

1. Liquid-liquid extraction (partition gravimetric method) : for 4 to 100 mgIL oil

and grease concentration in water and wastewater.
2. Soxhlet extraction (20 to 200 mgIL in water and wastewater).

Environmental Significance
• Oil and grease determination on raw and settled wastewater gives a measure of
the effectiveness of primary settling tanks.
When discharged in wastewater, they may cause surface films and shoreline
deposits leading to environmental degradation.

Estimation of Oil and Grease

PRINCIPLE (Liquid-liquid extraction)
• In this method an acidified water sample is extracted with petroleum ether solvent
in a separatory funnel. •
• In the gravimetric portion of the procedure, the petroleum ether solvent
Analysis o/Water and Eflluents 127

containing the extractable material is evaporated and the residue is determined

Oil and grease may be recovered as a substance soluble in petroleum ether,
hexane or Freon (1,1,2 trichloro-l,2,2 trifluroethane). The oil and grease content
of domestic and certain industrial waste is an important consideration.
1. Hydrochloric acid, HCI, 1+ 1
2. Petroleum ether (boiling temp.65 "C)
3. Beaker; filter paper
4. Separating funnel- 500 mL
5. pHmeter
6. Water bath or drying oven
1. Take 250 mL of sample in a 500-mL beaker.
2. Add 5 mL of concentrated (1+ I) HCI to make pH 2 (acidification is needed to
maintain the integrity of the sample) ..
3. Transfer the sample to separating funnel. Add 30 mL of petroleum ether and
shake vigorously for 5 to 10 minutes. Invert the separatory funnel and vent with
stopcock to relive pressure building during extraction. Allow the layer to
separate. If a clear solvent layer cannot be obtained due to emulsion,with water,
add up to 100g ofNaCI to separatory funnel. Shake to dissolve the salt frequently.
This will break the emulsion.
4. The upper one petroleum ether and lower one is of water become distinct. Discard
the lower water portions from separating funnel. The systematic procedure is
shown in Fig. 3.21.


Fig. 3.21; Extraction with solvent

128 Handbook of Methods in Environmental Studies

5. Then 'filter the solution in the weighted beaker (WI)'

6. Repeat twice /thrice according to 3 to 4.
7. Evaporate the solvent from the beaker by putting in a hot water bath and take
the final weight of the beaker (W2).
8. Try to maintain temperature in water bath at 60 ± 5°C.

(W2-W I ) x 1000
Oil and grease (mgIL) = - - - - - - - - -
Volume of sample taken, mL

Along with the results, mention the solvent used for extraction.

Check Your Confidence on Oil and Grease

1. Defme oil and grease?
2. What happens if oil and grease are discharged with eftluents in water bodies?
3. List the test methods used for oil and grease estimation.
4. Name the different solvent being used for oil and grease estimation.
5. What is partition gravimetric method?

3.25 pH (Potentia Hydrogenii)

Drinking water
WHO (1984) = 6.5 to 8.5;
MWH (1975) = 7.0 to 8.5 (Acceptable) and beyond 6.5 - 9.2 (Cause ofrejec-
IS: 10,500 (1983, 1991) = 6.5 to 8.5 (Beyond this range the water will affect
the mucous membrane and/or water supply system).
USEPA (1974) = 6.5 to 8.5.
Effluent discharge
IS: 2490 (1981) = 5.5 to 9.0
MOEF, Schedule - VI, (1993) = 5.5 to 9.0

pH of most natural water falls within the range of 4 to 9. The majority of waters are
slightly basic (Le. generally over 7.0) because of the presence of carbonate and
bicarbonates. The pH increases (acidic) during daytime due to photosynthetic activity
because of consumption of CO2, whereas it declines (alkaline) at night due to
respiratory activity. By defmition, pH is the negative logarithm of hydrogen ion
concentration, more precisely hydrogen ion-activity.
Analysis of Water and EfJluents 129

or, pH = log - -
10 [W]

~ Acidic range Alkaline range ~

o 7 14
pH scale

As pH is expressed in a logarithmic scale, a drop of 1 unit of pH value (e.g. from

pH 7.0 to pH 6.0) is equivalent to an increase of 10 times acid intensity. pH does
not measure total acidity or alkalinity. This can be explain by comparing the pH of
O.1N solution ofHCI and acetic acid, which give the same neutralizing value. The
pH of the 0.1 N HCI is 1.1 because of its high degree of ionisation and the pH of
0.1 N acetic acid is about 2.9 because of its low degree of ionisation. The pH of
O.IN solution of few standard acids and bases are given in Table 3.14.

Table 3.14:
pH of standard acids and bases (0.1 N solution).

Acids at 25°C pH Bases at 25°C pH

HCI O.lN 1.1 NaOH O.lN 13.0

H2 SO. O.lN 1.2 Ammonia O.lN 11.1
Acetic acid O.lN 2.9 Sodium biocarbonate O.lN 8.4
Boric acid O.lN 5.2

Environmental Significance
It is important in almost every phase of environmental engineering practice, such
as of water supplies, water softening, disinfection and corrosion control. Low pH
causes corrosion, high pH causes taste, soapy feel; pH < 8.0 is preferable for effective
disinfection with chlorine.

Example - 1
Calculate the pH values for two solutions (a) in which [OH-) is 0.001 moles; (b)
[OH-) is 2 .5 x 10-'° moles.

sdi:rtIon; , i

" lH.t) [OH-) =1.0 x 10-1•

" 1 .O)()O~J, ~ ,> 1.0 x ;, 10-14
-- ;;, " (OH-l . -- 10
-;:t'log[H+i 1 ~:..liJg (1 ' x l O-~') =-(- 1 1.00) = 1 1.00 (basic) (AI"l~)
1.0 x 10-"
= 4 .0x 10-5 M
,,2.5 x 10- 10
pH,;= -Jog (4.0x JO-5) = -(log4+ log 10-5 ) -(13.602-5.00) = 4.40 (acidic)
' (Ans) "
130 Handbook ofMethods in Environmental Studies

Estimation of pH
Detennination of pH of water should be made in situ. If this is not possible, for
example with well water or when access to a lake or river is very difficult, the
measurement should be made immediately after the sample has been obtained.
Two common methods used for pH measurement are:
Use ofpH indicator paper: Simple, inexpensive, inaccurate.
Use ofelectronic pH meter: Accurate and free from interferences, gives reading
with an accuracy of ± 0.05 pH.


The basic principle of electronic pH measurement is the detennination of activity

of hydrogen ions by potentiometric measurement using a standard sensing electrode
(glass-electrode) and a reference electrode (calomel electrode) ..
Glass electrode (sensing electrode): It consists of a thin glass bulb (special
quality) containing a fixed concentration of HCI solution, into which a Ag-AgCI
wire is inserted, serving as the electrode with a fixed voltage. When glass electrode
is immersed in a solution, a potential deference develops between the solution in
the glass bulb and sample solution. The potential difference E is fonnulated by
Nemst equation:

E=-- log-- .,. (1)
nF Mn+
E = Half-cell potential R Gas constant
T = Absolute temperature n Valence
F = Faraday constant K Constant
M = Activity of ions to be measured
The E (half-cell potential) can not be measured alone. If the glass electrode is
placed against a reference electrode (usually the calomel electrode), the potential
difference between two electrodes is measurable. (E - Ecal).
Reference electrode consists of a half cell that provides a constant electrode
potential. Commonly used reference electrodes are Calomel and silver: silver-chlo-
ride electrode. Calomel electrodes are of 3 types, nonnal, tenth nonnal and satu-
rated depending on the concentration ofKCI solution used in preparing them. The
potential develops on each electrode is a function ofKCI concentration. In general,
saturated type of calomel electrode is employed.
Before any pH measurement, two electrodes have to be placed first in a solu-
tion of known pH (e.g H+ concentration = 19!L). This is called standardization of
electrode and pH meter. The overall potential of the total cell Eo equals to E-Eca1'
and is given as:
Eo =/ ~ log -1- - Eca1 ... (2)
Analysis of Water and Effluents 131

If the two electrodes are now placed in the solution with the unknown H+ ion
concentration, the potential Eo is given as:

E=-- log (W) - Eca1 ... (3)
c nF
Subtracting eq.2. from eq 3 gives,

E-E = RT (.Og ~ -logJ

o ° F l (W) ]

RT 1
F log-W


For H- ions, RTIF = 0.0591 at 25°C; therefore

E -E
pH = _0_ _0

1. pH meter: It consists of a potentiometer, a glass electrode, a reference electrode,
and a temperature compensating device (Fig. 3.22)
2. Beakers
3. Stirrer: Use either magnetic, TFF- coated stirring bar or a mechanical stirrer
with inert plastic-coated impeller.

Buffer solutions of pH of 4.0, 7.0 and 9.2. Standard buffer tablets are commercially
available. Buffer of different pH can be prepared by dissolving standard pH tablets
in distilled water. The above buffer solution can be prepared as follows :
1. Pthalate buffer (pH 4.0 at 25°C): Dissolve 10.12 g of potassium hydrogen
phthalate (KHC 8HP4) in distilled water to make IL of buffer.
2. Phosphate buffer (pH 7.0 at 25°C): Dissolve 3.40 g ofKH2P04and 4.45 g of
Na2HP04.2Hp in distilled water to make IL of buffer.
3. Borax buffer (pH 9.18 at 25°C): Dissolve 3.81 g of sodium tetraborate
decahydrae (Na2Bp7.lOHP) in distilled water to make IL of buffer.

Calibration of pH meter
I. Read carefully the operational manual of pH meter to be used.
2. Calibrate the pH meter against the standard buffer solution of pH 4.0, 7.0, and
9.2 according to the requirement as follows .
l32 Handbook ofMethods in Environmental Studies

tltemlll HCL htWtdard [H+))

~tura.d yjth Agel

o 0

Fig. 3.22: pH measurement (Note: pH measures the electrical potential due to

charge imbalance caused by unequal [H+) across glass membrane) .

• Wash the electrode with distilled water and dip the electrode in buffer of pH
7.0 and move the temperature knob to the required or room temperature and
adjust the meter accordingly.
• Wash the electrode with distilled water and dip the electrode in buffer of pH
4.0 and move the temperature knob to the required or room temperature and
adjust the meter accordingly.
• Wash the electrode with distilled water and dip the electrode in buffer of pH
9.2 and move the temperature knob to the required or room temperature and
adjust the meter accordingly

Sample analysis
1. After calibration, rinse the probe with distilled water, blot dry with a soft tissue
paper, place in sample solution.
2. Establish equilibrium between electrodes and sample by stirring sample to ensure
homogeneity; stirring gently to minimize CO2 entrapment.
3. Keep the electrode dipped in storage solution when not in use.
Analysis of Water and Effluents 133


Report the pH with precision of ± O.Oland the temperature of the sample at the
time the measurement.

pH Electrode Maintenance

G lass electrodes (sensing electrode) fail because of scratches, deterioration or ac-

cumulation of debris on the glass surface. The electrode in rejuvenated by alter-
nately immersing it 3 times each in O.IN HCI and O.IN NaOH. If this fails, im-
merse tip in KF solution for 30 sec. After rejuvenation, soak in pH 7.0 buffer
overnight. Rinse and store in pH 7.0 buffer. Rinse again with distilled water before
use. The pH electrode actually requires little maintenance. The electrode should be
stored in water. If allowed to dry out, the electrode needs to be hydrated for a
couple of hours prior to use. The level of concentrated KCI must be monitored and
replenished by adding saturated KCI through the stoppered orifice in the side of the
electrode. The correct liquid level is approximately 5 mm below the bottom edge
of the hole. The electrodes used for measurement generally need replacing periodi-
cally (e.g. yearly). Old or poor quality electrode often shows a slow drift in the
Electrode should not be left out for long periods, and be always kept immersed
in distilled water or pH 7.0 buffer.

Check Your Confidence on pH Estimation

I. List the environment significance of pH measurement.
2. Calculate the pH ofN/10 and Nil 00 solutions ofHCl.
3. Calculate the pH ofN/lO and Nil 00 solutions ofNaOH.
4. What are sensing and reference electrodes?
5. How to rejuvenated the pH-electrode?
6. What solution is used in reference and sensing electrodes?

3.26 Phenol (Phenolic Compounds)

Drinking water
IS, 10500 (1991) = 0.001 mg/L (maximum), beyond this limit, it may cause
objectionable taste and odour - 0.002 mg/L (maximum permissible in the
absence of alternate source).
WHO (1984, 1993) = No guideline value set.
Effluents discharge
MOEF (1993) = 1.0 mgIL (inland surface water); 5.0 mgIL (public sewers);
and 5.0 mg/L (marine coastal areas)
134 Handbook of Methods in Environmental Studies

Phenols are the most important groups of aromatic compounds. It is commonly
know as carbolic acid. It has been used widely as a germicide and disinfectants.
Phenol can be treated biologically up to 700 mgIL aerobically and up to 200 mgIL
anaerobically. Phenolic compounds are sometimes found in surface waters from
natural and industrial sources.
Generally, the level of phenol in domestic wastewater is low. However, it has
been reported in the range of 10 to I 00 ~gIL in Mumbai sewage (Igole and Kadam,
1999). But many industries produces high concentration of phenol in their efflu-
ents, like Petrochemical (10-30 mglL phenol); Petroleum refineries (10-200
mglL); Coke oven effluent (30-1000 mgIL) etc. Phenol removal processes in water
treatment include superchlorination, chlorine dioxide or chloramine treatment,
ozonation and activated carbon absorption.

Environmental Significance
Phenols are highly toxic to aquatic organisms. For human, phenol is toxic by inges-
tion and inhalation. Above 1 ppb concentration of phenol are highly objectionable
with respect to taste and odour problems in water. They react with chlorine which
may produce chlorophenol that are odouriferous and are of noticeable medicinal
taste in the drinking water. Their presence in streams and other waterways will
cause off flavour in fish tissues and other aquatic food.

Estimation of Phenolic Compounds

Two methods are recommended (ASTM, 1995):
A. Chloroform extraction method: 0 to 100 mgIL
B. Direct photometric method: > 0.1 mgIL (100 mgIL)
1. Both test methods are photometric procedures based on the reaction of steam
distillable phenolic compounds with 4-arnminopyrine.
2. Test method (A) differs from (B) mainly in that the sample is extracted with
chloroform, thereby providing 20-fold greater sensitivity.
3. Both the procedures involve separation of phenolic compounds from the back-
ground matrix by distillation.


1. Phenols, in noteworthy concentration are usually encountered in wastewater.

Phenols are subject to biological and chemical oxidation. Preserve and store
samples at 4°C or lower unless analysed within 4h after collection.
2. Acidify with 2 mL conc. H2S04 ,
3. Analyse preserved and stored samples within 28 d after collection.
Analysis of Water and Effluents 13S


Phenols are distillate from non-volatile impurities. Because volatilisation of phe-

nol is gradual, the distillate volume must ultimately equal that of the original vol-

1. Distillation apparatus: All glass, consisting of l-L borosilicate glass distill-
ing apparatus with Graham condenser.
2. pH meter.

Prepare all reagents with distilled water free of phenols and chlorine.
1. Phosphoric acid, H 3PO. (1+9): Dilute 10 mL 8S% Hl0 4 to 100 mL with
water. .
2. Methyl orange indicator
3. Special reagents for turbid distillates:
a. Sulphuric acid, H 2S04, IN
b. Sodium chloride, NaCI
c. Chloroform, CHCl 3
d. Sodium hydroxide. NaOH, 2.5N: Dissolve 10 g NaOH pellets in 100 mL

• Preparation of distillation mixture
1. Measure SOO mL of the sample into a beaker.
2. Adjust pH of the sample to 4.0 with Hl0 4 solution using methyl orange or a
pH meter.
3. Transfer the mixture to the distillation apparatus.
4. Use a SOO-mL graduated cylinder as a receiver.
S. Omit adding Hl0 4 and adjust pH to 4 with 2.S N NaOH, if sample was pre-
served as described earlier.
• Distillation
6. Collect about 4S0 mL distillate. When distillation is finished, and, boiling is
ceased, add SO mL warm water to the distilling flask. Next, continue the distil-
lation until a total of SOO mL ha!: been collected.
7. Ifthe distillate is turbid, a second distillation may be helpful. Acidify the turbid
distillate with H2 S04 solution (1 +9) and repeat the previously described distil-
lation. The second distillate usually eliminates the turbidity.

1. Stock phenol solution (lmL = Img phenol): Dissolve 100 mg of phenol

(C6H~OH) in 100 mL of freshly boiled and cooled water. The fresh stock solu-
tion may be preserved up to 30 days for use.
2. Phenol solution, intermediate C mL = 10 /lg of phenol): Dilute 1 mL of the
136 Handbook o/Methods in Environmental Studies

stock solution to 100 mL with freshly boiled and cooled water. Prepare this
solution fresh on the day it is used.
3. Phenol solution, standard (1 mL = 1Jig of phenol): Dilute 50 mL of the inter-
mediate solution to 500 mL with freshly boiled and cooled water. Prepare this
solution fresh within 2 h of use.

This test method is applicable to water that contains more than 0.1 mg/L of phe-
nolic compounds.
This is a photometric test method, based on the reaction of steam-distillate phe-
nolic compounds which react with 4- aminoantipyrine at pH 10 ± 2 in the presence
of potassium ferricyanide [~Fe(CN)6] to form a coloured antipyrine dye. The an-
tipyrine colour formed in an aqueous solution is measured at 510 om. The concen-
tration of phenolic compounds in the sample is expressed in terms of mg/L of
phenol (C6HPH).
Photometric equipment: Spectrophotometer equipped with absorption cell
providing light paths 1 to 5 cm for use at 500 om.
Chemicals and Reagents
1. Ammonium chloride (NH4CI)
2. Ammonium hydroxide (NHPH)
3. 4-aminoantipyrine
4. Potassium ferricyanide [~Fe(CN)6]
1. Ammonium chloride (NH.CI) solution: Dissolve 20 g ofNH4CI in water and
dilute to I L.
2. Ammonium hydroxide (NHPH): Concentrated (sp. gr. 0.90)
3. 4-aminoantipyrine solution: Dissolve 2 g of 4-aminoantipyrine in water and
dilute to 100mL. Prepare the reagent fresh as used.
4. Potassium ferricyanide: Dissolve 8g of [~Fe(CN)6] in water and dilute to
100mL. Filter if necessary. Prepare fresh daily.

1. Calibration standards: Prepare a 100mL distilled water blank and series of
100-mL phenol standards containing 0.1, 0.2, 0.3, 0.4 and 0.5 mg phenollL.
2. Sample: Transfer 100-mL distillate, or a portion not containing more than 0.5
mg phenol diluted to 100mL, in a 250-mL beaker. Also prepare a blank consist-
ing 100 mL of water.
Analysis of Water and Effluents 137

Treat sample, blanks and standards as follows:

3. Add 5 L NHpH solution immediately. Adjust the pH between 9.8 and 10.2
4. Add 2mL of 4-aminoantipyrine solution, mix well; add 2mL ~Fe(CN)6 solu-
tion and mix well.
5. After 15 min, transfer the solution to absorption cells and read absorbance of
sample and standards against the reagent blank at 51 Onm.


Use of calibration curve: Estimate sample phenol content from photometric read-
ing by using a calibration curve.

mgIL phenol = - - x 1000
A = mg phenol in sample, from calibration curve
B = mL original sample.
Use of single phenol standard:

ex D x 1000
mgIL phenol = -------
C = mg standard phenol solution,
D = absorbance of sample
E = absorbance of standard phenol solution
Test data of photometric method (ASTM, 1995) as follows:

Amounts added Amount found Bias, Bias, Statistically

mgIL mgIL mgIL % significant

7.154 6.930 - 0.224 -3.1 yes

35.768 34.430 - 1.38 - 3.7 yes
71.535 68.777 - 2.758 - 3.9 yes

Check Your Confidence on Phenol Estimation

1. List the major sources of phenolic compounds that pollute the aquatic
2. Why presence of even trace amounts of phenolic compounds in drinking water
is totally undesirable?
3. What are the different methods of phenol estimation?
4. How phenolic compounds can be removed from drinking water?
5. Why distillation of industrial effluent sample is required for the estimation of
138 Handbook of Methods in Environmental Studies

3.27 Phosphorus
.of phosphorus
Drinking water
IS: 10500 (1983, 1991) = No standards.
WHO (1984) = Not issued any regulation or guidelines for phosphorus.
U.S.EPA (1974) = Phosphorus has not been regulated not even in secondary
drinking water standards
Effluent discharge
MOEF (Schedule-VI, \993) = Dissolved phosphate (as P), 5 mglL (max.) for
discharge in inland surface water bodies.

Phosphorus occurs in waters and wastewater almost solely as phosphates. These
are classified as (a) orthophosphate; (b) condensed phosphates and (c) organically
bound phosphates. They occur in solution, in particulates or detritus or in the body
of aquatic organisms.
Water supplies may contain phosphate derived from natural contact with min-
erals or through pollution from application offertiliser, sewage and industrial waste.
Groundwater, therefore, is more likely to have higher phosphate concentration.
In sewage, concentration o.f phosphorus (total as P) has been reported in the
range of4 mglL (organic- P, 3 mglL and inorganic- P, I mg/ L) to 15 mglL (organic-
P, 5 mglL and inorganic-P, 10 mglL).
The daily requirement of P is equal to that of Ca. The recommended dietary
allowance (RDA) is 800 mg for adults . Dietary deficiency is not expected because
of abundance of phosphorus in the diet.

Environmental Significance
The crucial level of phosphorus that causes eutrophication problems is somewhere
near 0.005 mglL in summer growing conditions. Studies suggest that 0.0 I mglL of
phosphorus is acceptable while 0.02 mglL is excessive.

The presence of phosphorus in large quantities in freshwater indicates pollu-

tion through sewage and industrial wastes.
Though phosphorus poses problems in surface waters, its presence is necessary
for biological degradation of wastewater.
Phosphorus can be classified as total-P, inorganic-P (orthophosphate,
HlO~, HP01-, PO; ·) and organic-P

Only inorganic compounds of phosphorus are of significant environmental

Analysis a/Water and Ejjluents l39

Estimation of Phosphorus
APHA (1995) lists three preliminary steps and the four methods for examination of

Preliminary steps
a. Preliminary filtration step.
b. Preliminary acid hydrolysis step for acid-hydrolyzable phosphorus.
c. Preliminary digestion step for total phosphorus.

a. Vanadomolybdo phosphoric acid colorimetric method
b. Stannous chloride method
c. Ascorbic acid method
d. Automated ascorbic acid reduction method.


A scheme for the differentiation of various phosphorus forms which can be deter-
mined in water is given in Fig. 3.23.

ISample I
Unfiltered E No sample digestion (Total orthophosphate)
Acid digestion (orthophosphate + hydrolysable phosphate)
Acid-persulfate digestion (Total recoverable phosphate)

Filtered through 0.45 micron -4 Residue (Particulate phosphorus)

No sample digestion -4 Dissolved orthophosphate
E Acid digestion -4 Dissolved orthophosphate + hydrolysable phosphate
Acid-persulfate digestion -4 Dissolved total recoverable phosphate

Fig.3.23: Scheme for differentiation of various forms of phosphorus in water.

3.27.1 Estimation of Inorganic Phosphate


Phosphorus occurring as orthophosphate can be measured colorimetric ally. In

acidic conditions, orthophosphate reacts with ammonium molybdate to form
molybdophosphoric acid. It is reduced by stannous chloride to a blue colour
complex. The intensity of the blue colour is measured, which is directly propor-
tional to the concentration of phosphate present in the sample.
140 Handbook of Methods in Environmental Studies

PO/"+ 12 [(NH4)2Mo04] +24W ~ [(NH4)104.l2MoOJ] + 21NH/+ 12 ~O

[(NH4)104.l2MoOJ] + Sn2+ ~ (molybdenum blue) + Sn4+


1. Potassium dihydrogen phosphate, KHl04

2. Phenolphthalein indicator
3. Stannous chloride, SnCl2
4. Ethyl alcohol
5. Ammonium molybdate
6. Conc H2S04
7. Glycerol


1. Stock phosphate solution: Dissolve 0.7111 g anhydrous KHl04 and dilute to

IL; I mL = 0.5 mg P0 4or 500mg PO/L.
2. Phosphate working solution: Dilute 100 mL of stock solution to IL; I mL =
0.05 mg P04or 50mg PO/L.
3. Ammonium molybdate solution: Dissolve 2.5 g ammonium molybdate in 17.5
mL of distilled water. Cautiously add 28 mL of conc. H2S04 to 40 mL of dis-
tilled water. Cool, add molybdate solution, and dilute to 100 mL.
4. Phenolphthalein indicator solution: Dissolve Ig of phenolphthalein in 100
mL of95% ethyl alcohol and add 100 mL of distilled water.
5. Stannous chloride solution-I: Dissolve 2.5 g offresh SnCI2.2Hp in 100 mL
glycerol. Heat in a water bath and stir with a glass rod to hasten dissolution.
This reagent is stable and requires neither preservative nor special storage.
6. Dilute stannous chloride reagent-II: Mix 8 mL stannous chloride reagent I
with 50 mL glycerol. This reagent is stable for at least 6 months.

1. Preliminary sample treatment (acidification):

• Take 100 mL sample containing not more than 200 mg P and free from
colour and turbidity.
• Add 0.05 mL (1 drop) phenolphthalein indicator. If sample turns pink, add
strong acid solution drop-wise to discharge the colour.
• Ifmore than 0.25 mL (5 drops) is required, take a smaller sample and dilute
with distilled water after first discharging the pink colour with acid.
2. Blank preparation: Use distilled water.
3. Calibration curve: Prepare a calibration curve for the range of 0.3 to 2mg PIL.
4. Colour development:
• Add, with through mixing after each addition, 4 mL of molybdate reagent
and 0.5 mL (10 drops) of stannous chloride reagent-I.
• Rate of colour development and intensity of colour depend on temperature
ofthe fmal solution. Each 1°C increase in temperature produces about 1%
Analysis afWater and E.IJluents 141

increase in colour. Hence, hold samples, standards, and reagent within 2°C
of one another and in the temperature range between 20 and 30°C.
5. Colour measurement:
• After 10 min, but before 12 min, measure absorbance of the colour
spectrometric ally at 690 nm and compare with a calibration curve using a
distilled water blank.

Calculate phosphate concentration as follows:

mg ofP (in approx. 104.5 mL fmal volume)

mg phosphate/L = x 1000
mL of sample

3.27.2 Estimation of Total Phosphorus

All the forms of phosphorus, whether dissolved or particulate are converted to
inorganic forms (phosphate) after digestion of sample. Many techniques such as
using perchloric acid, H2S04-K 2S04 , H2S04-HNO) etc can be employed for diges-
tion of the sample. The H2S04-HNO) digestion technique is discussed here.

1. Conc. sulphuric acid (H 2S04)
2. Phenolphthalein indicator
3. Conc. nitric acid (HNO))
4. Sodium hydroxide (NaOH), 1N: Dissolve 40 g NaOH in 1L of distilled water.
1. Take a suitable volume (50 mL) of sample in a Kjeldahl flask.
2. Add 1 mL H2 S04 and 5 mL HNOr
3. Digest the sample on a hot plate till the volume becomes nearly 5 mL and
continue the heating further until the solution becomes colourless after com-
plete removal of HNO) (or heat in a beaker covered with a watch glass to
prevent undue evaporation) .
Estimation of Phosphorus
1. Cool and transfer completely to a 100-mL volumetric flask.
2. Add 1 drop of phenolphthalein indicator.
3. Neutralise the acidity by adding 1 N NaOH. At the end the solution turns pink.
Make up the fmal volume to 100 mL.
Colour development
1. Take 20 mL of digested sample in a conical flask and add 4 mL of ammonium
molybdate reagent and 0.5 mL (10 drops) stannous chloride reagent II.
2. Rate of colour development and intensity of colour depend on temperature of
142 Handbook of Methods in Environmental Studies

the [mal solution. Each 1°C increase in temperature producing about 1% in-
crease in colour. Hence, hold samples, standards, and reagent within 2°C of
one another and in the temperature range between 20 and 30°C.
Colour measurement
1. After 10 min, but before 12 min, measure the absorbance of the colour
spectrometric ally at 690 nm and compare with a calibration curve, using dis-
tilled water blank.


mg ofP (in approx 104.5 mL [mal volume)

Phosphate (mgIL) = - - - - - - - - - - - - - - - - x 1000

3.27.3 Estimation of Particulate Phosphorus

Particulate phosphorus can be obtained as a difference between the total phospho-
rus concentration in unfiltered and filtered samples.

Particulate P = Total P in unfiltered sample - Total P in filtered samples

3.27.4 Estimation of Organic Phosphorus

Organic phosphorus can be obtained as difference between the total phosphorus
and inorganic phosphorus of the sample.

Organic phosphorus = Total phosphorus - Inorganic phosphorus

Check Your Confidence on Phosphorus

1. Discuss the environmental significance of phosphorus.
2. What is the difference between orthophosphate, polyphosphate and organic
phosphorus? Which form of the P is analysed colorimetrically?
3. What is the purpose of addition of SnCI2 during colorimetric analysis of P?
4. "For estimation of total P acid digestion is needed." Why?
5. How do particulate P and organic P are calculated?

3.28 Solids
Solids refer to matter suspended or dissolved in water or wastewater. Solids may
affect water or effluent quality adversely in a number of ways. Waters with high
dissolved solids generally are of inferior palatability. For this reason, a maximum
limit of 500 mg dissolved solidslL is permissible for drinking water. Three types of
solids are analyzed for water and wastewater.
Analysis of Water and Effluents 143

Total Solids (TS)

Total dissolved solids (TDS)
Total suspended solids (TSS)

3.28.1 Estimation of Total Solids (TS)


Total Solids (TS) is the measure of all kinds of solids i.e. suspended, dissolved and
volatile solids. Total solids can be determined as the residue left after evaporation
at 103 to 105°C of the unfiltered sample. It consists of two parts: Total Suspended
Solids (TSS) and Total Dissolved Solids (TDS).
Each fraction is again divided into volatile suspended solids (VSS) and fixed
solids after heating at 550°C. VSS is the organic fraction that lost as CO 2 and fixed
solids are the ash portion that remains.
Total solids

by filtration (1 micron pore size filter paper)

Total suspended solids (TSS) Total Dissolved solids (TDS)

Volatile solids Fixed solids Volatile solids Fixed solids
(VSS) (FS) (VSS) (FS)
Organic Inorgainc Organic Inorgainc


1. Evaporating dishes: Dishes of 100 mL capacity made up of either porcelain or

2. Desiccator, provided with a desiccant containing a colour indicator of moisture
(CuS04 • 5 HP).
3. Drying oven or hot air oven, for operation at 103 to 105°C.
4. Analytical balance, capable of weighing to 0.1 mg.


1. Take an evaporating dish or clean beaker (400mL capacity) of suitable size and
dry at 103 to 105°C for Ih. Store and cool the dish in desiccator until needed.
Weigh immediately before use. Note the initial weight (Wi) in mg.
2. Put 250-300 mL unfiltered well-mixed sample in it.
3. Put in hot air oven at j 03 °C to 105°C for 2 h up to dryness.
4. Cool in desiccator and take the fmal weight (Wt) in mg.
5. Repeat cycle of drying, cooling, desiccating and weighing until a constant weight
is obtained, or weight change is less than 4% of previous weight or 0.5 mg,
which ever is less. When weighing dried sample, be alert to change in weight
due to air exposure and lor sample degradation. Duplicate determination should
be within 5% of the average.
144 Handbook of Methods in Environmental Studies

(Wf - Wi) x 1000
Total Solids mglL = - - - -- - - -
Volume of sample, mL

3.28.2 Estimation of Total Suspended Solids


,Standards for Suspended Solids

Drinking Water IS: 10500 (1991): No standards

Effluent Discharge MOEF (1993): General discharge standards for
wastewater effluents:
In inland surface waters: 100 mgIL
In public sewers: 600 mgIL
On land for irrigation: 200 mglL
In marine coastal areas: 100 mglL (For Process Water) and for cooling water
effluents 10% above TSS of influents.
BIS standards for discharge of industrial and sewage effluents
(IS: 2490:1982)
In inland surface waters: 100 mglL
In public sewers: 600 mglL
In marine coastal areas: 100 mglL (For Process Water)
On land for irrigation: 200 mglL
Sewage farming: 300 mglL


What physical problems do you think may be caused by suspended solids?

They cut down light transmission through the water and so lower the rate of
photosynthesis in aquatic flora.
In less turbulent parts of river, some of the solids may sediment out, smothering
life of the riverbed.
Why TSS is determined for effluent samples?
The TSS determination is extremely valuable in the analysis of polluted water.
It is a major parameter used to evaluate the strength of domestic wastewater
and to determine the efficiency of treatment unit.


The term TSS applies to the dry weight of the material that is removed from a
measured volume of water sample by filtration through a standard filter. To achieve
reproducibility and comparability ofthe results close attention of procedural details,
especially filter characteristics and time and temperature of drying is required.
Analysis of Water and Effluents 145


1. Results may be questionable if the TSS include oils, grease or other volatile
2. Exclude large floating particles or submerged agglomerates of non-homogeneous
material from the sample.
3. In case of highly polluted water, sewage or wastewater, excessive residue on
the filter paper may form a water-entrapping crust, in such case, limit the sample
size so that yielding no more than 200 mg residue.
4. Prolonged filtration times resulting from filter clogging may produce high results
owing to increased colloidal materials captured on the clogged filter.


In addition to the ones listed in section 3.28.1 for total solids (page 143) the following
apparatus are required:
1. Glass-fiber filter paper
2. Filtration apparatus - membrane filter funnel
3. Suction flask
4. Aluminum weighing dishes.


Sample size

I . The TSS determination is subjected to considerable error. Sufficient sample

should be filtered, if possible, to yield an increase in weight of about 10 mg.
This often requires filtration of500mL or more of sample of biologically treated
wastewater or lightly polluted waters.
2. Choose the sample volume to yield 10 to 200 mg of dried residue. If more than
10 minutes are required for complete filtration, decrease the sample volume.

Sample analysis

I. Assemble filtration apparatus and begin suction.

2. Wet the filter paper with a small volume of reagent grade water (double-distilled
3. Stir sample with a magnetic stirrer and while stirring, pipette a measured volume
of sample onto the filter.
4. Wash with 3-successive 10-mL volume of distilled water, allowing complete
drainage between washing, and continue suction for about 3 minutes after
filtration is completed.
5. Carefully remove the filter paper from filtration apparatus and transfer it to an
aluminum-weighing dish as a support.
6. Dry for at least I hat 103 to 105°C in an hot air oven, "001 in a desiccator and
7. Repeat cycle of drying, cooling, desiccating and weighing until a constant weight
is obtained, or weight change is less than 4% of previous weight or 0.5 mg,
whichever is less. Duplicate determination should be within 5% oftheir average.
146 Handbook of Methods in Environmental Studies


(Wf - Wi) x 1000

Total Suspended Solids mgIL = - - - - - - - -
Volume of sample, mL
Report the results as: Total SS dried at .......... .."C, .. .......... mglL.
Total suspended solids can also be obtained as a difference between TS and


3.28.3 Estimation of Total Dissolved Solids (TDS)

Drinking water
ICMR (1963) = 500 mgIL (minimum) to 1500 mgIL (maximum)
WHO (1984) = 1000 mgIL (maximum)
WHO (1993) = 1000 mgIL (consumer complaint is taste)
IS: 10500 (1983,1991) = 500 mgIL (maximum) (beyond 500 mgIL
palatability decreases and may cause gastrointestinal irritation). 2000 mgIL
(permissible limit in the absence ofaltemate source)
MHW (1975) = 500 mgIL (acceptable) and 1500 mglL (cause for rejection)
Effluent discharge
IS: 2490 (1981) = 2100 mgIL (maximum).
MOEF, Schedule - VI (1993): 2100 mgIL - inland surface water. Omitted by
GSR 80 (E) dated 31st Dec, 1993 (w.e.f 31.12.93)

A large number of solids are found dissolved in natural waters, the common ones
are carbonates, bicarbonates, chlorides, sulphates, phosphates, and nitrates of
calcium, magnesium, sodium, potassium, iron, magnesium etc. In other words, TDS
is simply the sum of the cations and anions concentration expressed in mgIL.
The results of a determination of TDS can be used to check the accuracy of
analyses. This is accomplished by comparing the value of calculated TDS with the
measured value. TDS can be calculated by adding the ionic concentrations and
express the results as mgIL as follows:

Calculated TDS = 0.6 (alkalinity) + Na + K + Ca + Mg + Cl + S04 + SiO J

+NO J + F

The measured TDS should be higher than the calculated value, because a significant
contributor may not be included in the calculation. If the measured value is less than
the calculated value, all values are under suspicion. The acceptable ratio is:
A nalysis of Water and EjJluents 147

measured TDS
1.0 < <1.2
calculated TDS

A high content of dissolved solids elevates the density of water, influences osmo-
regulation of freshwater organisms, reduces solubility of gases (like oxygen) and
reduces utility of water for drinking, irrigation and industrial purposes. TDS
concentration beyond 500 mg/L, decreases palatability and may cause
gastrointestinal irritation.

A well mixed, measured portion of sample is filtered through a standard glass-fibre

filter and the filtrate portion is evaporated to dryness at 180 ± 2 °C and that gives
the amount of total dissolved solids. The reason for higher temperature used is to
remove all mechanically occluded water. Where organic matter is generally very
low in concentration, the losses due to higher drying temperature will be negligible.

Evaporating dishes, analytical balance

Desiccator, hot-air oven, filter paper (Whatman No. 41).

I. Take an evaporating dish of suitable size (or a 500-mL capacity beaker), make
it clean, dry at 103-105 °C, cool in a desiccator and note the initial weight (W I)'
2. Filter about 100mL to 250mL of sample through filter paper (Whatman No.
41) and take the filtrate in the evaporating dish. (Note: Choose the sample
volume to yield 10 to 200 mg of dried residue. If more than 10 minutes are
required for complete filtration, decrease the sample volume.)
3. Evaporate the sample in a hot air oven at 180 ± 2 °C and after the whole water
is evaporated, cool the evaporating dish in a desiccator and take the fmal

Total Dissolved Solids (IDS), mg/L = 2 I X 1000 x 1000

WI initial weight of the evaporating dish (g)
W2 final weight of evaporating dish (g)
V volume of the sample taken (mL)
148 Handbook ofMethods in Environmental Studies

Problem: Find out TDS with the following results.

An analysis of a sample of water with pH 7.5 has produced the following
concentrations (mg/L). Find the total dissolved solids (TDS) in mg/L.

3.28.4 Estimation of Settleable Solids

(Volumetric Method)

The term settleable solids are applied to the solids in suspension that will settle,
under quiescent conditions, because of the influence of gravity. Only the coarser
suspended solids with a specific gravity sufficiently greater than that of water settle.
Sludge is accumulation of settleable solids. Settleable solids may be determined
and reported on either a volume (mLlL) or a weight (mgIL) basis.

To determine the need for and design of primary settling tank.
To determine the efficiency of sedimentation units.
To study the physical behaviour of waste streams entering natural water bodies.
Analysis of Water and Effluents 149

The volumetric test requires only an Imhoff cone.

I. Fill an Imhoff cone to the l-L mark with a well-mixed sample (Fig. 3.24).
2. Settle for 4S-min, gently agitate sample near the sides of the cone with a rod or
by spinning, settle 15 min longer, and record volume of settleable solids in the
cone as mUL.
3. If the settled matter contains pockets of liquid between large settled particles,
estimate volume of these and subtract from volume of settled solids.
4. The practical lower limit of measurement depends on sample composition and
generally is in the range of 0.1 to 1.0 mLlL. where a separation of settleable and
floating materials occurs, do not estimate the floating material as settleable
5. Replicates usually are not required.

Report the results as mL of settleable solids IL (volumetric method).


Fig. 3.24: Imhoff cones used to measure settleable solids.

