CHAPTER ONE

INTRODUCTION

1.1 Background Of The Study

Water is indispensable and intricately connected to life, without which there is no life. This is the reason
for which water must be given the necessary attention at all times. Good drinking water is not a luxury; it
is one of the most essential amenities of life itself. The supply of safe drinking water to all has therefore
engaged the attention of many individuals, groups, governmental organizations and private organizations.
(Adetunde et al. 2010).

Drinking water free of pathogenic organisms is fundamental to breaking one of the principal transmission
routes of infectious disease. This fact has stimulated worldwide investment in the construction of water
systems that are designed to meet stringent water quality standards. (Trevett, 2004). Waterborne
pathogens, including a variety of viral, bacterial, algal and protozoan agents, account for much of the
estimated 4 billion cases and 2.5 million deaths from endemic diarrheal disease each year. (Kosek et al.
2003). Increase in human population has exerted an enormous pressure on the provision of safe drinking
water, especially in developing countries (Umeh et al. 2005). Unsafe water is a global public health
threat, placing persons at risk for a host of diarrheal and other disease as well as chemical intoxication
(Hughes et al. 2005). Unsanitary water particularly has devastating effects on young children in
developing world. Each year, more than 2 million persons, mostly children less than 5 years of age, die of
diarrheal disease (Kosek et al. 2003; Parashar et al. 2003). Nearly 90% of diarrheal-related deaths have
been attributed to unsafe or inadequate-water supplies and sanitation conditions affecting a large part of
the world’s population (Hughes et al. 2005; WHO 2004). An estimated 2.6 billion persons lack access to
adequate sanitation (Okonko et al. 2008).

The problem of environmental pollution due to toxic metals has begun to cause concern now in most
major metropolitan cities. The toxic heavy metals entering the ecosystem may lead to geo-accumulation,
bioaccumulation and bio-magnification. Heavy metals like Fe, Cu, Zn, Ni and other trace elements are
important for proper functioning of biological systems and their deficiency or excess could lead to a
number of disorders (Ward, 1995). Food chain contamination by heavy metals has become a burning
issue in recent years because of their potential accumulation in bio-systems through contaminated water,
soil and air. Therefore, a better understanding of heavy metal sources, their accumulation in the soil and
the effect of their presence in water and soil on plant systems seem to be particularly important issues of
presentday research on risk assessments (Rajesh et al., 2004). The main sources of heavy metals to
vegetable crops are their growth media (soil, air, nutrient solutions) from which these are taken up by the
roots or foliage (Ward, 1995). Most of our water resources are gradually becoming polluted due to the
addition of foreign materials from the surroundings. These include organic matter of plant and animal
origin, land surface washing, and industrial and sewage effluents (Karnataka State Pollution Control
Board, 2002). Rapid urbanization and industrialization with improper environmental planning often lead
to discharge of industrial and sewage effluents into lakes.

The lakes have a complex and fragile ecosystem, as they do not have self-cleaning ability and therefore
readily accumulate pollutants. Bellandur Lake, the largest one in Bangalore urban area, recently attracted
a lot of public attention because of the formation of froth during rainy season due to chemicals (soaps,
detergents, etc.) and biosurfactants. For the last few decades, the treated, partially treated and untreated
wastewater has been discharged to this lake and the lake water is being used for farming purposes (Pruss
et al., 2002). Individual rural homeowners are often responsible for providing and protecting their own
water supplies. Where safety of these sources is concerned, no “short-cuts” can be taken. Protecting the
quality of individual water supplies is a combination of controlling land use around the supplies and using
proper water treatment techniques where necessary. Rural homeowners must assume responsibility for
protecting their families from contaminated drinking water. Assistance in this regard can be obtained
from a number of agencies (Ward, 1995). Local health authorities can answer questions relating to
applicable local regulations; health hazards posed by contaminated water, and suggested procedures for
sampling and analyzing drinking water for contaminants. In some cases, local health officials will analyze
individuals‟ water samples for common pollutants at no cost or for a nominal charge. Complete well
water analysis is the homeowner‟s responsibility and is not free. State regulatory agencies charged with
water resource management can answer questions regarding water use. They usually also have
information regarding the availability and suitability of water sources in the State. Such agencies usually
administer safety regulations for dams as well (Ward, 1995

