MICROBIOLOGICAL PROFILE OF BOTTLEDWATER IN EGYPTIAN MARKET

By

AMIRA FATHY MOHAMED SAYDB. Sc. Agric. Sci., (Reclamation Technology and Culture Desert Lands),

Fac. Agric., Cairo Univ., 2008

THESISSubmitted in Partial Fulfillment of the

Requirements for the Degree of

MASTER OF SCIENCE

In

Agricultural Sciences(Agricultural Microbiology)

Department of Agricultural MicrobiologyFaculty of Agriculture

Cairo UniversityEGYPT

2015

Water is the most important resource for humans. Approximately 75% of the earth’ssurface is covered by water, but only 1% of that is drinkable. Twelve bottled waterbrands {9 domestic (A1, B2, A3, D4, H5, M6, N7, S8 and S9) and 3 imported (E10,H11 and Y12} were collected. Total bacterial count at 22oC and 35oC for all the testedbrands of bottled water are laying under the limits of the Egyptian law. The testedbrands of bottled water were almost free from indicator bacteria (coliform and fecalcoliform) and some other pathogenic bacteria. Unfortunately two brands (D4 and M6)were found to be contaminated with Pseudomonas aeruginosea, with counts of 285and 400 CFU/ 100 ml respectively. Protozoa detected in five tested bottled waterbrands B2, A3, D4, N7 and S8. All samples were under the recommendable limits forphysical characteristics, where the pH values ranged from 7.2 to 7.92 and the TDSvalues ranged from 137 to 485 mg /l. The samples showed that local brandscontaining bacteria at the start time and slight elevation in total count of bacteria after3 and 6 months, but the counts start to decline again at the 9th month and continued todecrease at the end of the year while the foreign brands are free of bacteria during thestudy. The unsuitable storing conditions (Sunny high from temperature ) has a greatthreat on the bacterial counts recorded in the selected bottled water brands as it ishighly increased during the study period.Key words: Bottled water, pathogenic bacteria, fungi, protozoa.

INTRODUCTION

Water is the most important resource for humans. It is an essential

element to life on the earth. Everyone needs water to survive, especially when

up to 60% of the human body is composed of it. Approximately 75% of the

earth’s surface is covered by water, but only 1% of that is drinkable (Soechtig,

2009). Therefore, clean drinking water is not as abundant as it may seem. With

water as a limited available resource and not as plentiful in some regions as it is

in others, it has recently become common for water to be bottled and sold.

Water forms 50% to 60% in weight of our bodies and play an active role in

all their vital processes. It allows digestion, food elaboration and waste

elimination. Every day we drink water or we eat watery food to replenish our

metabolic reserve.

Also, water is a very important element for a person so every day must

continuously and be sure to use clean water, Can we rely on drinking tap water

or it is preferable to buy bottled water?, especially now in Egypt we suffer from

ABSTRACT

water pollution in certain parts of our country and to maintain the health resort

many decide to use bottled water instead of tap water. The purity, portability

and the mineral content is important for consumption by humans. This behavior

is ideal when the bottled is pure water, when water companies use good

tracking measurements and specifications are for wells in terms of depths and

areas where these wells and ongoing analysis of the quality of this water,

especially the percentage of metals, especially heavy metals in the water?

The price of bottled water is extraordinarily higher than that of tap water,

but in most cases, the water quality is very similar. There is even a chance that

an expensive bottle of water is just purified municipal tap water.The increasing

dependency on bottled water may not have happened had our governments

neglected to preserve watersheds, monitor as well as update aging pipes and

infrastructure. There happens to be skeptics of both bottle and tap, but either

way, good quality drinking water is going to become harder and harder to find.

Overall, (Huber, 2010) wanted to prove that, bottled water is not all it is made

out to be, while noting that municipal systems also need to increase their

standards to provide clean drinking water to all .

Water is an essential requirement of all life forms. Satisfactory supply of

clean, safe and hygienic water is imperative for health. Drinking unsafe and

unhygienic water can cause high prevalence of waterborne diseases like

diarrhea, typhoid and cholera (Fawell and Nieuwenhuijsen, 2003 and Oyedeji

et al., 2010). As a preventive measure, consumption of bottled water has

increased in recent years in developing countries and elsewhere. Sale of bottled

water has gone to more than 35 billion US dollars (Raj, 2005) and by an

average of 12% increase in all over the world (Abd El-Salam et al., 2008).

