Journal of Science (JOS) ISSN 2324-9854
Vol. 4, No. 3, 2015, Pages: 390-395
Copyright © World Science Publisher, United States
www.worldsciencepublisher.org
390
Physicochemical studies and bacteriological assay of Sachet Water
samples marketed in Kogi state University Compound, Anyigba.
Onojah, P.K., Odin, E.M. and Ochala, A.U.,
Department of Pure and Industrial Chemistry Kogi State University, Anyigba.
Abstract- Fifteen samples made of five (5)brands of sachet packaged water samples labelled as A,B,C,D and E
commonly found in the Kogi State University, Anyigba were examined for bacteriological and physic-chemical
properties to determine the potability if the sachet water are to be used in the school. Standard conventional methods
were employed for the detection of coliforms and other bacteria. Physical examination for organoleptic qualitysuch as
taste, color, odour,microscopic examination for sediments and other debris and/or bacteria, protozoa and fungal hyphae
as well as chemical and biochemical analysis. Bacteriological examination of samples revealed the presence of the
following pathogens: Yeast, staphylococcus species, streptococcus species, E,coli, Bacillus species, micrococcus
species, pseudomonas species Actinomycetes, Klebsiell., Chemical analysis revealed the presence of metals ranging
from lead (Pb), iron (Fe), and chromium (Cr). Absence of nickel, cadmium and copper was apparent from the study.
Physical examination of samples showed a variable level of turbidity, colour, PH, hardness, acidity and alkalinity. The
result also revealed variable level of taste but none had objectionable odour. In conclusion, bacteriological and
physicochemical indices of contamination detected from majority of the samples are indications that sachet water
available in the University environment do not meet, neither the NAFDAC (2004), SON (2003) nor WHO (2012),
standard and so may not be suitable for drinking purposes.
Keywords: Sachet water, physicochemical, Biochemical and Bacteriological studies, organoleptic quality, coliform
bacteria.
1. INTRODUCTION
Water has always been a subject to great interest to
man since it is essential to human survival. Man needs
water for industrial development, navigation, irrigation to
grow food, generation of hydro-electric power, recreation
and enhancement of fish, wildlife and host of other
purposes. Water has found its wildest use in the industry
as a medium of heat transfer, heat exchangers; it also
functions as raw materials in the beverage and chemical
industry. The energy required to rapture the hydrogen
bonds and liberate a molecule of water to a vapour is
much greater than of other chemical compounds liquid
water consists of a continuous network of randomly
connected hydrogen bonds which form liquid molecules
that move freely. Water in its pure form is colourless,
tasteless and sparking in nature. (Egereonu, 2006).
The quest for cheap and readily available source of
potable water has led to the emergence of sachet water.
Packaged water is defined as any potable water processed
and offered for sale in sealed food grade bottles or other
appropriate containers for human consumption. (Food
and Drug Administration, 2002).Anne, (2002), reported
that with the significant increase in sachet or bottle water
consumption, there has arisen a growing concern over the
bacteriological and chemical quality of these products.
Bottle or sachet water like any other food product, must
be processed and packaged under aseptic conditions.
Packaged water however is generally not sterile, being
collected from almost every available water source,
ranging from rain water to tanker borne water most of
which are rusty and unwashed. Contaminants are also
introduced during manufacturing and consumer handling
(Warburton and Austin, 1997). Irrespective of their
sources, these products are susceptible to microbial
contamination. Again, the absence of sterilization
procedures such as pasteurization and thermal
sterilization for the treatment of pure water increase their
susceptibility to contamination by both autochthonous
bacterial flora, exogenous contaminating microbes, as
well as a variety of other contaminants including mineral
salts, organic pollutants, heavy metals and radioactive
residues.
The quality of drinking water is a powerful
determination of health. Assurance of drinking water
safety is a foundation; for the prevention and control of
water borne diseases. In many developing countries,
availability of water has become a critical and urgent
problem. Because of the magnitude of health hazards
associated with water, water sold to the public must be
whole some and must meet World Health Organisation
(WHO) Standards. (Oyeku, et al, 2001). Safe drinking
water is a basic need for human development, health and
well being; it is an internationally accepted human right.
(WHO, 2001). The chemical contaminants of drinking
water are often considered a lower priority than microbial
contaminants, as adverse health effects from chemical
contaminants are generally associated with long term
exposures, whereas the effects from microbial
contaminants are usually immediate. Nonetheless,
chemicals in water supplies can cause very serious
problems. (WHO, 2007).