The Need For Water Analysis

If water is badly polluted with raw sewage for example, it might be obvious from its appearance or odour.
It might be coloured or turbid, have solids or oil floating on it. It might have a rotten odour or smell like
industrial chemical. Many harmful and beneficial materials in water are invisible and odourless. In order
to determine what materials are in water, we need to conduct chemical and microbiological analyses
Water analysis involves the qualitative and quantitative determination of substances suspended or
dissolved in water. Since the human body is approximately 70 percent water by weight, it is essential
from a municipal water supply viewpoint that water delivered to consumers be free of pathogenic
microorganisms, free of toxic components and aesthetically acceptable (WHO, 2004). Analysis of water
is therefore necessary as it enables the determination of the nature of impurity and contamination and thus
helps to determine their suitability for human consumption. Welcher (1963) says that chemical analysis is
performed on water to ascertain its physiological or technological acceptability. In order to be used as
healthful fluid for human consumption, water must be free from organisms that are capable of causing
diseases and from minerals and organic substances that could produce adverse physiological effects. The
only way to achieve this is by carrying out analysis on the water and consequently giving it adequate
treatment.

1.2 Statement Of Problem

Water pollution has been documented as a contributor to a wide range of health problems and disorders in
humans. It has also been shown to have drastically negative impacts on wild animals and the environment
as a whole. Water has been life sustaining substance which exist in liquid form, it is widely use in
household activities and in production firm for production, a little contamination of water will lead to
several health issues if not properly checked.

1.3 Aim Of The Study

This study is aimed at the bacteriological analysis of water tanks in ATAP female hostel Bauchi State

1.4 Objectives Of The Study

1. To attain the total bacterial analysis of water tanks in ATAP female hostel, Bauchi State.
2. To determine the coliform counts (Most Probable Number) of the water bacterias in ATAP Female
hostel Bauchi State.
3. To determine the species of bacteria present in the water.

1.5 Research Question

1. What is the total bacterial analysis of water tanks in ATAP female hostel?
2. What is the total coliform counts of the water bacterias in ATAP Female hostel?
3. What kind of species of bacterial is present in the water?

1.6 Scope Of Study

The study will cover the entire water tanks in all the female hostels in Abubakar Tatari Ali Polytechnic
Bauchi, Bauchi State.
 CHAPTER TWO

LITERATUER REVIEW

2.1 Importance of water:

Water is essential to sustain life and satisfactory supply must be made available to consumer.

The acceptable quality of water is defined by the WHO guidelines as that which is suitable for all usual domestic
purposes, including personal hygiene (WHO, 1993). It should be potable, wholesome, attractive to sense of sight, taste, and
hygienically safe. There is an urgent need for simple, effective and low-cost methods for the production of water free of
pathogenic and harmful chemical substances (John, 1997).

Every effort should be made to maintain drinking water quality as high as practicable. Protection of water supplies
from contamination is the first line of defense. Source protection is almost invariably the best method of ensuring safe
drinking water and is to be preferred to treating a contaminated water supply to render it suitable for consumption. Once a
potentially hazardous situation has been recognized, the availability of suitable remedial measures must be considered
(Ahmed, 2005).

As far as possible, water sources must be protected from contamination by human and animal waste. Failure to provide
adequate protection and effective treatment will expose the community, to the risk of water-borne diseases.

2.2 Water Sources and Resources:

In nature water is constantly changing from one state to another. The sun constantly evaporates water into the atmosphere.
Some of that water is returned as snow or rain. Part of this water, rapidly evaporates back into the atmosphere. Some drains
into lakes and rivers to commence a journey back to sea. Some infiltrates into the soil to become soil moisture or ground
water. Under natural condition, the ground water gradually works it way back into surface waters and makes up the main
source of dependable river flow (Elrofaei, 2000).

Although Sudan is the largest country in Africa and lies mostly in the arid region where water is a scarce commodity, it is
considered to be rich in water recourses (Ginawi, 1994).

2.3 Water Treatment:

For small communities, it is generally preferable to protect a groundwater source that requires little or no treatment than to
treat surface water that has been exposed to faecal contamination and is usually of poor quality. In many circumstances,
however, surface water is the only practicable source of supply and requires affordable treatment and disinfection.