People from all over the world drink about 13x1010 liters of bottled water

annually.

A comparison between the water composition and the maximum

contaminant levels imposed by the Egyptian standards and several other

international guidelines for all parameters was reported and discussed. They

1

varied in composition among the brands and from lot to lot for a particular

brand.

Therefore, the aim of this study is studying the effect of storing

conditions on the microbial community of each bottled water brand collected

from Egyptian markets.

REVIEW OF LITERATURE

Water is an essential element to life on planet earth. Everyone needs

water to survive, especially when up to 60% of the human body is composed of

it (USGS, 2009). Approximately 75% of the earth’s surface is covered by

water, but only 1% of that is drinkable (Soechtig, 2009). Therefore clean

drinking water is not as abundant as it may seem. With water as a limited

available resource, and not as plentiful in some regions as it is in others, it has

recently become common for water to be bottled and sold. Representative

Dennis Kucinich (D) of Ohio claims that, water is a basic human right, it’s

necessary for survival of life. When you start modifying the necessities of life

in such a way as to make it more difficult for people to gain access, you have

the basis for serious political instability (Soechtig, 2009).

There are four main reasons why people buy bottled water: fear of their

tap water, taste, style, and convenience (Gleick, 2010).

We no longer see water as a basic human right but as a product with an

enlarged price sticker that we pick up in the store, rather than get from our

kitchen sink, or water fountain. We no longer drink from public water fountains

and having easy accessibility to bottled water has decreased their demand. The

more we buy bottled water, the more we are convinced that, bottled water is not

a luxury, but rather a necessity (Gleick, 2010). We have been persuaded by big

businesses such as Nestlé, Coca-Cola and Pepsi to drink more bottled water.

Our nation has been brainwashed to believe that paying a thousand times more

for individual plastic bottles and not taking advantage of our readily available

tap water is a rational idea (Gleick, 2010).

We all try to avoid the things that we fear. Some people fear that, the

water that comes out of their tap will harm them. Fear of sickness and of

invisible contamination is an effective tool (Gleick, 2010).

Due to the fear of tap water, many drink bottled water assuming it is the

healthier option. “Some people have gone to drink bottled water literally

because they are concerned about their water, and the problem is they are

unaware of the fact that buying bottled water is not necessarily safe, that you

end up being exposed to other chemical compounds. (Soechtig, 2009). The

information label lists all the nutritional aspects that water lacks, but there are

still lots of things in our water (Gleick, 2010).

On contrary, bottled water can actually lead to health concerns for those

with a weak immune system, such as the elderly, infants, and cancer,

transplant, and HIV/AIDS patients (NRDC, 1999).

Bottled water consumption has been steadily growing in the world for

the past 30 years. It is the most dynamic sector of all the food and beverage

industry. Consumption in the world increases by an average of 12% each year,

in spite of its high price compared to tap water (Rosmann, 2005). Consumers

may have various reasons for purchasing bottled drinking-water, such as taste,

convenience, or fashion, but for many consumers, safety and potential health

benefits are important considerations because they believe bottled water is safer

than tap water. There are concerns about chlorine by-products, contaminants

such as lead, nitrates, and microorganism's contamination in municipal water

supplies. However, some microorganisms, which are normally of little or no

public health significance, may grow to higher levels in bottled waters (World

Health Organization, 2008).

Although certain mineral waters may be useful in providing essential

micro-nutrients, such as calcium and magnesium, World Health Organization

(WHO, 2000) is unaware of any convincing evidence to support the beneficial

effects of consuming such mineral waters and has no scientific information on

the benefits or hazards of consuming bottled waters with very low mineral

content (The Chartered Institution of Water and Environmental Management

CIWEM, 2006.)