The abundance of toxic chemicals in drinking water
may cause adverse effects on human health such as
391
cancer and chronic illness (Al-Saleh and Al-Doush,
1998).
Epidemiological studies have indicated a strong
association between the occurrences of several diseases
in human, particularly cardiovascular diseases, kidney
related disorders, neurocognitive effects and various
forms of cancer. (Ikem, et al, 2002).
Water may contain toxic metals like mercury, lead,
cadmium, Arsenic and Selenium. These metals can cause
acute or chronic poisoning and should be eliminated from
drinking water if possible. Several metal ions such as
sodium, calcium and magnesium are essential to sustain
biological life. Other additional metals are also essential
for optimal growth, development and reproduction like
zinc which is in small enough quantities to be considered
trace elements. Trace metals function mostly as catalyst
for enzymatic activity in human bodies; however their
accumulation; in the human body causes harmful effects.
(Skeat, 1969).
Water quality refers to the physical, chemical and
Biological characteristics of water. (Diersing, 2009). It is
a measure of the condition of water relative to the
requirement of one or more biotic species and/ or to any
human need or purpose. It is most frequently used by
reference to a set of standard against which compliance
can be assessed. The most common standard used to
assess water quality relate to health of ecosystems safety
of human contact and drinking water (US.EPA,20002005).
The assessment of water quality has beenclosely
monitored by many international bodies such as World
Health Organization (WHO), Environmental Protection
Agency (EPA), European Community (EU) American
Public Health Association (APHA), American Water
Works Association (AWWA), and also Federal
Environmental Protection Agency (FEPA). In Nigeria,
National Environmental Standard Regulation and
Enforcement Agency (NESREA) and National Agency
for Food and Drug Administration Commission
(NAFDAC) have prescribed their own standard.
Analysis of water samples for presences of
microorganisms in relation to human health requires
determining principally the pathogenic organizations.
(Gordon, 1990), the most significant of which are faecal
bacteria. The organisms most commonly used as
indicator of faecal pollution are the coliform bacteria.
Coliform bacteria occur in high numbers in human faeces
and can be detected at occurrences as low as one
bacterium per 100ml; therefore they are sensitive
indicators of faecal pollution. Organisms found in water
are involved in food poisoning and frequent outbreaks of
water- borne infections (Cholera, Salmonellosis,
gastroenteritis shigellosis, etc). Among the heavy metals
of medical importance found in water are: aluminum,
copper, lead, chromium and iron (Dibua, et al, 2007).
The US Environmental Agency Health-based
Standards (Maximum contaminant levels or MCLS) in
Miligrams per litre (mg/L), approximately equivalent to
parts per million (ppm) is as follows: Aliminum: 0.2 mg/l;
chromium; 0.05 mg/l; Lead: 0.01 mg/l; Iron: 0.03 mg/l.
minimum standards are generally accepted for the
coliform bacteria. However, the stipulated criteria
indicate that:
1) In any year, 90% of samples taken at any point
of the system should be coliform free.
2) Maximum count in any positive test should be
10 coliforms per 100 ml.
3) No sample should contain more than two E. coil
per 100 ml.
4) No sample should contain 1 or 2 E. coil per 100
ml in conjunction with a total count of 3 or more
per 100 ml.
5) Coliform should not be detectable in 100 ml of
any consecutive samples.
It is against this background that this work was caused
out with the view of determining the microbiological
standards as well as physic chemical compositions of
locally available sachet water with the campus resultant
safety and potability indices. Despite the large market for
bottled water today, there have been relatively few
investigations into the public health aspects of these
products. (Hunter and Burge 1987).Vonwiesenberger
(2004), pointed out that some water have very short
transit time making them potentially susceptible to
contaminations. Previous analysis have reported that
some microorganisms are associated with drinking water
and that sachet water cannot undergo any treatment such
as pasteurization and thermal sterilization for the
elimination of these microbes (Cruickshank, 1968), and
when these natural water are sources of industrial water
for the manufacture of packaged sachet water, there may
be the possibility of these packaged water becoming
contaminated with these heavy metals which may have
serious effect on health.
Consequently, this study is aimed at ascertaining the
physico-chemistry and microbiology of sachet water sold
within the University campus with the view of finding
out whether these meet the standard to be considered safe
for drinking.
2. Material and Methods
Material:
The material and reagents used includes: pH meter,
Beakers, thermometers, turbidity meter, conical flasks,
measuring cylinders, Burettes, Retort stand, Atomic
Absorptionspectrophotemetre (AAS), 4.0 pH buffer
solution, 7.0 PH buffer solution, 9.2 pH buffer solution,
4MHCL, Erichrome black T indicator, 0.01MEDT
solution, MC cradys statistical table, petri dishes, sample
bottles, culture media, counters, pipette and graduated
glassware, dilution bottles.