The range of treatments available for small community supplies is necessarily limited by technical and financial
considerations: the most appropriate and commonly used treatments are summarized below. Installation of packaged
treatment plants is not a suitable means of dealing with the typical water-quality problems that prevail in rural areas (WHO,
2004).

2.3.1 Chlorination:

Chlorination can be achieved by using liquefied chlorine gas, sodium hypochlorite granules and onsite chlorine
generators. The gas is supplied in pressurized containers. The gas is withdrawn from the cylinder and is dosed into water by
chlorinator, which both controls and measures the gas flow rate. Sodium hypochlorite solution is dosed using a positive-
displacement electric dosing pump or gravity feed system. Calcium hypochlorite has to be dissolved in water, and then
mixed with the main supply. Chlorine, weather in the form of chlorine gas from a cylinder, sodium hypochlorite or calcium
hypochlorite, dissolved in water to form hypochlorous acid (HOCI) and hypochlorite ion (OCI) (WHO, 2004).

Chlorination is employed primarily for microbial disinfection. However, chlorine acts an oxidant and can remove or
assist in the removal of some chemicals for example, decomposition of easily oxidized pesticides such as aldicarb;
oxidation of dissolved species (e.g, manganese (II)) to form insoluble products that can be removed by filtration, and
oxidation of dissolved species to more easily removable forms (WHO, 2004). A disadvantage of chlorine is its ability to
react with natural organic matter.
 However, byproduct formation may be controlled by optimization of the treatment system (WHO, 2004).

2.3.2 Ozonation:

Ozone is a powerful oxidant and has many uses in water treatment, including oxidation of organic chemicals. Ozone can be
used as a primary disinfectant. Ozone gas (O3) is formed by passing dry air or oxygen during a high-voltage electric field.
The resultant ozone-enriched air is dosed directly into the water by means of porous diffusers at the base of baffled
contactor tanks. The contactor tanks, typically about 5m deep, provide 10 - 20 min of contact time. Dissolution of at least
80% of the applied ozone should be possible, with the remainder contained in the off-gas, which is passed during an ozone
destructor and vented to the atmosphere (WHO, 2004).

Ozone reacts with natural organics to increase their bio-degradability, measured as assimilable organic carbon. To avoid
undesirable bacterial growth in distribution, ozonation is normally used with subsequent treatment, such as filtration to
remove biodegradable organics, followed by a chlorine residual, since it does not provide a disinfectant residual. Ozone is
effective for the degradation of a wide range of pesticides and other organic chemicals (WHO, 2004).

2.3.3 Filtration:

Particulate matter can be removed from raw waters by rapid gravity horizontal, pressure or slow sand filters. Slow sand
filtration is essential biological process, whereas the others are physical treatment processes (WHO, 2004). Rapid gravity,
horizontal and pressure filters can be used for direct filtration of raw material, without pretreatment. Rapid gravity and
pressure filters are commonly used to filter water that has been pretreated by coagulation and sedimentation. An
alternative process is direct filtration, in which coagulation is added to the water, which then passes directly onto the
filter where the precipitated flock (with contaminants) is removed, the application of direct filtration is limited by available
storage within the filter to accommodate solids (WHO, 2004).

2.4 Drinking Water Standards:

No single standard for drinking water suffices for all countries but there is a considerable degree of agreement on
contaminants and their allowable concentration (Sayre, 1988). However different conditions in countries will maintain
differences in standards currently enforced. The first priority of water supplies in all countries is to ensure that drinking
water is bacteriologically safe (Craun, 1986).

Drinking water must contain no impurity that would offend sight, taste, or smell and substance with deleterious physiologic
effects must be eliminated or not introduced (Smith, 1981).

The World Health Organization (WHO) publishes Guidelines for drinking- water quality which many countries use as the
basis to establish their own national standards. The Guidelines represent a scientific assessment of the risks to health from
biological and chemical constituents of drinking-water and of the effectiveness of associated control measures. WHO
recommends that social, economic and environmental factors be taken into account through a risk-benefit approach when
adapting the Guideline values to national standards. As the WHO Guidelines for Drinking-water Quality are meant to be
the scientific point of departure for standards development, including bottled water, actual standards will sometimes vary
from the Guidelines (WHO, 2000).