Water is one of the most abundant and essential commodities of man,

occupying about 70% of the earth's surface, yet a greater percentage of the

world's population, particularly developing countries live without access to safe

water (Adriano and Joana, 2007). Water forms about 50 to 60% of our body

weight and plays an active role in our entire vital body metabolism: It allows

digestion, food elaboration and waste elimination. Although natural mineral

waters have been consumed since Roman times, only the 20th century has seen

the emergence of natural mineral water industry and the drinking of these

products on a large scale as an alternative to tap water and non-alcoholic

beverages (Stickler, 1989).

In recent years, coping with the modern human lifestyle, there has been a

tremendous increase in consumers demand for bottled mineral waters

worldwide (Warburton, 1993 and Venieri et al., 2006).

The United States Environmental Protection Agency (EPA) is the

regulatory agency behind the public water supplies of surface water through the

Safe Drinking Water Act. Bottled drinking water on the other hand, mainly

comes from groundwater, but since many municipalities already use surface

water as their source, and some bottlers use municipality sources, bottled water

can come from surface water as well.

Additionally, the Food and Drug Administration (FDA) regulates

bottled water as a packaged food under the Federal Food, Drug, and Cosmetic

Act. The EPA then creates the standards for tap water that is supplied by public

suppliers, while the FDA creates standards for bottled water based off the EPA

standards (EPA, 2005).

Until 1993, there were no proposed standards for bottled water. In 1996

Safe Drinking Water Act (SDWA) amendments require bottled water to meet

many of the same regulations as tap water for the first time (Knoxville Utilities

Board 2002). However, manufacturers of bottled water, unfortunately, do not

undergo the same rigorous quality standards and municipal water treatment

facilities bottled water is not regulated by the Environmental Protection

Agency (EPA) which is responsible for regulating public tap water supplies but

by the Food and Drug Administration (FDA) because it is considered a food

product not drinking water (National Sanitation Foundation International, NSF,

2004). Despite the FDA attempts to follow the EPA's tap-water standards, it is

not required to do so, thus allowing a greater range of bacterial contaminants to

be present in bottled water. (INC. 2008).

If Americans are buying so much bottled water, there has to be good

reasons why they are spend in substantial amounts of money on it (Gleick,

2010). A lot of Americans are afraid of waterborne diseases, microbes, and

dirty pathogens they do not really know anything about. The fear of tap water

could come from considerable media coverage about illness due to drinking

municipal tap water rather than drinking bottled, or because bottled water

advertisers inadvertently suggest that tap water is inferior to bottled. The

percentage of people who drink bottled water based on the fact that they think

it is safer than tap water is 35% according to the American Water Works

Association Research Foundation’s Consumer Attitude Survey on Water Quality

Issues. Huber, M. K. (2010).

Bottled water consumption in North America registered, in the last decade,

an annual growth rate of 25% (Bharath et al., 2003). European Federation of

Bottled Water (EFBW, 2006) estimated the consumption of bottled water

during 2003 as 45,000 ml. Bottled water is often expected to be purer

compared with tap water, although this is not necessarily the case.

Public awareness about waterborne diseases and poor quality control of

drinking water has been increased (Rosenberg, 2003). Both bottled and tap

water can be oligotrophic environments with sufficient nutrients to maintain

bacterial growth, releasing organic matter and providing additional substrates

for the microbial growth. The presence of opportunistic pathogens such as

Pseudomonas aeruginosa in mineral waters underscores the importance of

caution regarding the safety of these products, especially for health

compromised individuals.

The water quality index for drinking and bottled water by World Health

Organization (WHO, 2004 and 2006), Ministry of Health (Egypt) and United

Stated Environmental Protection Agency (USEPA) summarized below at Table

(1).

Table 1. Water quality standards and guidelines.