Methods:
Sample Collection: The water sample used for these
work were sachet water samples sold within Kogi State
University campus, Anyigba. They are; Erigray, Aditi,
KSU, Ayes Oasis and Ramboil sachet water.
Sample preservation: Sachet water samples from
five (5) different sources were filled in 100ml amount
into sterile plastic disposable bottles and preserved in
aseptic conditions as recommended by the standard
392
methods of Greenberg (1992). Samples were refrigerated
at 40C and analysed within 24hr of collection.
pH Measurement: The PH value of each sample was
carried out using a micro-pHmetre (pH 600-Milwanked)
standard with buffer solution to pH 7.0, 9.2 and 14 as
described by Walter (1981).
Physical Examination: Physical examination was
aimed at detecting odour, taste, colour and turbidity in
water and the measures of its acceptability or
attractiveness to consumers. This involves careful
examination of samples for some organoleptic parameters:
taste, odour as well as colour, turbidity/presence of
sediments.
Odour: five 50ml wide-mouthed glass-stoppered
bottles were rinsed with 4M hydrochloric acid until
completely odourless, and then re-rinsed with distilled
water. The bottles were half-filled with each sachet water
sample, stoppered and shaken vigorously for 3 seconds.
The stoppers were then removed and bottles observed for
odour by putting the nostril near the mouth of the bottle.
Taste: Stoppered bottles were rinsed with
4MHydrochloric acid and then re-rinsed with distilled
water. Aliquots of each sample were then poured into
each bottles and the taste noted immediately.
Colour: Colour profile of each sample was measured
by comparing the water samples against a standard
prepared from potassium chloroplatinate (k2PEcl2) tinted
with small amount of cobalt chloride. (C0Cl2.6H2O)
which yielded colour very much like water. The standard
and test samples were each dispensed into nessler tubes
to 50ml volume and then placed on white surfaces
(whitetiles) to match the colour of standards against
samples. The colour of the sample closest to the standard
was selected and the corresponding units recorded.
Acidity profile: 100ml of the sachet water samples
were dispensed into 250ml conical flasks and 3 drops of
phenolphthalein indicator was added and titrated against
the standard 0.02NNaoH until faint pink colour
development atpH 8.3. Acidity was calculated.
(Greenberg, 1992).
Alkali Profile: 100ml of the water sample was poured
into a 250ml conical flasks. 3 drops of methyl orange
indicator was added. The burette was rinsed with 10ml
0.1MHCl which was done before the first titration. The
burette was filled with the acid and the initial volume
recorded. The sample was titrated with 0.1MHCl to the
end point (orange to red) and the final volume was
recorded. The alkalinity of the sample in ppm (mg/l) was
calculated.
Microscopy: Microscopic examination of the
specimens was carried out to check for ova, cysts, worms,
and trophozoites of protozoa. Ten milliliters (10ml) of
each sample was concentrated by centrifugation and a
loop of the deposits then view under X40 objective of the
light microscope.
Viable Bacterial count: One milliliter (1ml) of each
sample was serially diluted (10-fold) to thin out the
microbial population; in previously sterilized Ringers
solution and 0.1ml of 10-3 and 10-4 of each sample was
dispensed into the nutrient agar and MacConkey plates,
spread-inoculated (to ensure even distribution) and then
incubated at 370o C for 24h. Viable bacterial count was
carried out using the hand lens, following which discrete
colonies were transferred into slants for Gram staining
reaction and other biochemical tests according to the
method of Cheesbrough (1991).
Presumptive Coliform Test: Coliform test was
performed to detect coliform bacteria (using E. coli as the
indicator organism) in the water samples according to the
method described by Cheesbrough (1991).
Determination of Most Probable Number (MPN) of
Coliforms: Numbers of positive tubes with acid (Yellow
coloration) and gas production were match with the
Mecrady’s statistical Table, and the most probale number
(MPN) of coliforms present in 100ml of each samples
was thus determined. For the confirmation test, a loopful
of cultures from the presumptive test was inoculated into
brilliant green broth containing Durham tubes acid
inoculated for 48hrs at 370C. Gas production confirmed
presence of E. coli. Cultures were further inoculated into
eosin methylene blue medium and incubated at 370C for
24hrs. A positive test was indicated by purple-green
metallic sheen on the surface of the colonies.