2.5 Contamination of Drinking Water:

Drinking water may be contaminated by a range of chemical, microbial and physical hazards that could present risks to
health if they are present at high levels. Examples of chemical hazards include lead, arsenic and benzene. Physical hazards
include glass chips and metal fragments. Microbial hazards include bacteria, viruses, and parasites (Elrofaei, 2000).

Bacteria are single-celled organisms commonly found in soil, on our bodies, on leaf materials and in water. There may be
over a million cells per gram of soil. Bacteria have many functions in nature, they help breakdown matter (decomposition)
and transform it through chemical reactions. Pathogenic bacteria carry diseases such as typhoid, dysentery and cholera
(Geldrich and Litsky,, 1992). Bacteria are the most common contaminant in drinking water (Wright, 1984).

Any of several thousand types of bacteria (both non-pathogenic and pathogenic) can contaminate water supply. While
surface water commonly supports bacteria, most ground supplies don’t. This is because the conditions for bacterial growth
(food, oxygen, temp, ph) are not often found in ground water. Yet, many well water samples still show that bacteria are
present (Wright, 1984). Here are some ways whereby contaminated water may enter a well:

1 Water may leak in through a loose or cracked cover.

2 Water may flow down the side of the well casing without being filtered by the soil.
3 Water may come through the site of a dug well (without adequate soil filtration).
4 Bacteria may be added when the well is repaired.
The ground water supplying the well may contaminated with bacteria (Ahmed, 2005).
The movement of animal wastes into the surface water is often cited as a major factor contributing to the pollution of
available water in many rural areas (Fernandez-Alverez et al., 1991). Untreated excreta carrying disease organisms wash or
leach into fresh water sources, contaminating drinking water and food. The extents to which disease organisms occur in
specific fresh water source depend on the amount of human and animal excreta that they contain. Diarrheal disease, the
major water-borne disease, is prevalent in many countries where sewage treatment is inadequate. An estimated 4 billion
cases of diarrrheal diseases occur every year, causing 3 million to 4 million deaths, mostly among children. Using
contaminated sewage for fertilizer can result in epidemics of such diseases as cholera. These diseases can even become
chronic where clean water supplies are lacking (Brock, et al., 1994). EL Shazali and Erwa (1971) reported that studies in
the Sudan have clearly demonstrated the close association of biological contamination of drinking water with the high
prevalence of diarrheal diseases causing by certain enteric pathogens.

Hammad and Dirar (1982) found that zeers were faecally contaminated, with faecal coliforms in 70% and faecal
streptococci in 92% of samples examined.

2.6 Characteristics of Indicator Organisms:

Different bacterial indicators are used. The most common ones include the coliform group, the Alfa-hemolytic streptococci
of fecal origin and the Entrococci.

2.6.1 Coliform Bacteria:

Coliform bacteria considered to be members of genera or species within the family Enterobacteriaceae, capable of growth
in presence of bile salt and able to ferment lactose at 35-37ºC with the production of acid, gas and aldehyde within 24-48
hours. They are oxidase negative and non spore- forming. They display B-galactosidase activity (WHO, 1993).

The coliform group includes: Citrobacter, Enterobacter, Escherichia coli and Klebsiella spp (WHO, 1995). Most coli form
bacteria don’t transmit diseases but they are a very common group of bacteria. They are used as an indicator that harmful
bacteria may be present.

If the samples come back with high coli form counts, this means that the water entering the well is not being filtered
enough by the soil and the presence of coliform organisms in drinking water is used as an indicator of faecal contamination
since they are the most sensitive indicator demonstrating excremental contamination (Packer et al., 1995).

2.6.2 Escherichia coli (E. coli):

These bacteria conform to the criteria for coliform organisms but are capable of growth at 44°C. Escherichia coli is
regarded as an essential indicator of faecal pollution of human or animal origin (Colee et al., 1996). According to Bergey’s
manual of systematic bacteriology (1984). E. coli is gram-negative, straight rod, motile or non motile and facultatively
anaerobic. Tests of coliform bacteria and E coli are the most important routine microbiological examination. They provide
the most sensitive means of assessing the effectiveness of water treatment and disinfection, detecting faecal contamination
and for monitoring water quality in distribution.