3.28.5. Estimation of Settleable Solids

(Gravimetric Method)
1. Determine the total suspended solids (TSS) as in section 3.28.2 (page 144).
2. Determination of non-settleable solids is as follows.
• Take lL measuring cylinder and pour about 1L ofsample (Note: the minimum
depth of sample should be 20 cm).
• Let stand quiescent for Ih and, without disturbing the settled or floating
material, siphon 2S0mL from centre of measuring cylinder at a halfway
between the surface of the settled material and the liquid surface.
• Determine total suspended solids (mgIL) of this supernatant liquor. These
are the non-settleable solids.
ISO Handbook ofMethods in Environmental Studies

Settleable Solids, mgIL = TSS, mgIL - Non-settleable Solids, mgIL

Check Your Confidence on Solids Estimation

1. Why analyses of the following solids are of interest in water quality control?
• TOS in municipal water supplies?
• Total and volatile SS in domestic wastewater?
• Settleable solids in domestic wastewater?
2. What significant information is furnished by the determination of volatile solids?
3. Discuss the sources and impacts of suspended solids?
4. How suspended solids are measured?
S. The analysis for SS is run as follows:
(a) A glass-fibre filter is dried to a constant weight ofO.1372g;
(b) 100 mL of sample is filtered;
(c) the filter along with residue are placed in a hot air oven at 104 "C;
(d) the weight of the filter along with residue is 0.1834g. Determine the SS
concentration in mgIL.
6. What are the sources and impacts of dissolved solids in water supplies?
7. Why TOS are measured at higher temperature (I80 "C)?
8. List three environmental significances of determination of settleable solids?
9. Discuss the determination of settleable solids by gravimetric method?

3.29 Sodium and Potassium

(Flamephotometric method)

Drinking water
IS: 10500 (I991) =No standards for sodium
USEPA (1974) = No MeL
USEPA (I980) = 20 mgIL recommended.
WHO (1984, aesthetic quality) = No maximum contaminant level has been set
for Na but 200 mgIL was fixed based on taste
Ee (1980) = 20 mgIL guide level
Emuent discharge
MOEF (1993) = No standards for Na

Sodium (Na) ranks 6th among the elements in order of abundance and is present in
most natural waters. Sodium is generally found in lower concentration than cal-
Analysis of Water and Fffluents 151

cium and magnesium in freshwater. The salts ofNa are highly soluble in water and
impart softness (in contrast to hardness). In food, USEPA recommends daily intake
levels of 1600-9600 mg for adults and 69-92 mg/kg/day for infants based on mea-
surement of sodium excretion from urine (sodium is mostly excreted in urine).
Food intake of sodium averages 90% or more of the total sodium, estimated at 2-
• Na levels may vary from < I mg/L to more than 500 mg/L.
• In ground water, it varies widely but normally range between 6 mg/L and 130
• In surface waters, sodium concentration may be less than 1 mg/L or may ex-
ceed 300 mg/L depending upon the geographical area.
• The average level ofNa is > 100 mg/L.

Environmental Significance
1. High Na content, in the form of chloride and sulphate, make the water salty in
taste and unfit for human consumption (i. e. unpalatable).
2. High amount ofNa wiII render the boiler operations difficult.
3. High Na concentration also leads to hypertension.
4. A water with high Na content is not suitable for agricultural use as it tends to
deteriorate the soil.
5. As such there is no standards ofNa in drinking water. But for the effluent to be
discharged on land for irrigation purpose, maximum allowable percent ofNa is
60. (However, the standard of sodium has been omitted by GSR, 80 (E), 31.12.93
MOEF - schedule - VI).

3.29.1 Estimation of Sodium

The estimation of Na and K is based on the emission spectroscopy, which deals
with the excitation of electrons from ground state to higher energy state and com-
ing back to its original state with the emission of light.
Trace amount of sodium can be determined by flame emission photometry at a
wave length of 589 om. The solution is sprayed as a fme mist into a non-luminous
flame, which becomes coloured according to the characteristic emission of the
metal. The intensity of the light is measured by a photo-detector. The output of the
photo-detector is connected to an electronic metering unit to provide the readout,
before analyzing the unknown solution. A schematic diagram of a flame photom-
eter is shown in Fig. 3.25. The system is standardised with solutions of known
concentrations. The calibration curve may be linear but has a tendency to level off
at higher concentrations.

I. Flame photometer, with flame accessories: The total flame-photometer system

consists of four units (Fig. 3.26).
152 Handbook of Methods in Environmental Studies

Slit Phototube

Fig. 3.25: Schematic diagram of a flame photometer

Fig. 3.26: Different units of a typical Flame photometer.

I. Burner unit: The main unit, consists of an atomizer (for aspiration of solution)
mixing chamber, burner, optical lens, light filter, photo-detector and control
2. Compressor unit: Compressed air from the compressor unit is applied to the
3. Electronic metering unit to read out readings.
4. LPG (liquid petroleum gas) or laboratory gas.
II. Working principle of flame photometer
1. The sample enters in the mixing chamber as a fine atomized jet.
2. LPG is also injected into the mixing chamber at a controlled rate.
3. The mixture of gas and atomized sample is passed on to the burner and is ignited.
4. The emitted light from the flame is collected by a lens and is passed through an
appropriate filter (selected for different elements).
5. The filtered light is then passed on to a photo detector and the output is applied
to the electronic metering unit for read out.
Analysis of Water and EfJluents 153

1. Deionised distilled water: Use deionised distilled water to prepare all reagents
and calibration standards, and as dilution water.
2. Stock sodium solution: Dissolve 2.5418 g NaCI dried at 140 "C and dilute to
1000 mL with water; 1 mL = I mg Na.
3. Intermediate sodium solution: Dilute 10 mL of stock solution with water to
100 mL; 1 mL= 100 Ilg Na. Use this intennediate solution to prepare calibration
curve in sodium range of 1 to 10 mgIL.
4. Standard sodium solution: Dilute 10 mL intennediate sodium solution with
water to 100 mL; ImL = 10 Ilg Na. Use this solution to prepare calibration
curve in sodium range of 0.1 to 1 mgIL
Pretreatment of Sample
I. For non-polluted samples, filter the sample to remove any suspended particles
which otherwise may clog the capillary of the instrument.
II. For highly polluted samples and wastewater samples, pretreatment is required.
For the pretreatment of samples following materials are needed:
• Hot plate
• Nitric acid (HN03) conc.
Procedure of nitric acid digestion of sample
1. Take (50-100 mL) mixed sample in a beaker and 5 mL conc. HN03•
2. Bring to slow boil and evaporate on a hot plate to lowest possible volume (about
10 to 20 mL).
3. Add 5 mL conc. HN03 cover with a watch glass, heat to obtain gentle refluxing
4. Continue heating and adding conc. HN03 until a light coloured clean solution
is obtained.
5. Add 1-2 mL conc HN03 and warm slightly to dissolve any remaining residue.
6. Wash down the walls of the beaker and watch glass with distilled water and
then filter.
7. Transfer the filtrate to a lL volumetric flask, cool and makeup the volume.
Operation of flame photometer
1. Follow the instruction manual given by manufacturer.
2. Set the filter for reading at 589 om for Na and 769 om for K.
3. Start the compressor and ignite the burner of flame photometer.
4. Keep the air pressure at 0.5 kglcm2 and adjust the gas feeder knob so as to have
a blue sharp flame.
5. Feed the standard Na solution of the highest value in the range and adjust the
flame photometer to read full value of emission on the scale.
6. Aspirate the distilled water and adjust the meter reading to zero.
154 Handbook of Methods in Environmental Studies

7. Now feed different standard sodium solutions within the range (i.e. 0-1, 0-10
or 0-100 mg NaiL) as desired.
8. Plot a standard curve between concentration and emission of standard sodium
9. Put off the fuel supply first followed by air and then main switch.

For direct reference to the calibration curve:

mg NaIL = (mg NaIL in portion) x D

mL sample + mL distilled water

D = dilution ratio =
mL sample

3.29.2 Estimation of Potassium

(Flame photometric method)
There are no specific standards of potassium for either drinking water or effluent

1. It is not present in high concentration and ranks 7th among the elements in
order of abundance and constitutes 2.4% by weight of the earth's crust. It is
never found free in nature. Most potassium minerals are insoluble.
2. Its concentration in drinking waters seldom reaches 20 mgIL. As a rule ratio of
Na to K is 10:1 or 20: 1.
3. The method of analysis is flame photometry at 770 nm.
4. It is a naturally occurring element, however concentration remain quite lower
compared to Na, Ca and Mg.
5. It has got more or less similar chemistry like Na and remains mostly in solution
without undergoing any precipitation.

APHA (1995) recommend four methods for the determination of potassium. All
are rapid, sensitive and accurate, and selection depends on instrument availability
and analyst choice.
1. Atomic absorption spectrometric method
2. Inductively coupled plasma spectroscopy method
3. Flame photometric method
4. Potassium-selective electrode method.
Analysis of Water and Effluents ISS

Interference may occur at sodium to potassium ratios of 5: 1 or greater. Calcium
may interfere if calcium-to-potassium ratio is 10: 1 or more. Magnesium begins to
interfere when the magnesium-to-potassium ratio exceeds 100: 1.
Flame photometer

1. Deionised distilled water: Use this water for preparing all reagents and cali-
bration standards, and as dilution water.
2. Stock potassium solution (1.907g1L): Dissolve 1.907 g of dry (11 OOC) KCI in
1000 mL of distilled water; 1 mL = 1 mg K.
3. Intermediate potassium solution: Dilute IOmL of stock K solution with water
to 100 mL. ImL = IOOllg K. Use this intennediate solution to prepare calibra-
tion curve in K range of 1 to 10 mgIL.
4. Standard potassium solution: Dilute 10 mL intennediate potassium solution
with water to 100 mL; I mL = 10 Ilg K. Use this solution to prepare calibration
curve in K of 0.1 to 1 mgIL.
To minimize potassium pickup, store all solutions in plastic bottles.


Same as sodium. Use potassium filters instead of sodium. Minimum detectable
concentration of potassium is approximately 0.1 mgIL. '

Check Your Confidence on Sodium and

Potassium Estimation
I. List four important environmental significances of sodium estimation.
2. Enumerate the principle of flame photometer.
3. List the important units ofa flame photometer.
4. Discuss the operation of flame photometer.
S. How sodium is estimated in highly polluted samples and wastewater?

3.30 Sulphate

Drinking water
USEPA, SMCL (1974) = 2S0 mgIL
Canadian MAL (1987) = 500 mgIL
WHO (1984) = 400 mgIL (aesthetic quality)
156 Handbook ofMethods in Environmental Studies

ICMR (1963) = 200 mWL - 400 mWL (max. pennissible)
IS: 10500 (1991) = 200 mWL (max) - 400 mWL (max pennissible in the
absence of alternate source)
MWH (1975) = 200 mWL (acceptable - 400 mWL (cause of rejection)
Effluent standards
MOEF (1993) = 1000 mWL (maximum limit) is for discharge in inland
surface water, p~blic sewer and water for irrigation.

1. The sulphate ion is one of the major anions occurring in natural waters.
2. It is of importance in public water supplies because of its cathartic effect upon
humans when present in excessive amounts.
3. Sulphate is one of the least toxic anions and WHO (1984) does recommend
guideline value of 400 mWL in drinking water. However, catharsis, dehydration
. and gastrointestinal irritation have been observed at high concentration in
drinking water and WHO therefore suggests the health authorities should be
notified when concentration of sulphate in drinking water exceeds 500 mWL.
4. Waters with about 300-400 mWL sulphate have a bitter taste and those with
1000 mWL or more of sulphate may cause intestinal disorders (laxative).

Environmental Significance
• Water containing appreciable amounts of sulphates fonns hard scale in boilers
and heat exchangers.
Sulphate also causes odour and corrosion of sewer in anaerobic conditions
because it gets converted to hydrogen sulphide.

Estimation of Sulphate
Sulphate can be estimated by either gravimetric or turbidimetric methods (APHA,
1. Gravimetric method is suitable for SO:- concentration above 10 mWL.
2. Turbidimetric method is applicable in the range of 1-40 mg SO:-IL.

Sulphate ions are precipitated in a hydrochloride acid medium as barium sulphate

(BaSO4) by the addition of barium chloride (BaCl). The precipitation is carried out
near the boiling temperature, after a period of digestion the precipitate is filtered,
washed with water until free of chlorides, dried and weighed as BaSO."
Baelz (excess) + SO:- ~ BaS04 (precipitate)
Analysis of Water and E.fJluents 157

The samples are acidified to eliminate the possibility of precipitation ofBaC03,

which might occur in highly alkaline water maintained near boiling temperature.
Excess BaClz is used to produce sufficient common ion to precipitate sulphate
ion as completely as possible. Because of the high insolubility of barium sulphate,
there is a considerable tendency for much of the precipitate to form a colloidal
condition that can not be removed by ordinary filtration process.
Thus digestion of the sample at temperature near the boiling point for a few
hours usually results in a transfer of the colloidal to crystalline forms and filtration
can then be accomplished.
The gravimetric determination of sulphate is subjected to many errors, both positive
and negative. Interferences that lead to high results are suspended matter, silica,
BaClz precipitant, NO;, SO:- and water occluded in the precipitant. Interferences
leading to low results due to alkali metals, sulphates, heavy metals (especially
chromium and iron) and the solubility of the BaS04 precipitant, especially in acid
solution. In drinking water where the mineral concentration is low, these are of
minor importance.
I. Drying oven, equipped with thermostat control Muffle furnace with heat
2. Desiccator, Crucible
3. Analytical balance, capable of weighing upto 0.1 mg.
4. Filter paper: Acid -washed, ash-less hard-frnish filter paper sufficiently retentive
for frne precipitates.
Chemicals and Reagents
1. Methyl red indicator
2. Hydrochloric acid, HCI
3. Conc. HN03
4. Barium chloride, BaClz
5. Silver nitrate, AgN03
1. Methyl red indicator solution: Dissolve 100 mg methyl red-sodium salt in
distilled water and dilute to 100 mL.
2. Hydrochloric acid, HCI 1+1: Dilute conc. HCI with distilled water in 1: 1
3. Barium chloride solution: Dissolve 100 g BaCI2' 2Hp in 1000 mL of distilled
water. Filter through a membrane filter before use. 1 mL ofthis reagent is capable
of precipitating approximately 40 mg S04'
4. Silver nitrate-nitric acid reagent: Dissolve 8.5 g AgN03 and 0.5 mL conc
HN03 in 500 mL distilled water.
158 Handbook ofMethods in Environmental Studies

I. Removal of silica
1. If the sample contains more than 25 mgIL silica, it has to be removed prior to
the sulphate analysis. Forremoval ofsilica, evaporate suitable volume of sample
near to dryness in a platinum dish on a water bath. Add 1 mL HCI , tilt, and
rotate dish until the acid comes in complete contact with the residue. Continue
evaporation to dryness. If organic matter is present dry in hot air oven at
180°C. Moistened the residue with 2 mL of distilled water and 2 mL of HCl.
Transfer the residue in hot water and filter it. Wash insoluble silica with several
small portions of hot distilled water. Combine filtrate and washings. Discard
II. Precipitation of barium sulphate (BaSOJ
1. Adjust the volume of clarified sample (treat if necessary to remove interfering
substances) which contains approximately 50 mg sulphate ion in a 250-mL
2. Acidity with HCI to pH 4.5 to 5.0, using a pH meter or orange colour of methyl
red indicator.
3. Then add an additional 1 to 2 mL HCl.
4. Heat the solution to boiling and with stirring gently, and warm BaC~ solution
slowly precipitation appears to be complete; then add 2 rot in excess.
5. If the amount of precipitate is small, add a total of SmL BaC~ solution.
6. Digest the precipitate at 80 to 900c, preferably overnight but not less than 2 h.
IlL Preparation of filter
Place a filter on a watch glass and dry in a conventional oven of 103 to 105°C. Cool
in a Desiccator and weigh the filter paper.
Iv. Filtration and weighing
1. Mix a small amount of ash-less filter paper pulp with the BaSO. and filter at
room temperature. The pulp aids filtration and reduces the tendency of the
precipitate to creep.
2. Wash the precipitate with small portions of warm distilled water until the
washings are free of chlorides, as indicated by testing with silver nitric acid
3. Dry the filter with precipitate by the same procedure used in preparation of
filter. Cool in a desiccator and weigh.
mg BaSO. x 411.5
mgIL SO.
Used successfully for drinking water, ground and surface water.
Analysis of Water and Effluents 159

Sulphate ion (SO;-) is precipitated in an acetic acid medium with barium chloride
(BaCI 2) so as to form barium sulphate (BaSO4) crystals of uniform size.
BaCI2 (excess) + SO:- --+ BaS04 (precipitate)
The crystal formation is enhanced in the presence of an acetic acid buffer solu-
tion containing magnesium chloride, potassium nitrate, sodium acetate and acetic
Light absorbance of the BaSO4 suspension is measured by a photometer and
sulphate concentration is determined by comparison of the reading with a standard
curve. Minimum detectable concentration is approximately 1 mg sulphate IL.
• Colour or suspended matter in large amounts will interfere. Some suspended
matter may be removed by filtration.
• Make determination at room temperature; variation over a rage of 10°C will not
cause appreciable error.
1. Magnetic stirrer: Use a constant stirring speed. Use magnets ofidentica1 shape
and size. The exact speed of stirring is not critical, but keep it constant for each
run of samples and standards and adjust it to prevent splashing.
2. Nephelometer or Spectrophotometer (use at 420 nm, providing a light path of
2.5 to 10 cm).
3. Stopwatch.
4. Measuring spoon, capacity 0.2 to 0.3 mL.
Chemicals and Reagents
1. Magnesium chloride (MgClr 6HP)
2. Sodium acetate (CH3COONa.3Hp)
3. Sodium sulphate (Na2S04)
4. Potassium nitrate (KN03)
5. Acetic acid (CH3COOH, 99%)
6. Barium chloride crystals (BaCl r 2HP)

1. Buffer solution A: Dissolve 30 g magnesium chloride (MgClr 6H 20), 5 g

sodium acetate (CH3COONa.3Hp), 1 g potassium nitrate (KNO) and 20 mL
acetic acid (CH3COOH, 99%) in 500 mL distilled water and make up to 1000
2. Buffer solution B (required when the sulphate concentration is less than
10 mgIL): Dissolve 30 g magnesium chloride (MgClr6~O), 5 g sodium acetate
(CH3COONa.3Hp), 0.111 g sodium sulphate (Na2S04) and 20 mL acetic acid
(CH3COOH, 99%) in 500 mL of distilled water and make up to 1 L.
160 Handbook ofMethods in Environmental Studies

3. Barium chloride, BaClz.2HzO: Crystals of 20 to 30 mesh size. In

standarisation uniform turbidity is produced with this mesh range and the
appropriate buffer. To prepare in the laboratory, spread crystals over a large
watch glass, desiccate for 24h. Screen to remove any crystals that are not 20 to
30 mesh size. Store in a clean dry jar.
4. Standard sulphate solution: Dissolve 0.1479 g anhydrous Na2SO4 in distilled
water and dilute to 1000 mL. 1 mL = 100 J,lg sulphate.
1. Formation of barium sulphate turbidity
• Filter the sample, if it is turbid, adjust the temperature between IS to 30°C.
• Measure 100 mL sample, or a suitable portion made up to 100 mL, into a 250
mL erlenmeyer flask.
• Add 20 mL buffer solution and mix in stirring apparatus.
• While stirring, add a spoonful of BaCl2 (0.3 g) crystals and note the timing
Stir for 60 ± 2 seconds at constant speed. Stirring should be at a constant rate in
all detenninations. The use of magnetic stirrer has been found satisfactory for
this purpose.
2. Measurement of barium sulphate turbidity
• After stirring period has ended, pour solution into absorption cell of photom-
eter and measure turbidity at 5 ± 0.5 min.
3. Preparation of calibration curve
• Estimate sulphate concentration in sample by comparing turbidity reading with
a calibration curve prepared by carrying sulphate standards through the entire
• Space standards at 5 mg/L increments in the 0 to 40 mglL sulphate range.
• Above 40 mg/L accuracy decreases and BaSO4 suspensions lose stability. Check
reliability of calibration curve by running a standard with every three or four
4. Correction for sample colour and turbidity
• Correct for sample colour and turbidity by running blanks to which BaCl2 is not
mg SO~- x 1000
mg SO~-IL = - - - - - -

Removal of Sulphate from Water

Conventional treatment does not remove sulphate. Ion-exchange can be used for
sulphate removal from municipal water supplies. If chemical coagulation process
is used with aluminum sulphate, concentration of sulphate in raw water is increased
Analysis o/Water and Effluents 161

in the purification process with finished water containing an additional 20-5,0

mglL of sulphate. Corrosion of metals (distribution system or cooling system) may
be aggravated by high sulphate concentration.

Check Your Confidence on Sulphate Estimation

1. What are the effects of high concentration of sulphate on human health when
present in drinking water?
2. List three environmental significances of sulphate estimation.
3. List two important precautions that must be observed to ensure an accurate
gravimetric determination of sulphate concentration.
4. Why excess BaCl2 is added?
5. Why precipitation ofBaS04 is done at boiling point temperature in gravimetric
6. For estimation of low level of sulphate concentration which method to be
7. What is the purpose of adding buffer solution in the turbidimetric determination
of sulphate concentration?
8. How sulphate is removed form municipal water supplies?

3.31 Sulphide
(Iodometric Method)

Drinking water
WHO, (1984) = No guideline value set for drinking water, however, it should
not be detectable by consumers
IS: 10500 (1991) = No guideline value set for drinking water.
Effluent discharge
MOEF (1993) = Max 2mgIL and 5mgIL has been set for discharge to inland
surface water and marine coastal areas respectively

1. Sulphide is often present in groundwaters, especially in hot springs.
2. Its common presence in wastewater comes partially from the decomposition of
organic matter, sometimes form industrial wastes, but mostly from the bacterial
reduction of sulphate.
3. Hydrogen sulphide escaping into the air from sulphide-containing wastewater
causes odor nuisance. The threshold odour concentration ofH2 S in clean water
is 0.025 and 0.25 mgIL.
162 Handbook ofMethods in Environmental Studies

Environmental Significance
1. Gaseous H2S is very toxic in sewers.
2. H2S attacks metals directly and indirectly and causes serious corrosion of
concrete sewers.
3. Dissolved H2S is toxic to fish and other aquatic organisms.

Estimation of Sulphide
In acidic medium excess iodine is added which oxidizes H2S. The remaining iodine
is titrated against standard thiosulphate solution using starch as an indicator.
H2S + 12 ~ 2W + 21- + S
12 + Sp:' ~ 21- + S.o:·
(lmL 0.025N 12 = 0.4 mg H2S)
1. Hydrochloric acid
2. Iodine
3. Starch
4. Potassium iodide, KI
5. Sodium thiosulphate, Na2SPr5HP
6. Formaldehyde as preservative
1. Hydrochloric acid; Hel, 6N: Dilute conc. HCl (I2N) two times
2. Standard iodine solution, O.025N: Dissolve 20 to 25 g KI in a little water and
add 3.2 g iodine. After iodine has dissolved, dilute to 1000 mL and standardize
against 0.025N Na2Sp) using starch solution as indicator.
3. Standard sodium thiosulphate solution (O.02SN): Dissolve directly 6.025 g
Na2Sp).5Hp in a previously boiled 1L cooled distilled water, which will give
0.025N. Add 1.5 mL 6N NaOH or 0.4 g solid NaOH per litre . . Store in
brown bottle. This solution will have to be standardised against standard
potassium dichromate (K2Crp,) solution for each set of titration.
4. Starch indicator, 1%: Dissolve Ig of starch in 100 mL of distilled water and
heat. Add few drops of formaldehyde solution after cooling.
PROCEDURE (Fig. 3.27)
1. Measure from a burette into a 500-mL flask an amount of iodine solution
estimated to be an excess over the amount of sulphide present.
2. Add distilled water, if necessary, to bring volume to about 20 mL.
Analysis o/Water and Effluents 163

3. Next, add 2 mL 6N HC!.

4. Pipette 200 mL sample into flask, discharging sample under solution surface. If
iodine color disappear, add more iodine until colour remains.
5. Back titrate with Na2Sp3 solution, adding few drops of starch solution as end
point is approached, and continuing until blue colour disappears.
Precision: The precision of the end point varies with the sample. In clean waters it
should be determinable within 1 drop, which is equivalent to 0.1 mgIL in a 200-mL

Stlnd ..d

Scheme of sulphide determination

200 IIIL S• ..,.,
~O.02S N Std. Iodint Sotution
+2 .. L6N HCL
+SIa~ ind\Q1ot


(End PoInt,

Fig. 3.27: Estimation of sulphide.


1 mL 0.025 N iodine solution reacts with 0.4 mg S2-

[(A x B)-(C x D)] x 16 x 1000
Sulphide (S2-) mgIL =
mL sample

A = mL iodine solution
B = normality of iodine solution
C = mL Na2Sp3 solution
D = normality of Na2Sp3 solution

Check Your Confidence on Sulphide Estimation

1. What are the sources of sulphide in wastewater?
2. List three important environmental significances of sulphide.
164 Handbook of Methods in Environmental Studies

3.32 Thiocyanate

Drinking water: No guideline value set (IS: 10500, 1983, 1991; WHO, 1984,
Emuent discharge: No guideline value set (MOEF, 1993).

Environmental Significance
Many natural waters contain thiocyanate from organic decomposition products and
wastewater discharges. Some industrial waste, such as those from the steel indus-
try, petroleum refining, and coal gasification, may contain significant concentra-
tion of thiocyanate. Thiocyanate is not recognised as a toxic chemical compound.
However, when chlorinated, thiocyanate is converted to the highly toxic and vola-
tile cyanogen chloride.

Estimation of Thiocyanate
This test method covers the detennination of dissolved thiocyanate in water, waste-
water, and saline water in the range from 0.1 to 2.0 mgIL. For higher concentra-
tions, use an aliquot from the diluted sample.


1. Thiocyanate reacts with ferric ions at a pH of <2. to fonn a coloured complex

which is detennined colorimetrically at 460 om and adheres to Beer's Law.
2. Industrial waste may be highly coloured and contain various organic compounds
which must be removed by absorption on macroreticular resin prior to analy-

Many samples contain cyanide. Because of the toxicity of cyanide, great care must
be exercised in its handling. Acidification of cyanide solutions produces toxic
hydocyanic acid (HCN). All manipulations must be done in the hood so that any
HCN gas that might escape is safely vented. Residual sample remains could be
toxic hence these should be disposed off properly.

I. Thiocyanate is stable in both the acid and alkaline pH range.
2. If the sample is to be preserve for cyanide, remove the sulfide before stabilisation
at a high pH. Cyanide can be converted into thiocyanate in the presence of
sulfide at a high pH.
3. Thiocyanate is biodegradable. Samples that may contain bacteria should be
preserved at pH < 2 by the addition of mineral acid and refrigerated.
Analysis o/Water and EfJIuents 165


1. Spectrophotometer: Suitable for absorbance measurement at 460 nm and

using a 5-cm cell.
2. Column: Chromatographic glass, 12-mm inside diameter, by 600-mm length,
equipped with a reservoir and stopcock or a 50-mL burette with a glass wool
plug and a funnel attached with a short piece of tubing.


I. Acetone
2. Hexane
3. Methyl alcohol
5. Macroreticular resin, 18 to 50-mesh or equivalent
6. Ferric nitrate solution
7. HPz solution
8. Conc HN03 , sp. gr. 1.42
9. NaOH

1. Ferric Nitrate Solution: Dissolve 404 g of ferric nitrate [Fe(NO)).9HPJ in

about 800 mL of water. Add to this solution 80 mL of conc HNOr Mix and
dilute to 1 L with water.
2. Hexane
3. Hydrogen Peroxide Solution (RzOz), 30%.
4. Methyl Alcohol.
S. Nitric Acid: Concentrated HNO), sp. gr. 1.42.
6. Nitric Acid (O.IM): Mix 6.4 mL of conc HNO) in about 800 mL of water.
Dilute to I L with water and mix.
7. Thiocyanate Solution, Stock (1 mL =lmg SCN-): Dissolve 1.673 g ofpotas-
sium thiocyanate (KSCN) in water and dilute to lL.
8. Thiocyanate Solution, Standard (1 mL = 0.01 mg SCN-): Dilute 10 mL of
the stock thiocyanate solution to I L with water. Prepare fresh for each use.
9. Sodium Hydroxide Solution: Dissolve 4 g ofNaOH in about 800 mL ofwa-
ter. Mix and dilute to 1 L with water.
10. Macroreticular Resin, 18 to SO-mesh or equivalent: The available resin may
not be purified. Sometimes resin may be contaminated with waxes and oils,
which gives poor permeability and absorption. Purification of res in can be car-
ried out as follows:
a. Place sufficient resin to fill the column or columns in a beaker and 5 times the
resin volume of acetone. Stir gently for I h.
b. Pour off fines and acetone from settled resign and add 5 times the resign vol-
ume of hexane. Stir for lh.
c. Pour off fines and hexane and add 5 times the resin volume of methanol. Stir
for 15 min.
166 Handbook a/Methods in Environmental Studies

d. Pour off methanol and add 3 times the resin volume ofO.IN NaOH. Stir for 15
e. Pour offNaOH solution and add 3 times the resign volume ofO.IN HN03 • Stir
for 15 min.
t: Pour off HN03 solution and add 3 times volume of distilled water. Stir for 15
min. Drain excess water and use purified resin to fill the column. Store excess
purified resin after converting it with distilled water. Keep in a closed jar.
Preparation of calibration curve
l. Prepare a series of thiocyanate standards containing 0.0 to 2 mg SCN-IL by
pipetting 0 (blank) to 40 mL aliquots of standard thiocyanate solution into
200 mL volumetric flask. Dilute to volume with water and mix thoroughly.
Follow step 2, 3 and 4 as follows.
Method of estimation In water sample
2. Acidify 150 mL of sample (or an aliquot of sample diluted to 150 mL) to pH 2
by dropwise addition of cone HN03 and pass it through the resign column at a
flow rate not exceeding 20 mUmin.
3. Pour the 50 mL of collected elute into a beaker, add 2.5 mL of ferric nitrate
solution, and mix.
4. Within 5 min., determine the absorbance of the solution at 460 om in a 5-cm
cell using water as a reference.
Calculate the concentration of thiocyanate (SCN-) in mgIL as follows:
Slope and Intercept of Standard curve:
Thiocyanate, mgIL = (ma + b) x (dilution factor, ifany)
a = absorbance of sample solution
b = intercept of c axis, and
m = slope of standard curve
(b and m are constants in regression equation used for making the calibration

Check your Confidence on Thiocyanate

I. Name a few industrial processes from where thiocyanate is discharge in
2. Discuss the principle involved in thiocyanate estimation.
3. How resins are purified in the laboratory?
4. Thiocyanate is biodegradable (True/false).
Analysis of Water and EjJ1uents 167

3.33 Transparency (Sec chi Disc Method)

The Secchi disc is named after its Italian inventor Pietro

Angelo Secchi. Transparency is a water-quality
characteristic of lakes and reservoirs and can be
measured quickly and easily using simple equipment.
This characteristic varies with the combined effects of
colour and turbidity. Some variation may also occur
with light intensity and with the apparatus used.
Secchi disk attached to a string
The disc is made of rigid plastic or metal, but the details
of its design are variable. It may be 20 to 30 cm or
even larger in diameter and is usually painted white.
Alternatively, it may be painted with black and white
quadrants. The disc is suspended with a light rope or
chain so that it remains horizontal when it is lowered
Fig. 3.28: The Secchi disc. into the water. The suspension rope is graduated at
intervals of 0.1 and 1 meter from the level of disc
itself and usually does not need to be more than 30 m length. A weight fastened
below the disc helps to keep the suspension rope vertical while a measurement is
being made. Fig. 3.28 shows a typical secchi disc. The same size and pattern of
disc should be used at any given sampling station so that a series of measurements
made over a number of years are free as possible from distortions arising from
differences in apparatus.


Suspended algae, microscopic animals, suspended matter (silt, clay and organic
matter) and water-colour are factors that interfere with light penetration into the
water column and reuce Sec chi disc transparency.


Combined with other field observations, Secchi disc reading may furnish
informations on:
1. Suitable habitat for fish and other aquatic life;
2. The lake's water quality and esthetics;
3. The state of the lake's nutrient enrichment; and
4. Problems with and potential solutions for the lake's water quality and recreational
use impairment.
168 Handbook ofMethods in Environmental Studies

1. The observation should be made early in the morning or late in the afternoon.
2. Lower the Secchi disc, where possible, through a shaded area of water surface.
As the disc is lowered, note the depth at which it just disappears from view.
Note the point on string at upper surface of water (L.).
3. Lower the disc a little further, then lift up slowly till it just reappears. Note the
point on the string at upper surface of water (L2). Calculate as follows:

L. +L2
Secchi disc Transparency = - - -
The report must also state the diameter of the disc and the pattern, if any, on the
upper surface of the disc.

Sec:chi disc (SO) transparency value is used to state the lake tropic status index
(TSJ) as below.
Lou tropic state SD transperancy, m Chl-a (pg/L) Total P (pg/L) 1'81
Oligotropic <4 <2.6 < 12 <40
Mesotropic 2-4 2.6-7.2 12-24 40-50
Eutropic 0.5-2 7.2-55.5 24-96 50-70
Hypereutropic <0.5 > 55.5 >96 >70

The TSI can be calculated for SO transparency, Cha a (ChI) in Ilg/L and total P
(TP) in Ilg/L as follows:
On the basis of SO tranaparency; TSI = 60-14.4 In (SO)
On the basis of Chi: TSI = 9.81 In (Chi)) + 30.6
. On the basis ofTP, TSI = 14.42 In (TP) + 4.15

Example - 1
Lake monitoring shows that the Secchi disc transparency is 1.96m, total P 33 mg/L
and chi-a 3.4 mg/L. Calculate TSI using the above equation.

Check Your Confidence on Transparency

1. List the important water quality parameters that affect transparency
2. When transparency should be measured?
3. List two applications of transparency data.
Analysis of Water and E.IJluents 169

3.34 Turbidity (Nephelometric Method)

Drinking water:
WHO (1984) = 5 NTU (preferably <1 for disinfection efficiency).
WHO (1993) = 5 NTU (median turbidity <1 NTU, single sample <5 NTU)
USEPA (1974) = 1 to 5 NTU
Canada (MAC, max allowable conc, 1987) = 5 NTU
IS: 10500 (1983) = 10 NTU (max);
IS: 10500 (1991) = 5 NTU (above 5 NTU consumers acceptance decreases),
10 NTU (maximum permissible in the absence of alternate source.
MHW (1975) = 5 NTU (acceptable) - 25 NTU (cause of rejection).
Emuent sample
MOEF (1993) = No guideline value set.

Sources of Turbidity
Turbidity is caused by wide variety of suspended and colloidal materials. Run-off
from barren areas during rain is the most natural contributor of turbidity, particularly
silt and clay. The discharge of untreated industrial and domestic effluents also adds
great quantities of turbidity. Organic material reaching water bodies serves as food
for bacteria, resulting the enhancement of bacteria and other microorganisms feed
upon bacteria. Effluents from sewage treatment plants, rich in nitrogen and
phosphorus, as well as agricultural run-off stimulate growth of algae and also
contribute to turbidity.

Environmental Significance
Turbidity is an important consideration in public water supplies for three major
reasons -
• Aesthetics - turbidity in drinking water is automatically associated with possible
wastewater pollution.
• Filterability - Filtration of water is rendered more difficult and costly when
turbidity increases.
• Disinfection - many ofthe pathogenic organisms may be encased in the particles
and protected from the disinfectant.

Estimation of Turbidity

Turbidity may be defmed as interference to the passage of light by suspended par-

ticles in water. It can be measured by its effect on the transmission of light, which is
termed as Turbidimetry or by its effect on the scattering oflight, which is termed as
170 Handbook of Methods in Environmental Studies

Nephelometry (Fig.3.29). Turbidimeter can be used for sample with moderate tur-
bidity and Nephelometer for samples with low Turbidity. Higher the intensity of
scattered light higher the turbidity.
The original standard chosen for turbidity was silicon dioxide (Si02).
1 mg Si02 1L = 1 unit of turbidity.
The Si02 was originally used to calibrate the Jackson candle turbidimeter. But
Jackson candle turbidimetric unit (JTV) has been removed as standard method of
turbidity measurement in 17th edition of Standard methods (1989). The formazine
standard of 40 NTV is equivalent to 40 JTU. The lowest turbidity measured by
Jackson candle turbidimeter is 25 JTV.

Li .... "'' .7
Sht c:::::I c:::o

Pl'Iototube \.:.J'r N~ph~lorn~ .r
Fig. 3.29: Schematic diagram of a turbidimeter and a nephelometer.

Dissolved material that imparts a color to the water may cause serious errors in
nephelometric readings. Floating or suspended large particles and entrained air
bubbles will give false or unstable readings. Certain turbulent motions also create
unstable reading conditions of nephelometer.


Scratches, fmger marks, or dirt on the wall of the sample cell may give erroneous
readings. Cells should be kept scrupulously clean both inside and outside and discard
when their is a scratch.
Determine the turbidity on the day the sample is taken. If this is not feasible, store
the sample in the dark for up to 24 h and refrigerate at 4"C if possible, but do not
freeze. Prolonged storage is not recommended because of irreversible changes.

Bring the sample to room temperature and shake sample vigorously for at least one
.min. Let the sample stand 2 to 3 min to allow air bubbles to disappear, then gently
invert the sample several times or swirl, mix before examination.
Analysis o/Water and EjJ1uents 171


1. Nephelometer: Range 0.05 - 40 NTU to as low as 0.05 - 1 NTU.
2. Formazine Turbidity Suspension, Stock: A stock turbidity suspension for
fonnazine polymer is prepared by reacting hydrazine sulfate with hexamethylene
tetramine under carefully controlled conditions.
a. Solution - I: Dissolve 1.0 g hydrazine sulfate [~)lHzS041 and dilute to mark
in a lOO-mL volumetric flask. (caution: Hydrazine sulfate is carcinogenic, avoid
inhalation, ingestion and skin contact. Fonnazine suspension can contain
residual hydrazine sulfate).
b. Solution - II: Dissolve 10.0 g hexamethylene tetramine and dilute to mark in a
100-mL volumetric flask.
Co Solution - III: Mix 5mL of Solution - I with 5mL of Solution - II. Allow to
stand for 24 h at 25 to 28 "C and dilute to 100mL after reaction. This mixed
solution will have turbidity of 400 units (NTU). This 400 NTU stock has to be
prepared monthly.
3. Formazine turbidity suspension, standard (40 NTU): Pipet 10 mL of 400
NTU stock into a 100-mL volumetric flask and dilute to 100 mL with water.
The turbidity of this suspension is defined as 40 NTU and has to be prepared
4. Diluted Formazine turbidity suspension, standard: - Prepare diluted turbidity
suspension below 4 NTU daily. Those above 4 NTU have a useful life of one
1. Preparation ofcalibration curve: Prepare turbidity solution from 0 to 10 NTU
as follows: . .
Stock turbidity solution Distilled water, mL Total volume. mL NTU
40NTU. mL
2.5 97.5 100 1
5 95 100 2
10 90 100 4
12.5 87.5 100 5
15 85 100 6
20 80 100 8
25 75 100 10
2. Calibration o/instruments (Fig. 3.30):
NTU standard value NTU instrument reading
10.0 10.0-
8.0 8.1
6.0 6.3
4.0 4.7
2.0 2.8
-Instrument is adjusted to read this value.
172 Handbook ofMethods in Environmental Studies


o2 4 6 • ~

Fig. 3.30: Linear calibration of Instrument.

3. Turbidity measurement ofsample: Gently agitate the sample and wait till all air
bubbles disappear.
4. The Nephelometer reading is noted and NTU value is determined from the
standard curve (Fig. 3.30).
5. If turbidity is exceeded 40 NTU, dilute the sample. In such case, apply correction
factor. (for example, if the sample is diluted to 10 times and the Nephelometer
reading is 9.5 NTU, the turbidity of unknown solution is 9.5 x 10 = 95 NTU).