Determination of heavy metals:Heavy metal
analysis described by Standard Methods for the
Examination of Water and Waste water (1998) was
performed to ascertain the level of these in the test water
samples as these have been reported to cause serious
health problems such as cancer, learning inability, gastric
irritation, etc when found in concentration higher than the
tolerant concentration. The heavy metals include: Pb, Ni,
Cd, Fe, Cr, Cu,.
Determination of Total Hardness by EDTA
Titration: 50ml of the water sample was taken into a
250ml conical flask, 5ml of 0.1MHCl was added and
heated. When cooled, 0.1MNaOH was added and 2ml of
PH 10 Ammoniacal buffer was added. A drop of
Eriochrome black T indicator was added and the solution
was titrated with 0.01MEDTA solution until the reddish
colour turned to clear blue. Total hardness as CaCO3 was
calculated through the formula:Total hardness as CaCO3 (ppm) = volume of
0.01MEDTA X 1000
Ml of sample taken
Determination of chlorides
1ml of Al(OH)3 was added to 50ml of the water
sample, filtered and washed with distilled water. The PH
was adjusted between 7-10 with sodium hydroxide
solution (NaOH) by adding 2ml of 0.02MNaOH. 0.5ml
of potassium dichromate indicator was added and it was
treated with standard silver nitrate solution until pinkish
yellow end point was observed. The standard silver
nitrate was titrated with blank in the same way as
outlined above. Samples were calculated using the
reagent blank by the formula:
Cl mg/l = A-B X N X 35.50 X1000
ml of sample taken
393
A = ml of sample taken
3. Results
B = ml of titre for the blank
Results of the microscopic view of analysed sachet
water samples revealed the absence of Ova, cysts or
trophozoites of protozoa in the test samples.
N = concentration of the titre
Table 2. Present the results of physical examination of analyzed samples. Trace of chlorine and rancidity were apparent
in Ramboil Sachet water. Odour and colour was not detected in any of the sample.
Parameters
samples
A
PH (at 250C)
Temperature (0C)
Turbidity (NTU)
Colour (TCU)
Taste
OdourHardness (mg/l)
Acidity (mg/l)
Alkalinity (mg/l)
Chloride (mg/l)
- = negative,
B
7.47
28
0.01
15
-
C
7.07
28
0.16
13
-
-
7.12
28
0.20
13
-
28
4.0
45
7.09
D
E
6.33
28
0.18
14
20
3.0
17.5
23
32
2.0
5.5
27.5
42.5
7.09 7.09
6.61
28
0.21
13
13
2.5
40.0
7.09
7.09
+ = positive
NTU :Nephelometric Turbidity unit
TCU:
True colour unit.
Colour however met the acceptable standard which should not exceed 15 TCU.
Result of chemical analysis of samples for heavy metals is presented in table 3. Samples exceeded the NAFDAC,
SON (2003) and WHO (2012) tolerant concentrations for lead, iron, chromium. However, all samples met the tolerant
concentrations for Nickel, cadmium and copper.
Table 3: Heavy metal analysis of sachet water samples (ppm) or (mg/ml)
Elements
A
B
C
Lead (Pb)
0.13
0.11
0.15
0.10
Nickel (Ni) ND
NDNDNDND
Cadmuim (Cd)
ND
NDNDNDND
Iron (Fe)
0.76
1.00
0.92
Chromium (Cr)
0.51
0.30
0.38
Copper (Cu)
ND
NDNDNDND
D
E
0.12
0.84
0.27
0.79
0.19
ND = Not Detected
In table 4, results of the viable bacterial counts obtained from bacteriological assay of tested sachet water were
shown. Significant bacterial counts (in colony forming units – CFU/ml) was observed (p = 0.05). The result obtained
showed significant amount of gas production in four of the samples tested. In Eosin methylene blue medium, there was
also pure purple metallic sheen on the colonies, thus showing the presence of E.coli in the water samples.
Table 4: Total viable bacterial count of isolates from test samples.
394
Isolate
E. coli
Klebsiellaspp
Pseudomonas spp
Strephylococussaureus
Bacillus species
Steptococus species
Micrococus species
Yeast
Actinomycetes
4. Discussion
Results of the physicochemical and bacteriological
analysis on the sachet water of common use within the
Kogi State University environment indicate that some of
the sachet waters did not meet the required NAFDAC,
SON, OR WHO standards for portable water. From the
physicochemical analysis objectionable taste was
observed in one of the samples. This could be attributed
to severable factor such as the presence of both inorganic
compounds (ammonia, sulphides, chlorides and cyanides);
organic substances including unsaturated hydrocarbons
as well as bacteria contaminants which could have been
introduced during processing, packaging or distribution,
storages as well as long storage of the sachet water. The
Standard Organization of Nigeria (SON) and NAFDAC
Maximum Allowed Units (TCU) are 100mg/L; while
WHO maximum permissible unit is 250mg/l (see table 1).