2.6.3 Escherichia coli O157:H7:

The recovery of E.coliO157:H7 from environmental samples is often difficult because of the altered physiological state that
bacteria sometimes developed in order to survive hostile environments. Infections involving E.coliO157:H7 have
occasionally been implicated with contaminated water, but food borne infections are more common. E.coliO157:H7 is a
recognized cause of haemorrhagic colitis, an illness characterized by bloody diarrhea and severe abdominal pain but little
or no fever. It is also on of the causes of haemolytic uramic syndrome. Strains of E.coliO157:H7 produce a toxin which is
similar to that produced by Shigella dysenteriae type 1 which is cytotoxic to vero cells in cell culture (Colee et al., 1996).

2.6.4 Faecal streptococci:

The presence of fecal streptococci is an evidence of fecal contamination. They are tend to persist longer in the environment
thermo tolerant or total coliform and are highly resistant to drying (Bartram and pedley, 1996).

2.7 Water- borne diseases:

Polluted water, tainted food and malnutrition accounted for high infant mortality when sanitation measure are lacking.
Throughout history water borne diseases have an important restraint on population growth (Deming, 1975).

Water- borne diseases are "dirty- water'' diseases those caused by water that has been contaminated with human, animal or
chemical wastes. World wide, the lack of sanitary waste disposal and of clean water for drinking, cooking and washing is to
blame over 12 million deaths a year (Alcamo, 1997).

2.7.1 Cholera:

The disease is caused by Vibrio cholera. The infection is caused by ingestion of water contaminated by infected human
faecal material. It gives rise to sudden and profuse diarrhea which leads to severe dehydration and death within 1 or 2 days
if fluids are not replaced (Carincross et al., 1980).

2.7.2 Typhoid fever:

The disease is caused by Salmonella typhi. The infection usually contracted by ingestion of material contaminated by
human feaces or urine, including water and food (Twort et al., 1985).

2.7.3 Paratyphoid fever:

Paratyphoid fever is caused by Salmonella paratyphi A, B or C. Infection may exceptionally be via contaminated water, but
is more commonly due to ingestion of contaminated food (Twort et al., 1985).

Typhoid and paratyphoid are again specific severe fevers sometimes with intestinal symptoms, which have a high mortality
if un treated (Cairocross et al.,1980).

2.7.4 Dysentery:

Dysentery is caused by Shigella spp. Infection is occasionally contracted via water contaminated by human faeces, but
more commonly it is due to ingestion of food contaminated by flies or by unhygienic food handlers who are carries (Twort
et al., 1985). The disease is characterized by severe bloody diarrhea accompined by abdominal pain. A severe illness ia
affected by access to domestic water and its quality (Cairocross et al., 1980).

2.8 Bacteriological Quality Standards:

Supplies of drinking water contaminated with sewage or other excreted matter from man and animal may cause diseases. In
the interest of public health, supplies should be tested regularly to confirm their freedom from such contamination (Collee
et al., 1996). Improving public sanitation and providing a clean water supply are the two steps needed to prevent most
water-borne diseases and deaths. In particular, constructing sanitary latrines and treating waste water to allow for
biodegradation of human wastes will help curb diseases caused by pollution (Johns Hopkins School of Public Health USA,
1998).

A standard demanding that coliform bacteria be absent from each 100-ml samples of water entering the distribution system
whether the water be disinfected or naturally pure from at least 90% of the samples taken from distribution system can be
applied in many parts of the world. For each individual sample, coliform density is estimated in terms of the most probable
number (MPN) in 100ml of water (WHO, 1963).

The following bacteriological standards are recommended for treated and un treated supplies for present throughout the
world.
a) Treated water:

In 90% of the samples examined throughout any year, coliform bacteria shall not be detected or the MPN index of coliform
microorganisms shall be less than 1.0 per 100ml. None of the samples shall have an MPN index of coliform bacteria in
excess of 10-100 ml samples (WHO, 1964).

b) Untreated water:

In un treated water which is naturally pure, the standards are the same as above but it is stated that none of the samples
should show an MPN index greater than 20 per 100ml (WHO, 1963).