Turbidity Summary
Caused by: Suspended and colloidal matter such as clay, silt, organic and inor~
ganic particulate, plankton and other micro-organisms.
Concentration expected: From almost zero (0.05 NTU) in distilled water to over
1000 NTU in highly turbid water.
Indicator of: Necessity of treatment, potential contamination by pathogen; poor
treatment plant efficiency - Coagulant dosage, filter run timing, contamination in
distribution system.
Test: A physical and microbiological parameter, simple, inexpensive, mandated
by public health regulation; expressed in the Nephelometric turbidity unit (NTU).
NTU: Turbidity unit measurd by Nephelometric standard method.
Regulation: 0.1 NTU as a goal; less than INTU as a Standard; 5 NTU as an
exception for potable water.

Application of Turbidity Data

Turbidity measurements are used to determine the effectiveness of the treatment
produced with different coagulants and the dosages needed.
Turbidity measurements of the filtered water are needed to check on faulty
filter operation.
Analysis o/Water and EfJluents 173


• To determine the effectiveness of suspended solids removal.
• Chemical dosages can be adjusted to use the minimum amount of chemical
while producing a high-quality effluent.

Check Your Confidence on Turbidity

1. List the various sources of turbidity in water bodies.
2. High turbid water is a indicator of -----------, -------------, ---------.
3. What are the differences between NTU and JTU.
4. Why turbidity measurement by Jackson Turbidimeter . has been rejected?
5. List two interfering substances that cause error in nephelometer reading.
6. Why turbidity measurement is important in drinking water?
7. "Turbidity should be determined on the day the sample is collected! reached to
the laboratory" - Why? J~tify your answer.
8. As per WHO (1993) "5NTU is permissible but <1 NTU is preferable for
disinfection efficiency"- Why?
Analysis of Metals in
Water and Effluents

4.1 Introduction

Metals constitute an important portion of drinking water and wastewater. Metals
with specific gravity greater than 5 or often more are termed as heavy metals. The
term simply used to denote metals that are toxic. The most important route for
elimination of metals is via kidney. In fact kidney can be considered to be complex
filter whose primary purpose is to eliminate toxic substances from the body.
The kidney contains million of excretory units called nephrons. Chemicals that are
toxic to the kidney are called nephrotoxins. Metals like Cd, Pb and Hg are
1. Heavy metals: Cd, Cr, Cu, Mn, Hg, Pb, Ag, Zn
2. Non-metallic: As, Se
3. Others: Na, K, Ca, Mg, Fe

4.2 Standards of Heavy Metal

About 12 important major elements occasionally present in drinking water are
generally monitored. The limits of permissible concentrations as suggested by
IS:I0500 (1991), WHO (1984) and USEPA (1974) are given in Table 4.1.
Table 4.1:
Limits of heavy metals in drinking water (mg/L).

Elements in IS 10.500 WHO USEPA Remarks

mg/L (1991) (1984) (1974)

1. Arsenic 0.05 (no 0.05 0.05 Dermal and nervous sys-

(As) relaxation) tem toxicity. To be
tested when pollution is
2. Copper 0.05 (may be 1.0 1.00" Desirable.
(Cu) relaxed upto (permis-
1.5) sible)1.5
(excessive) Cont...
Analysis ofMetals in Water and Effluents 175

... Cont.
3. Cadmium 0.01 ( no 0.005 0.01 Kidney effect.
(Cd) relaxation)
4. Chromium, 0.05 (no 0.05 0.05 Liver/kidney effect.
crS· relaxation)
5. Iron (Fe) 0.3 (may be 0.3 0.30· Taste/appearance are
extended up to affected.
1.0 in absence of
alternative sources)
6. Lead (Pb) 0.1 (no 0.05 0.05 Central and PNS; kidney.
••. cont.
7. Manganese 0.1 (may be 0.1 0.05· Hardness,taste,
(Mn) extended up to gastrointestinal irritation
0.5 where altern- in presence of sulfate.
ate source is not
8. Mercury 0.001 (no 0.001 0.002 Central nervous system
(Hg) relaxation) disorder.
9. Silver NVS NVS 0.05 Skin discoloration
(Ag) (Argyria).
10. Selenium 0.01 (no 0.01 0.01 Gastrointestinal effect.
(Se) relaxation)
11 . Zinc (Zn) 5 (may be 5 (permis- 5· Desirable.
extended upto sible)

·Secondary drinking water standards (USEPA, 1974): NVS = No value set.

PNS = peripheral nervous system
Cu, Fe, Mn and Zn are put under Aesthetic quality by WHO (1984). Rest 8 metals are
toxic even in trace amounts.
Guidelines for Drinking water Quality, Vol 1, and Recommendation. (Source: The
Water Encyclopedia, Lewis Publishers, 1990.pp 441)


In effluent discharge, about 16 elements are monitored as per IS: 2490 (Part-I,
1981) and 13 elements are mentioned as per MOEF, Schedule - VI, Part A, 1993
(Table 4.2).

Table 4.2:
General standards of effluent discharge for the metals pollutants,
(MOEF, Schedule - VI, Part A, 1993)

Elements Inland Public Discharge on Marine

All in mg/L surface sewers Land for coastal
water irrigation areas

1. Arsenic (as As). Max 0.2 0.2 0.2 0.2

2. ·Boron (as B), max 2 2 2
3. Copper (as CuI, Max 3 3 3
4. Cadmium (as Cd), Max 2 1 2
5. Chromium,Cr·', Max 0.1 2 1
6. Total chromium (as Cr),Max 2 2 2
176 Handbook ofMethods in Environmental Studies

... Cont.
7. Iron (as Fe) 3 3 3
S. Lead (as Pb), Max. 0.1 1 2
9.Manganese (an Mn) 2 2 2
10.Mercury (as Hg), Max. 0.Q1 0.01 0.01
11.· Nickel (as Nil, max. 3 3 5
12.· % sodium (% Nal, max· 60
13.Selenium (as Se), Max. 0.05 0.05 0.05
14. Zinc (as Znl, Max. 5 15 15
15. Vanadium (as V) 0.2 0.2 0.2

·Boron,Nickel and % Sodium have been omitted by G.S.R. SO(EI, dated 31st
December,1993 (w.e.f. 31.12.1993).

Na x 100
• % Na =
Ca + Mg + Na + K
Where concentration of all the metals is in meq./L.

4.3 Sampling and Preservation of Samples

Preserve samples immediately by acidifying with concentrated nitric acid (RN03)
to pH < 2. Filter samples for dissolved metals before preserving. Usually 1.5 mL
conc. RN0 3 per litre sample is sufficient for short-term preservation.
1. Preliminary treatment of sample: Sample containing particulate or organic
material generally require pretreatment before analysis. Total metals in-
clude all metals, inorganically and organically bound, both dissolved and
2. For drinking water analysis: Can be analyzed directly without acid diges-
tion. However, the sample need acidification with RN0 3 to pH <2 before
3. Waste water/ effluent samples: Digest before analysis. Nitric acid will di-
gest most samples adequately.

Those constituents (metals) of an un-acidified sample that pass through 0.45 ~m

membrane filter.


Those constituents (metals) of an un-acidified sample that are retained by a 0.45

~m membrane filter.


The concentration of metals determined on an un-acidified sample after vigorous

digestion. or, sum of the concentration of metals in both dissolved and suspended
Analysis ofMetals in Water and Effluents 177


The concentration of metals in solution after treatment of an unfiltered sample with

hot dilute mineral acids.

4.4 Digestion of Metals with Nitric Acid


1. Hot plate
2. Conical (erlenmeyer) flasks, 125-mL, or Griffin beakers, 150-mL, acid-washed
and rinsed with water.
3. Volumetric flasks, 100-mL.


Nitric acid, HN03 Conc.

1. Mix samples and transfers a suitable volume (50 to 100 mL) to a beaker. Add 5
mL conc. HN03 , cover with glass.
2. Bring to a slow boil and evaporate on a hot plate to the lowest volume possible
(about 10 to 20 mL) before precipitation occurs.
3. Continue heating and adding conc. HN03 as necessary until qigestion is com-
plete as shown by a light-coloured, clear solution.
4. Do not let the sample dry during digestion. Wash down the walls of the beaker
and watch -glass with distilled water and then filter.
5. Transfer the filtrate to a 100 mL volumetric flask, cool and makeup the volume.
Use portions of this solution for required metal analysis.

4.5 Digestion of Metals with HCI and HN03


1. Hot plate
2. Conical (erlenmeyer) flasks, 125-mL, or Griffm beakers, 150 mL, acid-washed
and rinsed with water.
3. Volumetric flasks, 100-mL.

Nitric acid, HN03 Conc.

Hydrochloric acid, 1 + 1

Samples are acidified with HN03 and HCI and heated on a hotplate to reduce the
volume to a defmed level. After filtration, the samples are ready for analysis by
atomic absorption spectrophotometer (AAS).
178 Handbook ofMethods in Environmental Studies

1. Measure 100 mL ofa well-mixed sample into a ISO mL beaker or flask. Add
0.5 mL of conc. HN03 (sp. gr. 1.42). (Note: If the sample has been preserved at
the recommended level of 5 mL of conc. HN03 per liter of sample, the addition
of acid at this step may be omitted).
2. Add 10 mL ofconc. HCl (l + I) to the beaker or flask.
3. Heat the sample in a steam bath or hot plate in a well ventilated hood until the
volume has been reduced to IS to 20 mL, making certain that the sample does
not boil.
4. Cool and remove solids. Transfer sample to 100 mL volumetric flask. Adjust to

4.6 Analysis of Heavy Metals by AAS

Atomic Absorption Spectrophotometer (AAS)

The concentration metals are easily determined by atomic absorption spectropho-
tometer (AAS). The atomic absorption uses essentially monochromatic radiation
to excite vaporized atoms in their ground state. The instrument consists of a light
source, a cell (consisting of the aspirated sample), a monochromator, and a detec-
tion system. The instrument is shown in Fig. 4.1.
The light source, usually a hollow cathode tube, emits essentially line radiation
of the same wavelength as that being absorbed by the element under study. This is
accomplished by making the source out of the sample element. Thus, ifiron is to be
determined, a lamp having an iron cathode is used.
51it-s M.t.r

. . "'"" . .-'-.- D- ·o.t.ttor

Oxidiz.r Gas

Samplf Cup

Fig. 4.1 : A basic Atomic Absorption Spectrometer (AAS)

Analysis ofMetals in Water and EfJluents 179

Wavelength and Detection Limits for Various

Table 4.3 lists the atomic absorption detection limits and wavelength used for sev-
eral environmentally important elements.

Table 4.3:
Wavelengths and detection limits for the various elements in AAS.

SINo. Element Wavelength (nm) Instrument detection

limit (mg/L)

1. Silver (Ag) 328.1 0.01 (A-Ac)

2. Aluminum (AI) 309.3 0.1 (N-Ac)
3. Calcium (Ca) 422.7 0.003 (A-Ac)
4. Cadmium (Cd) 228.8 0.002 (A-Ac)
5. Cobalt (Co) 240.7 0.03 (A-Ac)
6. Chromium (Cr) 357.9 0.02 (A-Ac)
7. Copper (Cu) 324.7 0.01 (A-Ac)
8. Iron (Fe) 248.3 0.02 (A-Ac)
9. Magnesium 285.2 0.0005 (A-Ac)
10. Manganese (Mn) 279.5 0.01 (A-Ac)
11. Molybdenum (Mo) 313.3 0.1 (N-Ac)
12. Sodium (Na) 589.0 0.002 (A-Ac)
13. Nickel (Ni) 232.0 0.02 (A-Ac)
14. Lead (Pb) 283.3 0.05 (A-Ac)
15. Tin (Sn) 224.6 0.8 (A-Ac)
16. Zinc (Zn) 213.9 0.005 (A-Ac)

A-Ac = Air-acetylene flame; N-Ac = Nitrous oxide-acetylene flame

Standard Solutions of Metals

AAS method is applicable for the following metals, those are generally monitored
in drinkiilg water and wastewater. Prepare the standards solution as describe be-
1. Aluminum (AI) solution, standard (I mL = 0.1 mg AI): Dissolve 1.758 g of
aluminum potassium sulfate (AIK(S0.JrI2HP) in water. Add 1 mL of con-
centrated HNO] and dilute to I L.
2. Arsenic (As) solution, stock (ImL= Img As): Dissolve 1.320 g of Arsenic
trioxide (Asp]), dried for at least 1h at II 0 DC, 10 mL ofNaOH solution (420 gI.
L) and dilute to 1 L with distilled water. This is 1000 mg AsIL stock solution.
• Arsenic solution, intermediate (I mL = 10 p.g As): Dilute 5mL of Arsenic
stock solution to 500mL with water.
• Arsenic solution, standards (I mL=1 p.g As): Dilute lO mL of arsenic inter-
mediate solution to 100 mL with water. Prepare fresh before use.
3. Cadmium (Cd) (I mL = 100 p.gCd): Dissolve 0.1 g cadmium metal in 4 mL
conc. HNO]" Add 8 mL conc. HNO] and dilute to 1 L with water.
180 Handbook ofMethods in Environmental Studies

4. Chromium (Cr) (I mL = 100 "g Cr): Dissolve 0.1923 g crOJ in water, dis-
solved completely, acidify with 10 mL conc. HNOJ and dilute to 1 L.
• Chromium stock solution (I mL = 1 mg Cr): Dissolve 2.828 g of primary
standard potassium dichromate (K2Crp7) in 200 mL of water and dilute to 1 L.
• Chromium solution, standard (I mL = 0.1 mg Cr): Dilute 100 mL of Cr
stock solution and 1 mL conc. HNOJ to 1 L with water.
5. Copper (Cu) (ImL = 100 "g Cu): Dissolve 0.1 g copper metal in 2 mL conc.
HNOJ , add 10 mL conc. HNOJ and dilute to 1 L with water.
6. Iron (Fe) (ImL = 100 mg Fe): Dissolve 0.1 giron wire in a mixlure ofl0 mL
(1+1) HCI and 3 mL conc. HN0 3. Add 5 mL conc. HN03 and dilute to 1 L.
7. Lead (Pb) (I mL = 100 "g Pb): Dissolve 0.1598 g lead nitrate, Pb(N03)2' in a
minimum amount of (1 + 1) HN0 3, add 10 mL conc HN03, and dilute to 1 L
with water.
8. Manganese (I mL = 100 "gMn.): Dissolve O.1gMn metal in 10 mLconc HCI
mixed with 1 mL conc HNOJ • Dilute to 1 L with water.
• Manganese solution, stock (I mL = ImgMn): Dissolve 3.076 g of Manga-
nous sulfate monohydrate (MnS04 • HP) in a mixture of 10mL of conc. HNOJ
( 1.42) and 100 mL of water. Dilute to 1 L with water.
• Manganese solution, standard (I mL = O.lmg = 100 "g Mn): Dilute 100
mL ofMn stock solution to 1 L with water.
9. Silver (Ag) solution, stock (I mL = 100"gAg): Crush approximately 2 g of
silver nitrate (AgN03) crystals and dry to constant mass at 40°C. Dissolve
0.1575 g of AgN0 3 in water containing 5 mL of conc HNOJ dilute to 1 L. Store
in a amber glass bottle.
• Silver (Ag) solution, intermediate (I mL = I"g Ag): Dilute 10 mL of stock
silver solution and 5 mL of conc HNOJ to 1 L with water. Store in an amber
glass bottle.
• Silver solution, Standard (1 mL = 0.1 "g Ag): Dilute 100 mL of silver inter-
mediate solution and 5 mL of conc. HN03 to 1 L with water. Prepare fresh
before use.
10. Zinc (Zn) ( 1 mL = 100 "g Zn): Dissolve 0.1 g zinc metal in 20 mL 1+ 1 HCI
and dilute to 1000 mL with water.
• Zinc stock solution (1 mL = 1 mg Zn): Dissolve 1.245 g of Zinc oxide (ZnO)
in a mixture of 10 mL conc. HNOJ and 10 mL of water. Dilute to 1 L of water.
• Zinc solution, standard (1 mL = 100"g Zn): Dilute 100 mL ofZn stock solu-
tion and 1 mL ofconc. HN0 3 to 1 L of water.
The Arsenic (As) and Selenium (Se) are analyzed by hydride generation atomic
absorption spectrometric method.

Data Analysis in AAS

Report the results as follows: (Liquid sample)

Metal concentration, mg/L = A x --
Analysis of Metals in Water and Effiuents 181

A = Concentration of metal in digested volume, mgIL
B = Final volume of digested solution, mL and
C = Sample size, mL.

Report the results as follows: (Solid sample, i.e soil,sludge, etc)

Metal concentration, mg/kg (wet - weight basis) = - - -
g sample

A = Concentration of metal in digested volume, mgIL
B = Final volume of digested solution, mL

4.7 Aluminum (AI)

Drinking water
IS: 10500 (1991) = 0.03 mgIL (0.2 mgIL is permissible in the absence of
alternate source).
WHO (1993) = 0.2 mgIL
EC (1980) = 0.05 mgIL (guide level) and 0.2 mgIL (MAC, max admissible
Emuent discharge
MOEF, (Schedule VI, 1993) = No Standards.

There is little information available regarding the toxicological effects of alumi-

num of human health. Al is ljI1onitored in boiler make-up water, where alum has
been used, to determine whether aluminum is present after pretreatment. Residual
Al may consume ion exchange capacity. Al is monitored in cooling water make-up,
since its presence may result in deactivation of anionic substances in scale or cor-
rosion inhibitor treatment chemicals or both.

Estimation of Aluminium
1. AAS, direct - 0.3 mgIL to 5.0 mgIL
2. AAS, Chelation-extraction - 0.01 mgIL (10 flgIL) to 0.3 mgIL (300 flgIL)
3. Colorimetric: Eriochrome Cyanine R method.
182 Handbook of Methods in Environmental Studies

4.8 Arsenic

Drinking water
WHO (1984); USEPA (1974); EC (1980) = 0.05 mgIL
WHO (1993) = 0.01 mgIL.
IS:I0500 (1991); ICMR (1963); MHW, (1975) = Indian standard: 0.05 mgIL
Effluent discharge
MOEF, Schedule VI (1993) = 0:2 mgIL max. (for inland surface water, public
sewers, land of irrigation and marine/costal areas).

Herbicides, insecticides, and many industrial effluents contain arsenic and are po-
tential sources of water pollution. Arsenic is significant because ofits adverse physi-
ological effects on human.

Estimation of Arsenic
Preserve the sample with HNO) (sp. gr. 1.42) to a pH of2 or less immediately at the
time of collection, normally about 2 mLIL is required. If only dissolved arsenic is
to be determined, filter the sample through a 0.45 J,lm membrane filter before acidi-
fication . Arsenic in most waters and wastewater is determined by three test meth-
ods as given in Standard Methods (1995):
1. Colorimetric silver diethyldithio carbamate: 5 to 250 Ilg As/L
2. AAS, Hydride generator (I to 20 Ilg As/L)
3. AAS, Graphite furnace (5 to 100 Ilg As/L).



Organic arsenic containing compounds are decomposed by adding sulfuric and

nitric acid and repeatedly evaporating the sample to fumes of sulfur trioxide .
The arsenic (V) so produced, together with inorganic arsenic originally present,
is subsequently reduced to arsenic (III) by potassium iodide and stannous chloride,
and finally to gaseous arsine by zinc in HCI solution.
Alternatively, the arsenic is converted to arsine by sodium borohydride (NaBH 4 )
in HCI solution. The arsine is removed from solution by aeration and swept by a
flow of nitrogen into a hydrogen flame where it is determined by AAS at 193.7 nm.

Determination of Arsenic with sodium borohydrate

I. Clean all glassware before use by rinsing first with hot HNO) (1 +.1) and then
with water.
2. Pipette a volume of well-mixed acidified sample containing less than 1.0 Ilg of
Analysis of Metals in Water and Ejj1uents 183

arsenic (50 mL maximum) into a 200-mL Berzelius beaker (or similar appara-
tus) and dilute to approximately 50 mL.
3. To each beaker, add 7 mL~SO,,(1 + 1) and 5 mLofconc. HN03• Add a small
boiling chip and carefully evaporate to fumes ofS03, maintaining an excess of
HN03 until all the organic matter is destroyed. This prevents darkening of the
solution and possible reduction and loss of arsenic. Cool, add 25 mL of water,
and again evaporate to fumes ofS0 3, to expel oxides of nitrogen. .
4. Cool, and adjust the volume in each beaker to approximately 50 mL with water.
5. Add 8 mL conc. HCI and mix.
6. Attach one beaker at a time to the rubber stopper containing the gas dispersion
7. Fill the dropper or syringe with 0.5 mL of sodium borohydrate solution (Dis-
solve 4 g of NaBH4 in 100 mL of water. Prepare fresh before each use) and
insert into the hole in the rubber stopper.
8. Add the sodium borohydrate solution to the sample solution. After the absor-
bance has reached a maximum and has returned to the baseline remove the
beaker. Rinse the gas dispersion tube with water before proceeding to the next
sample. Treat each succeeding sample, blank, and standard in a like manner.
Removal of As
There are following treatment options under evaluation for treatment of arsenic
(De Zuane, 1977). These are:
• Coagula!ion with filtration
• Lime softening
• Activated alumina
• Reverse osmosis

4.9 Cadmium (Cd)

Drinking water
WHO (1971) = 0.01 mgIL
WHO (1984); EC (1980); Canadian MAL; (i987) = 0.005 mgIL
WHO (1993) = revised to 0.003 mgIL
IS: 10500 (1991); ICMR (1963); MHW (1975) = 0.01 mgIL
Emuent discharge
MOEF (Schedule VI, 1993) = 2 mgIL (Inland surface and marine/coastal
areas) and 1 mgIL public sewer.
184 Handbook ofMethods in Environmental Studies

Cadmium is extensively used in the manufacture of batteries, paints and plastics. In

addition, it is used to plate iron product, such as nuts and bolts, for corrosion pre-
vention (electroplating industries). It is from plating operations that most of the
cadmium reaches the water environment.
At extreme levels, it causes an illness called "Itai-Itat' disease, characterized
by brittle bones and intense pain. At low level of exposure over prolonged period,
it causes high blood pressure, sterility among males, and kidney damage.
It has recently been discovered that significant amount of Cd is contained in
cigarette smoke.

Estimation of Cadmium
1. AAS, Direct - 0.05 to 2 mgIL
2. AAS, Chelation-extcaction: 5 to 200 J.lg/L
3. AAS, Graphite furnace: 0.5 to 10 J.lgIL
4. Colorimetric: Dithizone method.

4.10 Chromium (Cr)

Drinking water
IS: 10500 (1991) = 0.05 mgIL (may be carcinogenic above this limit)
ICMR (1963); MHW (1975) = 0.05 mgIL.
WHO (1984); WHO (1993); EC (1980); USEPA (1974) = 0.05 mgIL
Emuent discharge
MOEF (l993) = 2 mgIL Total Cr (max) (as Cr) - public sewers; inland surface
water; and marine/costal areas.

Hexavalent chromium (VI) salts are used extensively in metal finishing and plating
application, in anodizing aluminum, and in the manufacture of paints, dyes, explo-
sives and ceramics. Cr (VI) is toxic to human, animals and aquatic life. It can
produce lung tumor when inhaled and readily induces skin sensitization. There is
no evidence to indicate that Cr (III) form is detrimental to human health.

Estimation of Chromium
1. AAS, Direct - 0.1 to 10 mgIL
2. AAS, Graphite-furnace: 5 to 100 J.lglL
3. Colorimetric Diphenylcarbohydrazide method - 0.01 to 0.5 mgIL
Analysis ofMetals in Water and Effluents 185

4.11 Copper (Co)

Drinking water
IS: 10500 (1991) = 0.05 mgIL (may be relaxed up to 1.5 mgIL in the absence
of alternate source).
ICMR (1963) = 1 mgIL (desirable) - 3 mgIL (max. permissible).
WHO (1984, 1993) = 1 mgIL (health -based provisional guideline value is
2 mgIL).
USEPA (1974) = I mglL.
EC (1980) = 0.1 mgIL (at plant) and 3 mgIL at supply point. Above 3 mgIL,
astringent taste, discolouration, and corrosion may occur.
Emuent discharge
MOEF (1993) = 3 mgIL (Inland surface water, public sewer and marine coastal

Copper salts are used in water supply systems to control biological growth in reser-
voirs and distribution pipes and to catalyze oxidation of manganese. Corrosion of
copper-containing alloys in pipe fitting may introduce measurable amounts of cop-
per into the water in a pipe system.
In small amount, it is not detrimental to health, but it will impart an undesirable
taste to the water. It causes Wilson's disease. Copper is essential to human, the
adult daily requirement has been estimated at 2 mg.

Estimation of Copper
1. AAS, direct - 0.02 to 5 mgIL.
2. AAS, Chelation-extraction - 2 to 500 ~gIL
3. AAS, Graphite furnace - 5 to 100 ~gIL.
4. Colorimetric: Neocuproine method

4.12 Iron (Fe)

Drinking water
WHO (1984, 1993) =Permissible limit of filterable Fe is 0.3 mgIL;
IS: 10500 (1983, 1991) = 0.3 mgIL (may be extended up to 1 mgIL in absence
of alternative sources);
ICMR (1963) = 0.3 mgIL (desirable conc) - 1.0 mgIL (max. permissible)
186 Handbook ofMethods in Environmental Studies

... cont.
USEPA (1974) = 0.3 mgIL
Effluent discharge
MOEF (l993) = 3 mgIL (for inland surface water, public sewers and marine
costal areas).

Surface water generally contain < 1 mgll of Fe. Some groundwaters contain much
higher level of Fe. Water containing Fe >2 mgIL, causes staining of clothes (while
washing) and sanitary wares and imparts bitter astringent taste. Taste and odour
problems may be caused by filamentous organisms that prey on iron compounds
(Frenothrix, Galionella and Laptothrix are called "iron bacteria") originating an-
other consumers objections (red water).
It is a known fact that iron in trace amounts is essential for nutrition. In case of
Fe deficiency anaemia, larger doses are taken for therapeutic reason. The daily
requirement is 1-2 mg, with dietary ranges of 7-35 mglday, with an average of 16
mglday. Fe from ambient air constitutes a negligible exposure. With a daily food
contribution of 16 mg and water contribution of 0.14 mg, it is clear that water
contributes to the diet between less than 1-15%.

Estimation of Iron
1. AAS, direct - 0.05 to 5.0 mgIL
2. AAS, Chelation-extraction - 5 to 500 ~gIL
3. AAS, Graphite furnace - 5 to 100 ~gIL
4. Colorimetric: Phenonthroline method - 40 to 1000 ~gIL

Fe removal can be accomplished using aeration, sedimentation/filtration with pH
adjustment, ozone or chlorine treatment, Chemical precipitation/filtration, ion ex-
change (softening), potassium permanganate oxidation with precipitation/filtration,
well water pretreatment with sequestering agents like polyphosphate and sodium
silicates and lime softening.

4.13 Lead

Drinking water
IS: 10500 (l991) = 0.05 mgIL (maximum); no relaxation.
WHO (1971) = 0.1 mgIL
WHO (1984) = 0.05 mgIL
WHO (1993) = 0.01 mgIL
Analysis ofMetals in Water and Effluents 187

... cont.
WHO (European, 1984) = 0.10 mgIL - 0.05 mgIL (max. for running water);
Netherlands and Germany (in pipes) = 0.3 mgIL (water still for 24h).
USEPA (1974) = 0.05 mgIL.
Emuent discharge
MOEF (1993) = 0.1 mgIL (Inland surface water), 0.1 mgIL (public sewers) and
2 mgIL (marine/coastal areas)

Lead concentration in surface and ground raw water range from traces to 0.04
mgIL, averaging about 0.01 mgIL. Industrial and mining sources may contribute to
some localized Pb pollution. Use ofPb pipes in water supplies may provide high
Pb at consumers' points. In most cases, higher Pb concentration is expected at the
consumers tap than that of water treatment plant.
The health effects of Pb are neurotoxic and three human systems are most
affected: blood-forming system, nervous system (which include irreversible brain
damage) and renal system. Such a toxic level is reached when the blood level ex-
ceeds 100-120 JlgIL.

Estimation of Lead
1. AAS-Direct: Range 1 to 10 mg PblL. This test method is applicable for the
determination of dissolved and total recoverable Pb in most waters and waste-
2. AAS, Chelation -extraction: 100 to 1000 JlgIL
3. AAS, Graphite furnace: 5 to 100 JlgIL.
4. Colorimetric: Dithizone method - 1.0 Jlg Pb/l 0 mL dithizone solution.

4.14 Manganese (Mn)

Drinking water
WHO recommended (1993) = 0.05 mgIL (maximum acceptable); 0.50 mgIL
(maximum allowable)
WHO (1984) = O.lmgIL (under aesthetic quality- staining oflaundry and sani-
tary ware)
USEPA (1994 and 1991) = 0.05 mgIL (under Secondary drinking water stan-
EC (1980) = Guide value of 0.02 mgIL and max of 0.05 mgIL
IS: 10,500 (1983, 1991) = 0.10 mgIL (max) (0.3 mgIL maximum permissible
limit in the absence of alternate source)
MHW (1975) = 0.05 mg/L (acceptable)- 0.5 mgIL (causes of rejection)
ICMR (1963) = 0.1 mgIL (desirable) - 0.5 mgIL (maximum permissible)
188 Handbook ofMethods in Environmental Studies

Emuent discbarge
MOEF (1993) = 2.0 mgIL (for Inland surface water, public sewers, land of
irrigation and marine/coastal areas».

The concentration ofMn in water is less than that of Fe. A mean expected value is
around 0.06 mgIL. Mn is an essential trace nutrient for plants and animals. Nutri-
tional deficiency in man has not been evaluated as a health hazard. Also minimum
requirements for nutrition have not yet been established. WHO estimates an aver-
age daily requirement for normal physiological function of 3-5 mg. Undesirable
effects may be taste, staining of laundry and discoloration of water at 0.15 mgIL.

Estimation of Manganese
1. AAS, Direct: 0.02 to 5 mgIL
2. AAS, Chelation-extraction: 2 to 500 JigIL
3. AAS, Graphite furnace: 5 to 50 JigIL.
4. Colorimetric: Persulfate method - 43 to 210 Jig MoIL
Mn removal: Aeration, hyperchlorination, pH adjustment and chemical precipita-

4.15 Mercury (Hg)

Drinking water
WHO guidelines (1984) = 0.001 mgIL
WHO (1993) = 0.001 mgIL (Hg total)
USEPA (1974) = 0.002 mgIL
EC (1980) = 0.001 mgIL.
IS: 10500 (1983, 1991) = 0.001 mgIL (no relaxation).
Emuent discbarge
MOEF (1993) = 0.01 mgIL (for inland surface water, public sewers and marine
coastal discharge)

Mercury detected in drinking water is predominantly in the form of inorganic mer-

cury. Maximum concentration reported by USEPA (1978-80) are as follows: (i)
30% groundwater had level above 0.5 JigIL; (ii) 40% had above 2 JigIL; (iii) 32%
surface water had level of 0.5 JigIL and (iv)14% had 2 JigIL.
Analysis ofMetals in Water and Effluents 189


The principal target organ of inorganic Hg is kidney with neurological and renal
disturbance. Methyl mercury compounds are very toxic to the central nervous sys-
tem; they are also the major source of environmental contamination. Fatal dose for
man varies between 3-30 g. Four to 12 mg of mercury per day may be safe, and
fatal doses of mercury would be 75-300 mgIL.

Estimation of Mercury
Standard Methods (1995) advises to preserve samples by treatment with HNO) to
reduce the pH to less than 2. For all samples the Cold vapour Atomic Absorption
Spectroscopy is the method of choice, with the "Dithizone method" as the method
to be used in potable waters for high levels of mercury.

The specific technique is based on room temperature reduction of Hg to Hgo (ele-
ment) by SnCll in solution, followed by sweeping ofHgo by air into an absorption
cell. In absorption cell, liberated Hg is irradiated by a low pressure mercury lamp,
which emit 253.7 nm wavelength. Now the Hg vapour, in atomic form, absorbs the
253.7 nm radiation and causes a change in transmittance, which can be correlated
to the total mercury content in the sample solution.
Chemicals and Reagents
1. Potassium permanganate (KMnOJ
2. Conc. HNO)
3. Stannous chloride (SnCll)
4. Potassium dichromate (KzCrl0 7)
5. Conc. HlSO.
6. NaOH pellets
7. HgCll
I. Potassium permanganate solution (KMnOJ, 1% (w/v) ): Dissolve 5 g of
KMnO. in water and carefully add to it 50 mL of conc. HlSO. make up a vol-
ume of500 mL using distilled water. (Fill this solution in RI and R5 vessels).
2. Sodium hydroxide, (NaOH), 200/. solution: Dissolve 50 g ofNaOH pellets
in distilled water and make up a volume of 250 mL. (Fill this solution in R3
vessels and function is to absorbed acid vapour).
3. Cone. H1SO.: Filled in R4 vessels. Function is to absorb moisture.
4. SnC11 20% solution (w/v): Take 20 g ofSnCll in a clean beaker. Add 10mL
190 Handbook ofMethods in Environmental Studies

conc. HCl and dissolve while it over a burner. Boil for one min, cool and dilute
with distilled water to make 100 mL.
5. RN03 , 10% solution: Add 20 mL ofHNO) to water and make up 200 mL.
6. Stock mercury solution (1 mL = 1000 pg): Dissolve 0.1354 g mercuric chlo-
ride (HgCI2) in about 70 mL water, add 1 mL conc. HNO), and dilute to 100 mL
with water; ImL = Img Hg or 1000 ~g.
7. Intermediate stock solution: Dilute 1mL of stock mercury solution to 100 mL
with distilled water. 1mL = 10 ~g.
8. Standard mercury solution: Prepare a series C?f standard mercury solutions
containing 0 to 500 nanogram ImL by appropriate dilution of stock mercury
solution with water containing 10 mL conc. HNO/L (dilution water). Prepare
standards daily.

Preparation of Calibration curve of Mercury

Preparation of standard mercury solution with stock solution (1 mL = 10 ~g).

Stock solution Dilution water, Final volume, Concentration of

mL mL mL Hg, nglmL

0.2 99.8 100 20

0.5 99.5 100 50
1 99 100 100
2 98 100 200
3 97 100 300
4 96 100 400
5 95 100 500

Analytical method used with the FAAS method involves three important steps:
a. Sample preparation,
b. Vapour generation, and
c. Absorption measurement.
A typical mercury analyzer is shown in Fig. 4.2.
1. Fill the RI and R5 vessels with 1% KMn04 solution (16 to 20 mL),just suffi-
cient enough to dip the inlet. R3 contain 4 mL of20% NaOH. R4 contain 4 mL
of conc. H2 S04 ,
2. Standards or sample solution is placed in the reaction vessel R2.
3. Add the required amount ofHNO) to maintain a volume of 10 mL + 2 mL
Snel2 solution and replace the stopper immediately (Max volume 12 mL).
4. Switch on the magnetic stirrer immediately and stir vigorously for about 5 min.
(purpose is to generate mercury vapour).
5. Pump the resulting Hg vapour through 20% NaOH solution (R3) (act as acid
trap i.e. absorb acid) and 50% H2 S04 solution (R4) (act as moisture trap) and
then absorption cell.
Analysis ofMetals in Water and E.fJluents 191


MA 5800 DIE

R, to Purify Air
R2 to Generate to Merwry Vapour from Sample Solution
R3 to Absorb Acid Vapour
Rio to Absorb Moisture
RS to Absorb (Gtnerated)twrcury Vapour

Signal ~ui ...l~nt

to 100 ng 01 Hg

o 1() 20 30
(Tim~ (5~t»)

Fig. 4.2: Mercury analyzer.

6. Measure the absorbance at 253.7 run using Hg-hollow cathode lamp as the light
7. At the end of measurement absorb Hg vapour in 1% KMnO4 in 10% H2 S04 in
the outlet.
Repeat the measurement for standard 20, 50, 100, 200, 300, 400 and 500 nano-
gram ofHglmL. Prepare calibration graph by plotting the absorbance Vs con-
centration ofHg in nanogram/mL.
• Note: Never fill the traps with more liquid than specified above, otherwise the
liquid may be carried over the next trap or to the absorption cell.
Start the pump and allow the air to flow through for a minute.
The RI and R5 traps should be replaced at least once in 3 days.
Clear R3 (alkali) and R4 (acid) traps and refill then with respective reagents
192 Handbook ofMethods in Environmental Studies

4.16 Selenium (Se)

Drinking water
IS 10,500 (1983, 1991)= O,ol mgIL (maximum)- no relaxation
WHO (1984, 1993) = 0.01 mgIL
EC (1980) = O,ol mgIL
USEPA, 1974 (MCL, max contaminant level) = 0.01 mgII.
ICMR. (1963) = 0.01 mgIL (maximum pennissible)
MWH (1975) = 0.01 mgIL (maximum pennissible)
Emuent discharge
MOEF, Schedule VI (1993) = 0.05 mgIL (Inland surface water, public sewers
and marine/costal areas).

Selenium is a priority pollutant and all public water agencies are required to moni-
tot its concentration. In animals Se prevent bone formation and causes "alkali dis-
ease" and "blind staggers". The problem is critical with animals because they are
dependent upon the local plants for food.

Estimation of Selenium
1. AAS, Gaseous hydride (applicable: 1 to 20 JigIL).
2. AAS, Graphite furnace (applicable: 2 to 100 JigIL).
3. Colorimetric: Minimum detectable quantity: 10 Jig SelL.
When determining only dissolved selenium, filter the sample through a 0.45 J1M
membrane filter as soon as possible after sampling. Add HNOl to the filtrate to
bring the pH to < 2. .
When determining total recoverable selenium, add HNOl to the unfiltered
sample to a pH of < 2.
AAS, Gaseous hydride (applicable: I to 20 J1IIL)
1. The determination consists of conversion of selenium from its various forms to
gaseous selenium hydride (hydrogen selenide), with the subsequent analysis of
the gas by flame.
2. The conversion consists of (a) decomposition and oxidation of selenium (VI), '
(b) reduction to selenium (IV), and (~) fmal reduction to selenium hydride.
3. The absorbance is determined at 196.0 om in a hydrogen-argon (air-entrained)
Analysis ofMetals in Water and Effluents 193

4.17 Silver (Ag)

Drinking water
IS: 10500 (1983, 1991) = No standard is set for Ag.
WHO (1984) = No guideline set for silver.
WHO (1993) = guideline states that" It is unnecessary to recommended a health-
based guide line value for Ag because it will not cause health hazards to human
at concentration normally found in drinking water".
Canada (MAL, 1987) = 0.05 mgIL;
USEPA (1974) = 0.05 mgIL;
EC (1980) = O.oI mgIL.
Emuent discharge
No standard is set for Ag (MOEF, 1993).

The principal adverse effect of Ag in the body is cosmetic. It causes argyria, a

permanent blue-gray discoloration of skin, eyes, and mucous membrane. Relatively
small quantities of Ag are bactericidal or bacteriostatic and fmd limited use in both
disinfection of swimming pool waters and point-of-use water filter.

Estimation of Silver
1. AAS, Chelation-extraction: 1 to 10 J.lgIL.
2. AAS, Direct: 0.1 to 10 mgIL.
3. AAS, Graphite furnace: 1 to 25 J.lgIL.
4. Colorimetric: Dithizone method.

4.18 Vanadium (V)

Drinking water
IS: 10500 (1983, 1991) = No guideline value sets.
USEPA (1974) = No standards.
WHO (1984, 1993) = No standards.
Emuent discharge
MOEF (Schedule - VI, 1993) = 0.2 mgIL (Inland surface water, public sewers
and Marine coastal areas)

Vanadium has an atomic number of23, an atomic weight of50.94, and valances of
2, 3,4, and 5. The dominant form of vanadium in natural waters is V$+. It is consid-
194 Handbook of Methods in Environmental Studies

ered as nonessential for most higher plants and animals. Vanadium complexes have
been noted in coal and petroleum deposits. Vanadium is used as a catalyst in the
production of sulfuric acid and synthetic rubber. Vanadium can be found in waste
that result from chemical cleaning of components in which the metals is alloyed.
Vanadium pentaoxide dust causes gastrointestinal and respiratory disturbances. The
United Nations Food and Agriculture Organization (UNFAO) recommended maxi-
mum level for irrigation waters is 0.1 mgIL.