These permissible values donot impact chlorine taste or
odour to potable water, though results showed that
chlorine taste was apparent.
The pH value ranges from 6.33-7.47 with sachet water
E having the highest pH. (7.47). In comparison with the
NAFDAC, SON and WHO standard for pH, all the
sachet water samples were in line with the standards
except that of samples D (6.33). The institute of public
analysts of Nigeria (IPAN) indicated that pH of water is
one of the most important water parameters. It is a
measure of the acidity or alkalinity of water (IPAN,
2005). An optimal pH range D of immense necessity for
the clarification and disinfection of potable water, while a
range outside the acceptable could enhance the rancidity
and subsequently presence of malodour and objectionable
taste as observed in the results. One of the samples (pH
6.33) deviates from the WHO, NAFDAC and SON
permissible pH range 6.5 -9.5. From visual observation,
the tested samples were however free of particles.
Hardness of the sachet water samples was observed
between 13 mg/l and 32 mg/l. The hardness of all the
sachet water samples were within the permissible level of
NAFDAC, SON (100mg/l), WHO highest desirable
(100mg/l) and WHO maximum permissible had (500,g/l).
Results of the heavy metal analysis indicated the
presences of chromium, lead and iron. Samples A,B, C,
D, and E had higher values of the above than others. This
result conforms to earlier report by Pocok et al, (1984),
that water found in nature generally contains a variety of
contaminants such as mineral salts, heavy metals, organic
chemicals, and radioactive residues. Therefore when this
counts (log 10 cfu/ml).
2.10
2.19
3.20
5.30
5.13
5.00
4.70
4.50
3.21
natural water serve as industrial water for the
manufacture of packaged water, there may be the
possibility of their packaged water becoming
contaminated with these chemicals and heavy metals
which may have deleterious effects on health. According
to Goyer (1991), drinking water containing heavy metals
like chromium, aluminum and lead in concentrations
higher than the tolerant concentration have detrimental
effect on health resulting to cancer, learning inability and
behavioral problem in children.
The presence of pathogens including streptococcus
species, staphylococcusaureus, micrococcus species,
klebsiella species, Bacillus species, pseudomonas species,
E.coli, yeast and Actinomycetes is an indication of
serious contamination of samples. The presence of these
pathogens could account for the incidence of diarrhoea,
food poisoning and gastro enteritis common in the
University environment especially among the
undergraduate students. According to Hunter and Burge
(1987), food intoxication could occur due to the presence
of bacterial pathogens such as staphylococcus and
Bacillus in drinking water. E.coli is an example of faecal
coliforms which are indicators of coliform contamination
of water and these occurs in high numbers in human
faeces bacterium per 100ml. From table 4, the presences
of the coliforms at values above the maximum accepted
standards, an indication of high fiscal contamination and
consequently the danger of food poisoning and other
related gastrointestinal
disorders arising from
consumption of these sachet waters. The result also
indicates that these water do not undergo appropriate
sterilization techniques and also do not conform to either
NAFDAC or WHO standards for potable water as
supported by the work of Cruickshank (1968) showing
that some microorganisms are associated with drinking
water and since sachet water cannot undergo any
treatment such as pasteurization and thermal sterilization
for the elimination of these microorganisms, they are
never free from bacteria (Guilot and Lecters, 1993). The
reason being that most sachet water are sourced from
shallow wells or tanker-delivered waters which are never
washed or sterilized.
The market of sachet and bottle water is national with
a population of over one hundred and forty million
(140,000,000) people and an estimated national
population growth rate of 5.7% per annum, average
economic growth rate of 3.5% per annum in the past five
(5) years. Nigeria is a large expanding and sustainable
market for sachet and bottle water. The quest for quick
395
money has resulted in sachet water business and the
associated inability to pass through treatment processes
to remove all pathogenic organisms and heavy metals
that have caused serious health problems.
In conclusion, it is necessary that the sachet water
business be critically reviewed by NAFDAC to ensure
that producers comply with standards at every stage of
the production and distribution processes. It is of utmost
importance that regulatory authorities intensify their
inspection activities in this ever growing area of sachet
water production as they should ensure that producers
adhere strictly to the stipulated standards to avoid public
health hazards. Finally, NAFDAC should also educate
both the producers as well as consumers on the health
hazards of untreated or contaminated sachet waters.
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