Drinking water guidelinesBottledwater

guideline

UnitPARAMETER

USEPAWHOEgyptEgyptPhysical Examination

11NTUNTUTurbidity80017002000SμElectrical Conductivity

6.5-8.516.5-9.2PH151520-30CUColor

acceptableacceptableAcceptableTaste and OdorChemical Examination

0.05-0.210.20.20.2mg ̸ 1Aluminium (AL)1.5mg ̸ 1)3Ammonia, total (NH

0.0060.0050.005mg ̸ 1Antimony0.010.010.050.01mg ̸ 1Arsenic (As)

7 MFLUmg ̸ 1Asbestos20.70.7mg ̸ 1Barium

0.004NADmg ̸ 1BeryliumNANA6.0NAmg ̸ 1Biological Oxygen

)2Demand (BOD)(OO.3mg ̸ 1Boron(Bo)

0.0050.0030.0050.003mg ̸ 1Cadmium(Cd)200mg ̸ 1)+

2Calcium (Camg ̸ 1)2Carbon Dioxide (CO

NANA10.0mg ̸ 1Chemical Oxygen)2Demand (COD) (O

250250500250mg ̸ 1)-Chloride (CL0.10.050.050.05mg ̸ 1Chromium, total (Cr)1.3212mg ̸ 1Copper (Cu)0.20.070.050.07mg ̸ 1Cyanide (Cn)NANA5.0NAmg ̸ 1Dissolved Oxygen

)2(O41.50.80.3mg ̸ 1Fluoride (F)

0.05mg ̸ 1Hydrogen SulfideS)2(H

0.310.30.30.3mg ̸ 1Iron (Fe)0.0150.010.050.01mg ̸ 1Lead (pb)

150mg ̸ 1)+2Magnesium (Mg

0.0510.50.10.1mg ̸ 1Manganese (Mn)0.0020.0010.0010.001mg ̸ 1Mercury "total" Hg

0.070.07mg ̸ 1Molybdenum (Mo)0.020.02mg ̸ 1Nickel (Ni)

45504450mg ̸ 1)-3Nitrate (NO3.330.0160.02mg ̸ 1)-2Nitrite (NO

mg ̸ 1)3+4Phosphate (PO

0.050.010.010.01mg ̸ 1)+Potassium (K0.050.010.010.01mg ̸ 1Selenium (Se)

mg ̸ 1)2Silica (SiO0.11Umg ̸ 1Silver (Ag)

200200200mg ̸ 1)+Sodium (Na2501250400250mg ̸ 1)4Sulfate (SO0.0020.01mg ̸ 1Thallium

Umg ̸ 1Tin (T)mg ̸ 1Total Alkalinity

)3(CaCo

Cont.

Table 1. Continued.

USEPA, United Stated Environmental Protection Agency; TT, treatment technique is mandated in lieuof a parameter concentration limit (treatment goal value); NR, not currently regulated or norecommended value; U, unnecessary a health-based guideline value as these compounds are nothazardous to human health at concentration normally found in drinking-Water ; CU, color unit; NTU,nephlometric turbidity unit; TON, threshold odor number; MFL, million fibers per liter; HPC,heterotrophic plate count; 1. In case of large supplies, where sufficient samples are examined, must notbe present in 95% of samples taken throughout any 12- month period; NA, not available.

(WHO, 2004 and 2006)

1. Bottled water characteristics

The market is inundated with a large number of brands of bottled water.

Various countries have enforced drinking water standards for the maximum

permissible levels of different constituents (Misund et al., 1999). Due to

increased demand and consumption of bottled water there has been a growing

concern about the quality of these products. In recent times concerns have been

Drinking water guidelinesBottledwater

guideline

UnitPARAMETER

USEPAWHOEgyptEgypt51353mg ̸ 1Zinc (Zn)

Disinfectants and Disinfectant by- product455mg ̸ 1Chlorine

325 } 4 as Cl

33mg ̸ 1Monochloramine55mg ̸ 1Di and tri- chloramines

10.20.20.2mg ̸ 1Chloritezs0.0100.0250.0250.025mg ̸ 1Bromated

Biological ExaminationNRNRFreeFreecell ̸ 1Blue green algae

Bacteriological ExaminationFreefreeNRFreeCells ̸

mlE. coli (thermotolerantcoliform)

FreeNRFreeFreeCells ̸ml

Fecal Coliform

95% of>40

routinesample are

free

free3cells ̸100

FreeCells ̸ml

Total Coliformbacteria

HPC;TT(free)

<50 cell ̸NR ml 24hrs at 37 ͦC 48 hrsat 22 ͦC

Cfu ̸ ml<50 cell ̸ NRml 24 hrs at37 ͦ C 48 hrs

at 22 ͦC

Cfu ̸ mlTotal bacteria (Platecount )

TT,99%killed or

inactivated

NRNRNRCfu ̸ mlGiardia lamblia

TT,99%killed

NRNRNRCfu ̸ mlLegionella

expressed about the increase in poor quality of well water due to the nitrate

pollution through continuous and liberal use of organic manure and inorganic

fertilizers (Mahler, et al., 2007). Therefore, recently consumption of bottled

water has been increas. But, the quality of bottled water used for human

consumption is not subjected to any stringent quality control measures.