Estimation of Vanadium
1. Graphite furnace: ASS: 10-200 IlgIL of V based on a 20-IlL sample size. (Mini-
mum detection limit: 4 IlgIL).
2. Colorimetric: Gallic acid method - 21lg VIL.
3. ASS, ICP and Gallic acid methods are suitable for potable water sample, where
as ASS and ICP preferred for polluted water sample.

4.19 Zinc (Zn)

Drinking water
IS: 10,SOO (1983, 1991) = S mgIL and may be relaxed upto IS mgIL in the
absence of alternate source.
WHO (1984, Aesthetic quality) = S mgIL;
WHO (1993)= 3 mgIL (appearance and taste);
USEPA (1974)= SmgIL.
Emuent discbarge
MOEF (1993) = SmgIL (inland surface water) and IS mgIL (for public sewers
and marine/coastal discharge)

Zinc (Zn) is an essential and beneficial element for human bodies. However, above
S mgIL, cause bitter taste and opalescence in alkaline waters. The Zn concentration
of U.S. drinking water ranges from 0.06 to 7 mgIL with a mean of 1.3 mgIL. Zn
enters the domestic supply from deterioration of galvanized Fe and de-zincification
of brass, besides industrial waste.

Estimation of Zinc
1. AAS, Direct: O.oI to 2 mgIL
2. ASS, Chelation-Extraction: 1 to 20 IlgIL
3. Colorimetric: Dithione method-I (Potable water). Dithione method-II (polluted
water) and Zincon method (both).
Analysis of Metals in Water and EfJIuents 195

4. 20 Check Your Confidence on Heavy Metals

1. Defme heavy metals. Why heavy metals are known as nephrotoxin elements ?
2. For heavy metals analysis why samples must be preserved at pH <2.
3. How to determine dissolved metals, suspended metals, total metals and acid
extractable metals?
4. List the metals that are put under aesthetic quality guidelines by WHO (1984).
5. What are the sources of arsenic in environment? How Arsenic is determined
and which method is most sensitive? List a few arsenic removal processes.
6. What are the sources of Cd in drinking water? Which method is most sensi-
tive? What are the symptoms ofItai-Itai disease?
7. List the sources of chromium in environment. Which form of chromium is toxic
to human health ?
8. Write the sources ofCu in drinking water.
9. Write the environmental significance ofFe determination. "Fe causes test and
odour problems also"- How? How Fe is removed?
10. List the undesirable impacts of manganese.
11 . Discuss the health impacts of lead.
12. Discuss the Selenium estimation method by Gaseous hydride generation.
13. What are the adverse health effects of silver? Has any standard been set for
Silver either for drinking water or effluent discharge in India?
14. What are the sources of zinc in environment?" Znhas been put under aesthetic
quality by WHO guidelines (1984)"- Why?
15. List the adverse health effect of Hg. What are the sources of Hg in environ-
ment? Which form ofHg is most harmful to human health?
16. Discuss the principle ofFlameless or cold vapour AAS.
17. What are the purposes of use ofKMno4, H2S04, NaOH and SnCl2 solution for
analysis of Hg in cold vapour AAS method.
18. The metals are also analyzed by colorimetrically. Match the following colori-
metric methods against the respective metal.

Metals Colorimetric method

Aluminium (AI) Dipheny lcarbohydrazide method
Boron (B) Neocuproin method
Chromium (Cr) Eriochrome Cyanin R method
Copper (Cu) Curcumin method
Iron (Fe) Dithione method
Lead (Pb) Phenoanthroaline method
Manganese (Mn) Gallic acid method
Silver (Ag) Dithione method
Zinc (Zn) Persulfate method
Vanadium (V) Diethyldithio-carbamate method
196 Handbook ofMethods in Environmental Studies

19. The heavy metals are known to be nephrotoxin elements and in addition cause
diseases in nervous system in human. Match the following diseases with the
respective metal.

Metals Types ofDiseases/symptoms

1. Chromium (Cr) a. Itai-Itai disease
2. Cadmium (Cd) b. Wilsons's disease
3. Copper (Cu) c. Argyria (darkening ofthe skin and eyes)
4. Iron (Fe) d. Minamata disease
5. Lead (Pb) e. "Alkali disease" and "blind staggers"
6. Mercury (Hg) f. Anemia
7. Manganese (Mn) g. Taste and staining of clothes and
discoloration of water
8. Selenium (Se) h. Nervous system and kidney
9. Silver (Ag) i. Skin disorder and lungs tumor
10. Zinc (Zn) j. Gastrointestinal and respiratory
11. Vanadium (V) k. Bitter stringent test and an opalescence
in alkaline water.

Hints: l(i); 2(a); 3(b); 4(f); 5(h); 6(d); 7(g); 8(e); 9(c); lQ(k); 110>.
Treatabili 4y Studies of

5.1 Coagulation-Flocculation Jar Test

of WaterlEffluents

Coagulation is defmed as "a unit process that destroys the mutual repulsive forces
between particles" and Flocculation as "a unit operation that induces particles to
coalesce and form bigger aggregate of particles". The most commonly use coagu-
lants are salts of Aluminiwn and Iron. Some of the common coagulants are discuss

1. Alum [Aluminium sulphate, AI1(S04)3.18HP1: When alwn is added in wa-

ter containing Ca and Mg bicarbonate alkalinity, the reaction is:

Al2(S04)3.l8H20 + 3Ca(HCO)2 ~ 2Al(OH)3,j, + 3CaS04+ 6Hp + 18Hp

... (1)
The insoluble aluminiwn hydroxide is a gelatinous floc that settles slowly through
the water, swiping out the suspended materials. Most effective in the pH range of
5.5 to 8.0
2. Ferrous sulphate (Copperas): Effective for clarification of turbid water at
high pH value.

... (2)

3. Ferric Sulphate and lime:

Fe2(S04)3 + 3Ca(OH)2 ~ 2Fe(OH)3,j, + 3CaS04 ... (3)

When natural bicarbonate alkalinity is present in water:

Fe 2(S04\ + 3Ca(HC03)2 ~ 2Fe(OH)3,j, + CaS0 4 + 6Hp ... (4)

4. Ferric chloride (FeCI3.6HP): Ferric chloride is used primarily in the coagu-

lation of wastewater and industrial waste.

2FeC13 + 3Ca(OH)2 ~ 3CaCl 2+ 2Fe(OH)3,j, ... (5)

198 Handbook of Methods in Environmental Studies

Alkalinity-Coagulation relationship: When metal coagulants are added in water

they releases hydrogen ion (Eq. 6), which decrease pH value by consuming
alkalinity. As effectiveness of each coagulant is depended on pH, sufficient
alkalinity is maintained by adding lime [Ca(OH 2)].

AP+ + 2Hp ~ Al(OH)JJ.. + 2W ...(6)

The consumption of alkalinity (as mglL CaCO J) can be ca1cuiated by taking

example of eq. I .
666.7 glmol reacts with 3 x 100 glmol ofCaCOJ.
ImglL alum consumes = 3 x 100 g.mol- 1/666.7 g.mol- I x 1 mglL = 0.45 mgIL

Example - 1
What amount of alkalinity is required to react with 10 mg/L alum?

' Solution;
}O mg/l x (3 x 100 g.mol- 1 /666 .7 g.mol-1 ) = 4 .5 mg/L CaC0 3
.If alkalinity is le~s than 4 .5 mglL CaC0 3, then additional lime is to be added.

,. ,.. ' .

Example - 2
What is the amount of natural alkalinity required for coagulation of raw water
with dosage of 15.0 mg/L of ferric chloride?

Step-l: Write the reaction equation and calculate molecular weight (MW) .
.2FeCl z+ 3Ca(HC0 3)z ~ 2Fe(OH)3 + 3CaCI z + 6CO z
2[55 .85 + (2 x 35 .45)] 3[40.08 + 2(1 + 12 +48))
I 253.5 486.2

The above equation suggests that 2 moles of ferric chloride react with 3 moles of
calcium bicarbonate.

Step-2: Determine the alkalinity needed for X

mg/L Ca(HC0 3 )z 486.2
~---- = --= 1.92
mg/l FeCl z 253 .5

Therefore, X = 1.92 x dose of ferric chloride, mg/L

= 1.92 x 15.0 mg/L
. = 28.8 mg/L as Ca(HC03 )2
icc Assume Ca(HC0 3 )z represents the total alkalinity of the naturalwater. However.
, alkalinity concentration is usually expressed in terms of mg/L asCaC0 3• We need
to convert ·X" to a concentration of mg/L asCaC0 3.

MW of CaC0 3 = 40.08 + 12 + 48 = 100.1

MW of Ca(HC0 3 )z = 162.1
X = 28.8mg/L x (100.1/162.1) = 17.8 mglLas CaC0 3. (Ans).
Treatability Studies of Wastewater 199

Example - 3
Given that liquid alum is used as a coagulant. Sp . gravity of alum is 1.33. One
gallon (3 .785L) of alum weights 5.03 kg and contains 2 .44 kg of dry alum .
(a) mL of liquid alum required to prepare a 100 mL solution of 20,000 mg/L alum
(b) The dose concentration of 1 mL of stock solution in a 2000 mL jar sample.

Step-l: Determine alum concentration in mg/L
Alum concentration = 2.44 kg/3.785 L = 0 .644 kg/L = 645 mg/L.
Step-2: Prepare 100 mL stock solution having 20,000 mg/L alum concentration.
Let X = mg of alum required to prepare 100mL stock solution.
X 20,OOOmg
= 2000 mg
100mL 1000mL

Step-3: Calculate mL (Y) of liquid alum required to give 2000 mg alum.

YmL 2000
- - - Y = 3 .10 mL (Ans. a) .
lmL 645
Step-4: Find lmL of alum concentration (Z) in 2000 mL sample (Jar sample).
(Z mg/L) (2000 mL) = (20000) (1 mL)
Z = --- = 10 mg/L. (Ans).
Note: Actual final volume in the Jar after addition of alum is 2001 mL, using
2000 mL sample, which is reasonable.

Coagulant aids

Coagulant aids are used sometimes to produce tougher and better settling floes.
Some common coagulant aids are: polyelectrolytes, activated silica and bentonitic

Polyelectrolytes: These are classified according to their type of charge on the

polymer chain. There are three forms.
Cationic (Positive): Magnaflocs LTlI ; Chitosan, Zetag 63
Anionic (Negative): Setlyte AP50, EA 1533, Sufloc, Magnafloc ISS
Non-ionic (no-charge): Setyle.
Activated silica and clays: These offer advantages of increased rate of chemical
reaction, reduced coagulant dose, extended optimum pH range and production of a
faster settling and tougher floes. Activated silica is normally used with aluminium
coagulants at a dosage between 7 and II percent of alum dose, expressed as mgIL
ofSi0 2•
Bentonitic clays: These have been used as coagulant aids in conjunction with Fe
and Al primary coagulants in treating waters containing high colour, low turbidity
and mineral content.
200 Handbook ofMethods in Environmental Studies


This practice covers a general procedure for the evaluation of a treatment to reduce
dissolved, suspended, colloidal, and non-settleable mater from water/effluent by
chemical coagulation- flocculation, followed by gravity settling. The procedure
may be used to evaluate colour, turbidity, and hardness reduction.
• This practice permits the evaluation of various coagulants and coagulant aids
used in the treatment of water and wastewater.
• The effects of concentration of the coagulants and coagulant aids and their
order of addition can also be evaluated by this practice.

The coagulation-flocculation test is carried out to determine the chemicals, dos-
ages, and conditions required achieve optimum results. The primary variables to
be investigated using the recommended practice include, but-are not limited to:
1. Chemical additives
2. pH
3. Temperature
4. Order of addition and mixing conditions
APPARATUS (Fig. 5.1)

Multiple Stirrers - A multiposition stirrer with continuous speed variation from

about 20 to 150 rpm should be used. The stirring paddles should be oflight gaging
corrosion-resistant material all of the same configuration and size. An illumination
base is useful to observe the floc formation. Precautionary measures should be
taken to avoid heat being imparted by the illumination system, which may counter-
act normal settling.
Beakers: All of same size and shape; 1500 mL bakers may be used (1000 mL
recommended minimum size).

Fig. 5.1: Jar test apparatus.

Purity ofReagents: Reagent grade chemicals shall be used in all tests. The chemi-
cals and additives typical of those used for test solution and suspensions are given
in Table 5.1.
Treatability Studies of Wastewater 201

Table 5.1:
Prime chemicals use in coagulation process

Name Chemical formula Dosage required, Remarks,/most

ppm effective pH

1.Alum AI2(SO.'3·1SH20 40-90 Commonly use water

treatment pH:6-S.5
2. Ferric Fe 2(SO.'3. x Hp 35-40 with lime Sewage treatment
sulphate pH:S-S.5
3. Ferric FeC13~6H20 25-30 Sewage treatment
chloride pH:5.5-7
4. Ferrous FeSO•. 7H 2O - -
5. Lime, Ca(OH'2 - -
Coagulants aids - Polyelectrolytes (anionic, cationic, non-ionic'

1. Nonnally 6 beakers are used, one Jar is used as control and rest 5 beakers are
filled with sample.
2. Measure equal volwne (500 mL) of sample into each of the beakers. Place the
beakers such that the paddles are in centre.
3. Record the sample temperature at the start of the test. In addition, record the
initial pH, turbidity, COD, and colour (in case of coloured eflluents).
4. Start the multiple stirrer operation at the ''flash mix" speed of approximately
5. Add different chemical dosages, e.g. 200 mg/L, 300 mg/L, 400 mg/L, 700 mg/
L etc. Flash mix for approximately 1 min after the addition of chemical. Records
the flash mix time and speed.
6. The flash mixing is followed by slow mixing for about 20 minutes with a speed
• Record the time for the first visible floc fonnation.
• Every 5 minutes (during the slow mix period), record relative floc size and
mixer speed.
• If coagulant aids are used, mixing speed is critical because excessive stirring
tends to break up early floc fonnation and may redisperse.
7. After the slow mix period, withdraw the paddles and observe settling of floc
particles. Record the time required for the bulk of the particles to settle. In most
cases this time will be that required for the particles to settle to the bottom of
the beaker; however, in some cases there may be interfering convection cur-
rents. If so, the recorded settling time should be that at which the unsettled or
residual particles appear to be moving equally upward and downward. .
8. After 15 minutes of settling, record the appearance of floc on the beaker bot-
tom. Record the sample temperate. By means of a pipette or siphon, withdraw
an adequate sample volwne of supernatant liquor from the jar at a point one
half of the depth of the sample.
202 Handbook ofMethods in Environmental Studies

9. The supernatant liquid is tested for colour, turbidity, pH, COD and other re-
quired analysis.

It has been well recognised that reproducibility of results is important. To demon-
strate reproducibility, the so-called 3 and 3 procedure is suggested. In this proce-
dure, duplicate sets of 3 jars are treated simultaneously with the same chemical
Jar test data-sheet (ASTM, 1995)

1. Date.................... ..
2. Sample No. and name:
3. Location: ............ ..
4. Sample size: .............. mL
5. Initial pH: ............ ..
6. Initial Turbidity (NTU): ............ ..
7. Initial Colour (Hz): ............ ..
8. Initial Temperature ("C): ............ ..
9. Initial COD (mgIL): ............ ..

Parameters Jar Number

Chemicals, mgIL * 1 2 3 4 5 6
Flash Mix Speed, rpm
Flash Mix Time, min
Slow Mix Speed, rpm
Slow Mix Time, min
Temperature, °C
Time First Floc, min
Size Floc
Settling rate
q'urbidity, NTU

* Indicates order of addition of chemicals.

Treatability Studies a/Wastewater 203

Observation sheet of Jar-test.

Coagulants pH COD (mg/L) Turbidity, NTU Colour, Hz
dose, (mg/L) Initial Final Initial Final Initial Final

Test Your Confidence on Jar-Test

1. What is the difference between coagulation and flocculation?
2. Most of the metal coagulants are salt of .................... and ................... .
3. Why alkalinity is consumed during coagulation process?
4. How to maintain alkalinity in coagulation process, if water has low alkalinity?
5. What are advantages of use of coagulant aids in coagulation process?
6. Discuss the use of Jar-test data.
7. What is flash mixing and slow mixing?
8. How to measure the RPM of stirrer?
9. List important parameters that are analysed in the supernatant.
10. Name few coagulants commonly used in water/wastewater treatment process.
11. A dose of 50 mg/L of alum is used in coagulation of turbid water. How much
alkalinity is consumed? How much mg/L of aluminium hydroxide floes are
produced? [(Ans: 25 mg/L CaC03 consumed; 13 mg/L Al(OH)3]

5.2 FoodlMicroorganisms Ratio (FIM Ratio)

The FIM ratio is an important loading criteria used for operation of biological
treatment process and express as kg BODs appliedlkg of In other
words, FIM ratio is one of the processes controlling parameters as well as design
parameter. The values ofFIM ratio for some of the conventional biological treat-
ment system are given in Table 5.2.

Table 5.2:
F/M ratio of some of the conventional biological treatment process.

Name of the treatment process F/M value

Conventional ASP 0.6 to 0.9 (average 0.7) - warm climate

0.4 to 0.6 (average 0.5) - temperate climate
0.2 to 0.5 (average 0.3) - cold climate
Oxidation ditch 0.05 to 0.30 (average 0.1)
Sequential batch reactor (SBR) 0.05 to 0.30 (average 0.1)
204 Handbook o/Methods in Environmental Studies


So.Q So
FIM ratio =

So = Influent BODs or COD concentration, mgIL
e = (V/Q) = hydraulic retention time (HRT), days
V = Volume of aeration tank, m3
Q = Influent wastewater flow rate, m3/day
FIM = Food-to-microorganisms ratio, dayl.
Measure the influent BODs concentration by standard method. Also calculate
MLVSS in the aeration tank. Calculate the HRT of the system. The FIM ratio can
be calculated by using the above formula and express in per day (i.e., l/day).

Check Your Confidence on F/M Ratio Estimation

1. Defme FIM ratio? How it is calculated?
2. What is the unit ofFIM?
3. "Higher the FIM ratio, higher the BOD loading in the system": (TIF)
4. "FIM is a process controlling parameter": (TIF)
5. Is FIM ratio is constant for all biological treatment system?
6. Why FIM ratio is changed in different climatic conditions?
7. (a) An activated sludge process (ASP) receives influent BODs of300 mgIL in
aeration tank with a flow of 5 MGD. The detention time is 6hand MLSS concen·
tration is 3000 mgIL (MLSS: MLVSS = 0.8). Calculate FIM ratio (Ans: 0.5/
(b) As F1M ratio is exceeded the prespecified design value of 0.3 day, how to
control it?

5.3 Mixed Liquor Suspended Solids (MLSS)

and Mixed Liquor Volatile Suspended
Solids (MLVSS)
The MLSS and MLVSS terms are used to express the microbes concentration
in biological wastewater treatment process. The MLSS contains microbes mixed
with inorganic substances like, clay, silts, fine sands etc, whereas MLVSS
measure true microbes concentration. The MLVSS value is always less than
MLSS. In practice, the MLVSS concentration is generally maintained 80% of
Treatability Studies of Wastewater 20S

5.3.1 Estimation of MLSS

The MLSS is detennined by filtering the sample through a glass fibre filter disk.
The residue retain in the filter is dried to constant weight at 103°C.
Filtration apparatus, Vacuum pump, Hot air oven 103°C, Analytical balance, Glass-
fibre filter disc, Desiccator.
1. Place a glass fibre filter disk, rough side up, in a filter container and place in
filtration apparatus.
2. Wet the filter with a small volume of reagent grade water.
3. Depending upon the MLSS content, filter 2S, SO or 100 mL portion ofa well-
mixed sample. Filter the measure volume by applying suction for about 3 min
after filtration is complete.
4. Remove the filter paper from the filtration apparatus and transfer to an alu-
minium-weighting disk as a support.
S. Dry for at least lh in the range 103 to 10SoC in Hot air oven, cool in a desicca-
tor to balance temperature and weigh. Repeat the cycle of drying, cooling, des-
iccating and weighing until a constant weight is obtained.
(A-B) x 1000
sample volume, mL
A = Weight of filter paper + dried residue, mg
B = Weight of filter paper, mg.

5.3.2 Estimation of MLVSS

The residue ofMLSS is ignited to constant weight at SSOoC. The determination is
useful in controlling of wastewater treatment plant operation because it offers a
rough approximation of the amount of organic matter present in the solid fraction
of activated sludge (Le. microbes fraction as microbes mass are organic).
The ratio of MLSS: MLVSS should be around 0.80 for satisfactory perfor-
mance of wastewater treatment plant.
For example, if MLSS concentration is 3500 mgIL, then what is the MLSS
MLVSS = 3S00 mgIL x 0.8 = 2800 mgIL
The MLVSS value is used for calculation of design parameters of biological
treatment system, like substrate utilisation rate, u (So-S)/9X; FIM ratio (So/9X);
solid retention time (9c); recirculation ratio (Qr/Q), etc.
206 Handbook ofMethods in Environmental Studies


Muffie furnace, 550°C in addition to the apparatus used for MLSS detennination.

1. Put the filter paper containing MLSS residues in a muffie furnace at a tempera-
ture of 550°C. Allow furnace to raise the temperature before inserting the sample.
Usually, 15 to 20 min ignition are required for 200 mg residues.
2. Let dish or filter disk cool partially in air until most of the heat has been dissi-
3. Transfer to a desiccator for final cooling in a dry atmosphere.
4. Weight disks or dish as soon as it has cooled to room temperature.
5. Repeat cycle of igniting, cooling, desiccating and weighing until a constant
weight is obtained or until weight change is less than 4% or 0.5 mg, which ever
is less. Duplicate detennination should agree within 5% of their average.

(A- B) x 1000
sample volume, mL

A = weight of residue + dish before ignition, mg
B = weight of residue + dish after ignition, mg

Check Your Confidence on MLSS and MLVSS

1. Define MLSS and MLVSS?
2. Why MLVSS is always less than MLSS?
3. Discuss the steps ofMLSS measurement in Laboratory.
4. Generally the ratio ofMLSS: MLVSS is ------.
5. How MLVSS data's are useful for design, operation and control of biological
wastewater treatment plant?

5.4 Sludge Volume Index (SVI)

The SVI is use to monitor the settling characteristics of sludge. The SVI is defined
as " the volume in millilitre occupied by 1g of activated sludge (MLSS dry weight
basis) after 30-min settling in a 1-L graduated measuring cylinder". The SVI value
also depends on MLSS concentration.
Treatability Studies of Wastewater 207

Settled Sludge volume (mLlL)

SVI, mLig = - - - - - - - - - -

[mLlL x Llmg = mL/mg x 1000 = mL/g]

As SVI is reported in mL/g; this mLlmg unit is converted into mL/g by multi-
plying with 1000. The usual adopted range of SVI is between 50 -150 mL/g. Any
value obtained in this range indicates good settling characteristics of sludge.

Note: If the solids did not settle at all, but occupied the entire 1000 mL at the end
of 30-min, the maximum SVI value obtain would vary from 1000 for a MLSS
concentration of 1000 mgIL to 100 for a MLSS concentration of 10,000 mgIL.

Application of SVI Value

The SVI vale is used to control the pumping rate ofretum sludge from secondary
settling tank (SST) to aeration tank. If SVI is know, the % of sludge in terms of
recirculation ratio (Qr/Q) is calculated by the following empirical relationship:

100 Qr/Q = - - - - -
1001Pw. SVI - 1
Pw = MLSS in %.; SVI = Sludge volume index, mL/g.
Sludge density index is used in a way similar to the sludge volume index to indicate
the settleability of sludge in a secondary clarifier. The weight in grams of millime-
ter of sludge, after settling for 30 minutes, is calculated as:

SOl = 100/SVI
SOl = sludge density index, g/mL
SVI = sludge volume index, mLig.

A sludge with good settling characteristics has an SDI of between 1.0 and 2.0,
where as an SOl of 0.5 indicates a bulky or non-settleable sludge (CheremisinojJ,
PN., 1995. Handbook ofwater and wastewater treatmenttechnology, NY, Marcel

To maintain 3000 mg/l (0.3%) MlSS concentration in aeration tank with SVI value
of 100; what should be percent sludge that be returned?

------- = 43%
100/(0.3 x 100) - 1
208 Handbook of Methods in Environmental Studies

To maintain 2000 mg/L of MLSS in aeration tank with SVI of 100 mL/g, calculate the
recirculation ratio.

Estimation of SVI

1. Settling column: Use lL graduated cylinder equipped with stirring mechanisms.

2. Stopwatch


1. Determine the MLSS concentration of a well-mixed sample of the suspension.

2. Pour the suspension in 1L graduated measuring cylinder.
3. Allow settling for 30 minutes.
4. Note the volume occupied by suspension in mL.


Settled sludge volume, (mLlL)

SVI, mL/g = - - - - - - - - - - - x 1000
[mLlL x Llmg = mL/mg x 1000 = mL/g]

Check Your Confidence on SVI Estimation

1. Defme SVI.
2. How SVI is measured in the laboratory?
3. The SVI value is closely related with settling characteristics of sludge. (TIF).
4. What should be the ideal value of SVI?
5. What is sludge density index (SDI)? How SDI is determined? What should be
the acceptable value of SDI?
6. "Rate of return activated sludge (RAS) calculation is based on SVI value"-
justify your answer.
Treatability Studies of Wastewater 209

7. The MLSS concentration in the aeration tank is 2800 mgIL; sludge volume
(SV) is 285 mgIL. Calculate SVI and estimate MLSS concentration in RAS
and required returned sludge ratio. (Ans. SVI = 120 mUg, MLSS in RAS =9804
mg/L; return sludge ratio = 0.4)
8. Compute the return activated sludge (RAS) flow rate in m3/day as a percentage
of influent flow of 10 MGD (37,850 m3/day). The laboratory result shows that
SVI = 110 mUg and MLSS = 2500 mgIL (Ans. 37.9%).
9. Determine the RAS flow as a % of influent flow of 10 MGD. The sludge set
tling volume in 30 min is 255 mL. (Ans: 34% 12,900 mJ/day).

5.5 Volatile Fatty Acids (VFAs)

The VFAs are produce as an intermediate compound during anaerobic treat-
ment process by fermentative organisms (commonly known as acidogens or
acid formers).
Complex substrate ~ simple substrate ~ VFAs ~ Acetic acid ~ CH4 +
CO 2
Some common VFAs produced during anaerobic treatment process are:
Propionic acid (C3), Butyric acid (C4), Lactic acid (C3) etc. The VFA lowers
the pH of the system, which has an inhibitory effect on the growth of methane
bacteria. The VFAs should be less than 250 mg/L for satisfactory performance
of anaerobic process. To control VFAs, alkalinity is maintained in the range
from 1,000 to 5,000 mg/L.
VFAs are produced more in wastewater rich in carbohydrates (sugar), such
as effluents from sugar cane industry. If VFAs production is more, pH is
neutralised by increasing alkalinity by addition of lime. A ratio of VFAs to
total alkalinity (as CaCO) should be less than 0.1. When VFAs increases, pH
drops, in such case, feeding of new materials is stopped until pH and VFAs are
stabilized. If any day, it is observed that VFA: ALK ratio is less than 1:2,
feeding should be stopped for the day and add bicarbonate alkalinity to bring
the ratio to 1:2.
A drop in pH, results increase in total VFA/ALK ratio, thereby VFA in-
crease in effluents, which is an indicator of process failure. Thus, high VFA in
effluent indicates methanogenesis is incomplete, and low VFA indicates
completion of methanogenesis process.
Importance ofVFA in biological phosphorous removal: VFAs are impor-
tant substrate for the Acitinobacter. The Acitinobacter is one of the primary
organisms responsible for the removal of phosphorus. These organisms re-
sponded to VFAs in the influent wastewater under anaerobic condition by re-
leasing stored cell phosphorous (Metcaff & Eddy, 1993).
210 Handbook of Methods in Environmental Studies

Estimation of Volatile Fatty Acids

Volatile fatty acids (VFAs) are classified as water-soluble fatty acids that can be
distillate at atmospheric pressure. This teclmique recovers acids containing up to
six- carbon atoms. The VFAs are determined by heat distillation followed by titra-
tion of the condensate with a strong base such as NaOH using phenolphthalein as
an indicator. Calculation and reporting on the basis of acetic acid.

1. Centrifuge, with head to carry 50-rnL tubes or 250-rnL tubes.
2. Distillation assembly: Distillation flask, 500-rnL capacity; condenser; adapter
(Fig. 5.2).

100 mL Sample
+ 100 ml water (ollect
+ I) mL H2S04 150 mL
di!> till ate

Fig. 5.2: Distillation assembly for VFA estimation.

1. Sulphuric acid (1+1)
2. Standard NaOH titrant, O.IN
3. Phenolphthalein indicator
1. Take 200 rnL sample and centrifuge for 5 minutes (or filter it).
2. Collect supernatant, and place exactly 100 mL supernatant liquor in a 500 rnL
distillation flask.
3. Add 100 rnL distilled water, few glass beads or similar materials to prevent
bumping, and 5 rnL H 2S04 •
4. Mix well so that acid does not remain in the bottom of the flask.
5. Connect distillation flask to a condenser and adapter and distill at the rate of
about 5 rnL/min.
6. Discard first 15 ml distillate (Note: H 2S and CO 2, liberated during distillation
and titration, will give a positive error. Discarding first 15 rnL of distillate elimi-
nates this error).
Treatability Studies a/Wastewater · 211

7. Collect exactly 150 mL distillate in a 500 mL conical flask.

8. Titrate the distillate with 0.1 N NaOH, using Phenolphthalein as an indicator.
At the end point, the colour changes to pink.

mL NaOH x N x 60 x 000
VFA as acetic acid, mgIL = - - - - - - - - - -
mL of sample

N = Normality ofNaOH

Note: The distillation method is empirical and gives incomplete and somewhat
variable results. Factors such as heating rate and proportion of sample recovered as
distillate affect the results. Therefore, recovery factor is 4:alculated for each distill-
ing apparatus and set of operating condition. The recovery factor is calculated by
diluting approximate volume of acetic acid stock solution to 250 mL' in a volumet-
ric flask to approximate the expected sample concentration and distil as for a sample.

Recovery factor can be calculated as follows:

a = volatile acid concentration recovered in distillate, mgIL
b = volatile acid concentration in standard solution used, mgIL
If recovery factor is consider, the VFA is calculated as:
mL NaOH x N x 60,000
VFA as acetic acid, mgIL = - - - - - - - - -
mL of sample x f
f = recovery factor.

Check Your Confidence on VFA Estimation

1. Define VFAs.
2. How VFAs are formed?
3. Write two importance ofVFA analysis.
4. Why correction factor is necessary for VFA estimation?
5. VFAs are monitored in the effluents of ............. treatment process (aerobic/
6. Why level ofVFAs must be controlled and how to control?
7. What is the satisfactory level ofVFA in effluents?
8. VFAs increase in etl1uent is an indicator of process failure (True/ false).
9. Name three important VFAs produce during anaerobic treatment process.
10. VFAs are converted to .... ........ . and is used as substrate for methane production
by methanogens.
Analysis of Water

Standards for Coliform Bacteria

Drinking Water
1. Should not contain more than 1 coliformllOO mL (USEPA, 1974).
2 The U.S.EPA also recommended use of 10 replicate tubes each containing 10
mL or 5 replicates tubes each containing 20 mL, or a single bottle containing
a 100-mL sample portion.
3. As per IS: 10500(1983, 1991) E.coliin 100 mL ofariysamples should be zero,
or coliforms not more than 11100 mL shall be present in any sample.
• Coliform organisms should not be detectable in 100 mL of any two consecu-
tive samples or more than 50% of the samples collected per year. This is
done by presence-absence (P-A) test.
• If coliform exceeds 3/100 mL the sample should be disinfected.
Standards of WHO (1993)
Organisms Guideline value
All waters intended for drinking:
E. coli or thermo tolerant coliform bacteria Must not be detectable in any
1OO-mL sample.
Treated water entering the distribution
E. coli or thermotolerant coliform bacteria Must not be detectable in any
l00-mL sample.
Total coliform bacteria Same as above
Treated water in the distribution system:
E. coli or thermotolerant coliform bacteria Must not be detectable in any
l00-mL sample.
Total coliform bacteria Same as above
In the case oflarge supplies, where sufficient samples are examined, must not
be present in 95% of samples taken throughout any 12-month period
MOEF (2000) notification for bathing water
• Fecal coliform (MPN/IOO mL) - 500 (desirable) 2500 (Max. permissible).
• Fecal streptococci (MPNII 00 mL) 100 (desirable) 500 (Max. permissible).
Microbiological Analysis of Water 213

6.1 Introduction
Pathogenic bacteria, pathogenic protozoan cysts, and viruses have been isolated
from wastewater and natural waters. The sources of these pathogens are the faces
of human and of wild and domestic animals. Identification and enumeration of these
disease causing organisms in water and wastewater are not recommended because
no single technique is currently available to isolate and identify all the pathogens.
In fact, concentrations of these pathogens are generally low in water and wastewa-
ter. In addition, the method for identification and enumeration of pathogens are
labour intensive and expensive. Instead of direct isolation and enumeration of
pathogens, total coliform (TC) has long been used as indicator of pathogens con-
tamination of a water that poses a public health risk.
Fecal coliform (FC), which is more fecal specific, has been adopted as a stan-
dard indicator of contaminations in natural waters. Both TC and FC tests are used
in standards for drinking water and natural water. Fecal streptococci (FS) is used as
a pollution indicator in Europe. The FCIFS ratios have been employed for identify-
ing pollution sources. The FS are present in the intestines of warm-blooded animals
and of insects, and they are present in the environment (water, soil and vegetation)
for long period of time. E. coli bacteria have also been used as an indicator.
The concentration of pathogenic microorganisms in untreated municipal waste-
water are presented in Table 6.1

Table 6.1:
Microorganism concentration in untreated municipal waste water.

Organisms Concentration (number/100 mL)

1. Total coliforms 107 - 10'0

2. Fecal coliforms 104 - 109
3. Fecal streptococci 104 -10 8
4. Protozoan cysts 103 - 105
5. Shigella 1 - 103
6. Salmonella 102 - 104
7. Helminth ova 10 - 103
8. Enteric virus 102 - 104
9. Giardia lamsdia cysts 10 - 104
10. Entamoeba histolytica cysts 1 - 10
11. Cryptosporidium oocysts 10 - 103

6.2 Diseases Associated with Polluted Water

Important diseases associated with polluted water are often caused by bacteria,
virus and protozoa.
1. Cholera: Vibrio cholerae
2. Bacillary dysentery (Shigellosis): Shigella dysenteriae
3. Typhoid and paratyphoid fever: S Ylmonella typhi and S. paratyphi
214 Handbook of Methods in Environmental Studies

4. Gastroenteritis (Diarrhoea): Escherichia coli

5. Yersinosis (Diarrhoea): Yersinia enterocolitica
6. Acute respiratory illness (Legionellosis): Legionella pneumophila
7. Leptospirosis: Leptospira


Viruses that are excreted by human beings may become a major hazard to public
health. For example, form experimental studies, it has been found that from 10,000 to
100,000 infections doses of Hepatitis virus are emitted from each gram offaces of a
patient ill with this disease. A number of outbreaks of infectious hepatitis have
been attributed to transmission of the virus though water supplies. Few common
diseases are:

1. Infectious hepatitis A (Jaundice): Hepatitis virus A

2. Gastroenteritis: Reovirus
3. Poliomyelitis: Polio virus
4. Gastroenteritis: Rotavirus
Enteric protozoan diseases are spread though fecal-oral rout.!, all produces envi-
ronmentally stable oocysts or cyst. For examples:
1. Amoebic dysentery (Amoebiasis): Entamoeba histolytica
2. Giardiasis (Giardia lamblia) and
3. Balantidiasis (Diarrhoea, dysentery): Balantidium coli.
4. Cryptosporidiosis (Cryptosporidium). Cryptosporidium is the most disinfec-
tant resistant waterborne microorganism.

6.3 Coliform Group of Bacteria

The coliform group of bacteria comprises all the aerobic and facultative anaerobic,
gram negative, non-spore forming rod-shaped bacteria, which ferment lactose with
gas formation within 48 h at 35°C. Coliform bacteria include species of genera,
Escherichia, Enterobacter, Klebsiella and Citrobacter. These bacteria inhabitate
the intestines of all warm blooded animals. Escherichia and Klebsiella are called
fecal coliforms as they are essentially multiply only in intestines. The other two
bacteria besides intestines, can also multiply in organic matter and hence desig-
nated as nonfecal coliforms.

The discharge of~dstewater from municipal sewers is one of the most important
sources of coliform in drinking water. Municipal sewage contains human feces and
water contaminated with these effluents may contain pathogenic (disease-causing)
organisms and consequently, may be hazardous to human health ifused as drink-
ing water. In rural areas, open-defecation in the field, bathing and washing of clothes
Microbiological Analysis of Water 215
etc. are some of the common sources of coliform contamination. Fecal contamina-
tion of water is routinely detected by microbiological analysis. Pathogenic microor-
ganism are transmitted by water include bacteria, viruses and protozoa.
The intestinal tract of man contains countless rod-shaped bacteria known as
coli/arms. Each person discharges from 100 to 400 billion coliform organisms per
day, in addition to other kind of bacteria (Metcaff and Eddy, 1985). The coliform
bacteria include the genera Escherichia and Aerobacter.


Since the number of pathogenic organisms present in waste and polluted waters are
few and difficult to isolate, the coliform organisms, which are more numerous and
more easily tested for, are used as indicator organisms.
The presence of coliform organisms is taken as an indication that pathogen
organisms may also be present, and absence of coliform organisms is taken as an
indication that the water is free from disease producing organisms.
Techniques generally used to evaluate the microbiological quality of water are
based on detection of indicator organisms. These indicator organisms are 'coli/arms'
(enteric bacterial species from colon) residing normally in the human/animal intes-
tine are excreted in large numbers in feces irrespective of whether the person suf-
fers from a waterborne disease or not. Accordingly, coliforms do not necessarily
relate to the popUlation of pathogens but their occurrence detected in water is
suggestive of contamination of water resource by feces (and hence a likelihood of
occurrence of enteric pathogens).

Some desirable characteristics of indicator organisms

1. They should be harmless to humans.
2. They should be present in water in large numbers.
3. They should be easy and quick to identify and one should able to enumerate
them though simple laboratory tests.
4. They should survive unfavorable environmental conditions longer than
5. They should not mUltiply under conditions where pathogens do not multiply.
Problems related to the use of E. coli as an indicator organisms include:
1. E. coli is not a single species.
2. Other organisms found in water have the ability to ferment lactose.
3. E. coli identical to those in humans are in the intestinal tract of other warm
blooded animals. In spite of all these shortcomings, E. coli continues to be
used as an indicator organism.

6.4 Estimation of Coliform Bacteria in Water

1. Coliforms are colon bacteria (Escherichia coli) residing normally in the human
or other warm blooded animals' inte~tine, which are used as an indicator organ-
216 Handbook of Methods in Environmental Studies
ism in microbiological examination of water.
2. E.coli is, as such, completely hannless. Its presence in drinking water is not
dangerous, though some strains are enteropathogens and cause diarrhoea.
3. Colifonn counts of any potable sample should be zero.
4. To satisfy the standard "in 100 mL sample colifoml should be absent"- use
directly 100 mL sample or use P-A test.
5. No colifonn standards set for effluent discharge (MOEF, 1993).
6. Colifonn is expressed as MPN index/lOO mL of water.
7. Sampling bottle must be sterilized and use dechlorinating agents when sample
is taken from chlorinated effluents/chlorinated potable water.
8. Maximum time pennissible in 24 h between collection and analysis.
9. For Total colifonn (TC) test, use lauryl tryptose broth for presumptive test and
brilliant green lactose broth for confinnatory test. In completed test nature of
colony and morphology of E. coli are studied.
10. Fecal coliform (FC) test is used to distinguished between total coliform and
fecal colifonn.
11. Fecal streptococci (FS) test is used to distinguish the sources of contamina-
tion, whether from human faces or warm-blooded animals.
12. Membrane filtration techniques are more rapid but have limitations.
13. For rapid coliform estimation use 7-h fecal coliform test.