When samples are taken from the distribution system they should be free from coliform organisms. In practice, this cannot
always attained and the standard applied should ensure that throughout the year 95% of the samples should not contain any
coliform organisms or E. coli in 100 ml (Dart and Stretton, 1980)

2.9 Monitoring of drinking water:

The monitoring of drinking-water quality ideally consists of two components:

1. Continual control of quality on a routine basis to ascertain that treatment and distribution comply with the given
objectives and regulations.

2. Periodic microbiological and public health surveillance of the entire water supply system from source to consumer.

Also the frequency of sampling, the more frequently the water is examined; the more likely it is that chance
contamination will be detected.

Care must be taken to ensure that samples are representative of the water to be examined and that no accidental
contamination occurs during sampling. Surveillance is the continuous and vigilant public health assessment and over-view
of the continuous and acceptability of drinking-water supplies (Ahmed, 2005)
 CHAPTER- 3
METHODOLOGY

3.1. SAMPLE COLLECTION, TRANSPORTATION AND PROCESSING

Samples of nine brands of jar water of 20 litres capacity were collected randomly from
all the water tanks at Abubakar Tatari Ali Polytechnic Female hostel. For analysis of
physicochemical parameters, water sample was collected in PVC sampling tank. Some
parameters such as temperature, pH, chloride, dissolved oxygen (DO), hardness, alkalinity
and free carbon dioxide were determined in site while other parameters were determined in
the laboratory of Central Department of Environment Science T.U. For determination of iron
contents, about 1ml conc. HCl was kept in the samples tanks before the collection of water
sample, in order to preserve the samples in reduced state. Samples for bacteriological analyses
were collected in sterilized tank, stored in ice cold box and transported to laboratory and were
processed within 6 hours of collection.

3.2. SAMPLING FREQUENCY:

Water quality was analyzed twice for each brand of tank water during months of September,
October and November when the difference in daily temperatures and change in season
proceeds. The methodologies used to analyze various parameters are described below.

3.3. ANALYSIS OF WATER SAMPLES

3.3.1. Analysis of physicochemical parameters of water samples:

"Standard Methods for the examination of water and wastewater", (APHA, 1998) was
followed to analyze most of the physicochemical parameters of water.

3.3.2. Analysis of microbial variables of water sample:

Microbial analysis was carried out following the "Standard Methods for the examination of
tank water at ATAP Female hostel ", (APHA, 1998) and "Chemical and Biological
methods for water pollution studies", 18th edition, R.K. Trivedi and PK. Goel 1984".
 Table 3.3.1.: Methodology of Physico-chemical parameters and microbial analysis

S. No. Parameter Equipments/Methods

1 Temperature Mercury Thermometer
2 pH pH meter
3 Conductivity Conductivity meter
4 DO Winkler’s iodimetric titration
5 Iron Spectrophotometer
6 Total Hardness EDTA method
7 Total Alkalinity Titration method
8 Chloride Titration method
9 Free CO2 Titration method
10 Phosphate Spectrophotometer
11 Nitrate Spectrophotometer
12 Ammonia Spectrophotometer
13 Total Coli form & MPN method
Faecal coliform

1. Temperature
For the determination of the temperature, water was collected in a beaker. Mercury filled
Celsius thermometer was inserted into the beaker and reading was noted.

2. pH
pH was measured by automatic digital pH meter. The pH meter was first calibrated with a
standard buffer solution. The glass electrode was washed with distilled water. Then glass
electrode was dipped in the beaker containing water sample until the reading stabilized at a
certain point. Then pH reading was noted down.

3. Conductivity
The instrument used was digital conductivity meter. The conductivity meter was first
calibrated with standard Potassium chloride solution of 0.01N. Then reading was noted.