Recent study by the International Bottled Water Association (IBWA)

revealed that 25% of all bottled water is simply tap water placed in a bottle

which is a valid method of bottling water by the FDA under certain Good

Manufacturing Practices (GMPs) regulations. Wikipedia, The Free

Encyclopedia (Bottled Water Information, 2008).

In addition, the quality of bottled water can also substantially vary

among brands as well as with time and with different production runs

depending on its source, treatment technology, manufacturing operation,

packaging material, and shelf-life before use (NRDC, 1999). Although, bottled

water should have a shelf life of 30 days unopened, most bottled water

companies' label showed that their water is valid for 1 to 2 years. On the other

hand, bottled water is most commonly disinfected with ozone, which provides

a residual disinfection for a limited time and subsequently does not leave a

residual taste like tap water, which uses chlorine as a final disinfectant. The

length of time chlorine and ozone remains active in water depends on many

factors, including temperature. However, bottled water may be in distribution

and storage conditions for several weeks which may adversely affect its quality

(Kendall, 2007).

2. Microbiological quality of bottled water

Water should be free from any organisms. But unfortunately water is

not always found pure. The contamination of natural water with fecal material,

domestic and industrial sewage as well as agricultural and pasture run off may

result in an increased risk of disease transmission to humans (Medema et al.,

2003).

The microbiological quality of bottled water is of great interest as

many consumers use it as an alternative to tap water and consider it to be better

and safer. The quality of water is determined largely by bacteriological

analysis. In bottled waters, the bottling process may be a source of additional

contamination. In addition, the common sources of contamination of bottled

water are equipment, bottles and caps, exposure to air and contact with humans

during the bottling process (Warburton et al., 1998).

Although the microbial quantity levels in processed water are often

initially low, they can evolve rapidly to high levels during storage (Stickler,

1992). This rapid growth of bacteria after the water is bottled may be due to

oxygenation of the water during processing, the increase in surface area

provided by the bottle, the increase in temperature and the amount of nutrients

arising from the bottle (Warburton et al., 1992; Guerzon et al., 1994 and

Warburton, 2000). They reported that, some bacteria can multiply on polyvinyl

chloride of ultra marine blue dye in bottle plastic material. Another factor to be

taken into account is whether the water is carbonated, Since the decrease in pH

resulting from carbonation acts to prevent bacterial growth (Moreira et al.,

1994).

Bottled drinking water is defined as natural water packaged in bottle that

has definite physical, chemical and microbiological specifications. These

specifications are met through physical treatment. The source from

underground must be far from any source of contamination and is not treated

by chlorine. Bottled water must be at least as good in bacteriological quality as

potable water and thus the total bacterial count should not exceed 50 cells ml-1

at 37°C after 24h. It should also be free from coliform organisms, E. coli, E.

faecalis, P. aeruginosa and C. perfringens (WHO, 1984). Several methods are

used for bottled water to meet the required specification. Among these methods

are filtrations by using special kinds of filters and ultraviolet exposure.

Although modern treatment processes and stringent regulations mean

that tap water is generally safe to drink in developed countries, microbial

pathogens remain the major public health risk associated with drinking water.

Major waterborne pathogens include bacteria (e.g, pathogenic Escherichia coli,

Campylobacter and Salmonella spp.), protozoa (e.g., Cryptosporidium parvum

and Giardia lamblia), and viruses (e.g., adenoviruses, entero - viruses and

Rota-viruses) (Karin Leder et al., 2002). In addition to that many filamentous

fungi appear in various surface water and underground water, but during the

last years they have been found in various drinking water as well (Arvanitidou

et al., 2000; Warris et al., 2001; Zacheuset al.,2001; Cabral and Fernandez,

2002; Gottlich et al., 2002 and Canovas et al ., 2003).