The testing for colifonns includes:

1. Total coliforms (TC) test: It can be done by
Multiple tube fennentation test or Most Probable Number (MPN) test
Membrane filtration (MF) techniques
Standard plate count (SPC) test: Not commonly used as routine coliform estima-
2. Fecal coliform (FC): Positive tubes from presumptive tubes are inoculated in
3. Fecal streptoccoci (FS) test: Use Azide dextrose broth.
To differentiate colifonn groups IMViC test (Indol, Methyl red, Voges-Proskauer
and Citrate) are conducted.


Sample bottle: Use sterilized bottles of glass or plastic of any suitable size and
shape (preferably use 250 mL capacity). Plastic bottles are sterilized in autoclave at
121°C for 15 min. The non-plastic bottles (glass) sterilized in hot-air oven at 170°C
fOF Ih.

Dechlorination of Sample
Chlorinated effluent discharge: Add sufficient sodium thiosulphate solution to a
sterilized sample-bottle to give a concentration of 100 mg/L of sample. For example,
in a 120-mLcapacity sample-bottle, add 0.1 mL oflO% Na2 Sp3' thatwiIl neutralized
a sample containing 15 mg/L residual chlorine.
Microbiological Analysis of Water 217

Chlorinated drinking water: Add 0.1 mL of3% Na2SPJ solution in a 120-mL

capacity sample-bottle, which will give a final concentration of 18 mg/L. It will
neutralize up to 5 mg/L of residual chlorine.
Sample preservation: If sample can not be analysed within 1h after collection, use
an ice cooler for storage during transport to the laboratory. In any case, the time
elapsed between collection and analysis should not exceed 24 h.


1. Autoclave: Used for sterilisation ofculture media. Keep 121°C for 15 min at 151b
2. Hot-air sterilising oven: Able to give uniform sterilising temperature of
170 ± 10°C for at least I h, equipped with thermometer; used for sterilisation of
3. Bacteriological incubator: Used for maintaining optimum temperature for bac-
terial growth.
4. Laminar flow chamber: Used for bacteriological work under sterilized condi-
5. Culture tubes: (Test tubes, Petri dish) used to keep culture media.
6. Fermentation vials or Durham's vials: Used for collection offermentation gas,
i.e. COl'
7. Inoculation loop: Needed for transferring inocula
8. Burner or spirit lamp
9. pH meter, buffer tablets, balance, pipet container; refrigerator
10. Dilution bottle or tubes, pipettes, spirit, cotton, etc.


The common procedure for MPN determination involves the use of sterile pipettes
calibrated in 0.1 mL increments, sterile screw-top dilution bottles containing 9 mL
of water and a rack containing 3 sets of 5 lauryl tryptose broth fermentation tubes.
Fig. 6.1 is a sketch showing test preparation.
The most probable number (MPN) technique has been conveniently used for
drinking water and effluent samples for many years with satisfactory results. The
tests for fecal contamination are carried out in three stages. Schematic outline of the
MPN test is given below:

Stage I Presumptive test Lauryl Tryptose Broth (LTB)

Stage-II Confirmatory test Brilliant Green Lactose Bile Broth (BGLB)
Stage-III Completed test Endo or Eosin Methylene Blue (EMB)

Presumptive test (Stage-I)

The first step in coliform testing is the presumptive test. A set of tubes of lauryl
tryptose broth (LTB) (Table 6.2) is inoculated with samples of water and then
incubated. Lauryl sulfate is a surface-active detergent which inhibits the growth of
gram-positive organisms while encouraging the growth of coliforms. Coliform use
any oxygen present in the broth and then ferment the lactose there by producing
218 Handbook of Methods in Environmental Studies

Stage-I: Presumptive test

Inoculate Lauryl tryptose broth tubes and incubate 24 ± 2 hat 35 ± 0.5°C
J; 'l-
Gas produced, Positive No gas, or gas production doubtful.
Presumptive test Incubate for another 24h (total 48 ± 3 h)
(I) (2) J,
J; 'l-
Gas produced, No gas produced, Negative
Positive test test. Coliform group absent

Stage-II: Confirmed test

Transfer one loopful of medium (1) to confirmatory brilliant green lactose bile
broth. Incubate for 48 ± 2 h at 35-37°C.
Gas produced."" Coliform No gas produced. Negative test.
group confirmed + ve test. Coliform group absent.

Stage-III: Completed test

Transfer inoculum from each positive tubes of confirmatory test (2) to Endo
or EMB plates. Incubate for 24 ± 2 hat 35 ± O.5°C.
"" with a dark centre
Typical well isolated Negative colonies
coliform colonies having a metallic watery colonies
surface sheen. (positive colonies) Coliform group absent.
Coliform group present.

acid and gas under anaerobic conditions. Gas formation in 24 h or 48 h is a positive

test. If the medium has been refrigerated after sterilisation, incubate overnight at
35°C before use. All dehydrated ingredients are added to water, mix thoroughly and
heat to dissolve. Next, the media is sterilized in autoclave.
Procedure of Presumptive test
For non-potable water/emuent/domestic wastewater sample
1. For wastewater five replicates tubes having decimal dilution of 0.1, 0.01, 0.001
mL or more are used.
2 Take 3 beakers and keep 5 test-tubes in each beaker and mark the beaker as
O.lmL, 0.01 mLand O.OOlmL. It is better to mark the test tubes also.
3. Fill the test tubes and Durham's tube with sterilised lauryl tryptose broth (Table
6.2). Durhams tubes are placed in inverted manner.
Microbiological Analysis of Water 219

Media composition of Lauryl tryptose broth (L T8).

1. Tryptose 20.00 9
2. Lactose 5.00 9
3. K2 HPO. 2.75 9
4. KH 2 PO. 2.75 9
5. NaCI 5.00 9
6. Sodium lauryl sulfate 0.1 9
7. Distilled water 1.0 L
Total ingredients 35.06 g
Final pH after sterilisation 6.8

/\ I
10"'l I mL lmL

Amount p.petted onto termentahon tube

I ml

IO"'L lmL I mL ·Olml ·001 ml

A mount ot wat... .n tach II rmlnt.t.on tub'

Fig. 6.1: Procedure for decimal dilution techniques used for MPN
count in wastewater or domestic sewage.

4. Care should be taken that no air bubbles should remain inside the inverted
Durham's tubes. The air bubbles trapped in the Durham's tubes are removed by
gently inverting the test tubes after closing their mouth by thumb or a fmger.
For drinking water
In case of drinking water, use:
1. Five replicate tubes each containing 20 mL of water, or
2 Ten replicate tubes each containing 10 mL of water, or
3. One single bottle containing 100 mL of wa!er.
Prepare the Lauryl tryptose broth (LTB) media as per Table 6.3, because by
adding the sample portion of 100-mL, 20-mL or 1O-mL to the medium, it will reduce
ingredient concentration below those of standard medium.
When 5 replicates are used: Pour 10 mL medium (106.8g!L) in each test tube and
add 20 mL of sample.
220 Handbook of Methods in Environmental Studies

When 10 replicates are used: Pour 10 mL medium (71.2 gIL) in each test tubes
and add 10 mL of sample.
When 100 mL portion is used: Pour 50 mL medium (1 06.8g1L) and add 100 mL of
sample in one bottle.

Table 6.3:
Preparation of Lauryl tryptose (L TB) broth.

Inoculum, Amount of medium Volume of medium Dehydrated lauryl

mL in tube, mL + inoculum,mL tryptose broth
required, gIL

10 or mo~·e 11 or more 35.6

10· 10 20 71.2
10 20 30 53.4
20·· 10 30 106.B
100 50 150 106.B
100 35 135 137.1
100 20 120 213.6

• For drinking water, when 10-mL portion sample is used in each of 10 test tube or
fermentation tube (10 mL water sample + 10 mL medium) , prepared LT broth as
per Table -4.1 but dissolve 35.6g ingredients in 500 mL of distilled water instead
of 1000 mL of distilled water .
•• For drinking water, when 20-mL portion sample is used in each of 5 test tube or
fermentation tube (20 mL water sample + 10 mL medium), in such case prepare
LT broth as per Table -4.1, but dissolve 35.6g ingredients in 334 mL of distilled
water instead of 1000 mL of distilled water.

Incubate the inoculated fennentation tube at 35 ± O.soC. At the end of 24 ± 2 h
shake each tube gently and examine it. If no gas is fonned repeat this for another 48
± 3 h. Record the gas fonnation.

The appearance of gas bubble must not be confused with actual gas production. If
the gas is fonned as a result of fennentation, the broth medium becomes cloudy.
The absence of gas fonnation at the end of 48 h of incubation constitute negative
Since this reaction may also be produced by the organisms other than the
colifonns, the positive tubes from the presumptive test are subjected to confinna-
tory test.
Record the number of positive tubes and refer Table 6.10 or 6 .11 for drinking water
or Table 6.12 for effiuent.

Confirmatory test (Stage-II)

Transfer one loopful of medium from positive (+) tubes of presumptive test to
Brilliant Green Lactose Bile (BGLB) broth medium (Table 6.4). The BGLB broth, in
Microbiological Analysis of Water 221
addition to containing lactose, also contains two components inhibitory to gram-
positive bacteria. Brilliant green is a dye related to crystal violet and belongs to the
triphenylmethane dye series. Oxgall is a surface active agent which also inhibits the
growth of gram-positive bacteria. Gas formation in 24 or 48 h confirms the results of
the presumptive step.

Table 6.4:
Media composition of BGLB broth

1.Peptone 10.0 9
2.Lactose 10.0 9
3.0xgall 20.0 9
4.Brilliant Green 13.3 mg
5.Distilled water 1.0 L
Final pH after sterilization 7.2
Note: 5 g sodium taurocholate can also be used in place of 20 g oxgall

Procedure of Confirmatory test

1. Diagram ofMPN technique for determination of the coliform group in water by
presumptive and confirmed test is given in,Fig. 6.2.
2 Add dehydrated ingredients to water, mix thoroughly, aIld heat to dissolve.
3. Prepare fermentation tubes with 10 rnL BGLB medium along with Durham's
tubes. The numbers of tubes to be prepared are equal to all positive tests in the
presumptive test.
4. Shake gently, the fermentation tube with positive results and transfer one loopful
of medium to BGLB broth
5. Incubate the tubes at 35-37°C for 482 h. and record the tubes with gas formation
as + ve number. The wrong concentration ofBGLB or the exposure of the media
to excessive heat or light may give false positive tests.
6. To confirm presence of coliform bacteria, the completed test is carried out. For
this test, inoculum from each positive tube of the conflrmatory test is streaked
on a plate ofEMB (Eosin Methylene Blue) or Endo agar plate.
Record the number of positive tubes and refer Table 6.10 or 6.11 for drinking water
or Table 6.12 for effluent.
Completed test (Stage-III)
In completed test the characteristics of coliform colony and morphology of E. coli
are observed.
Since some of the positive results from the confirmatory test may be false, it is
desirable to do occasional completed test.
Use at least 10% of positive confirmed tubes.
For this, inoculum from each positive tube of the confirmatory test is streake1
on a plate of eosin-methylene blue (EMB) agar or endo agar. In EMB agar, two
dyes, eosin and methylene blue, inhibit the growth of gram positive organisms.
222 Handbook of Methods in Environmental Studies

§ No9rowttl
or grow til

Growth tr_,.rrotl
Inc.ubation tMgabv. tItt by wlr. loop . or growttl
&1 3S'C· Without

~I 3SoC

gr ••n
bi .. brolh

:':. ~i\h g..'

. In th.
:" ¥.a''W' •• s.t

Fig. 6.2: Total coliform by MPN technique (Presumptive test and confirmed test).

Procedure for Completed Test

Prepare Endo agar or Eosin Methylene Blue (EMB) agar petri-plates (Table 6.5). The
number of petri-plates to be prepared is the same as of tubes showing gas produc-
tion in BGLB medium. Label the plates corresponding to the tubes of confirmatory

Table 6.5:
Media composition of EMB and Endo agar


1. Peptone 10.0 g 1. Peptone 10.0 g

2. Lactose 10.0 9 2. Lactose 10.0 g
3. K2HPO. 2.0 9 3. K2HPO. 3.5 9
4. Agar 15.0 9 4. Agar 15.0 9
5. Eosin Y 0.4 9 5. Sodium sulphate 2.5 9
6. Methylene blue 0.065 9 6. Basic fuchsin 0.5 9
7. Distilled water 1 L 7. Distilled water 1 L
pH after sterilisation 7.1-7.2 pH after sterilisation 7.4
Decolourization of the medium The medium should be light pink when
occurs during sterilisation, hot and almost colourless when cool.
but colour returns after cooling.

1. Incubate these plates at 37°C ± 2°C for 24 h.

2. Now examine the plates for bacterial growth and colony appearance. Well-
isolated colonies with a dark center (nucleated) are typical coliform colonies.
They may have a metallic surface sheen. Clear, watery colonies are not of coliform
and are reported as negative in the completed test.
3. Then inoculate an isolated coliform colony (avoid picking up a mixture) form
Microbiological Analysis of Water 223

'each plate into the tubes of MacConkey broth and record the gas production (a
repetition of presumptive test but with the colonies) within 48 h. at 37°C.
4. Also examine the colonies by gram-staining. For this transfer the colonies to a
nutrient agar slant. Subject the colonies from agar slants to the gram staining.
S. If organisms appear rod (bacilli) shaped, red stained, occurring singly, in pairs
or in short chains, the test is confirmed. Since the coliform organisms are gram-

6.5 Presence-Absence (P-A) Coliform Test

The presence-absence (P-A) test for the coliform group is a simple modification of
the multiple-tube procedure. Simplification, by use of one large test portion (100
mL) in a single culture bottle to obtain qualitative information on the presence or
absence of coliform, is justified by the theory that ''no coliform should be present in
100 mL of a drinking water sample." A flow diagram ofP-A test is given in Fig. 6.3.


P-A broth medium is commercially available in dehydrated and in sterile concen-
trated form. The culture medium may be prepared by mixing ingredients as per
Table 6.6.
1. Make this formulation triple strength (3X) when 100-mL sample is examined.
(e.g. dissolve triple the quantities of ingredients in 1000 mL water or the
quantities as per Table 6.6 in 334 mL water).
2 Dissolve the P-A broth medium in water without heating, using a stirring de-
3. Pour SO mL prepared medium into a screw-cap 2S0-mL dilution bottle. A fermen-
tation tube insertion is not necessary.
4. Autoclave for 12 min at 121°C with the total time in the autoclave limited to 30
minor less.
S. The pH should be 6.8 ± 0.2 after sterilisation.

Table 6.6:
Media composition for P-A test.

1. Beef extract 3.0 9

2. Peptone 5.0 9
3. Lactose 7.46 9
4. Tryptose 9.83 9
5. K2 HPO. 1.35 9
6. KH 2 PO. 1.35 9
7. Sodium chloride 2.46 9
8. Sodium lauryl sulfate 0.05 9
9. Bromocresol purple 0.0085 9
10. Reagent grade distilled water 1.0 L
224 Handbook of Methods in Environmental Studies
IP-A Hedia -3X I

Dlutian bottle 250 ml

Ster,liud fer 30 min. \
adJU.t pH

add 100 III umplel

Incubate at 35 'f S·C \
for 1 to 2 days

Contents turn to
di.tinct yellow colour

gas production
Po ..m-. Cont,
t ••t

Fig. 6.3: Flow-diagram for conducting P-A test.

1. Shake sample vigorously for 5 sec (approximately 25 times) and inoculate 100
mL sample into a P-A culture bottle.
2 Mix thoroughly by inverting bottle once or twice to achieve even distribution
of the triple-strength medium though out the sample. Incubate at 35 ± 5°C and
inspect after 24 and 48 hour for acid reactions.


1. A distinct yellow colour forms in the medium when acid conditions exist follow-
ing lactose fermentation.
2 If gas is also produced, a gentle shake of the bottle will result in a foaming
3. Any amount of gas and/or acid constitutes a positive presumptive test requir-
ing confirmation.

Culture medium: Use brilliant green lactose bile (BGLB) broth fermentation tube.
Transfer all cultures that show acid reaction or acid and gas reaction to BGLB broth
for incubation at 35 ± O.soC.
Microbiological Analysis of Water 225

Gas production in the BGLB broth culture within 48 h confirms the presence of
coliform bacteria. Report result as presence-absence test positive or negative for
total coliforms in 100 mL of sample.

The completed test is same as for total coliforms as given in earlier section.

6.6 Fecal Coliforms (Thermotolerant Coliforms)

The test is used to distinguish between total coliforms and fecal coliforms. Fecal
coliform test using EC medium (Table 6.7) is applicable for investigation of drinking
water, stream pollution, raw water source, wastewater treatment systems, bathing
waters and general water quality monitoring.
The term "fecal coliforms" has been used in water microbiology to denote
coliform organisms that grow at 44 to 44.5°C and ferment lactose to produce acid
and gas. So, thermotolerant coliform is more correct term and is becoming more
commonly used.

Table 6.7:
Composition of EC medium.

1. Tryptose 20.0 9
2. Lactose 5.0 9
3. Bile salts mixture or bile salt No.3 1.5 9
4. K2 HPO. 4.0 9
5. KH 2 PO. 1.5 9
6. NaCI 5.0 9

Before sterilisation, dispense fermentation tubes with sufficient medium to cover

the inverted vials at least partially after sterilisation.


1. Transfer all positive tubes from the total coliform MPN test (i.e. presumptive
test) to EC medium. This examination may be performed simultaneously with
the confirmatory procedure using brilliant green lactose broth.
2 Use a sterile metal loop with a minimum 30-mm diameter to transfer the positive
fermentation tube to EC medium containing fermentatiJn tube.
3. Inoculated tubes are incubated at 44.5 ± 0.2 DC for 24 ± 2 h.
4. Place all EC tubes in the water bath within 30 minutes after planting.
5. The water depth in the incubator should be sufficient to immerse tubes to the
upper level of the medium.
226 Handbook of Methods in Environmental Studies


Gas production with in 24 h or less is considered a positive reaction indicating fecal

origin. Failure to produce gas constitutes a negative reaction indicating a source
other than the intestinal tract of wann-blooded animals.
Record the number of positive tubes and refer Tables 6.10 or 6.11 for drinking water
and Table 6.12 for efiluent.

Select all positive tubes of presumptive test
from total coliform MPN test

Transfer one loopful of inoculum to
EC medium. Incubate at 45.5± O.2°C for 24 ± 2 h

INo gas (- ); negative test I Gas formation (+); positive test
(Record the no ofpositive tubes
and calculate the MPN index)

Fig. 6.4: Flow-diagram for conducting fecal coliform test.

6.7 Fecal Streptococci (FS) Test

by MPN Method
The test is used to identify whether fecal contamination was from human origin or
from wann-blooded animals origin. The presence offecal streptococci is evidence
offecal contamination.
1. The normal habitat for fecal streptococci is the intestine of man and animals;
thus, these organisms are indicator of fecal pollution.
2 FS tend to persist longer in the environment than thermotolerant or total coliforms
and are highly resistant to drying.
3. The fecal streptococcus group consists of a number of species of the genus
Streptococcus, such as S. faecalis, S. faceium, s. avium, S. bovis, S. equinus,
Microbiological Analysis of Water 'I27

and S. gallinarum and all have been isolated from the feces of warm-blooded
4. The normal habitat of fecal streptococci is the gastrointestinal tract of warm-
blooded animals.
5. Fecal Streptococci - two stains of fecal streptococci, namely - S. faecalis and
S. faceium are the most human specific members of the FS group. The fecal
streptococci are frequently used in identification of fecal pollution in water.
They are unable to multiply significantly in open water and do not survive long,
thus their presence in high numbers indicates recent pollution.
6. The fecal streptococci test have been used with fecal coliforms test to differen-
tiate human fecal contamination from that of other warm-blooded animals. The
ratio offecal coliforms (FC) to fecal streptococci (FS) could provide information
about the sources of contamination.


It has been observed that the proportions in which FC and FS are discharged by
human beings are significantly different from the quantities discharged by animals.
Therefore, it has been suggested that, the ratio of FC to FS counts in a sample can
be used to show whether the suspected contamination derives from human or from
animal wastes.
The FCIFS ratio for domestic animals is less than 0.7, whereas the ratio for
human beings is more than 4.0 (Table 6.8) (APHA, 1995). The 20th edition of APHA
(1998), stated that FC/FS ratio can not be recommended, and should not be used as
a means of differentiating human and animal sources of pollution. The reason
stated is that the different fecal Streptococcus species have different survival rates
in aquatic environment.

Table 6.8:
Ratio of FCI FS

Animals FC(10 6 /g feces) FS(10 6 /g feces) Ratio (FC/FS)

Chicken 1.3 3.4 1.3/3.4 = 0.4

Cow 0.23 1.3 0.23/1.3 =0.2
Human 13.0 3.0 13/3= 4.4
Sheep 16.0 38.0 16/38 = 0.4

If the ratios are obtained in the range of 1 to 2, interpretations are uncertain. Use
ofFCIFS ratio can be very helpful in establishing the source of pollution in rainfall-
run-off studies and in pollution studies conducted in rural areas, especially where
septic tanks are used. In many situation where human pollution is suspected on the
basis of coliform test results, the actual pollution may, in fact be caused by animal

The FS can be estimated by multiple tube MPN techniques and membrane filter
techniques. The multiple tube techniques is discussed here. The multiple-tube test
228 Handbook of Methods in Environmental Studies
consists of preswnptive test and confmned test. The FS grown in a mediwn con-
taining sodiwn azide, at a temperature of 35°C. Routine methods may gives false
positive and additional confmnatory tests may be required. A flow diagram of fecal
streptococci (FS) test is given Fig. 6.5.

Presumptive FS-test by using azide dextrose broth
1. Inoculate a series of tubes of azide dextrose (AD) (Table 6.9) broth with appro-
priate graduated quantities of sample.
2 Sample size for drinking water: Use sample of 10 mL portion or less. Use double-
strength (2X Le. 69.4g1L) broth for 10-mL inocula.
3. Sample size for wastewater: Use only decimal multiples oft mL (1 .. 0, 0.1 and 0.01
mL) for wastewater.
4. Prepare AD broth according to the following compositions.

Table 6.9:
Media composition of Azide dextrose broth for FS test.

1. Beef extract 4.5 9

2. Tryptone 15.0 9
3. Glucose 7.5 9
4. NaCI 7.5 9
5. NaN 3 • 0.2 9
6. Distilled water 1.0 L
7. pH a iter sterilisation 7.2 ± 0.2 at 25°C
Total ingredients 34.79

1. Incubate inoculated tubes at 35 ± O.5°C.
2. Examine each tube for turbidity at the end of24 ± 2 h.
3. If no definite turbidity is present, re-incubate, and read again at the end of
48± 3h.
4. Calculate the fecal streptocotci density from the MPN index table.

The test is used to observe the bacterial colonies.
1. All the positive tubes after 24 h may be subjected to confirmatory test. Streak a
portion of growth from each positive AD broth tube on PSE (Pfizer selective
enterococcus) agar.
2 Incubate the inverted dish at 35 ± O.5°C for 24 h. ± 2 h.
3. Brownish-black colonies with brown halos confirm the presence of fecal
Microbiological Analysis of Water 229


Azide dellOse
broth 12 III
lI )ric ubate 35:!: 5· (
for 24 to 4& h

No gas: FS absent
Gas formahon: PoSlt.V@ F S tut


inocula from
positive tublils

Coo'" •• d , .. ,



cobni" with
brown hole -® 0

Fig. 6.5: Procedure for performing FS test.

6.8 Calculation of Most Probable

Number (MPN)
Unless a large number of sample portions are examined, the precision of the fennen-
tation tube test is rather low. The calculation ofMPN of colifonn is done by combi-
nation of positive and negative results in the multiple tube tests. The value can be
230 Handbook of Methods in Environmental Studies
• When 5 test tubes are used and each contains 20 mL of water sample - Table
6.10 (for drinking water).
When 10 test tubes are used and each contain 10 mL of water sample - Table
6.11 (for drinking water)
For decimal dilution (10mL, ImL and 0.1 mL) - Table 6.12 (efiluent).
When the series of decimal dilution is different from that in the Table 6.12, select
the MPN value from Table 6.12 for the combination of positive tubes and com-
pute the MPN index using the following formula:
MPNIl 00 mL = MPN value from table x
largest volume tested in dilution
series used for MPN determination

If the MPN comh;nation does not appear in Table 6.12, or for other combina-
tions of tubes or dilution or when MPN tables are not available, in that case Thomas
formula can be used.
No. of positive tubes x 100
..J (mL sample in negative tubes) x (mL sample in all tubes)

Table 6.10:
MPN index and 95% confidence limits for various combinations of positive and
negative results when five 20-mL portions are used.

No. of tubes giving positive MPN index/ 95% confidence

reaction out of 5 of 100 mL limits (approximate)
20 mL each Lower Upper

0 < 1.1 0.0 3.0

1 1 .1 0.05 6.3
2 2.6 0.3 9.6
3 4.6 0.8 14.7
4 8.0 1.7 26.4
5 >8.0 4.0 infinite

Table 6.11:
MPN index and 95% confidence limits for various combinations of positive and
negative results when ten 10-mL portions are used.

No of tubes giving positive MPN indexl 95% confidence

reaction out of 5 of 100 mL limits (approximate)
20 mL each Lower Upper

0 < 1.1 0.0 3.0

1 1 .1 0.03 5.9
2 2.2 0.26 8.1
Microbiological Analysis of Water 231
.. . Cont.
3 3.6 0 .69 10.6
4 5.1 1.3 13.4
5 6.9 2.1 16.8
6 9.2 3.1 21.1
7 12.0 4.3 27.1
8 16. 1 5.9 36.8
9 23.0 8.1 59.5
10 >23.0 13 .5 infinite

Table 6 . 12:
MPN index and 95% confidence limits for various combinations of positive results
when five tubes are used per dilution (10 mL. 1.0 mL • 0.1 mL) .

Combination MPN 95% confidence Combination MPN 95% confidence

of positive index! limits of positive index! limits
tubes 100 mL Lower Upper tubes 100mL Lower Upper

0-0-0 <2 - - 4-3 - 1 33 15 77

0-0-1 2 1.0 10 4-4-0 34 16 80
0-1-0 2 1.0 10
0-2-0 4 1.0 13 5-0-0 23 9 86
5-0-1 30 10 110
1-0-0 2 1.0 11 5-0 -2 40 20 140
1-0-1 4 1.0 15
1-1 -0 4 1.0 15 5-1-0 30 10 120
1-1-1 6 2.0 18 5-1-1 50 20 150
1-2-0 6 2.0 18 5-1-2 60 30 180

2-0-0 4 1.0 17 5-2-0 50 20 170

2-0-1 7 2.0 20 5-2- 1 70 30 210
2-1 -0 7 2 .0 21 5-2-2 90 40 250
2-1-1 9 3.0 24
2-2-0 9 3.0 25 5-3-0 80 30 250
2-3-0 12 5 .0 29 5-3-1 110 40 300
5-3 -2 140 60 360
3-0-0 8 3.0 24 5-3-3 170 80 410
3-0- 1 11 4 .0 29
3-1-0 11 4 .0 29 5-4-0 130 50 390
3-1-1 14 6 .0 35 5·4-1 170 70 480
3-2-0 14 6.0 35 5-4-2 220 100 580
3-2-1 17 7 .0 40 5-4-3 280 120 690
5-4-4 350 160 820
4-0-0 13 5.0 38
4-0-1 17 7 .0 45 5-5-0 240 100 940
4 - 1-0 17 7 .0 46 5-5- 1 300 100 1300
4- 1-1 21 9.0 55 5-5-2 500 200 2000
4-1 -2 26 12 63 5-5-3 900 300 2900
4-2-0 22 9 56 5-5 -4 1600 600 5300
4-2- 1 26 12 65 5-5 -5 > 1600 - -
4-3-0 27 12 67 Cont ...
232 Handbook of Methods in Environmental Studies

Example - 2
The following combinations of positive and negative tubes were found for treated
sewage. Calculate tilt: MPN index.
Size of sample portion No of positive tubes No of negative tubes
10 4 1
1 4 1
0.1 2 3
0.01 5

Estimate the MPN index for the following six samples.

Sample No. of positive tubes/5 tubes, mL used Combination MPN indexl

1 0.1 0 .01 0.001 positive tubes 100 mL.
A. raw 5/5 5/5 3/5 1/5 5-3-1 11,000
B x 10-3 5/5 5/5 3/5 1/5 5-3-1 11,000,000
C 5/5 3/5 2/5 0/5 5-3-2 1,400
0 5/5 3/5 1/5 1/5 5-3-2 1,400
E 4/5 3/5 1/5 0/5 4-3-1 330
F 0/5 1/5 0/5 0/5 0-1-0 20
Microbiological Analysis of Water 233

•.;~Confi ,. '
~/;For S~~J,~ 13: SalTle as sample, - Mt1owe~~r, it is >poJlJ't-ed water ~nd {0:3
5.~ilutioni~ used ••;rhus: ' ),)~<$Y\.: . ,
tMP'N. ~,*,JJi£~;;;J 0'3= 11,000,000 (Ans).
?(3.:~I\S~.' ' ofnbinationof 5-3-2froni Table 6:12, MPN 140..
': Sa~ple x T;iio'" '; = 1400 (Ans).
" 4.,:For Samp ; $,ihce' Of')~<is positive at' O.OOl mL, add 1 positive ioto 0.01 mL
- dilution"there ore, the combination of positive is 5-3-2 and MPN is 1400 (Ans).
,. (Note: If a positive occurs in a dilution higher than the three, choose according to
the rule, i~6rporate it In the' results for the highest chosen dilution (APHA, 1998).
. --d;..

5. For Sample' E: FOr,' ccimbination of 4-3-1 from Table 6.12"MPN

.1: .~ = 33 .

, sa~f)I~'Mf \33;;~ il~ , = 330 (Ans) •

. ::>~~0-':"9"'(f') -(~ _ . ,h, _.-:V
·\6 .. ~r s~'rpleJ~<F9.rcombl'1allo". 6!,' 0-1-0.tr0mTabli'l6;12, MPN 2.
, , .'~~~,.•'.. .. JO
Sample MPNk =Y2 xf ' 20(Ans).

6.9 Membrane Filter (MF) Technique


The membrane filter (MF) technique can be used to test relatively large number of
samples and it yields more precise numerical results and more rapid than the
multiple fermentation tube technique. The MF technique is extremely useful in
monitoring drinking water and variety of natural waters.

The MF method gives a direct count of total coliform and fecal coliform present in
the given sample. A measured volume of water sample is filtered though a mem-
brane filters with a pore size of about 0.4,5 l1m, which traps the bacteria on its
surface. The membrane filter is then placed on a suitable medium in a sterile con-
tainer and incubated at an appropriate temperature. Ifcoliforms and/or fecal coliforms
are present in the water sample, characteristic colonies are formed and that can be
counted directly. The standard volume to be filtered for drinking water samples is
The success of this method depends on using effective differential or selective
media that will enable easy identification of colonies. The method has advantage
over the traditional MPN water analysis because it is more direct and quicker (given
results in 18-24 h) and can easily test large volumes of water (hence yielding more
accurate results).
Limitations: Unsuitable for water containing very high levels of suspended
materials, sludge and sediments, all of which could block the filter.
234 Handbook of Methods in Environmental Studies
When small quantities of sample (for example, of sewage effluent or of grossly
polluted surface water) are to be tested, it is necessary to dilute a portion of sample
in sterile diluent to ensure that there is sufficient volume to filter across the entire
surface of the membrane (Table 6.13).
If the quality of water is totally unknown, or there is doubt concerning the
probable bacterial density, it is advisable to test two or more volumes in order to
ensure that the number of colonies on the membrane will be in the optimum range
for counting (i.e. 20-80 colonies per membrane filter).
MF and plate count techniques assume that each bacterium, clump of bacteria,
or particle with bacteria attached, will give rise to a visible colony. The result is
expressed as colony forming units (CFU) per unit volume.

Table 6.13:
Typical sample volume for membrane filtration analysis.

Sample type Sample volume, mL

100 10 l' 0.1'·2 0 .01'·2 0.001' ·2

Treated drinking water x - - - - -

Partially treated drinking water x x - - - -
Recreational water - - x x - -
Protected water source x x - - -
Surface water - - x x - -
Wastewater - - x x - -
Discharge from sewage - - x x x -
treatment plant
Pond, rivers, storm water runoff - - - x x x
Raw sewage - - - x x x

'Small volumes should be added to the filtration apparatus together with a mini-
mum of 9 mL of sterile diluent to ensure adequate dispersal across the surface of
the filter membrane.
21 .0, 0.1, 0.01, 0.001-mL volumes are filtered after first preparing serial dilu-
tions of the sample. To filter:
1.0 mL of sample, use 10 mL of 1/10 dilution.
0 . 1mL of sample, use 10mL of 1/100 dilution.
0.01mL of sample, use 10 mL of 1/1,000 dilution.
0.001 mL sample, use 10mL of 1/10,000 dilution.


1. Autoclave for sterilising prepared culture media.
2 Incubators with thermometer for checking calibration of incubator
3. Membrane Filtration unit: Complete with vacuum source and suction flask.
4. Membrane filter: 0.45 Jllll size and of diameter appropriate for the filtration
apparatus being used and complete with absorbent pad (if pads are used in-
stead of agar-based media). IfMF are to be sterilised in laboratory, autoclave for
1Omin at 121 °C. At the end of sterilisation period, let the steam escape rapidly to
minimize accumulation of water of condensation of filters.
Microbiological Analysis of Water 235
5. Forceps, blunt-edged: sterilised before use by dipping 95% ethyl alcohol and
6. Ethyl alcohol for flame sterilisation
7. Petri dishes and wax pencil for labeling them.
8. Pipette and pipette cans for sterilising
9. Measuring cylinders, capacity 100 mL and 250 mL.
10. Flask for containing culture media.
11. Digital colony cOWlter or magnifying lens.

For total coliform estimation: Use M-Endo mediwn (Table 6.14) and incubate the
petri plates at 35°C for 24h ± 2.
For Fecal coliform estimation: Use M-FC mediwn (Table 6.14) and incubate the
pertiplates at 44SC for 24h ± 2.

Table 6.14:
Composition of M-Endo and M-FC media .

M-Endo media composition * M-FC media composition *

1. Tryptose or polypeptone 10.0 9 1. Typtose or biosate 10.0 9

2. Thiopeptone or thiotone 5.0 9 2. Polypeptone 5.0 9
3. Casitone or trypticase 5 .0 9 3. Yeast extract 5 .0 9
4. Yeast extract 1 .5 9 4. Sodium chloride 5 .0 9
5. lactose 12.5 9 5. lactose 12.5 9
6 . Sodium chloride,NaCI 5.0 9 6. Bile salts mixture 1.5 9
7 . K2HPO. 4.375 9 7. Aniline blue 0.1 9
8 . KH 2PO. 1.375 9 8. Agar (optional) 15 .0 9
9. Sodium lauryl sulfate 0.05 9 9. Reagent grade water 1.0 L
10. Sodium desoxycholate 0.10 9
11 . Sodium sulfite, Na 2 S0 3 2.10 9
1 2 . Basic fuchsin 1 .05 9
13 . Agar (optional) 15 .0 9
1 4. Reagent grade water 1.0 L
*M -Endo Broth-MF and M-FC Broth-MF may be obtained from market .


Fecal coliform bacterial densities may be determined either by multiple-tube proce-
dure or by the MF techniques. The fecal coliform MF procedure uses an enriched
lactose mediwn and an incubation temperature of 44.5 ± 2°C for selectivity and
gives 93% accuracy in differentiating between coliforms fOWld in the feces of
worm-blooded animals and those from other environmental sources.


As applied to the MF technique, the coliform group may be defmed as comprising

236 Handbook of Methods in Environmental Studies

all aerobic and facultative anaerobic, gram-negative, non-spore fonning, rod-shaped

bacteria that develop a red colony with a metallic sheen within 24 h at 35°C on an
Endo-type medium containing lactose. Generally, all red, pink, blue, white or
colourless colonies lacking sheen are considered non-coliforms by this technique.
PROCEDURE (Same for both Total coliforms and Fecal coliforms)
l. Add absorbent pads to sterile petridi$hes for the number of sample to be pro-
cessed. Sterile pads may be placed in the petridishes with sterile forceps or with
an automatic dispenser (Fig 6.6a).
2 Soaking pad with nutrients: Pipette 2.5 mL of liquid nutrient media (Use mEndo
broth for total coliform and MFC broth for Fecal coliform) on to each pad and
replace the cover (Fig. 6.6b).
Note: Absorbent pads soaked in liquid medium may be replaced by medium
solidified by agar. In this cases, petridishes should be prepared in advance and
stored in a refrigerator.
3. Assemble the filter funnel on the flask.
4. Sterilise the tips of the blunt-ended forceps in a flame and allow them to cool
5. Carefully remove a sterile membrane filter(MF) from its package, holding it only
by its edge as shown in Fig. 6.6d.
6. Place the MF in the filter apparatus (Fig. 6.6e) and clamp it in place. If the
apparatus has been disinfected by boiling, ensure that it has cooled down
before inserting the MF.
7. Mix the sample by inverting its container several times. Pour or pipette the
desired volume ofsample into the filter funnel (Fig. 6.60. This volume should be
chosen in the light of previous experience, but suggested volumes are given in
Table 6.13. If the volume to be filtered less than 10 mL, it should be made up at
least 10 mL with sterile diluent so that sample will be distributed evenly across
the filter during filtration. Alternately, the sample may be diluted as suggested
in footnote of the Table 6.13.
8. Apply the vacuum to the suction flask gently and draw the sample though filter.
Just as the liquid level approaches the filter, rinse the sides with a small amount
of the sterile distilled water, and let the vacuum draw aU of the water though
filter (Fig. 6.6g).
9. Unclamp the funnel top with vacuum still applied. With a sterile forceps remove
the filter (Fig. 6.6h), taking care to touch only edge of the filter. Place the filter in
a previously prepared petridish, grid side uppermost (Fig. 6.6i).
lO. Replace the lid of the Petridish and mark it with the sample number and sample
volume. (i.e. 10 mL). Use wax pencil or waterproof pen for writing on Petridishes.
Pipette 1 mL, 10 mL and 100 mL sample respectively in filter as described in
3 replicates.
11. Incubate the M-Endo-MF plated at 35°C for 24 h.
12. Insert the M-FC plates inverted (bottom up) into a watertight, resalable plastic
bags and incubates at 44.5°C for 24 h immersed in a thermostatically controlled
water bath. If incubator is used, put a small container with a moist pad in the
Microbiological Analysis of Water 137
base, humid atmosphere will maintain inside the incubator. This ensures that
the pad does not dry out during the incubation period.
1. Incubated plates
2 Simple microscope (1 0-15x) or magnifying glass.
3. Examine the M-Endo broth-MF plates using a low power microscope. Coliforms
colonies are red or pink showing a bright golden-red metallic sheen. Colonies
without the golden sheen are non-coliform. Count the coliform colonies and
record the results.
4. Examine the M-FC plates in the same manner. Fecal coliform colonies are blue
regardless of shade. All others are not coliforms. Count and record the results.
Note: that coliform results are usually reported "per 100 mL" rather than ''per
milliliter" .
No. of colonies
No. ofcoloniesll 00 mL = x 100
Volume filtered, mL

6.10 Seven-Hour Fecal Coliform Test

This method is similar to the fecal coliform membrane filter procedure but uses a
different medium and incubation temperature to yield results in 7 h that generally
are comparable to those obtained by the standard fecal coliform method.
M-7 h Fe agar: This medium may not be available in dehydrated form and may
require preparation from the basic ingredients (Table 6.15).