4. Chloride
Chloride was measured by titration method. 50 mL of sample in a conical flask was taken.
2 mL of Potassium chromate was added to the sample solution. It was titrated against
0.02N silver nitrate until a persistent brick red color was appeared which was the end point
of the titration. A blank by placing 50 mL of chloride free distilled sample water was also
conducted.
 Calculation
Chloride (mg/L) = (a-b) × N ×35.5 × 1000
V
Where, a = Volume of titrant (silver nitrate) for sample
b= Volume of titrant (silver nitrate) for blank
V = Volume of the sample in mL
N = normality of silver nitrate

5. Total hardness
Hardness is caused by the calcium and magnesium ions present in water. Total hardness
was determined by EDTA method. This was done by titrating 100mL of sample in a
conical flask and adding 1mL of buffer solution with Erichrome Black-T indicator against
standard EDTA (Ethylene diamine tetra acetic acid). The solution was changed from wine
blue at the end point. Total hardness might be caused by the sum of all metallic cations
other than alkali metals and expressed as equivalent calcium carbonate concentration.
Total hardness (as CaCO3), (mg/L) = mL of EDTA used×100
mL of sample

6. Calcium hardness
Calcium hardness was determined by the same procedure as total hardness. Taking 50mL
sample in a conical flask with 2mL of NaOH solution of 1N was titrated against EDTA
solution using murexide indicator. At the end point, pink color changed to purple.
Calcium, mg/L (as CaCO3) = Vol. of EDTA×N × 40.08 × 1000
Vol. of sample

7. Magnesium hardness
Magnesium salts occur in significant concentration in natural waters which may be
calculated as the difference between total hardness and calcium hardness.
Magnesium hardness, mg/L (as CaCO3) = Total hardness - Calcium hardness

8. Free CO2
Free CO2 in water can be determined by using titration method. For this 100 mL of sample
was taken in a conical flask and 2 drops of phenolphthalein indicator was added. Then it
was titrated against 0.05N of NaOH from the burette until pink color was just appeared.
 Calculation
Free CO2 (mg/L) = A × Normality of NaOH × 44 ×1000
Volume of sample in mL

Where, A = Volume of NaOH used in mL

9. Dissolved Oxygen ( Winkler’s Method )

DO was measured by using APHA, (1998) method. The sample was collected in a 300 mL
BOD tank carefully, avoiding any kinds of bubbling and trapping of air bubbles in the
tank after placing the stopper. To the 50 mL sample taken in the conical flask 2 mL of
manganese sulphate (MnSO 4) and 2 mL of Sodium azide solution was added well below
the surface from the wall of the tank. A precipitate was appeared. Then the stopper was
placed tightly and the tank was shaken by inverting the tank repeatedly to insure proper
mixing of the contents. The tank was kept for some time to settle down the precipitate, 2
mL of conc.H2SO4 was added to it and shaken well to dissolve all the precipitate. Then 50
mL of sample were taken in a conical flask and titrate against sodium thiosulphate
(Na2S2O3) of 0.025N using starch as an indicator. At the end point the initial blue color
changed to colorless.

Calculation:
DO (mg/L) = (mL× N) of titrant ×8 ×1000
V2× (V1-V)
V1
Where, V1= volume of sample tank after placing the stopper
V2= volume of part of content titrated
V= volume of MnSO4 and KI added

10. Total Alkalinity

Total Alkalinity is the measure of the capacity of the water to neutralize a strong acid. The
alkalinity in water is generally imparted by the salts of carbonates, bicarbonates,
phosphates, nitrates, borates, silicates etc. together with hydroxyl ions in free state.
Total alkalinity of water was determined by titrimetric method. 100mL sample in a conical
flask with 2-3 drops of methyl orange was titrated against standard, 0.02N H 2SO4. At the
end point, yellow color was changed to pink color.
 Total Alkalinity (mg/L) = a ×N×1000×50
mL of sample

where, a= Volume of standard H2SO4 consumed in titration

N= Normality of H2SO4 used

11. Phosphate - P
Phosphate content in the given water sample was determined as inorganic phosphate by
calorimetric method. In this method, 50mL of the filtrate clear sample was taken in a
conical flask. 20 mL of ammonium molybdate was added to it. 5 drops of SnCl 2 solution
was added to it. The solution becomes blue and the reading was taken at 690 nm on the
spectrometer within 10-12 minutes. Same procedure was repeated for the standard solution
of different concentration for distilled water. The concentration was determined with the
help of standard curve obtained by plotting standard values against absorbance.