Table 6.15:
Media composition of M-7h Fe agar.

Polypeptone 5.0 9 Yeast extract 3.0g

Lactose 10.0 9 d-Mannitol 5.0g
Sodium chloride 7.5 9 Sodium lauryl sulfate 0.2g
Sodium desoxycholate 0.1 9 Bromocresol purple 0 .35 9
Phenol red 0.3g Agar 15.0 9
Reagent grade water 1.0 L

1. Heat in boiling water bath. After ingredients are dissolved heat additional 5 min.
Cool to 55 to 60°C and adjust the pH to 7.3 with O.IN NaOH (0.35
mLlI.. usually required).
238 Handbook of Methods in Environmental Studies

An unknown water sample is analyzed for the estimation of coliform organisms.
Three sample portions, 1 mL, 10 mL and 100 mL are used for filtration. Each of
these portions is filtered through five filter membranes using the membrane filtra-
tion technique. After incubation, in each petridish, the .colonies are counted. The
counts are as follows:

SI. No. Sample portion Number of colonies

1 1-mL 5, 7, 5, 8, 6
2 10-mL 20, 22, 27, 31, 25
3 100-mL 350, 340, 360, 370, 340
What is the number of coliforms per 100 mL of sample?

Counts with acceptable limits: Assume that filtration of volumes 100, 50, 25,
10, 3 and 1 mL produced FC colony counts of 200, 110, 75, 35, 11, 6, and 3
respectively. What is the FC density for the sample?

Filtration volume 40, 15, 6, 2 mL produced FC colony counts of 1, 0, and .0
respectively. Estimate the FC density for the sample.
~~ @ So_ )ad
in nutriMt ® OisintKt tips CIt blunt-.nded
t.((~S and coot
@)Rl'mCIIIl' m~bran<!
_". pac.k~

® hlt.r w,th
(!) Apply va,u\lm tl) .u(t,on <D Add .amp'. to (!) Plac. m.mbran. t,It.,
~'.rll. lortpp,
flaSlc f,ltrat,on apparatus In f,ltratlOl"l apparatll!

..... CD
® U~avp to rpsu(ltatP
CD Count colon,.s "ft...
and then inc.ubate full in(.ubat'on

Fig. 6.6: Steps in membrane filtration techniques.

240 Handbook of Methods in Environmental Studies
2 Cool to about 45°C and dispense in 4- to 5-mL to perti dishes with tight-fitting
3. Store at 2 to 10°C. Discard after 30 days.
1. Filter an appropriate sample volume though a membrane filter, place filter on the
surface of a plate containing M-7 h FC agar medium.
2 Incubate at 41.5°C for 7 h. Fecal coliform colonies ere yellow (indicative of
lactose fermentation).

6.11 Check Your Confidence on Coliform Test

1. Why coliform organisms are used instead of directly testing pathogens for
microbiological testing of water?
2 Pathogens are not always bacteria. Name two pathogenic bacteria, two viruses
and one protozoan sometimes found in water supplies.
3. "Coliform bacteria are found in densities roughly proportional to degree offecal
pollution". TruelFalse.
4. What are the important characteristics of coliform bacteria?
5. Culture media is sterilized by autoclaving and glasswares are sterilised by Hot-
air oven. Why?
6. Presence of high number offecal streptococci indicate recent pollution. Why?
7. Why presumptive test is not sufficient for coliform test?
8. What is the purpose of adding Lauryl sulfate in presumptive test?
9. Name two important ingredients that inhibit the growth of non-coliform bacteria
(gram-positive bacteria) during confirmatory test.
10. What are thermotolerant coliform?
11. What is the difference between a coliform and fecal coliform?
12. "No coliform should be present in 100 mL of a drinking water". Which test is
preferred for compliance monitoring of this requirement.
13. How to differentiate between the human fecal contamination from fecal con-
tamination of warm-blooded animals?
14. During analysis in one sample the ratio of FCIFS was found to be 0.4 and in
another sample the ratio was found to be 4.4. What do the results indicates?
15. MPN index tables are based on the assumption of Poisson distribution (i.e.
random dispersion) Why ?
16. During a coliform test, the positive tubes combination were found to be 4-4-2,
which is not found in MPN table. How would you calculate the MPN index?
17. What are the limitations of the membrane filter method?
18. Do coliforms or even fecal coliforms, always mean fecal pollution?
19. What are the advantages of the MF method over the MPN method?
20. How test results are reported in MF techniques?
21. "In case of turbid water, MF technique can not be used". Why?
22. What are the advantages of using 7-h fecal coliform test?
23. A sample ofwastewater is analysed for coliform organisms by the mUltiple tube
fermentation method. The results of the confirmed test are as follows:
Microbiological Analysis of Water 241
Sample size, No. of positive tubes No. of negative tubes
mL out of 5 tubes out of 5 tubes
0.01 5 o
0.001 4 1
0.0001 2 3
0.00001 2 3
0.000001 o 1
Detennine the MPN and range of coliform organisms per 100 mL at the 95
percent confidence level.
Biological Monitoring of


Physical and chemical characteristics of water bodies affect the abundance, spe-
cies composition, stability, productivity, and physiological condition of aquatic
organisms. Biological methods used for assessing water quality include the collec-
tion, counting, and identification of aquatic organisms; biomass measurement;
measurements of metabolic activity rates; measurement of the toxicity,
bioconcentration, and bioaccumulation of pollutants; and processing and interpre-
tation of biological data.

1. To explain the cause of colour, turbidity, odour, taste, or visible particulates in

2 To aid the interpretation of chemical analysis.
3. To determine optimum times for treatment of surface water with algicides and to
monitor treatment effectiveness.
4. To identify the nature, extent, and biological effects of pollution.
5. To indicate the progress of self-purification in bodies of water.
6. To document short-term and long-term variability in water quality caused by
natural phenomena and/or human activities.
7. To provide data on the status of an aquatic system on a regular basis.
8. To con:elate the biological mass or components with water chemistry or condi-

Site Selection and Sample Collection

1. Selection of sampling site and sampling frequency depend on the objective of
the monitoring programme. Consideration should be given to sites where other
physical and chemical measurements are made.
2 Typically, ecological methods are used for long-term evaluation of biological
water quality by sampling once a year in the same place and at the same time of
3. In stream and river monitoring, sampling stations are located in upstream and
downstream from suspected pollution sources.
Biological Monitoring of Waters 243

4. It is usually essential to find additional sites which can act as control, i.e. the
water is uncontaminated and the organisms found there are not subjected to
the pollutional stress (such as upstream of discharges to a river).
5. Recovery sites (beyond the mixing zone of contamination) should also be se-
lected, as well as sites which cover the full range of the suspected pollution
gradient, e.g. intervals downstream of a discharge to a river, or in a grid radiat-
ing outwards from a point source in a lake.
6. Water samples for bioassays should also be taken, whenever possible, espe-
cially when the environmental dilution of contaminants is at its lowest, such as
during low flow periods in river.
7. In rivers there is a vertical and horizontal mixing, so collect the sample at mid-
stream 0.5 to 1m below the surface.
8. In lakes, reservoirs and estuaries where plankton population varies with depth,
collect samples from all major depth zones or water masses. In shallow area of2
to 3 m depth, sub-surface samples collected at 0.5 to 1m may be adequate.


Once sampling locations, depths and frequency have been determined, prepare for
field sampling. Prepare label indicating:
1. Date of sampling:
2 Sampling station name and no.:
3. Study area (river, lake, reservoir):
4. Type of sample:
5. Depth of sampling:
6. Meteorological conditions:
7. Turbidity, water temperature, salinity or any other significant parameters:
Collect coincidal samples for chemical analysis to help define environmental
variations having a potential effect on plankton.
If samples are to be preserved immediately after collection, add preservative to
the container before sampling.

7.1 Study of Benthic Macroinvertebrates

Introduction to Benthic Macroinvertebrates

Macroinvertebrates are animals inhabiting the bottom of lakes or streams or at-
tached to stones or other submerged objects. For biomonitoring purpose the benthic
macroinvertebrates were found to be best among all other living systems present in
aquatic ecosystem. Benthic macro invertebrates are animal inhibiting the sediment
or in other available bottom surface of freshwater, estuaries and marine ecosystem.
As per definition, they should be visible with naked eye and retain in No. 30 sieves
244 Handbook of Methods in Environmental Studies

(0.595-mm opening or 0.60-mm opening). The clean water supports diverse

macroinvertebrate communities with reasonably balance distribution of species.
Four types of changes in the water environment cause change in macroinvertebrates
community structure; like increased inorganic micronutrients; increased organic
loading; substrate alteration; and increase in toxic chemical pollution. The
macroinvertebrate communities responses differently for these changes as shown

Quality of Taxonomic group Tolerance

water rating

Clean water Stonefly nymph (Plecoptera): Baetis, Brachytera. 2

(Class-I) Mayfly nymph (Ephemeroptera)
Caddisfly larvae (Trichoptera): Caddis hydropsyche,
C. ~alimnophilus.

Mild Dragon fly (Odonata) 4


Moderately Prawn (Crustacea) 6

polluted Beetles (Coleoptera) Riffle beetle (Stene/mis, Elmidae);
(Class-III) Dineutus (Gyrinidae); Hydrophilus (Hydrophilidae);
Dytiscus (Dytiscidae).
Bugs (Hemiptera)-Lethocerus (Belostomidae);
Notonecta (Notonectidae); Sigera (Corixidae);
Hydrometra (Hydrometridae); Gerris (Gerrldae).

Highly Chironomus larvae (Chironomidae-Diptera) 8

polluted Mollusca

Severely Chironomus 10
polluted water Tubificidae (Tubifex sp.-sludge worm); Tubifera
(Class-V) (Rat-tailed maggot).

7.1.1 Counting of Benthic Macroinvertebrates

1. Ascertain the physico-chemical characteristics of sampling sites.
2 Selection of area (stretches) to be sampled: Select at least following sampling
Biological Monitoring of Waters 245

a. One reference station (remote station in the extreme upstream (u/s) side).
b. Locate at least one station immediately downstream (dis) of discharge point or
affected zone; The station should be ecologically similar i.e. all the stations
should have similar bottom substrate, depth, stream width, flow velocity etc.

Collection of benthic macroinvertebrates in shallow flowing waters is done by

means of a standard hand net as shown in Fig 7.1 (a).

~ Net



Fig. 7.1: Various methods of sampling invertebrates:
a. Hand net, b. Surber sampler, c. grab (Ekman grab).
246 Handbook of Methods in Environmental Studies

Surber sampler (Fig. 7.1 b): Quantitative samples may be obtained in water with a
flow rate greater than 10 cm/sec and water depth up to 60 cm (2 feet). The sampler is
made up of a strong, closely woven fabric (0.595 mm opening), approximately 69 cm
long. Smaller mesh sizes cause backwash due to clogging. This net is held open by
a square-foot metal frame (30.5 cm x 30.5 cm). Place the net opening facing up-
stream, using the current hold the net open. Push the horizontal frame into the
stream bottom material.
Ekman grab sampler (Fig. 7.1 c): A grab has jaws which close beneath a known
area of sediment when triggered by a messenger released down through the sus-
pension wire from the boat. The entire content of the grab represents a quantitative
sample from a known area. Do not use grab sampler on rocky or sandy bottoms,
because small pebbles or grit may prevent closing of the jaws. The grab is made of
12 to 20 gauge brass or stainless steel and weighs approximately 3.2 kg. The grab is
made in three sizes: 15 xI::; em, 23 x 23 cm and 30 x 30 cm.
Artificial substrate sampler: This is applied where the sample can not be collected
by using traditional methods, examples include deep, fast flowing rivers or where it
is difficult to find suitable sites with similar physical characteristics. Two types of
artificial substrate sampler are shown in Fig. 7 .2a and 7.2b.

Hook for
Stones __itt! rtl":~

Wlremesh basket
Woodel"l plates

Fig. 7.2: Two types of artificial substrates samplers for

sampling aquatic organisms.


Collect bottom grab sample is transfer to a container.
Then the slurry is poured into the sieves: Top sieve is may be of l-cm to 0.5cm just
to holdback the larger materials like leaves, shell, gravels, and permitting organisms
to the bottom sieves. Carefully remove the gravels etc., because macro invertebrates
Biological Monitoring of Waters '2A7

may stick on the surface. In such case, soft-bristled toot brass may be used to
remove attached macro invertebrates.
Next a series of sieve are placed in decreasing order starting from 0.5 to 0.6 mm
and finer one of 0.2 mm. Sieving yields residue-a mixture of animals and sediments.
The organisms are picked by using forceps and pipettes. Macroinvertebrates can
be preserved in 10% formalin solution. Use 70% ethyl alcohol solution, if organisms
are having calcareous shells or exoskeleton..
For qualitative analysis, rocks, leaves, or other objects are put in a white pan
partially filled with water. Organisms may be float free from these objects or use
forceps to remove the organisms and identify them under stereo zoom microscoI'e
or compound microscope.


First step is to make a qualitative inventory of the benthic flora; like before and after
the suspected or known area of pollution by denoting the presence ''positive (+)"
and absence ''negative (-)". Count the organisms present in the slurry or fixed area.
Report the results as per m 2•
Next quantitative analysis of number of individuals, species and structure (abun-
dance and composition) of the aquatic community affected by pollution with re-
spect to reference station were done by using different types of indices (ex. MBI).

7.1.2 Macroinvertebrate Biotic Index (MBI)

The MBI was developed to provide a rapid stream-quality assessment. The MBI is
calculated at each stream section as a tool to assess the degree and extent of
wastewater discharge impacts. The MBI is an average of tolerance rating weighted
by macro invertebrates abundance, and is calculated from the formula:
MBI = L (niti)IN

ni = number of individuals in each taxon i;
ti = tolerance rating assigned to that taxon i;
N = total number of individuals in the sediment sample.

Example -1
The number of macroinvertebrates in a river sediment samples are 70, 30, 50,
150, and 30 organisms/m 2 for Elmidae, Gyrinidae, Hydrophilidae, Chironomidae
and Tubificidae respectively. The tolerance values for these organisms are 6,6,6,8
and 10 respectively . Compute MBI for this sample.
248 Handbook of Methods in Environmental Studies

7.2 Counting of Phytoplankton

(Microscopic Algae)

The phytoplankton occur as unicellular, colonial or filamentous fonn. Phytoplank-
ton have long been used as indicator of water quality. Some species flourishe in
highly eutrophic waters while others are very sensitive to organic and/or chemical

The algae divided into ::ight classes based on colour of pigments (chlorophyll).
The freshwater algae mainly belongs to Blue-green algae (Cyanophyta), Green
algae (Chlorophyta), the diatoms (Bacillariophyta), Yellow green algae (Xanthophyta)
and Flagillates (Euglenophyta).

1. Presence of algae in drinking water supply or sources causes the problems of
tastes and odours; imparts coloration of water and degrade the palatability of
2 Growth of algae in filter bed causes the hindrance of filter operation and
degrades the palatability of water.
3. Growth of algae in clariflocculator, reservoir wall etc. is totally unwanted, and
which is controlled by use of copper sulfate solution (CuSOJ
4. Algae are also used as indicator oflevel of water pollution.
5. Algae also used as source of oxygen supply for the bacterial oxidation of
sewage/wastewater in oxidation pond.

Environmental problems Type of algae

1. Taste and odour causing algae Pandorina, Volvox, Gomphosphaeria,

(habitat: Polluted water) Anabaena, Anacystis, Aphanizomenon,
Hydrodictyon, Species of Synedra,
2. Filter bed clogging algae Species of Chlorella, Synedra,
(habitat: Polluted water) Oscil/atoria, Spirogyra, Diatoms,
Palm ella, Anabaena,
Rivularia, Anacystis.
3. Growth of Reservoir wall. Cladophora, Tolypothrix, Chaetophora,
Oedogonium, Lyngbya, Stigeoc/onium,
Ulothrix, Batrachospermum, Chara,
Vaucheria, Draparnaldia, Rivularia.
Biological Monitoring of Waters 249

... Cont.
4. Indicator of water pollution Phormidium, Anabaena, Euglena,
Spirogyra, Chlorella, Gloeocapsa,
Stigeoc/onium, Chlamydomonas,
Lyngbya, Oscillatoria, Chlorococcum,
Spirulina etc. are the indicators of organic
5. Wastewater treatment pond Scenedesmus, Ankistrodesmus,
algae Spirulina, Closteridium, Chlamydomo-
nas, Schizothrix, Cloterium,
Chodatella, Closteriopsis.

The most suitable phytoplankton preservative is Lugol's solution. To preserve
samples with Lugol's solution add 0.3 mL Lugol's solution to 100 mL sample and
store in the dark. For long -term storage add 0.7 mL Lugol's solution per 100 mL
Preparation of Lugol 's solution: Dissolve 20g potassium iodide (KI) and 109 of
iodine crystal in 200mL distilled water containing 20mL glacial acetic acid.

Formalin: To preserve samples with formalin, add 40 mL buffered formalin (20g
sodium borate, N~BP7 + lL 37% formaldehyde) to lL of sample immediately after
Other commonly used preservative includes 95% alcohol, and 6-3-1 preserva-
tion (6 parts water, 3 parts 95% alcohol and 1 part formalin). Use equal volume of
preservative and sample.
To retain colour in preserve phytoplankton, store samples in the dark or add
ImL saturated copper sulfate (CuS0 4 ) solutionIL of sample.


Temporary mounts: Transfer 0.1 mL concentrated! sample to glass slide, place a
cover slip over the sample, ring the cover slip with an adhesive such as clear nail
polish to prevent evaporation.
For semi-permanent mount: Add few drops of glycerin to the slide. As the
sample ages the water evaporates, leaving the organisms imbedded in the Glycerin.
If cover slip is ringed with adhesive, the slide can be retained for a few years in the

As algae are green in colour due to the chlorophyll, staining is not required. How-
250 Handbook of Methods in Environmental Studies

ever, to stain the algae, methyl blue, gentin violet or acid fuchsin (up to 1% solu-
tion) can be used.


Some phytoplankton are unicellular while others are multicellular (colonial) and
some are filamentous (which may be broken or complete). So the variety of configu-
ration poses problems in counting. For example; should 4 celled Scenedesmus be
counted as one colony or four individual cell. Use this guidelines for counting and
reporting of phytoplankton (APHA, 1998). In case of filamentous algae like
Oscillatoria , a filament length of 10m can be counted as one unit.

Enumeration method Counting unit Reporting unit

Total cell count One cell CellslmL

Natural unit count One organism (any unicellular UnitslmL
or natural colony)
Areal std. unit count* 400mm2 Units/mL.

(* Areal standard unit equals area of 4-small squares in Whipple grid at a magnifi-
cation of200x).

Collection and Concentration of Samples

The organisms contained in water samples, if present in low density must be con-
centrated in the laboratory before counting. The initial concentration is carried out
by filtering a large volume of water through a plankton net at the site of collection.
The use of plankton nets, however, do not permit the study of nannoplankton as
they often pass through it. To study the complete phytoplankton the concentra-
tion should be carried out by centrifuging a known volume of sample to a fixed
lower volume in laboratory to achieve a desired concentration. For example, centri-
fuging a 100 mL sample to 10 mL will give a concentration of 1O.

Counting Methods of Plankton

For counting use a counting cell or chamber with a fixed volume and area for ready
calculation of population densities. Some commonly used techniques are
described here.

7.2.1 Sedgwick-Rafter (S-R) Cell Method

1. The technique is suitable only for low magnification up to 20Ox. This has a
disadvantage in counting smaller phytoplankton as they are not properly iden-
tifiable at lower resolutions.
1. The S-R cell is a slide with a rectangular cavity of 1-mL. The cavity dimension is
50 x 20mm with 1mm depth (Fig. 7.3).
Biological Monitoring of Waters 251

2 Filling of cell: Shake the concentrated plankton sample and quickly transfer
ImL of sample in the cavity of S-R cell with the help of a dropper. Cover the
S-R cell with a cover slip taking care to avoid trapping of air bubbles inside. It
would be better to keep the cover slip obliquely placed on the cell as shown in
Fig. 7.3 and then pour the sample through the opening in the side comer. Do not
overfill the cavity because this would yield a depth greater than 1 mm and
produce a invalid count.
3. Wait for at least 15 minutes and allow the plankton to settle.
4. Wait at least 15 min. and allow the plankton to settle.
5. Count the plankton. For counting the microscope is first calibrated with the
help of a Whipple grid (ocular micrometer) and a stage micrometer. Calibration
has to be done separately for each different microscope. The Whipple grid is
placed in the eyepiece of the microscope and count is made in a suitable strip
across the length of the cell, i.e. 50 mm. Alternatively, at low resolution, count
can also be made in the area of the Whipple grid. For lOx objective and lOx
eyepiece, the area of Whipple grid is usually 1mm2, but it has to be calibrated
exactly with the help of stage micrometer for the particular microscope. Study a
good number of replicates and calculate the average count.

When counting is made in a strip
Phytoplankton (unitslmL) = - - - - - - -

N = Number of organisms counted in 1 mL of concentrated sample
L = Length of strip of S-R Cell in which counting has been made, (5Omm)
D = Depth ofstrip in S-RCell,mm (Imm)
W = Width of strip counted by Whipple grid, mm
S = Number of strips counted
C = Total volume of concentrated sample (mL)
V = Total volume of sample concentrated (roL)
When counting is made in an area of Whipple grid

Nx CX 1000
Phytoplankton (units/mL) =
N = Number of organisms counted in 1 mL of concentrated sample
A = Area offield in Whipple grid in which counting has been made
D = Depth ofarea in S-RCell,mm (lmm)
F = Number of fields counted
C = Total volume of concentrated sample (mL)
V = Total volume of sample concentrated (mL)
252 Handbook of Methods in Environmental Studies

Fig. 7.3: Counting cell (Sedgwick Rafter) showing method of filling

7.2.2 Haemocytometer Method

It is generally used for counting of blood cells. This can also be useful for counting
of phytoplankton. Haemocytometer is a glass slide, which has a centrally located
"H-shaped" groove.

The central chamber ("+") is divided into 25 subchambers. Each subchambers is
further divided into 16 chambers. Thus total number of chambers is 400 (25 x 16).
Rest of the four subchambers are further divided into 16 each. The phytoplankton
are contained in the central cubical chamber having 400 sub-chambers.

Place a drop of well-agitated sample (natural or concentrated) onto the counting
chamber. Put the special type of cover slip provided with the Haemocytometer.
There should be any overflow, in case of overflow, fill the chamber again. Wait for
a few minutes just to allow the cells to settle. Use 40x objective or greater magnifi-
cation for phytoplankton count. Express the result as:

No. of organisms counted x 104

Volume of water sample x CF

Volume of concentrated water

WhereCF= - - - - - - - - - - - - - -
Volume of water used for concentration
Biological Monitoring of Waters 253

7.2.3 Lackey's Drop Method (Microtransect)

This is the one of the simplest method for COWlting phytoplankton when no other
measuring device is available. The cOWlting is fairly reliable when density ofphy-
toplankton is high.

1. Put exactly 0.1 mL volume of the sample by using a calibrated medical doser
onto a glass slide.
2 Place a coverslip of known area, avoiding any air bubbles inside of the
3. Put the slide Wlder microscope.
4. Measure the area of the microscopic field.
5. COWlt the no of species in each microscopic field and record as one
microtransect. COWlt the several field by moving the slide both vertical and
horizontal direction.
6. Note: COWlt must be quick to avoid drying of the sample (in case of water
mOWlt); therefore, to avoid the drying use semi-permanent mOWlting
(glycerin mOWlt).
7. Calculate the phytoplankton as follows:


C = Number of organisms cOWlted,
At = Area of coverslip, mm2
As = Area of one strip (microtransect), mm2
S = Number of fields cOWlted
V = Volume of water Wlder coverslip, mL.

7.3 Counting of Zooplankton by

Sedgwick-Rafter Cell Method
Zooplankton in the waterbodies belongs to four main taxonomic groups (Le. Proto-
zoa, Rotifers, Cladocerans and Copepods). They are abWldance in shallow areas of
reservoirs, but only few species are abWldant in the open water. They occupy an
intermediate position of food web in aquatic ecosystem, fed on algae and bacteria,
and in turn fed by numerous invertebrates and fish.
254 Handbook of Methods in Environmental Studies


Same as phytoplankton.

Several types of nets can be used for zooplankton collection. The size preferably is
8-mesh. In flowing or shallow water 20-30L of water is collected and filters through
the net. The volume of water filtered is noted.

Preserve zooplankton sample with 70% ethanol or 5% buffered formalin. Ethanol
preservative is preferred for materials to be stained in permanent mount or stored.
Formalin may be used for the 48h preservation with subsequent transferred to
70% ethanol. In turbid samples, differentiate animal and detrital material by adding
0.04% rose-bengal stain, which intensely stains the shell of zooplankton and is a
good general cytoplasmic stain.

For zooplankton analysis, withdraw 5-mL sub-sample form the concentrated and
dilute or concentrated further as necessary. Transfer sample to the counting cell or
chamber or ordinary glass slide for mounting.

Semi-permanent mount: Use Polyvinyllactyl phenol for preparing semi-permanent

mount. The mounts are good for about lyr. For long term storage, ring the coverslip
with clear lacquer (finger nail polish). For protozoan portion of the microplankton,
Protargal staining procedure is used, which not only provides a permanent mount
but also reveals the cytological details often necessary for identification.

Counting of Zooplankton
Zooplankton can be counted by using Sedgwick-Rafter cell just like phytoplankton
(see Section 7.2) and density is represented as organismslL or organismsll OOmL.

7.4 Diversity and Other Indices

Two organisms can not react identically to a pollutant (s) because of complex
relationship between genetic factor and environmental conditions. For example,
some macroinvertebrates like, stonetlies, mayflies and caddisflies are very sensitive
to organic pollution, while on the other hand pollution tolerant invertebrates like
Biological Monitoring of Waters 255

Chironomus larvae and Tubifex etc. are increased their number in organically enriched
conditions. As tolerant organisms may be found either in clean or polluted situations,
therefore their presence in not definitive. Thus, a population of tolerant organisms
combined with an absence ofintolerant ones is a good indication of the presence of
pollution (APHA, 1998).
Species diversity index is a ratio between the number of species and "impor-
tance value" (no. of biomass, productivity and so on) of individuals. Some of the
numerical indices are useful in characterising and describing the aquatic commu-
nity. These indices are based on structural and functional stability of the system.

7.4.1 Shannon Index (H)

This is also called Shannon-Weiner index or Shannon-Weaver index. It is most
widely used index for measuring biological diversity, which has been developed
from the information theory.

R =.L
P Inp

S = Total number of species.
PI = n/N = proportion of individuals of the total sample belonging to the ith
n , = Number of individuals (N) belonging to the ith species.
N = Total number of individuals of all the species.

This index can be used to calculate the diversity of phytoplankton, zooplank-

ton and benthic macro invertebrates. This formula assumes an infinite sample, but
as long as sample size is large, the bias is likely to be small if n/N is used to
approximate pi. The higher the value of R, the greater is the diversity. The maximum
value of R can be more than 1. The decline in the value of R is taken as an evidence
of pollution. According to Wilham and Dorris (1968), a value of this index above
3 will indicate clean water, whereas values fewer than this would indicate pollution.
However, the use of this index should be made with great care, as wrong interpreta-
tions can be frequently drawn in the conditions of mild pollution or when the
system is under transition from one season to another (Goel et al., 1989).

7.4.2 Evenness or Equitability Index

From the Shannon index, we obtain the Shannon equability (or evenness) index.

J ranges from 0 to 1.
256 Handbook of Methods in Environmental Studies

Evenness is to refer the absolute distribution of relative abundance of species

at a site, in which maximum "evenness" reefers to a uniform distribution of relative
abundance. The measurement of evenness alone requires a knowledge of the total
number of species in the community. Higher the pollution stresses lesser the even-
ness index.

7.4.3 Simpson's Index (D)

The Simpson's index measures the probability that two specimens picked at ran-
dom in a community belong to different species.
i=l '

= Simpson's index
S = Number of species
The Simpson's index ranges between a value of 0 (low diversity) and a maxi-
mum of 1 - 1/S.

7.4.4 Margalef Index of Species Richness

It is simple ratios between total species (S) and total numbers of individuals (N). It
can be used to compare one community with another. For making comparison one
must certain that the sample sizes are comparable.

D = Margalefindex
S = Number of species in sample
N = Total number of individuals in a sample

7.4.5 Mcintosh Index (MI)

The Mcintosh index is related to species richness. Higher the values of index (D)
indicate low level of pollution and vice-versa. The MI index is given by the follow-
ing formula:

D= r. (n)2
i=\ '

D = Mcintosh index
S = number of species
n, = number of individuals in the ith species
Biological Monitoring of Waters 257

7.4.6 Odum's Index

The Odum's index (Odum et al., 1960) is extremely useful for comparison of various
sites, and can be employed for both flowing and stagnant waters. The calculation
of the index is given below, and the values decrease with rise in the level of pollu-
Total no. of species in a sample
Odum's index = - - - - - - - - - - - - - - - x 100
Total no. of individuals of all the species

7.4.7 Goodnight and Whitley's Index

The Goodnight and Whitley's index (Goodnight & Whitley, 1960) is based on the
dominance of a group of benthic organisms, the Tubificidae, which is extremely
tolerant to organic pollution.

G & WIndex = _ _Number ofTubificidae

_ _ _ _ _ _ _ _ _ x 100
Total no. of benthic organisms

An index value of 80 above indicates high level of organic pollution; value

between 60-80, moderate pollution; and a value less than 60, no pollution.

7.4.8 Kothe's Species Deficit Index

The Kothe's species deficit index (Kothe, 1962) is suitable for flowing water and is
based on assumption that after the addition of pollutants, the species diversity
decreases in the downstream side. The index can be used to calculate the effect of
pollution on phytoplankton, zooplankton or macro invertebrates. An increase in the
level of pollution results in decrease in the value of index.

A I -A 2
Kothe's Species deficit Index = - - - x 100
A 1= Number of species in the upstream side before the discharge of pollutants
A2 = Number of species in the downstream side after the discharge of pollutants

7.4.9 Berger-Parkar Dominance Index or

Community Dominance Index (CDI)
The CD! is the percentage of abundance, which is contributed by the two most
abundant species within the community. Species abundance is determined as bio-
mass, productivity or density.
CD!= _1_2
258 Handbook of Methods in Environmental Studies

Y, = abundance of the most abundant species.
Y2 = abundance of the second-most abundant species.
Y = total abundance of all species.

7.4.10 Similarity Index

It is useful for a comparison of the species diversities between unpolluted and
polluted sites. If the level of pollution is low, similarity index tends to 1, and if
pollution level is high the index value tends toward O. The similarity index (S)
between two samples is given by the following:

A = Number of species in sample A
B = Number of species in sample B
C = Number of species common to both A and B

7.4.11 Autotrophic Index (AI)

The Autotrophic index (AI) is the ratio of total biomass to chlorophyll a and is a
water quality indicator. The ratio increases when the water is enriched with organic
matter, a situation that leads to an increase in the number of heterotrophic microor-
ganisms (e.g., bacteria, fungi, protozoa).

Total biomass (ash-free weight of organic matter) (mglm 3)

AI= -------------------------------------
Chlorophyll a (mglm3)

Some AI values are reported below. The normal AI values ranges from 50 to 200.

Sample AI

Algal culture 40-96

Marine phytoplankton 76-200
Pond water 44-221
Marine seston 40-146
Lake seston 457

Source: Weber, C.1. (1973) Recent developments in the measurement of the

response of plankton and periphyton to change their environment, 119-138.
In: Bioassay Techniques and Environmental Chemistry (G. Glass Ed.), Ann Arbor
Science, Ann Arbor, MI.
Biological Monitoring of Waters 259

7.5 Determination of Phytoplankton Biomass

The cell size and colonies of phytoplankton differ greatly and mere counting of
them is not always enough. Therefore, quantitative estimation of phytoplankton
biomass is considered to be a useful parameter for precise quantification.
The most accurate methods for estimation of biomass are dry weight, ash-free
dry weight, and volume of living organisms.

7.5.1 Chlorophyll a Extraction Method

Chlorophyll a is used as an algal biomass indicator. Assuming that Chlorophyll a
constitutes on an average, 1.5% of the dry weight of organic matter (ash-free weight)
of algae, estimate the algal biomass by mUltiplying the Chlorophyll a content by a

Biomass (mglm3) = Chlorophyll a (mglm 3) x 67

7.5.2 Gravimetric Method

1. The biomass ofthe phytoplankton community can be estimated from gravimet-
ric determination, although silt and organic detritus interfere.
2. Determine dry weight by placing 100 mg wet concentrated sample in a clean,
ignited, and preweighed tared porcelain crucible and dry at 105°C for 24 h. Take
final weight after cooling in a desiccator.
3. Alternatively, filter a known volume of sample through 0.45 !lm preweighed and
dried membrane filter or glass-fibre filter. Dry and cool the sample in a desicca-
tor and weigh.
4. The difference in the initiai and final weights of crucible or filter paper yields the
biomass of phytoplankton. Calculate on per mL basis after correcting for initial
concentration of the sample.

7.6 Phytoplankton Productivity

Light-Dark Bottle Method

Productivity is defined as the rate at which inorganic carbon is converted to an
organic form. Primary productivity can be determined by measuring the changes in
oxygen and CO 2 concentration. In this method, clear (light) and black (dark) bottles
are filled with water samples and suspended at regular depth interval for an incuba-
tion period of several hours. Alternatively, the samples may be incubated under
260 Handbook of Methods in Environmental Studies

controlled conditions in environmental growth chambers in the laboratory. The

dissolved oxygen (DO) concentration is determined at the beginning and the end of
the incubation period. Productivity is calculated on assuming that one atom of
carbon is assimilated for each molecule of oxygen released.

1. BOD bottles of 300 mL capacity (some clear colourless and some painted fully
2. Float, Rope, thermometer
3. Reagents for dissolved oxygen (DO) determination

1. Take the sample from the depth in question and fix the DO.
2. Fill the same sample in clear as well as dark bottles and suspend these paired
bottles at the depth in question for at least 2 h. DO will deplete in the dark bottle
and will increase in clear bottle.
3. On completion of incubation period, take out the bottles and measure DO in
both clear and dark bottles.

The increase in O 2 concentration in lighted bottle during incubation period is a
measure of net production, which is because of concurrence use of O 2 in respira-
tion. It is somewhat less than the total (or gross) production. The loss of0 2 in dark
bottle is used as an estimate of total plankton respiration.

Net photosynthesis = Light bottle DO - initial DO

Respiration = Initial DO - dark bottle DO
Gross photosynthesis = Light bottle DO - dark bottle DO

NPP = [(DO in light bottle - initial DO) x 0.375]IT
R = [(Initial 00- DO in dark bottle) x 0.375]/T
GPP = [(DO in light bottle - DO in dark bottle) x 0.3 75]1T

GPP = Gross Primary Productivity in gC/m) Ih or mgC/L1h
NPP = Net Primary Productivity in gC/mlfh or mgC/L1h
R = Respiration gC/mJIh or mgC/LIh
T = Time period ofincubation (h)

0.375 is a factor (i.e. 12/32 = 0.375) used to convert oxygen to carbon. Under ideal
conditions 1 mole of O 2 (32g) is released for each mole ofC (12g) fixed.
Biological Monitoring of Waters 261

1. As productivity is defined as the rate of production and generally reported in

grams of carbon fixed/m2/day, multiply the average per m3 value with the mean
depth (m) of the body of water.
2. Using the solar radiation profile and average photosynthetic rate during incu-
bation, the data can be adjusted to represent phytoplankton productivity for
the entire photoperiod. Because photosynthetic rates vary widely during the
die I cycle, do not attempt to convert data to other test circumstances.

7.7 Estimation of Chlorophyll a

Analysis of the photosynthetic chlorophyll pigment present in aquatic algae is an

important biological measurement, which is commonly used to assess the total
biomass of algae present in water samples.

Samples should be collected with an appropriate sampler, such as depth or grab
sampler. For nutrient-poor (high transparency) water up to 61itres will be required.
For eutrophic waters, 1-21itres are usually adequate.

1. Three types of chlorophylls (chlorophyll a, b, and c) are found in phytoplank-
ton and may be extracted with acetone. Each type has a characteristic light
absorption spectrum with a particular peak absorbance. The acetone extract is
analysed in a spectrophotometer at these peaks. The peak height indicates
chlorophyll concentration.
2. When samples are concentrated by filtration for the purpose of analysis, the
phytoplankton cells die. Consequently, the chlorophyll immediately starts to
degrade and its concentration is thus reduced. The degradation product of
chlorophyll a, phaeophytin, fluoresces in the spectral region, and this can lead
to errors in results. It is, therefore, essential to measure the concentration of
phaeophytin-a and to make appropriate correction to analytical results.

1. Spectrophotometer with a spectral width between 0.5 and 2 mm
2. Cuvettes, 1 cm or with longer path-length
3. Centrifuge, Centrifuge tubes, 15 mL, graduated, screw-tops
4. Tissue grinder or mortar and pestle
5. Filters, glass fibreGF/C, 4.7 cm diameter
6. Filtration cup and pump
Handbook of Methods in Environmental Studies

5 s


~o 350.0 550.0 750.8

weave length lmm) ....

Fig. 7.4: A typical absorbance spectrum of chlorophyll.

1. Magnesium carbonate suspension: Add Ig MgC0 3 in 100mL distilled
water. Shake before use.
2. Acetone solution, 90%: 90 mL acetone + 10 mL distilled water
3. Hydrochloric acid: 0.1 N

1. After recording the initial water volume, separate the cells from the water by
filtration. Filter continuously and do not allow the filter to dry during filtration
ofa single sample. As filtration ends, add 0.2 mL ofMgC0 3 suspension to the
fmal few millilitres of water in the filter cup. If extraction is delayed at this point.
filters should be placed in individual labelled bags or plastic petri dishes and
stored at - 20DC in darkness. Samples may be transported in this form.
2. Place the filter paper in the tissue-grinder, add 2-3 mL of 90% acetone, and grind
until the filter fibres are separated. Pour the acetone and put the ground filter
into a centrifuge tube; rinse out the grinding tube with another 2 mL of 90%
acetone and add this to the centrifuge tube. Make up the total volume in the
centrifuge tube to 10 mL with 90% acetone. Label, and store in darkness at 4 DC
for 10-! 2 h. Samples may also be transported in this form.
3. Centrifuge for 15 minutes at 3000 rpm to clarify the samples. Decant the clear
supernatant into a clean tube and record the volume.
4. Fill a cuvette with 900/0 acetone. Record absorbance on the spectrophotometer
at 750 nm and 663 nm. Zero on this blank ifpossible otherwise record the absor-
bance and subtract it from the sample reading. A typical absorbance graph of
chlorophyll is shown in Fig 7.4.
Biological Monitoring of Waters 263

5. Place sample in the cuvette and record absorbance at 750 run and 663 run (750a
and 663a)
6. Add two drops of 0.1 N HCI in sample in I-cm cuvette (increased acid in propoi'-
tion to volume for larger cuvettes). Agitate gently for Imin and record absor-
bance at 750 run and 665 nm (750b and 665b).
7. Repeat the procedure for all samples. Some preliminary samples may need to be
taken to assess the best sample volume.

1 Determination of Chlorophyll a in the presence of phaeophytin
i. Subtract absorbance: 663a - 750a = corrected 663a absorbance
665b - 750b = corrected 665b absorbance
ii. Use these corrected 663a and 665b absorbance to calculate the pigments:

26.73 (663a-665b) x Ve
Chlorophyll a, mg/m3
26.73 [l.7(665b)-663a] x Ve
Phaeophytin a, mg/m3 = - - - - - - - - - -
Vs x L
Ve = Volume ofacetone extract (L) or (mL)
Vs = Volume of water ~ample (m3) or (L)
L = Light path length of cuvette or width of cuvette (cm)
Note: rfVe in mL is taken then Vs must be in L.

Chlorophyll a concentration should be recorded. The ratio of chlorophyll a to

phaeophytin gives an indication of the effectiveness of sample preservation, as
well as of the condition of the algal population.

II. Determination of Chlorophylls a, b, and c by spectrometric method

The optical density of the extract is read at wavelengths of664, 647 and 630 nm for
determination of chlorophyll a, b and c respectively.

Chlorophyll a (Ca),mgIL= 11.85 (OD~-1.54 (OD647)-0.08 (OD63 J

Chlorophyll b(Cb),mgIL=21.03 (OD 647)-5.43 (OD~-2.66 (OD 63J
Chlorophyll c(Cc), mgIL= 24.52 (OD63 J-7.60 (OD647) - 1.67 (OD664)

The chlorophyll concentrations in a given water sample is given by the follow-

ing formula (chlorophyll a is given as an example). Use Cb for chlorophyll band Cc
for chlorophyll c.

Ca (mgIL) x extract volume (L)

Chlorophyll a (mg/m3) = - ---------
Volume of sample, m 3
264 Handbook of Methods in Environmental Studies

Note: Volumes of extract and sample can also used in (mL) and (L) respectively
in the above calculation.
Typical chlorophyll a concentrations in different waters are listed below:
Oligotrophic Mesotrophic Eutrophic

ChI a (mglml) l.7 (0.3-4.5) 4.7(3-11) 14.3(3-78)

ChI a peaks (mglml) 4.2 (1.3-10.6) 16.1(4.9-99.5) 42.6(9.5-275)
Source: Wetzel, R.G. (1983). Limnology, 2nd ed., Saunders College Pub.,

7.8 Biological Monitoring for Health of

Wastewater Treatment Plants

Usually single cell (unicellular), prokaryotic, motile, multiply by binary fission, con-
sume bacteria, polisher of effluents. They found in activated sludge, trickling filter
and oxidation pond treating wastewater as well in natural water. They act as scav-
enger i.e. tend to clean the excess bacteria from solution in wastewater treatment
plant. Generally in river water about 10,000 ImL of water. Sewage is particularly rich
in Protozoa and may have as many as 1 million! mL. Following protozoans are used
for monitoring the health of treatment plants.
Mastigophora: Flagellates protozoa (zoo-flagellates) are smallest type ranging from
10 to 50 mil. They moved by means offlagella and have definite cell wall. Trigonomous
is an indicator of anaerobic conditions resulting from decomposition of putrid
sludge or inadequate aeration.
Class-II: Sarcodina: Testate amoebae are more usually found in highly loaded
biological wastewater treatment plants. When shell are present, pseudopodia ex-
tended through distinct opening. For example, Difflugia.
Bare or naked amoeba: Occur in large number during start-up phase in heavy in
loaded plants. For example, Amoeba.
Class-III (Ciliatae): The ciliata are the most important protozoa for monitoring of
stream pollution and wastewater treatment system. Ciliates are typical coloniser of
ASP. Under optimal condition 20,000 to 1,00,000 individuals/mL in ASP are re-
corded. A sudden reduction in the number of individuals or occurrence of en-
cysted, inflated or dead ciliates is an indication of shock loading of toxic sub-
stances (industrial effluents) or case of overloading (02 deficiency). Major function
is not to stabilise the waste but to control the bacterial popUlation.
Biological Monitoring of Waters 265

Free Swimming Ciliates

Colpidium camphylum: Free swimming, 100 m~ size, egg shaped, with a tapering or
curved end, occurs in large number in overloaded plant with an indication of inad-
equate 02 supply.
Paramecium caudatum: Free swimming, important in wastewater treatment plants.
Size is about 200-300 m~, about 3 times as long as wide, has the shape ofa slipper
and swim freely around and among the activated sludge floes. It occurs in activated
sludge plants under normal loading conditions.
Aspidisca costata: Free swimming and about 40 m~ long. Moves principally with
aid of its clearly recognisable "feet" of cilia on the surface of the Activated sludge
floes. As it is sensitive to oxygen deficiency« 2mgO/L), its OCCUl'Fence is an
indication of good oxygen supply to the sludge biomass.
Euploes affinitis: Resembles Aspidisca in appearance but is 80-1 00 m~ in size and
more flattened in shape. It is too is an indicator of good oxygen supply to the
sludge biomass.
Attached ciliates: Vorticella is a stalked ciliata and is important in biological treat-
ment process, especially in the activated sludge process.

Rotifers are aerobic, heterotrophic and multicellular animals and metabolized solid
foods. Its name is derived from the fact that it has two sets of rotating cilia on its
head, which are used for mobility and capturing food. They are found in natural
waters, stabilisation ponds, and extended aeration basins under low organic load-
ing. Rotifers are very effective in consuming disperse~ and flocculated bacteria and
small particles of organic matter. Their presence in an effluent indicates a highly
efficient aerobic biological purification process. Two of the most common crusta-
ceans are of interest to the sanitary engineer. These are: Daphnia and Cyclops. The
crustaceans are strict aerobes, which feed on bacteria and algae. They are impor-
tant as a source of food for fish. Their presence indicates water has low organic
load content (low BOD) and high dissolved oxygen (DO).

7.9 Check Your Confidence on. Biological

1. Discuss the importance of conducting biological monitoring.
2. Discuss ihe site selection criteria for conducting biological monitoring.
3. How to collect samples from river and lakes for biomonitoring work?
4. What are benthic macro invertebrates? Why macro invertebrates are used as
indicators of aquatic pollution?
5. Presence of Stoneflies, Mayflies and Caddiesflies are indicator of.. ...............water;
and presence of Chironomus larvae and Tubifex are indicator of ............... water.
266 Handbook of Methods in Environmental Studies

6. How benthic macroinverbrate samples are collected from shallow water?

7. How macro invertebrates are isolated from the sediment sample? What is MBI?
8. What are the precautions to be taken for collection of sediment sample by using
Ekman grab sampler?
9. At what situations artificial substrate sampler is used for collecting sample from
river bottom sediments?
10. Discuss the importances of the study of phytoplankton with reference to
biomonitoring purpose.
II. How phytoplankton are preserved in the laboratory?
12. How phytoplankton are counted?
13. List the important groups of zooplankton used for biomonitoring purpose. How
zooplankton are preserve and counted in the laboratory?
J4. Tubifex worm can be used as an indicator of oxygen-poor condition? (Truel
15. How the phytoplankton biomass is determined in the laboratory?
16. How phytoplankton productivity is measured?
17. How chlorophyll pigment is extracted from algal biomass? What is the principle
of chlorophyll measurement? What is phaeophytin?
18. How the level of chlorophyll concentration is related to the tropic status of
19. Occurrence of protozoa Trigonomous is an indicator of development ofanaero-
bic conditions in aeration basin (T/F).
20. Difflugia (Protozoa: Sarcodina) are usually found in highly loaded wastewater
treatment plant (TIF).
21. Increase of ciliLited protozoa in aeration tank of ASP is an indication of oxygen
deficiency condition (TIF).
22. Match the following:
Occurrence of ciliated protozoa Condition of aeration basin
l.Colpidium camphylum a) Inadequate oxygen supply
2.Paramecium caudatum b) Indication of good oxygen supply
3.Aspidisca costata c) Running under normal loading
23. Presence of Daphnia and Cyclops indicates low BOD and high DO conditions

Answers:Q.19T; Q.20T;Q.2IF;Q.22 I (a),2(c),3(b);Q.23T

Toxicity Testing of Water
Pollutants and Effluents

8.1 Toxicity Test

Sgmdar~s for Toxicity

Effluent discharge
90% survival offish after 96 hours in 100% effluent (MOEF, Schedule VI, 1993)

8.1.1 Significance of Toxicity Tests

Toxicity text is useful for a variety of purposes like:
1. To assess the suitability of environmental conditions for aquatic ecosystems.
2. To establish acceptable receiving water concentration of conventional param-
eters, such as DO, pH, temperature, salinity or turbidity.
3. To assess the effects of various combinations of these environmental factors
on the toxicity of wastes.
4. To determine the relative toxicity of different waste to a selected species or a
number of species;
5. To determine the relative sensitivity of aquatic organisms to effluents.
6. To determine the amount of waste treatment needed to meet water pollution
permit requirement.
7. To evaluate the effectiveness of different wastewater treatment methods.
8. To formulate permissible discharge rate for effluents.
9. To determine water quality requirement for aquatic life.
10. To assess the ·compliance with water quality standards, effluent requirements
and discharge permits.

8.1.2 Classification of Toxicity Tests

Toxicity tests are classified according to:
1. Duration: Short term (Acute), intermediate and long term (Chronic).
2. Methods ofaddition ofsolution: Static, recirculation, renewal and flow-through.


a. Acute toxicity tests

Acute toxicity tests are short duration tests, typically representing a small fraction
268 Handbook of Methods in Environmental Studies

of the lifetime of the organisms. The concentration is higher and the impact on the
organisms is severe, usually death. The test is usually completed in less than
4 days (96 hours). It is expressed as Le lo (96h), i.e. that concentration of effluent,
which causes mortality to 50% of the test population after 96 hours of exposure.
Basically it is a short term, survival determination test. It involves exposure of a
selected test organism, such as fish, to a known dilution or concentration of sample
for a specific period, typically 48 hours or 96 hours, but occasionally as short as 24

b. Chronic tests
Test organisms are exposed to lower concentration for a long period of time, prefer-
ably for the entire reproductive life cycle of the organisms. Typically 7-day period
is assumed unless specified otherwise.


a. Static test
1. In this test, the test organisms remain in the same water for entire duration ofthe
2. The test water (effluent) and dilution water are mixed in a chamber to the desired
3. The test organisms are then placed in the chamber containing the diluted test
4. The static nature of the test may cause erroneous results under certain condi-
tions For example, a high BOD content may cause depletion of DO in the mix-
tures, which will result in the death of the organisms.
5. Also some toxic compounds may degrade or volatilize during the test, thus
distorting the toxicity effect on the test organisms.

b. Recirculation test
In this test, the mixture in which organisms are put is pumped through an apparatus,
such as filter, to maintain water quality but not to reduce the concentration of test
material. This test is not routinely used because it is expensive and results may be

c. Renewal test
The renewal test is similar to the static test except that the test solution and control
water are periodically renewed and the test organisms are transferred to the cham-
bers with freshly prepared test mixtures or by replacing the test mixtures in the
original chambers.

d. Flow through test

The test solution and control water flow into and out of the chambers in which test
organisms are maintained. The flow may be intermittent or continuous. Stock solu-
tion of the test material can be continuously mixed with the dilution water in
Toxicity Testing of Water Pollutants and Effluents 269

different proportions. Flow-through tests are desirable for high BOD samples and
for those containing volatile or unstable substances.

The prime considerations in the selection of the test organisms for toxicity tests are:

1. Their sensitivity to the material or environmental factors under consideration.

2. Their geographical distribution, abundance and availability throughout the year.
3. Their recreational, economic and ecological importance locally and nationally.
4. The availability of culture methods for their rearing in the laboratory and knowl-
edge of their environmental requirements.
5. Their general physical conditions and freedom from parasites and disease.
6. Their suitability for bioassay tests.
Other important considerations are:
a. Generally smaller organisms not over 5 to 8 cm long and heaving shorter life
cycle are desirable for toxicity tests.
b. The most sensitive locally available species and most sensitive life cycle stages.
c. Use test organisms that are nearly uniform in size.
d. Use organisms of the same age group or life stage.
e. Determine the past history of the test organisms including when and where
they were collected and method of collection, handling and transportation.
f Test organisms must not be collected from polluted areas where the organisms
are in poor condition or where they have usually high body burden of potential
g. Organisms are not to be collected from areas where disease and parasites are
prevalent or where deformed individuals are found.
h. Collect certain stage of life stages of selected organisms.
i Knowledge of environmental requirements and food habits is important in the
selection of test organisms.
The U .S.EPA (1991) recommends for a minimum of three species (for example,
a vertebrate like fish, an invertebrate and a plant) to be tested for evaluating toxicity,
but it should not include most sensitive species.

8.2 Measurement of Toxicity in Laboratory

Fish is commonly used as a test organism in toxicity tests. Common fishes
employed for toxicity testing in laboratory are common carps growing in freshwater,
rohu (Labeo rohita), catla (Catla catla) and lata fish (Channa punctatus).

1. Glass aqaria: Circular or rectangular glass aquaria of 10 to 25 L capacity

2 Acclimatization tank: Glass aqaria, 50-100 L capacity
3. Aerator
4. Fish nets for transferring fish in the laboratory
5. Fish food
270 Handbook of Methods in Environmental Studies



1. Collect fishes native to that area, preferably from the water bodies where the
eftluent is proposed to be discharged (i.e. select species representative to the
area impacted).
2. The fish species should have some economic importance. For pond and ditches,
lata fish is very common. For river, different species of carp could be selected.
The species must be sensitive to the eftluentltoxicant.
3. Chose fishes that are nearly uniform size, with largest individuals not more than
50% longer than the shortest.
Acclimatization in laboratory

1. The collected fish must be acclimatized to the laboratory conditions for at least
a week or even two weeks in a 100-200 L capacity glass aquaria.
2 Maintain the glass aquaria conditions similar to their original habitat. During
the acclimatization period remove those fishes showing any sign of diseases or
abnormal behavior. Feed them with any standard fish food.
3. After an acclimatization period of I to 2 weeks, expose the fish to the condition
to which they will be exposed during the toxicity test for one more week.
4. Stop feeding the fish before 24 h of the commencement of the test and do not
feed them during the test.
Different concentrations of eftluent are prepared for different objectives. There are
three types of acute toxicity tests that are readily applicable to wastewater
eftluents. They are:

a. Range finding toxicity test

b. Screening toxicity test
c. Defmitive acute toxicity test or short-term defmitive toxicity test

a. Range-finding test

A range fmding test may be used when the characteristics of an eftluent are essen-
tially unknown. This test is rarely used for regulatory purpose. A range finding test
is simple test, in which 5 or more organisms are exposed to 3 to 5 widely distributed
effluent concentration, such as 1%, 10%, 50%, 100% and a single control. (As per
standard methods, expose the test organisms to a wide range of concentration,
usually in a logarithmic ratio, such as 0.01, 0.1, 1, 10 and 100 of the sample). Test
duration is usually 8 to 24 hours.
b. Screening toxicity test
A screen test is usually conducted with a single 100% eftluent concentration.
Duration ofthe test is usually 24 hours. Time of death should be noted if acute toxic
is less than 24 hours. No control is required. If toxicity is observed, then a definitive
test may be required.
Toxicity Testing of Water Pollutants and Effluents 271

c. Definitive toxicity test

Acute definitive test is distinguished by its duration, usually 48 hours for inverte-
brates and 96 hours for fishes and use of multiple dilutions. At least S dilutions are
applied in a geometric series (x, 2x, 4x, 8x ,'16x ... ) for examples, 6.2S%, 12.5%, 2S%,
SO%, 100% effluent and appropriate controls.
1. Select a series of concentrations of effluent in a 10-L capacity aquaria.
2. It is advisable to keep replicate set of each concentration.
3. Measure the DO, pH, alkalinity and hardness of each dilution.
4. Put 10 fishes in each container. Fish of smaller size up to S cm (S-IS cm) are
suitable. All fish added in the container must roughly be of same size and
S. Monitor the DO level at frequent interval and correct it by immediate aeration.
6. Measure the temperature. Generally, variation of ± 1°C is allowable, but do not
exceed ± 2°C.
7. Counting: Count the number of dead or affected organisms in each container
daily throughout the test. It is advisable to count the number of dead organisms
at 1.S, 3, 6, 12,24 h on the first day and only once or twice a day afterwards to
get a well defmed shape of the toxicity curve. It may be possible that a proper
shaped curve may not come due to more death at prespecified times (i.e. 24, 48,
72 or96 h).
8. Also record changes in movement of fishes.
9. Light: Use cool white fluorescence tube as a light source. Measure light inten-
sity at the water surface.
10. Photoperiod: In short term test, a standard photoperiod of 16h light and 8 h
dark is suggested.


1. The endpoint for determining acute toxicity test to aquatic organisms is usually
death. The mean lethal concentration (LC), i.e. the concentration estimated to
killSO% of the test organisms, expressed as LC so ' The lower the LC so ' more the
toxic effects.
2. Death is plotted in a probit (probability scale) and concentration in a logarith-
mic scale. As probability scale never reaches 0 to 100%, the death can be
plotted in an arithmetic scale, but probability scale is preferred because it usu-
ally gives a straight line. A typical toxicity curve is shown in Fig. 6.1.
3. While reporting the bio-assay results, mention the species used and their length
and weight. Also mention the test conditions.
4. The concentration at which SO% test organisms are killed (LC so) can be ob-
tained directly from the toxicity curve.

8.3 Toxic unit

The toxic unit (TU) approach has become widely accepted for utilizing the toxicity
m Handbook of Methods in Environmental Studies

,00().()1 .1 S 10 :iO:t> 50 70 ~ !l) 9S 99 99.9 99.99
P.rt.ntAg. mortAlity

Fig. 6.1: A typical toxicity curve.

test results. In toxic unit approach, a TU concentration is established for the protec-
tion of aquatic life.
The Toxic unit acute (TUa) is the reciprocal of the effluent concentration in the
dilution water in % tenns that causes 50% of the organisms to die by the end of the
acute exposure period.

100 1
TUa=-- or, - - x 100
LC so LC so
For example, a waste with an acute toxicity of an LC so in 5% effluent is dis-
charged, then:

TUa = - - - - = 20
LC so (5%)
For comparative purpose, a waste discharge that contains 20 TUa is twice as
toxic as a discharge containing 10 TUa.

The Toxic unit chronic (TUc) is the. reciprocal of the effluent concentration
(in percent in dilution water) that causes no observable effects on the test organ-
isms by the end of chronic exposure period.

TUc= _ _- x 100
NOEC = Maximum no observable effect concentration
Toxicity Testing of Water Pollutants and Effluents 273

8.4 Check Your Confidence on Toxicity

I. What are the applications of toxicity test results?
2. What are acute and chronic toxicity tests?
3. "Static test is easy to carry out in laboratory". Why?
4. List the criteria to be followed for selection oftest organisms.
5. List some common fishes used for toxicity test in India.
6. How to acclimatize fish in laboratory for toxicity test?
7. To meet the MOEF (1993) standard, LC 50 96hr, which acute toxicity test wiII be
conducted? Range finding/screening/definitive.
8. Discuss the procedure of conducting toxicity test in laboratory.
9. How the toxicity test results are reported?
10. What is toxic unit?

9.1 Radioactivity
. StandardSohadidaclivity .. .... ;.

Drinking water Gross a-activity Gross b-activity

ICMR(1963) 3 pCi/L (max) 30 pCi/L (max).
MWH(1975) 3 pCi/L (max) 30 pCi/L (max).
IS-1O,500 (1991) 10-8 ~Ci!mL (max) 10-7 ~Ci!mL(max)
WHO (1984) 0.1 Bq/L IBq/L
WHO (1993) 0.1 BqIL 1 Bq/L
Remarks (WHO, 1993): If a screening value is exceeded, more detailed radionu-
clide analysis is necessary. Higher values do not necessarily imply that the
water is unsuitable for human consumption.
Effluent discharge
IS: 2490 (1981) 1O-7~Ci!mL(max) 1O.o~Ci!mL (max)
MOEF (1993) 10-7~Ci!mL (max) for discharg~ 1O.o~Ci!mL (max) for
into surface water, public discharge into surface water,
sewers and marine coastal public sewers and marine
areas; and 10-8~Ci!mL (max) coastal areas; and 10-7~Ci/
for irrigation. mL (max) for irrigation.

9.1.1 Introduction
The radioactivity in water and wastewater originates from natural sources and
human activities. The a- activity is mainly due to rocks and minerals and p-activity
is mainly due to potassium content in water.
Radiations have dangerous effects on living beings_ Low level exposure can
also cause somatic/genetic change. Somatic effects include risk of cancer, leukemia,
sterility, cataracts and a reduction in life span. Genetic damage is caused by increas-
ing the mutation rate in chromosomes and genes that will affect future generations.
The p-particles have more penetrating power than a-particles. The p-emitters
external to the body are more damaging than a-emitters, as p-radiation can pass
Radioactivity Measurement 275

through human skin. The a-radiation is the least penetrating type of radiations and
can be stopped by a thin layer of clothing or by a sheet of paper and barely
penetrates the human skin. The a -emitters are taken into human body by inhalation
or along with food or water and emit radiation inside the body.
Regular measurement of gross alpha and beta acti"ity in water is inexpensive,
can be completed quickly, and is useful to determine whether further analysis for
specific radionuclides is needed.

9.1.2 Units of Radioactivity

The unit of radioactivity is curie. One curie is equivalent to 3.7 x 10 10 atomic disin-
tegrations per second, which is considered as a standard curie (Ci). The curie
represents such a large number of disintegrations per second that the millicurie
(mCi), microcurie (/lCi), nanocurie (nCi), or picocurie (pC i), corresponding to 10.3 ,
10.6, 10.9 and 10. 12 curie respectively, are more commonly used.
The SI unit of radioactivity is becquerel (Bq), where lCi = 3.7 x 10 10 Bq.

1 Bq = 1 disintegration per second

Another SI unit, also sometimes used is Rutherford.

1 Rutherford unit = 106 Bq

Other units commonly used in radiation studies are rad and rem.

Rad (radiation absorbed dose): It corresponds to absorption of 100 ergs of

energy per gramme of any substance.
Rem (Roentgen equivalent man): A unit which takes into account the different
biological effects that the various forms of radiation may have on organisms. For
example, If a lO-rad dose of p-particles produces the same biological effects as
I-rad dose of a-particles, both doses have the same value when expressed in rems.

9.1.3 Sampling
Use plastic (polyethylene) or glass container for collection of sample. The sample
container may vary in size from 0.5 L to 18 L. Collect the sample and preserve in
radioactivity homogeneous state by addition of conc. HN0 3 to bring pH down

9.2 Measurement of p-Radioactivity in Water


It is applicable to p-emitters with maximum energies above 0.1 MeV and activity
levels above 0.5 pCi/mL of radioactivity homogenous water. It can be measured
276 Handbook of Methods in Environmental Studies

using a proportional or Geiger-Mueller counter. The test sample is reduced to mini-

mum weight of solid material having measurable B-activity by evaporation

Material interposed between the test sample and the instrument detector, as well as
increasing density in the sample containing beta emitters, produce significant losses
in sample counting rates. Liquid sample must be evaporated to dryness in dishes
that allow the sample to be seen directly by the detector. Most beta radiation
counters are sensitive to alpha, gamma and X-ray radiations.

1. Beta-particle counter: It consists of detector, detector shield and scalar (me-
chanical register, power supply and amplifier are contained in a single chassis).
2. Sample mounting dishes: (3.2 mm side wall, flat bottom, noncorrosive with
uniform surface density).
3. Alpha particle absorber: Aluminum or plastic, having a uniform density.
1. All reagents shall be of AR grade
2. Purity of water: Reagent water
3. Cesium -137 solution: Standard
4. Cone. HNOJ (sp. gr. 1.42)
For establishing counter control, manufacturer's instructions are followed and for
other aspects, control charts are used to assure uniform operation of the instru-
A known quantity of cesium-I 37 standard (approx. 5 x 10.3 Ilei) is placed into a
volume of water for counting. The procedure given below is followed for a length of
time to produce the desired statistical reliability. The efficiency factor is expressed
as a percentage of the disintegration rate of the reference standard.
1. Place an approximate volume of the test sample in a glass beaker, make 0.5 N
with HN0 3 and evaporate to 1-2 mL.
2. Transfer it to mounting dish and evaporate to dryness. (avoid spattering or
boiling) using a ring heater. Uniform spreading of residual salts is necessary for
reliable comparative data. Hygroscopic solids should be cooled in a dry atmo-
sphere and stored in a desiccator until the start of counting.
3. Place the sample in the counter and count beta-activity for a length of time
sufficient to obtain desired reliabilitY.
Radioactivity Measurement '2:77

4. Record the reading.

5. Precipitation methods may be used to quickly concentrated radioactive material
into small amounts of precipitate. The precipitate is separated and washed free
of precipitant by centrifugation/filtration.
Results may be expressed as counts per minute per mL (cpm) or in terms of equiva-
lent cesium-I 37 activity. Calculate the results as follows:
1. Beta activity, cpm = (Aft) - B
Beta activity, cpmlmL= (IN) [(Aft) - B]
A = Total counts accumulated
B = Background, cpm
t = Time of counting, min
V = Volume of test specimen, mL
2. Beta activity as equivalent to cesium-I 37, dpmlmL = ClEcs
dpm = Disintegration per minutes
C = Beta activity of test sample, cpmlmL
Ecs = efficiency of counter for cesium-I 37
3. Conversion ofdpm to microcurie
~CilmL=(dpmlmLY(2.22 x 1(6)

9.3 Measurement of a-Radioactivity of Water

The test as per standard methods (1995) covers the measurement of alpha particle
activity of water and is applicable to alpha emitters with energies >3.9 MeV and
activity levels of> 0.5 pCilmL. It is measured using a proportional and scintillation
counters. The test sample is reduced by evaporation to a minimum weight of mate-
rial having measurable alpha activity.
1. Solids content in sample containing thl! alpha emitters produces losses in count-
ing rates by 10-15% at 1 mglcm2. Liquid samples must be evaporated to dryness
onto dishes that allow the sample to be seen directly by the detector.
2 Most alpha counters are insensitive to beta, gamma, and X-rays.
278 Handbook of Methods in Environmental Studies

1. Alpha particle counter: Consisting of either a proportional detector or a scin-
tillation detector and a scalar.
2. Sample mounting dishes: Flat bottom having 3.2 rnrn high side wall, noncorro-
sive, with standard uniform surface density.
I. Same as described for beta-radioactivity in water. The radioactivity of the re-
agents may be considered as background and subtract from the test sample
counting rate.
2. Nitric acid (1 + 30): Mix 1 vol. of con. HN03 (sp. gr. 1.42) with 30 volumes of
For establishing counter control, follow manufacturer's instructions. Control charts
are used for assuring unifo~ operation of the instruments.
Place a known amount of alpha standard (approximately 5 x 10-3 IlCi) into a volume
of water sufficient to dissolve salts_ Throughout the experiment, the evaporation,
mounting, counting, and density of plate solids of this reference standard must be
identical with those of the test samples.
Counting is done for sufficient time to obtain reliable results. The efficiency
factor is expressed as % ofthe disintegration rate ofthe reference standard. Purified
natural uranium with specific activity of 1.50 disintegration per minute per micro-
gram (0.676 pCilllg) is considered satisfactory.
1. Place an appropriate volume of the test solution in a glass beaker, add 3 mL of
conc. HN03 (sp gr 1.42) for each 100 rnL of solution, and evaporate to 1 to 2 mL.
2. Quantitatively transfer to the mounting dish and evaporate to dryness (avoid
spattering/boiling) using a ring heater. After drying, heat the dish to dull
redness for a few seconds, using a burner.
3. Place the sample in the counter and count for a time interval sufficient till the
desired statistical reliability is obtained.
4. Record the reading.
5. Precipitation methods may be used expediently to concentrate the radioactive
material into sfall amounts of precipitate by centrifugation or filtration.
Results may be expressed as counts per minute per mL (cpm) or in terms of alpha
disintegration rate, using the efficiency determined by use of the calibration stan-
dard. Calculate the results as follows:
Radioactivity Measurement

1. Alpha activity, cpm = (Alt) - B

Alpha activity, cpmlmL = (IN) [(Alt) - B]
A = Total counts accumulated
B = Background, cpm
t = Time of counting, min
V = Volume of test specimen, mL
2. Alpha disintegration rate dpmlmL = CIE

dpm = Disintegration per minutes
C = Alpha activity of test sample, cpmlmL
E = Efficiency of counter (fraction)
3. Conversion of dpm to microcurie
J1CilmL = (dpmlmL)/(2.22 x 106)

9.4 Check Your Confidence on Radioactivity

1. What are the natural sources of radioactivity in water?
2 What is Curie? Name other two units of radioactivity.
3. What are rad and rem?
4. How to control ~-radiation in laboratory?
5. Name standard test material used for beta-radioactivity
6. What radioactive particles are most dangerous and why?
7. Discuss the procedure of alpha-radioactivity measurement?
8. Name the counters that are used in alpha and beta-radioactivity measurement.
9. What are the harmful effects of radiation?
References on VVater

1. APHA (1995) Standard methods for the examination o/water andwastewa-

ter. American Public Health Association, American Water Works Association
and Water Pollution Con~rol Federation, 19th edition.
2. APHA (1998) Standard methods/or the examination 0/water and wastewa-
ter. American Public Health Association, American Water Works Association
and Water Pollution Control Federation, 20th edition.
3. ASTM (American Society for Testing and Materials)( 1995): Water and Envi-
ronmental Technology, Vol. 11.02 Water (II). ASTM, 1916 Race Street, Philadel-
phia, PA, 19103-1187, USA.
4. ASTM (American Society for Testing and Materials)(1995) Water and Envi-
ronmental Technology, Vol. 11.01 Water (I). ASTM, 1916 Race Street, Philadel-
phia, PA, 19103-1187, USA.
5. Central Pollution Control Board (CPCB) Scheme/or zoning and classification
ofIndian rivers, estuaries and coastal areas, ADSORBS/3/78-79. CPCB, New
6. Choubisa, S.L. (1997) Fluoride distribution and fluorosis in some villages of
Banswara district of Rajasthan. IJEH, (39),4: 281-288
7. Handbook 0/ Environmental procedures and guidelines (1994) Ministry of
environment and forest (MoEF), GOI, New Delhi.
8. John De Zuane (1977) Handbook o/Drinking water quality (2nd Ed). P.E.Van
Nostrand Reinhold.
9. Indian Council of Medical Research (lCMR) (1963) Manual ofmethods for the
examination of water, sewage and industrial waste, Special report services,
No. 47,ICMR.
10. lSI Specification/or Drinking Water: 10,500 (1983). lSI, New Delhi (1983).
11. lSI Specification/or Drinking Water: 10,500 (1993). lSI, New Delhi (1993).
12. Khopkar, S.M (1994). Environmental pollution analysis. Wiley Eastern Lim-
ited, New Delhi.
13. Lal:S Commentaries on water and air pollution laws (1996). 3rd edition. Delhi
law house, Delhi, India.
14. Masters Gilbert, M. (1994). Introduction to Environmental Engineering and
Science. Prentice-Hall of India Private Limited, New Delhi.
15. Metcalf and Eddy Inc. (1991). (3rd Ed). Wastewater Engineering - treatment,
disposal and reuse. Tata McGraw-Hill Publishing Company Limited. New Delhi.
16. Peavy, Howard, S. Rowe, Donald R. and Tchobanoglous, George (1985). Envi-
References on Water Pollution 281

ronmental Engineering. McGraw-Hill Book Company, Singapore.

17. Pepper I.L, Gebra, C.P. and Brendecke (1995). Environmental Microbiology - A
~..1t;lboratory manual. Academic press, London.
18.~COPE (1975). Environmental pollutants -Selected analytical methods (SCOPE
6). Butterworths, London.
19. Sawyer, Clair N., McCarty Perry L and Parkin Gene F. (1994). Chemistry for
Environmental Engineering. 4th Edition, McGraw-Hill, Inc. New Delhi.
20. Sincero A.P. and Sincero, G.A (1999). Environmental Engineering - A design
approach. Prentice-Hall of India Private Limited, New Delhi.
21. Water quality monitoring. (Ed by). Jamie Bartram and Richard Ballance. UNEP/
WHO. E & FN SPON, U.K. an imprint ofChapman and Hall, 2-6 Boundary Row,
London SEI 8HN, UK. (1996).
22. Water Encyclopedia (2d Ed). (1990). Lewis Publishers, 121 South main Street,
Chelsea, Michigan, 48118.
23. WHO Guidelines for Drinking Water Quality, Vol. l Recommendations,
World Health Organization, Geneva. (1984).

List of the chemicals required for water analysis laboratory.

Name ofchemicals Availability Appox. Price


1. Acetic acid, conc. (glacial) SOOmL 103.4S

2 Acetone SOOmL 96.63
3. Aluminum hydroxide, AI(OH)3 2SOg 90.94
4. Aluminium potassium sulfate SOOg S1.16p
S. Alum, AlNH. (SO.)l.12~O SOOg 73.89
6. 4-Amino antipyrine 2Sg 454.74
7. Ammonium chloride ,NH.CI SOOg 140.00
8. Ammonium hydroxide, NH.OH SOOml 64.00
9. Ammonium molybdate (NH.)6.Mo7.4~O l00g 280.00
10. Ammonium fluoride, NH.F. SOOg 260.00
11. Ammonium acetate, C~COONH4 SOOg 15S.00
12. Ammonium perpurate (murexide) Sg 150.00
13. Boric acid, ~B03 SOOg 330.00
14. BuiTertablets, pH4.0, 7, 9.0 10 tablets 60.00
IS. Brucine sulfate, (C13 Hl6NP4)l .~SO•. 7~O 2Sg 25S.00
16. Barium chloride, BaCIl.2~O SOOg 14S.00
17. Calcium carbonate, Caco3 SOOg S6.00
18. Calcium oxide, Cao SOOg 7730
19. Cobaltous chloride,CoC~.6~O l00g 37S.00
20. Calcium chloride, CaC~.2~O SOOg 96.00
21. Chloroform, CHC~ SOOmL 170.00
22. Copper sulfate, CuSO4 SOOg 181.89
23. Diphenyl indicator, (C6H')lNH l00g 227.00
24. Deverda's alloy l00g 218.00
25. Ether SOOmL 170.52
26. Ethyl alcohol (MARK: Call0l076H) SOOmL S7S.00
27. EDTA- di sodium salt l00g 80.00
28. EDTA-Mg salt l00g 100.00
Appendices 283

29. Eriochrome black T 25g 90.00

30. Ferrous ammonium sulfate, 500g 160.00
31. Ferric chloride, FeC~. 6Hp 500g 72.00
32. Ferric nitrate, Fe(N03)3' 9Hp 500g 79.00
33. Ferric sulfate, Fe2(SOA x Hp 500g 185.00
34. Ferrous sulfate, FeS04. 7Hp 500g 205.00
35. Ferrion indicator (solution) lOOml 602.00
36. Formaldehyde (31-40%; w/v) 500mL 48.88
37. Glycerol 500mL 210.00
38. Hexane 500mL 85.00
39. Hexamethylene tetramine
40. Hydrochloric acid, HCl 500mL 89.00
2.5 L 300.00
41. Hydrogen peroxide, 30% (w/v) 500mL 118.00
42. Hydrazine sulfate l00g 175.00
43. Hydroxy amine hydrochloride
44. Iodine l00g 428.00
45. LAS
46. Macroreticular resign
47. Methyle orange indicator 125ml 40.00
48. Methyl red indicator 125ml 40.00
49. Methylene blue chloride 25g 125.00
50. Methyl alcohol 500mL 70.00
51. Magnesium sulfate, MgS04 500g 120.00
52. Magnesium chloride, MgCI 2.6Hp 500g 74.00
53. Magnesium carbonate, MgC03 250g 95.00
54. Mercuric oxide, HgO l00g 245.00
55. Mercuric iodide, HgI 2 l00g 312.00
56. Mercuric sulfate, HgS04 250g 420.00
57. Petroleium either 500mL 145.00
58. Phenolphthalein indicator- Powder l00g 154.00
Phenolphthalein indicator- Solution 125mL 50.00
57. Potassium bi-pthalate, KHC.H4 500g 278.00
58. Potassium chromate, ~Cr04 500g 278.00
59. Potassium permanganate, KMn04 500g 135.00
62. Potassium iodide, KI 250g 560.00
63. Potassium chloride, KCl 500g 120.00
64. Potassium dichromate, ~Crp1 500g 221.00
65. Potassium dihydrogen phosphate ~PO4 500g 233.00
66. Di-potassium hydrogen phosphate,~HP04 500g 433.00
67. Potassium sulfate, ~S04 500g 90.00
68. Potassium Sodium tartrate 500g 830.00
69. Potassium thiocyanide, KSCN l00g 250.00
70. Potassium nitrate, KN0 3 500g 130.00
71. Potassium hydroxide (pellets), KOH 500g 180.00
284 Handbook of Methods in Environmental Studies

12. Potassium chloroplatinate, K2PtCl6 Ig 1250.00

73. Potassium ferricyanide, K,Fe(CN)6 lOOg 140.00
74. Phenol 500g 170.00
75. Phosphoric acid, HlO4 500mL 185.00
76. Selenium powder 25g 273.00
n. SPANDS reagents Ig 227.36
5g 886.70
78. Silver nitrate 25g 454.12
79. Silver sulfate 500g 125.00
80. Sodium acetate, CHJCOONa.3Hp 500g 93.22
81. Sodium arsenite, NaAS0 2 lOOg 350.00
82. Sodium azide, NaN J lOOg 483.00
83. Sodium bi-carbonate. NaHCOJ 500g 130.00
84. Sodium carbonate, Na2CO] 500g 68.00
85. Sodium chloride, NaCI 500g 48.00
86. Sodium fluoride, NaF 500g 432.00
87. Sodium hydroxide, pellets, purified 500g 6821
88. Sodium oxalate 500g 170.52
89. Oi-sodium hydrogen phosphate, Na2HP04 500g 159.15
90. Oi-hydrogen sodium phosphate, NaH 2P04 500g 115.95
91. Sodium sulfate, Na 2S04 500g 125.00
92. Sodium sulfite, Na2 SOJ 500g 170.52
93. Sodium thiosulfate, Na 2SPJ.5HP, AR 500g 204.62
94. Sodium potassium terterate 500g 670.71
95. Sodium tetraborate decahydrate, (Borax) 500g 79.58
Na2Bp7· IOH p
96. Stach powder (soluble) 500g 426.00
97. Stannous chloride, SnCI 2 , AR lOOg 126.42
98. Sulphuric acid 500mL 98.67
2.5L 245.55
99. Sulfanilamide, C6HgNP2S lOOgl500g
100. Zinc sulfate, ZnS04 500g 165.00
JOI. Zironyl chloride octahydrate, ZrOCI 2.8Hp lOOg 455.00

List of important glasswires required for water analysis laboratory

(Price, 1997-1998; Borosil make)

Name Capacity Unit price (Rs)

I. Beakers, Griffin, Low Form, with 50mL 28.00
spout, Graduated lOOmL 31.50
(Borosil code-\ 000) 250mL 39.00
500mL 66.00
Appendices 285
, '0
l000mL 159.00
2. BOD bottles (Code -1250) 300mL 276.00
3. Bottles, aspirator for storing
of distilled water.
4. Reagents bottles l00rnr! 135.00
5. Burettes 25mL 80.00
50mL 120.00
6. Buchner funnel l00mL 250.00
7. Condensors (COD) medium 490.00
8. Conical flaks l00mL 41.00
250mL 64.00
500mL 89.00
lOOOmL 146.00
9. Crucible small 400.00
10. Desiccators 200mm 3390.00
11. Distilling apparatus 10mUmin 958.00
12. Durhams tubes 100nos. 30.00
13. Funnels 50mmdia 61.00
65mmdia 73.00
75mmdia 75.00
14. Measuring cylinder IOmL 210.00
50mL 290.00
lOOmL 328.00
250mL 519.00
500mL 710.00
l000mL 837.00
15. Nesslers tube 50mL 100.00
16. Pipettes ImL 44.00
2mL 48.00
5mL 55.00
lOmL 59.00
25mL 83.00
17. Petridishes 10crn 110.00
18. Test tube lOmm 350
19. Centrifuge tube 15mL
20. Separating funnel 125mL 436.00
21. Stands (burette, pipette unit price 300.00
and test tube)
22. Filter paper Whatman No.42 900.00
23. Glass slide and cover slip 100 nos. 300.00
24. Glass rods
25. Glass beads 500g 80.00
26. Rubber tubing (ordinary and pressure) -
27. Pestle and motor glass 800.00
28. Asbestos sheet
286 Handbook of Methods in Environmental Studies

Instruments for monitoring waterlwastewater.