12. Nitrate-N
Nitrate content in the water sample was determined by Phenol disulphonic acid method. In
this method, 50 mL of filtrate sample was taken in a porcelain basin and was evaporated to
dryness. It was cooled and residue was dissolved in 2 mL of phenol disulphonic acid and
was diluted to 50 mL. 6 mL of liquor ammonia was added to develop yellow color. Then
the reading was taken at 410 nm on spectrophotometer. Same procedure was repeated for
the standard solution of different concentration and for distilled water. Then the
concentration of Nitrate-N was determined from the standard curve obtained by plotting
standard value against absorbance.

13. Ammonia-N
Ammonia content in a water sample was determined by colorimetric method. In this
method 100 mL of water sample was taken in a volumetric flask. 1 mL of ZnSO4.7H2O
was added, 1 mL of 10% NaOH was added into it. Then it was stirred and filtered. One drop of
50% EDTA was added and well mixed. Then 2 mL of Nessler’s reagent (K2HgI4) was added.
Then the reading was taken at 420 nm on spectrophotometer. The same procedure repeated
for the standard solution of different concentration. Then the concentration determined with
the help of standard curve.
 14. Total Iron
Iron content in the water sample was determined by colorimetric method. In this method
50 mL of the sample was taken in a conical flask. 2 mL of concentrated HCl and 1 mL of
hydroxylamine hydrochloride solution was added. Then some glass beads were put in the
flask and boil till the content is reduced about half. It was cooled and 10 mL of acetate
buffer solution and 2 mL of Phenothroline solution was added, then orange red color was
appeared. Distilled water was added to make the volume 100 mL in a volumetric flask. Let
it stand for 10 minutes, and the absorbance of the color was measured by using
spectrophotometer at 510 nm using distilled water blank with the same amount of
chemical. The same procedure was repeated for standard solution of different
concentrations. Then the concentration was determined with the help of standard curve.

15. MPN Test:

The purity of drinking water regarding bacterial contamination is evaluated by testing the
presence of coliforms as these are designated as indicator organism of faecal
contaminations. Coliform bacteria in the water sample were determined by Most Probable
Number (MPN) or Multiple - Tube fermentation test. This test is performed sequentially in
3 stages:

1) Presumptive coliform test:

This is used to detect coliforms in a water sample. In this test lactose fermentation
tubes (Mac-Conkey Broth) were inoculated with different water volumes and
production of acid and gas from the fermentation of lactose within 48 hrs in any of the tubes
was the presumptive evidence of coliforms in the water sample.

a) 10 mL of water sample was inoculated in each of 3 tubes containing 10 mL of the

double strength of Mac-Conkey broth.
b) 1 mL of water sample was inoculated in each of 3 tubes containing 9 mL of the
single strength of Mac-Conkey broth.
c) 0.1 mL of water sample was inoculated in each of 3 tubes containing 9.9 mL of the single
strength of Mac-Conkey broth.

All the inoculated tubes were incubated at 37° C for 24 hours. After incubation, the
tubes were counted which had produced both acid and gas. The tubes showing
negative result were further incubated at 37° C for 24 hours. The tubes showing gas formation
and acid formation were further inoculated for confirmatory test.

2) Confirmed coliforms test:

This test is used to confirm the presence of coliforms and to determine the MPN
value in water sample. In this test water samples from all the positive presumptive
Mac Conkey broth tubes were inoculated into two sets of tubes of BGLB (Brilliant
Green Lactose Bile salt) broth and one set incubated at 37 oC for 24-48 hours- for total
coliform and another incubated at 44 0 C in water bath for 24 hours for fecal coliform.
Positive confirmed tubes were used to determine MPN/100ml by following statistical
Method (MPN table).

3) Completed coliforms test:

This test is used to establish the presence of Total Coliform as MPN/100 ml in a water
sample. Positive tube from the confirmatory was streaked on the plate of EMB Agar and
incubated it at 37° C for 24 hours. After 24 hours, the colonies that had typical growth (dark
centre with greenish metallic sheen) and atypical growth were transferred to nutrient agar slant
and Mac Conkey Broth and incubated at 37° C for 24 hours. The presence of total coliform and
faecal coliform bacteria was confirmed by Gram Staining and Spore Staining Technique.