Name Unit cost (Rs.) Remarks

I. Autoclave 25,000 Sterilization purpose
2. BOD incubator 30,000 BOD test
3. Bacteriological incubator 29,000 microbiological test
4. pH meter 8,000 pH measurements
5. Conductivity- meter 4,000 conductivity test
6. Centrifuge 35,000 miscellaneous purpose
7. Flame photometer 40,000 Na and K analysis
8. Heating mentle 6,000 COD test
9. Hot plate 2,000 miscellaneous purpose
10. Kjeldhal distillation 1,200 TKN and NH) estimation
II. Jar-test apparatus 15,000 Coagulation-flocculation test
12. Laminar flow chamber 60,000 Microbiology lab.
13. Muffle furnace 10,000 VSS estimation
14. Magnetic stirrer 3,000 miscellaneous purpose
15. Mechanical stirrer 2,500 miscellaneous purpose

16. Microscope (counting 12,000 - 2 lakhs miscellaneous purpose

and measuring device;
camera lucida)
17. Nephlometer 15,000 turbidity estimation
18. Precision analytical 40,000 miscellaneous purpose
balance (Metler)
19. Spectrophotometer 1,20,000 miscellaneous purpose
20. Secchi disk 500 per disk transparency measurements
21. Thermometer (0-1 OO'C) 500 miscellaneous purpose
22. Water distillation apparatus 5,000-15,000 production of distilled water
23. Water monitoring kit (field) 35,000 Field monitoring of water
quality parameters.
24. Water bath 5,000 miscellaneous purpose
25. Vacuum pump 8,000 - 20,000 miscellaneous purpose

List of sophisticated analytical Instruments.

Name Unit cost (Rs.) Remarks


I. UV -Visible spectrophotometer 4.5 lakhs miscellaneous purpose

Appendices 287

2. Atomic absorption spectrometer 20- 35lakhs metals analysis

3. Gas chromatograph (GC) 3.5 -5lakhs mIscellaneous purpose
4. Total organic carbon analyzer 61akhs organic carbon analysis
5. Mercury analyzer 55,000 mercury analysis
6. Ion selective electrode. 2.50 - 3 lakhs miscellaneous purpose

Characteristics of three most common acids.

Characteristics Hydroc/I/oric acid, Sulfuric acid, Nitric acid,

(HCI) (HtYO) (HNO)

1. Specific gravity 1.174-1.189 1.834-1.836 1.409-1.418

2. % of active ingredients 36-37 96-98 69-70
3. Normality 11-12 36 15-16
4. Molarity 11-12 18 15-16

Preparation of acid solution of 1000 mL.

Characteristics Hydrochloric acld,Sulfuric acid, Nitric acid,

(HCI) (HtYO) (HNO)

1.6N 500mlA.. 168mlA.. 380mlA..

2. IN 83mlA.. 28mlA.. 64mlA..
3. O.lN 8.3mUL 2.8mlA.. 6.4mlA..
Preparation 0.02N acid 20mLoflN 20mLoflN 20mLoflN
solution from IN stock solution in 1L solution in lL solution in lL
solution. distilled water distilled water distilled water

Preparation of uniform sodium hydroxide (NaOH) solutions.

Strength of Required weight of NaOH Required volume of 15N NaOH

NaOH solution for 1000 mL solution, (g) to prepare 1000 mL of solution,
15N or IS M 600
6Nor6M 240 400
INor 1M 40 07
O.lN orO.lM 4 6.7
288 Handbook of Methods in Environmental Studies

Stock sodium hydroxide solution: NaOH, 15N. (for preparing 6N, IN and O.IN
solution). Dissolve 600 g solid NaOH in 800 mL distilled water and dilute to 1000 mL.
Store NaOH in a polythene bottle with screw caps.
Ammonium hydroxide solution, NHpH: Prepare 5N, 3N, and 0.2N NHpH solution
by diluting 333 m, 200 mL, and 13 mL, respectively, of the concentrated reagent
(sp. gr. 0.90, 29%, 15N)to 1000 mL with distilled water.

Indicator solutions

a. Phenolphthalein indicator solution:

Use either the aqueous solution (1) or alcoholic solution (2).
1. Aqueous solution: Dissolve 5g phenolphthalein disodium salt in distilled water
and dilute to 100 mL.
2 Dissolve 5 g phenolphthalein in 500 mL 95% ethyl or isopropyl alcohol and add
500 mL distilled water.
If necessary, add 0.02N NaOH drop-wise until a faint pink colour appears in
solution 1) or 2).
h Methyl orange indicator solution:
Dissolve 500 mg methyl orange powders in distilled water and dilute to 1000 mL.

Quantities and units

7.1 Base units in the International system of units (SI).
Quantity Base SI unils Symbol
Length Meter m
Mass . kilogram kg
Time second s
Electric current ampere A
Temperature Kelvin K
Amount of current mole mol
Luminous intensity candela cd

7.2 Concentration
In environmental analysis whereever possible choose the units on the basis of:
1. Mass of analyte/ unit volume in case of water and air
2. Mass of analYlel unit mass in case of soils.
Typical units would be than:
Appendices 289

Water mgIL
Air mg'm3
Soils mg/kg
The alternative units sometimes found in environmental literature are based on:
• Parts per million (ppm)
• Parts per billion (ppb) and
• Parts per trillion (ppt).
For water and soil:
ppm = parts per million (mass/mass)
= mg/kg
- mg/L (assuming density of sample - IglmL)
ppb = ~gIL
ppt = ngiL
For gases:
ppm = parts per million (volume/volume) = ~LIL
ppb = nUL
ppt = pL/L
I million = 106
I billion = 109
I trillion = 1012

7.3 Conversion of ppm, ppb and ppt of chemicals to concentration ~

expressed in SI units.

Medium Conversion to Sf units

Water (4·C, 1 atm)
ppm I ppm I g'ml = I mg/L
ppb I ppb I mg'ml
ppt I ppt 1 ~g'ml
ppm I ppm I x Ml22.4 mg'ml
ppb I ppb I x Ml22.4 ~g'ml
ppt 1 ppt 1 x Ml22.4 ng'ml
ppm I ppm 1 mg/kgsoil
ppb I ppb 1 ~glkg soil
ppt 1 ppt 1 ng/kg soil
290 Handbook of Methods in Environmental Studies

7.4 Summary of alternative methods of expressing physical quantities.

1. Mass (SI unit: kg) Soils

g 10.1 kg.
mglkg 10-6g1g = ppm
mg 10.1 g = 10-6k~
mglkg 10.9 gig = ppb
mg 10-6 kg = 10.9 kg
nglkg 10.12 gig = ppt = cml/ml
ng 10.9 kg = 10.12 kg.
2. Length (SI unit: m)
mglml lO-6gIL
1O.2 m
mn = 10-1 m ppm(v/v) = 10-6 ml/ml
mn = lO-6 m 1O.9m3/m l = 10.1 cml/ml
ppb (v/v)
3. Volume (SI unit: ml)
S. Pressure
L = dml = 10.1 ml.
Pa = Nlm2
IlL = cml = 10-6 ml
atm = 101352N/m2
IlL = 10.1 cml =IO.9 ml.
bar = 10~/m2.
4. Concentration (SI unit: mol m-l) Torr = mmHg=133.322N/m2
M = mollL= mol/dml 6. Energy (SI unit: J = kg m1/s1)
cal = 4.184J
Water erg 100'J
mgIL 10.1 gldml = mglkg = ppm eV = 1.602 x 10.19 J
mgIL 10-6 gldml = mglkg = ppb
ngIL 10.9 gldml = nglkg = ppt

7.5 Derived SI units with special names

Quantity SIunit Name Units

Frequency Hz hertz lis
Force N newton kg.mls2
Pressure, stress pa pascal kglm.s2or N/m 2
Energy or work J Joule kg.m2/s2 or N.m
A quantity of heat J Joule kg. m2/s2 or N.m
Power, radiant tkux W watt kg. m2/ sl or J/s
Electric charge C Coulomb A.s
Electric potential V volt W/A
Potential difference V volt W/A
Electromotive force V volt W/A
Capacitance F farad CN
Electric resistance W ohm VIA
Conductance S siemens AN
... Cont.
Appendices 291

... Cont.
Magnatic flux Wb weber Y.s
Magnetic flux density T tesla Wb/m 2
Inductance H henry Wb/A
Luminous flux hl lumen
illuminance k lux or lmIm2
Activity (radionuclides) Bq becquerel lis
Adsorbed dose Gy gray m 2/s2 or J/kg

7.6 Useful conversion factors.

1. Length
1 gallon (U.S) = 0.134 ft3
1 inch = 2.540 cm
=0.003785 ft3
1 foot = 0.3048 m
1 yard=0.9144m
1 cubic meter = 8.11 x 10'" Ac-ft
1 mile = 1.6093 km
= 35,3147ft3
1 meter = 3.2808 ft = 39.37 in
= 264.172 gal (U.s)
1 kilometer = 0.6214 mile
2. Area = 106 cm3
1 square inch = 6.452 cm2 4. Pressure
= 0.0006452 m2
1 atmosphere = 76.0 cm ofHg
1 square foot = 0.0929 m2
= 14.696Ib/in2 (psia)
1 acre = 43,560 ft2
= 29.921 in ofHg (32 OF)
= 0.0015625 sq. mile
= 33.8995 ft of~O (32 OF)
= 4046.85m2
= 101.325 kPa
= 0.404685 ha
1 pound per square inch
1 square mile = 640 acre
= 2.604km2
= 2.307 ft of~O
= 2.036 in ofHg
= 0.06805 atm
1 square meter = 10.764ft2
= 1 N/m2•
1 hector =2.471 acre
1 Pascal (Pa) = 1.45 x 10'" psia
= 0.00386 sq. mile
=lO,OOOm2 1 inch of mercury (32°F) =3386.4 Pa
3. Volume
5. Energy
1 cubic foot = 0.03704 cubic yard
1 British thermal Unit (Btu)
= 7.4805 gal (U.S)
=0.02832 m3 =778ft-Ib
=28,32L =252 cal
1 acre foot = 43 ,560 ft3 = 1055J
= 1233.49m3
= 325,851 gal (U.s)
Handbook of Methods in Environmental Studies

... Cont. 9. Density

6. Linear Velocity I milligram! liter in water = 1.0 ppm
1 foot per second = 0.6818 mph (Specific gravity = 1.0)
= 0.3048m1s = 1000ppb
1 mile per hour = 1.467 ftls = 1.0 glml
= 0.4470mls = 8.34lb.lmillion gal
= 1.609 kmlhr I quadrillion Btu= 10 15 Btu
1 meter per second = 3.280 ftls = 1055 X lO l5 J
= 2.237 mph = 2.93 X 10" kWhr

7. Mass = 172 X 106 barrel (42-gal) of oil equv.

= 36 x 106 metric tons of coal equv.
1 pound = 0.453592 kg
= 0.93 x 10 12 cubic feet of natural gas
1kilogram = 2.205 lb. equivalent
= 35.273960z I joule = I N-m
1 ton (short) = 2000 lb. = 9.48 x 10-4 Btu
= 907.2kg
= 0.73756 ft-Ib
= 9072 ton (metric) I kilowatt-hour = 3600 kJ
1 ton (metric) = 1000 kg =3412Btu
= 2204.622 lb. = 860kcal
= 1.1023 ton (short). I kilocalorie = 4.185 kJ
8.Flowrate 10. Power
1 cubic foot/second = 0.028316 ml/s I kilowatt = 1000 J/s
=488.8 gal (U.S)/min (gpm)
= 3412 Btulhr
1 cubic foot/ minute = 4.72 x 104 ml/s
= 1.340hp
= 7.4805 gpm I horsepower =746W
1 gallon (U.S) Imin = 6.31 x 10-5 ml/s
= 550 ft-Ib/s
1 million gallon/day = 0.0438 ml/s I quadrillion Btu! year
1 million acre feet/year = 39.107 ml/s =0.471 million barrels
1 cubicmeterlsec=35.315 ftl/s(cfs) of oil per day
=2118.9 ftl/min (cfin)
= 22.83 x 106 gaVd
= 70.07 Ac-ftld

7.7 Values of useful constants

Acceleration due to gravity, g = 9.807 mls2
= 9807 crn/s2 (32.174 ftls 2)
Standard atmosphere = 101.325 kN/m 2 (14.696Ib£lin2)
= 101.325kPa(1.013 bar)
1 bar = 105 N/m 2 (14.504 Ibf/in 2)
Standard atmosphere = 10.333 m (33.899 ft) of water
1 meter head of water (20°C) = 9.790 Mlm 2 (1.420 Ib£lin2)
= 0.00979 N/mm2 (1.420 Ib£lin2)
= 9.790 kN/m2 (1.420 Ib£lin2)
Appendices 293

7.8 Atmospheric pressure (SI units)

Elevation above Atmospheric Atmospheric Specific weight
sea level, m pressure, kPa pressure expressed of air at 200 C,
as a column of: kN/IIf.
Water Mercury
(m) (mm)

o 101.3 10.33 760 O.oII8

500 95.6 9.47 717 0.0111
1000 90.1 9.19 676 0.0105
1500 84.8 8.64 636 0.0099
2000 79.8 8.13 598 0.0093
2500 73.3 7.47 550 0.0085
3000 70.3 7.17 527 0.0082
3500 66.1 6.74 496 0.0077

7.9 Units used in radiobiology.

Unit or quantity Symbol Application
Becquerel Bq SI quantity of radioactivity
Bq = 1 disintegrationls
Bq = 2.7 X 10.11 Ci
Curie Ci Quantity of radioactivity
1 Ci = 3.7 x 10 10 disintegrations/s
1 Ci=3.7 x IOIOBq
Electron volt eV Unit of energy
1 eV= 1.6 x 1O.12 erg
1 eV = 1.6 x 10.19 J
Linear energy transfer rEf Energy deposion per unit of path
length; usually in eVlnun
Quantity factor Q Biological effectiveness of radiations
Gray Gy SI unit of absorbed dose
1 Gy= 100rad= 1 J/kg
Rad rad Unit of absorbed dose
1 rad=O.OI Gy= 100ergig
Rem rem Unit of dose equivalent
rad x Q x other modifying factor
1 rem = 0.01 Sv
Sievert Sv SI unit of dose equivalent
rad x Q x other modifying factor
I Sv= 100 rem
294 Handbook of Methods in Environmental Studies

7.10 Light
a. Approximate wavelength ranges for the various region ofthe electromagnetic
Name of radiation Wavelength range

y-rays 0.003 -0.3 N

X-rays 0.3-100N
Far ultraviolet 100-20ooN
Ultraviolet 200400nm
Visible 400-800nm
Near infrared 0.8-2.5 11m
Infrared 2.5 - 15 !lID
Far infra red 15-200 11m
Microwave 0.2-7mm
radar 7-100mm
Very high frequency 1O-1000cm
Ultrahigh frequency 10-1oom
Radio waves 10-1O,OOOm

b. Conversions oflight
To convert Multiply by To obtain
einsteins 6.024 x 1023 quanta
footcandles 1.0 lumens/ft2
footcandles 10.764 lumens/m2
footcandles 10.764 lux
lumens 0.318 candellas
lumens/ft2 1.0 footcandelles
lux 0.0929 footcandles
quanta 1.66 x 10.24 einsteins
candella 3.l416 lumens

7.11 Physical Properties of water.

Properties Value
Density (25°C), kglm1 997.075
Density (20°C), kglm1 99820
Density (100°C), kglm1 958.40
Maximum density, kglm1 1000.000
Temperature ofmaximum density,oC 3.940
Viscosity (25°C), Pais 0.890 x 10.1
Kinematic viscosity (25°C), m2/s 0.89 X 10-6
Melting point (l01, 325 Pa),OC 0.0000
Boiling point(101, 325 pa),OC 100.00
Appendices 295

.. .Cont.
Latent heat ofice, kllmol 6.0104
Latent heat of evaporation, kJ/mol 40.66
Specific heat capacity (1 5°C), Jlkg."C 4186
Thermal conductivity (25°C)m, J/cm.s.oC 0.00569
Heat of vaporization, Jlkg 2.435 x 1()6
Surface tension (25°C), N/m 71.97 x 10']
Surface tension (20°C), N/m 72.75 X 1O.J
Surface tension (O°C), N/m 75.64 >~ 10']
Dielectric constant (25°C) 78.54
Vapour pressure (20°C), torr 17.535

7.12 Solubility of Oxygen in water (mgIL) at 1 atm pressure

Temperature (0C) Chloride concentration in water (mg/L)
0 5000 10000 15000
0 14.62 13.73 12.89 12.10
5 12.77 12.02 11.32 10.66
10 1129 10.66 10.06 9.49
15 10.08 9.54 9.03 8.54
20 9.09 8.62 8.17 7.75
21 8.99 8.57 8.14 7.71
22 8.83 8.42 7.99 7.57
23 8.68 827 7.85 7.43
24 8.53 8.12 7.71 730
25 838 7.96 7.56 7.15
26 822 7.81 7.42 7.02
Z7 8.07 7.67 728 6.88
28 7.CJ2 7.53 7.14 6.75
29 7.77 739 7.00 6.62
30 7.63 725 6.86 6.49
296 Handbook of Methods in Environmental Studies

Drinking water standards: IS: 10500 (1991).

S.No. Substances or Require- Undesirable effects Permissible Remarks

characteristics ment (desir- (outside the desira- limits in the
able limit) ble limit) absence 0/
1. Colour, Hazen 5 Above 5, 25 Extended up to 25
units, Max. consumer acceptance only if toxic
decreases substances are not
suspected, in absence
of alternate source
2. Odour Unobjec-
3. Taste Agree- Test to be conducted
able only after safety had
been established
4. Turbidity 5 Above 5, consumer 10
acceptance decreases
5. pH value 6.5 to 8.5 Beyond this range No relaxation -
the water will affect
the mucous membrane
and/or water supply
6. Total hardness 300 Encrustation in water 600
(as CaCO,), supply structure and
mg!L, max adverse effects on
domestic use
7. Iron (as Fe), 0.3 Beyond this limit 1.0
mglL, max taste/ appearance are
affected, has adverse
effect on domestic
uses and water supply
structures, and
promotes iron bacteria
8. Chloride (as CI) 250 Beyond this limit, 1000
mg!L, max taste, corrosion and
palatability are
9. Residual, free 0.2 To be applicable
chlorine, mglL, only when water is
max chlorinated. Tested
'at consumer end.
When protection
against viral infec-
tion is required, it
should be min. 0.5

10. Dissolved solids, 500 Beyond this palatab- 2000

mgIL, max ility decreases and
may cause gastroin-
testinal irritation
11. Calcium (as Cal, 75 Encrustation in water 200
mgIL, max supply structure and
adverse effects on
domestic use
12. Magnesium 30 Encrustation in water 100
(as Ca)* supply structure and
mglL, max adverse effects on
domestic use
13. Copper 0.05 Astringent taste, disc- 1. 5
(as Cu), oloration and corro-
mgIL, max sion of pipes, fitting
and utensils will be
caused beyond this. 1.5
14. Manganese 0.1 Beyond this limit 0.3
(as Mg), tastel appearance
mgIL, max are affected, has
adverse effect on
domestic uses and
water supply structure
15. Sulphate 200 Beyond this causes 400 May be extended up
(asSOJ gastrointestinal irrit- to 400 provided, as
ation when magnesium Mg does not exceed
or sodium are present 30
16. Nitrate 45 Beyond this metha- 100
(as NO) emo-globinemia
takes place
17. Fluoride (as F) Fluoride may be kept 1.5
as low as possible.
High fluorides cause
18. Phenolic 0.001 Beyond this, it may 0.002
compounds cause objectionable
(as C.HPH) tastes and odours
19. Mercury (as Hg) 0.001 Beyond this water No relaxation -
(mglL) becomes toxic
20. Cadmium 0.01 Beyond this, the No relaxation To be tested when
(as Cd) water becomes toxic pollution is
(mglL) suspected
21. Selenium 0.05 Beyond this, the No relaxation To be tested when
(as Se) water becomes toxic pollution is
(mg/L) suspected
22. Arsenic 0.05 Beyond this, the No relaxation To be tested when
(as As) water becomes toxic pollution is
(mg/L) suspected
23. Cyanide 0.05 Beyond this, the No relaxation To be tested when
(as CN) water becomes toxic pollution is
(mg/L) suspected
298 Handbook of Methods in Environmental Studies

24. Lead 0.05 Beyond this, the No relaxation To be tested when

(as Pb) water becomes toxic pollution is
(mglL) suspected
2S. Zinc (as Zn) S Beyond this limit, it I5 To be tested when
can cause astringent pollution is
taste and an opales- suspected
cence in water
26. Anionic 0.2 Beyond this limit, it I. 0 To be tested when
detergents can cause light forth pollution IS
(as MBAS), in water suspected
mg/L, max
27. Chromium 0.05 May be carcinogenic No relaxation To be tested when
(as C~) above this limit pollution is
mgIL, max suspected
28. Polynuclear May be carcinogenic -
(as PAH),
mg/L, Max.
29. Mineral oil, 0.01 Beyond this limit, 0.03 To be tested when
mglL, Max. undesirable taste and pollution is
odour after chlorin- suspected. Gas
ation take place chromatographic
mathod may be used.
30. Pesticides, Absent Toxic 0.001
mg/L, Max.
31. Radioactive 0.1
a. Alpha emiters,
Bq/L, Max.
b. Beta emiters,
pC ilL, Max.
32. Alkalinity, 200 Beyond this limit 600
mgIL, max taste becomes
33 Aluminium 0.03 Cumulative effect is 0.2
(as AI), reported to cause
mglL, max. dementia
34. Boron (as B) 5
mgIL max

• Magnesium (Mg) has been added in IS: 10500: 1991 Drinking water-specification after
amendment No. I January, 1993.

Biological Examination
1. Biological examination is of value in determining the causes of objectionable
tastes and odours in water and controlling remedial treatments, in helping to
interpret the results of various chemical analysis and in explaining the causes of
clogging in distribution pipes and filters. In some instances, it may be of use in
demonstrating that water from one source has been mixed with that from an-
Appendices 299

2 The biological qualities of a water are of greater importance when the conven-
tional flocculation and filtration processes, since increased growth of methane-
utilizing bacteria on biological slimes in pipes may then be expected, and the
development of bryzoal growths such as Plumatella may cause operational
3. Some of the animalcules found in water mains may be free-living in the water,
but others such as Dreissena and Asellus are more or less firmly attached to the
inside of the mains. Though these animalcules are not themselves pathogenic,
they may harbour pathogenic organisms or virus in their intestines, thus pro-
tecting these pathogens from destruction by chlorination.
4. Chlorination, at the dosages normally employed in waterworks, is ineffective
against certain parasites, including amoebic cysts, they can be excluded only
by effective filtration or by higher chlorine doses than can be tolerated without
subsequent dechlorination. Amoebiasis can be conveyed by water completely
free from enteric bacteria; microscopic examination after concentration is, there-
fore, the only safe methods of identification.
5. Strict precautions against back-syphonage and cross-connections are required
if amoebic cysts are found in a distribution system containing tested water.
6. The cercariae ofschistosomiasis can be detected by similar microscopic exami-
nation, but there is, in any case, no evidence to suggest that this disease is
normally spread through piped water supplies.
7. The cyclops vector of the embryos of Dracunculus medinensis, which causes
dracontiasis or Guinea-worm disease, can be found in open wells in a number of
tropical areas. They are identified by microscopic examination. Such well sup-
plies are frequently used untreated, but the parasite can be relatively easily
excluded by simple physical improvements in the form of curbs, drainage, and
apron surrounds and other measures, which prevent physical contact with the
water source.
8. The drinking water shall be free from microscopic organisms such as algae,
zooplanktons, flagillates, parasites and toxin-producing organisms. An illustra-
tive (and not exhaustive) Jist is given in the Table below.

Classijication Group and name of the orgallisms Habitat Effect ofthe of

microscopic organisms
organisms and signiji-

ALGAE a) Chlorophyceae
Species of Coelastrum, Gomphospherium, Polluted water, Impart
Micractimum, Mougeotla, Oocyst/s, Euastrum, impounded colouration,
Scenedesmus, Actmastrum, Gonium, sources
Eudorina,Pandorina, Pediastrum, Zygnema,
Chlamydomonas, Chlorella, Chroococcus,
Spirogyra, Chlorogonium, Stigeoclonium.

Species of Pandorina, Volvox, Polluted water Produce taste

Gomphosphaerium, Staurastrum, and odour
Hydrodictyon, Nitella

300 Handbook of Methods in Environmental Studies

Species of Rhlzoclonium. Calolhrix. Clean water Indicate clean

Ankislrodesmus. UlOlhrtx. Micraslerias. conditions

Species of Chlorel/a. Tribonema. Closlerium. Polluted water Clog tilters

Spirogyra. Palmel/a impounded and create
sources operational

b) Cyanophyceae
Species of Anacyslis and Cylmdrospermum Polluted waters Cause water
bloom and
impart colour

Sp,ecies of Anabaena. Phormidium. Lyngbya. Polluted waters Impart colour.

Arlhrosplra. Oscll/alOrla

Species of Anabaena. Anacystis. Polluted water Produce

Aphantzomenon impounded taste and odour
Species of Anacystts. Anabaena.
Coelospherium. Geolrlchia. Aphanizomenon Polluted watersToxin

Species of Anacystts. Rivularia. Oscil/aloria. Polluted water Clog filters


Species of Rivularia Calcareous Causes matted

waters and also growth

Species of Agmenel/um. Microcoleus, Clean waters Indicators of

Lemanea. purification

c) Diatoms (8acillariopbyceae)
Species of Fragillaria. Slephanodiscus. Cause
Slauroneis discolouration

Species of ASlerionel/a, Tabellaria Hill streams Taste and odour

higb altitudes, producing clog
torrential and filters

Species of Synedra and Fragi//aria Polluted waters Taste and odour


Species of Nilzchia and Gomphonema Moderately Cause

polluted waters discolouration

Species of Cymbela. Synedra. Melosira, Rivers and Clog

Navicula, Fragillaria, Diatoms. streams impo- tilters and
unded sources cause operatio-
nal difficulties

Species of Pinnularia, Cyclolella, Meridion, Clean water Indicators of

Cocconeis. purification

Appendices 301

d) Xanthophyceae
Species of Botryococcus Hill streams, Produce
high altitudes colouration
and temperate

PLANKTON a) Protozoa
Amoeba. Giardia. Lamblia. Arcolla. Difflugia. Polluted water Pollution
Actinophrys. Entamoeba histolytica Sewage and indicators,
activated Parasitic and
sludge pathogenic

b) Ciliates
Paramoccium. Vorticella. Carchesium. Highly Bacteria eaters
Stentor,Colpidium. Coleps. Euplotes. polluted waters,
Colopoda.Bodo sewage and
activated sludge

c) Crustacea
Bosmina. Daphnia. Cyclops Stagnant Indicator of
polluted water pollution.
Step wells in Carrier host
tropical of guinea
climate worm.

d) Rotifen
Anurea. Rotaria. Philodina Polluted and Feed on algae
algae laden

e) Flagellates
Cerallum. Glenodinium. Peridmium. Rocky strata, Impart colour
Dinobryon. iron bearing and fishy taste.
and acidic
Euglena. Phacus polluted water Impart colour

f) Miscellaneous organisms Fresh water Clog filters and

Sponges. Hydra render water
Plumatella Polluted Produces biolo-
waters gical slimes and
causes filter
Dreissena, Asellus Polluted Harbourpatho-
waters genic organisns

Virological Examination
1. It is theoretically possible that virus disease can be transmitted by water free
from coliform organisms.
2 Virus can be isolated from raw water and from springs. Enterovirus, rotavirus
and adenovirus have been found in water, the first named being the most
resistant to chlorination. If enterovirus are absent from chlorinated water, it can
302 Handbook of Methods in Environmental Studies

be assumed that the water is safe to drink. Some uncertainty still remains about
the virus of infectious hepatitis, since it has not so far been isolated but in view
of the morphology and resistance of enterovirus it is likely that, if they have
been inactivated, hepatitis virus will have been inactivated also.
3. An experimental relationship exists between the rate of virus inactivation and
the redox potential. A redox potential of650 mY (measured between platinum
and calomel electrode will cause almost instantaneous inactivation of even
high concentrations of virus. Such a potential can be obtained even a low
concentration of free chlorine, but only with an extremely high concentration
of combined chlorine. This oxidative inactivation may be achieved with a num-
ber of other oxidants also, for examples, iodine, ozone, and potassium perman-
ganate, but the effect of the oxidants will always be counteracted if reducing
components, which are mainly organic, are present.
4. In practice, 0.5 mg/L of free chlorine for one hour is sufficient to inactivate
virus, even in water that was originally polluted.

Bacteriological Examinatio~
1. Water ill distributioll system
Ideally, all samples taken from distribution system including consumer's premises,
should be free from coliform organisms. In practice, this is not always attainable,
and the following standard for water collected in distribution system is therefore
a) Throughout any year, 95percentage of samples should not contain any coliform
organisms in 100 mL.
b) No sample should contain E. coli in 100 mL.
c) No sample should contain more than 10 colifonn organisms per 100 mL.
d) Coliform organisms should not be detectable in 100 mL of any two consecutive
If any coliform organisms are found the minimum action required is immediate re-
sampling. The repeated or the appearance of higher numbers in individual samples
suggests that undesirable material is gaining access to the water and measures
should at once be taken to discover and remove the source of the polIution.
2. Ullpiped water supplies
1. Where it is impracticable to supply water to consumer's through a piped distri-
bution network and where untreated sources, such as wells, bore-holes and
springs which may not be naturalIy pure, have to be used, the requirements of
piped supplies may not be attainable. In such circumstances, disinfection al-
though is not always practicable, and considerable reliance has to be placed
on sanitary inspection and not exclusively on the results of bacteriological
2. Everything possible should be done to prevent pollution of water. Obvious
sources of contamination should be removed from the immediate catchment
area, special attention being given to the safe disposal of excrement.
Appendices 303

3. Wells and storage tanks should be protected by lining and covering, surface
drainage should be diverted, erosion prevented and the surrounding area paved.
Access of man and animals should be restricted by fencing, and should be so
designed that fouling is prevented when drawing water. Although not sup-
plied through pipes, water from such sources is likely to undergo further dete-
rioration in quality during transport or storage before drinking.
4. Containers used for water should be kept clean, covered and clean of the floor.
The most important factor in achieving these objectives is to ensure the coop-
eration of the local community, and the importance of education in simple
sanitary hygiene should be strongly stressed. In hospitals or medical clinics
with such supplies, the value of some form of treatment is stressed.
5. Bacteriologically, the objective should be to reduce the coliform count to less
than 10 per 100 mL, but more importantly, to ensure the absence offecal
coliform organisms. If these organisms are repeatedly found, or if sanitary
inspection reveals obvious sources of pollution which cannot be avoided,
then an alternative source of drinking water would be sought whenever pos-
sible. Greater use should be made of protected ground-water sources and
rainwater catchment which are more likely to meet requirements for potable
water quality.
6. Although private sources of drinking water may be outside the jurisdiction of
public health and water supply authorities, such supplies should still be of
potable quality. The results of bacteriological tests and those of sanitary sur-
veys should therefore be used to encourage improvement. Partial treatment
may be necessary to remove turbidity even when coliform counts are low; and
other quality criteria may dictate the need for treatment processes.

Acidity, 30 Checking correctness of analysis, 9

Measurement of, 31 Chemical oxygen demand, 60
Standards of, 30 Application of COD data, 60
Alkalinity, 33 COD standards, 60
Environmental significance, 34 Environmental significance, 60
Estimation of, 34 Estimation of, 63
Standards of, 33 Chlorine demand, 70
Aluminium, 181 Estimation of, 71
Estimation of, 181 Significance and use, 70
Standards of, 181 Chlorine (residual), 66
Ammonia nitrogen (see nitrogen Estimation of, 67
ammonical), 108 Standards of, 66
Anionic detergents (see methylene Chlorophyll, 261
blue active substances), 100
Estimation of, 261
Arsenic, 182
Chromium, 184
Estimation of, 182
Estimation of, 184
Standards of, 182
Standards of, 184
Artificial substrate sampler, 246
Coagulant aids, 1 99
Atomic absorption spectropho-
Coagulation-flocculation jar test, 197
tometer, 178
Application of, 200
Benthic macroinvertebrates, 243
Procedure of, 201
Counting of, 244
Cold vapour atomic absorption
Sampling method, 245
spectroscopy, 189
Biochemical oxygen demand, 37
Coliform group of bacteria, 214
Application of BOD data, 37
Estimation of, 215
BOD standards, 37
Presence-absence coliform
Procedure of, 40 test, 223
Biological monitoring of waters, 242 Coliform standards, 20
Significance of, 242 Colour, 57
Boron, 48 Environmental significance, 58
Estimation of, 48 Estimation of, 58
Standards of, 48 Standards of, 57
Cadmium, 183 Conductivity, 52
Estimation of, 184 Environmental significance, 53
Standards of, 183 Estimation of, 53
Carbon dioxide, 50 Standards of, 52
Estimation of free CO 2 , 50 Copper, 185
Standards of free CO 2 , 50 Estimation of, 185
Index 305

Standards of, 185 Flame photometer, 1 52

Cyanide, 74 Fluoride, 85
Estimation of cyanide: Estimation of, 87, 89
Colorimetric method, 77 Removal of fluoride from water, 90
lon-selective electrode, 78 Standards of, 85
Titration, 76 F/M ratio, 203
Total cyanide, 74 Hardness (Calcium), 94
Standards of, 74 Estimation of, 95
Description of monitoring area, 2 Standards of, 94
Digestion of metals, 177 Hardness (Magnesium), 96
Diseases caused by bacteria, 213 Estimation of, 97
Diseases caused by protozoa, 214 Standards of, 96
Diseases caused by viruses, 214 Hardness (Total), 91
Dissolved oxygen, 79 Environmental significance, 92
Alum flocculation method, 84 Estimation of, 92
Environmental significance, 80 Standards of, 91
Estimation of, 80 Heavy metals, 174
Standard of, 79 Iron, 185
Diversity and other indices, 254 Estimation of, 186
Autotrophic index, 258 Standards of, 185
Berger-Parker dominance Lead, 186
index, 257 Estimation of, 187
Community dominance index, 257 Standards of, 186
Equitability index, 255 Light-Dark bottle, 259
Evenness index, 255 Macroinvertebrate Biotic Index, 247
Goodnight and Whitley's Manganese, 187
index, 257
Estimation of, 188
Kothe's species deficit index, 257
Standards of, 187
Margalef index, 256
Membrane filter (MF) technique, 233
McIntosh index, 256
Mercury, 188
Odum's index, 257
Estimation of, 189
Shannon index, 255
Standards of, 188
Similarity index, 258
Methylene blue active substances,100
Simpson's index, 256
Environmental significance, 100
Drinking water standards, 1 5
Estimation of, 100
Indian, 15
Standards of, 100
International, 15
Microbiological water analysis, 212
Effluent standards, 22
Mixed liquor suspended solids, 204
Effluent standards for coal mines, 27
Estimation of, 205
Ekman grab, 246
Mixed liquor volatile suspended
Fecal coliforms, 225 solids, 204
Membrane filter technique, 235 Estimation of, 205
MPN test, 225 Most probable number (MPN), 229
Seven-hour fecal coliform test, 237 Calculation of, 229
Fecal streptococci, 226 Nitrogen (ammonical), 108
MPN test, 226 Estimation of, 109, 111
Flameless atomic absorption spec- Standards of, 108
trometry, 189
306 Handbook o/Methods in Environmental Studies

Nitrogen (nitrate), 113 Phytoplankton productivity, 259

Environmental significance, 114 Preservation of, 249
Estimation of nitrate, 114 Sedgwick-Rafter cell method, 250
Devarda's alloy method, 116 Potassium, 1 50
Phenol disulphonic acid Environmental significance, 1 54
method, 117 Estimation of, 154
UV screening method, 114 Preservation of water samples, 9
Standards of, 113 Primary water quality criteria for
Nitrogen (nitrite), 120 bathing waters, 27
Procedure of, 122 Radioactivity, 224
Standards of, 120 Measurement of:
Nitrogen (organic), 123 a radioactivity, 277
Nitrogen (total), 104 ~ radioactivity, 275
Environmental significance and Standards of, 274
use, 104 Units of, 275
Estimation of TKN, 105 Removal of hardness, 98
Standards of TKN, 104 Return activated sludge, 207
Odour (TON), 123 Calculation of, 207
Environmental significance, 124 Sampling, 3
Estimation of, 1 24 Composite samples, 3
Standards of, 123 Grab samples, 3
Oil and grease, 125 Integrated samples, 4
Estimation of, 126 Planning of sampling, 3
Environmental significance, 126 Sampling frequency, 4
Standards of, 125 Sample container, 4
pH (Potentia hydrogenii), 128 Sample collection, 6
Environmental significance, 1 29 Transportation of sample, 7
Estimation of, 130 Secchi disc (see transparency), 167
Standards of, 128 Selection of sampling location, 2
Phenol (Phenolic compounds), 133 Selenium, 192
Environmental significance, 134 Estimation of, 1 92
Estimation of, 134 Standards of, 192
Standards of, 133 Settleable solids (see solids), 142
Phosphorus, 138 Setting of water analysis
Environmental significance, 138 laboratory, 13
Estimation of phosphorus, 139 Silver, 193
Inorganic phosphorus, 139 Estimation of, 193
Organic phosphorus, 142 Standards of, 193
Particulate phosphorus, 142 Sludge density index, 207
Total phosphorus, 141 Calculation of, 207
Standards of, 138 Sludge volume index (SVI), 206
Phytoplankton, 248 Application of, 207
Counting of, 248, 250 Estimation of, 208
Determination of biomass, 259 Sodium, 150
Haemocytometer method, 252 E!1vironmental significance, 151
Importance of, 248 Estimation of, 151
Lackey's drop method, 253 Standards of, 150
Index 307

Solids, 142 Toxicity test, 267

Estimation of: Classification of, 267
Total solids, 143 Measurement of, 269
Total suspended solids, 144 Significance of, 267
Total dissolved solids, 146 Standards of, 267
Settleable solids, 148, 149 Toxic unit, 271
Standards of, 144 Transparency, 167
Specific conductance (see Application of, 167
conductivity), 52 Procedure of, 168
Standards for effluents from textile Turbidity, 169
industry, 28
Application of turbidity data, 172
Standards of coliform bacteria, 212
Environmental significance, 169
Standards of heavy metals, 174
Estimation of, 169
Standard solutions of metals, 179
Sources of, 169
Structuring of monitoring report, 10
Standards of, 169
Suber sampler, 246
Vanadium, 193
Sulphate, 155
Estimation of, 194
Environmental significance, 1 56
Standards of, 193
Estimation of sulphate, 1 56
Volatile fatty acids (VFAs). 209
Gravimetric method, 1 56
Estimation of, 210
Turbidimetric method, 1 58
Wastewater treatment plants, 264
Removal of sulphate from
water, 160 Biological monitoring, 264
Standards of, 155 Health of, 264
Sulphide, 161 Water quality monitoring, 1
Environmental significance, 162 Objectives of, 1
Estimation of, 162 Water quality in field, 7
Standards of, 161 Water quality standards, 1 5
Thiocyanate, 164 Water samplers, 5
Environmental significance, 164 DO sampler, 5
Estimation of, 164 Depth sampler, 6
Standards of, 1 64 Zinc, 194
Total dissolved solids (see solids), 142 Estimation of, 194
Total Kjeldahl nitrogen (see nitrogen Standards of, 194
(total), 104 Zooplankton, 253
Total solids (see solids), 142 Counting of, 253, 254
Total suspended solids(see solids),142
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