Epidemiological studies have reported the occurrence of disease

including problems with reproduction (Aschengrau et al., 1989) cancer (Shy et

al., 1982), Congenital malformations of the central nervous system (Morton,

1976 and Sherman et al., 1988), cardiovascular disease (Morris et al., 1961 and

Anderson, 1971) and even death (Eisenberg,1986 and 1992). Due to exposure

to trace elements and mineral contents of water.

Other routes, including food and person-to-person spread, may transmit

these organisms and drinking water is thought to be a minor contributor to

disease in most Australian communities. For example, a recent randomized

controlled trial in Melbourne found no difference in gastro-enteritis rates

between people who drank regular tap water and those who drank filtered,

ultraviolet-treated water, suggesting that drinking water does not contribute

significantly to background rates of gastroenteritis in that city (Hellard et al.,

2001).

In general, people are concerned about the poor quality of tap water that

is why they have switched over to bottled water perceiving it to be clean and

safe. Although, people consider bottled water safe but it can be also be

contaminated with chemical and biological agents (Khaniki et al., 2010).

Presence of coliform bacteria, E.coli or Pseudomonas in bottled water can pose

a great threat to the public health. Infants, young children, debilitated and

immuno-compromised people are at high risk of waterborne diseases, even at

lower infective doses (WHO, 2005).

Water borne diseases are one of the major health problems especially in

developing nations. The high prevalence of disease such as diarrhea, typhoid

fever, cholera and bacillary dysentery has been linked to the consumption of

unsafe water produced under unhygienic production practices. Water quality is

generally defined as the physical, chemical and bacteriological characteristics

of water in relation to the requirements to human need (Johnson et al., 1997

and Diersing, 2009). In Ghana, the required physical, chemical, microbial and

radiological properties of drinking water is adopted from the World Health

Organizations (WHO, 1993, 1996, 2001and 2002).

Pseudomonas aeruginosa has been found in some mineral waters in

various countries such as Brazil, Canada, France, Germany, Spain, United

States and others counters. Also, examination of drinking water for

Pseudomonas aeruginosa is not recommended as a routine procedure, but it

can be used as an indicator of good manufacturing processes and suitability for

drinking water. Bacteria belonging to the genus Pseudomonas are widely

spread in the environment and are often opportunistic bacteria for many

episodes of infections (Schindler et al., 1995). During the period of storage the

growth of Pseudomonas aeruginosa may lead to a risk for consumers

especially the immunologically weak persons, as well as very young or elderly

ones (Legnani et al., 1999).

In addition, Mardani (2007) reported that, the initial microbial counts of

the examined bottled water moderately increased during the increase of storage

time at room temperature. Improvement of the quality of bottled water will be

achieved only by improving the manufacturing processes and subsequent

storage condition (Warburton et al., 1986).

A survey of bottled water conducted in the United Arab Emirates,

where about 90% of the populations drink bottled mineral water; showed that

out of 2.0-1.5 liters bottles, 40% were bacteriologically contaminated (Nsanze

et al., 1999).

However, new researches claimed that; not all bottled water are pure,

for example Abed and Al-wakeel (2007) found that, of the thirty bottled

samples tested in Saudi Arabia, 2 of 9 bottled samples showed contamination

with Bacillus cereus and Pseudomonas spp., mineral contents of 9 branded

bottled water showed differences in levels and traces of aluminum were

recorded in all bottled water samples. Yamaguchi et al. (2007) found that 35%

and 20% of the total number of tested samples of mineral bottled water were

contaminated with fungi and total coliform bacteria, respectively.

3. Chemical characters of bottled water

The chemical quality of drinking water during recent years has

deteriorated considerably due to the presence of toxic elements, which even in

trace amounts can cause serious health hazards (Ikem et al., 2002).

a. Calcium and Magnesium

Calcium (Ca) and Magnesium (Mg) are essential elements for our body.

They can be provided to our organisms by food, but even diets rich in calcium

and magnesium intake may not be able to compensate their absence in drinking

water. Calcium is important for fetal growth, pregnancy and lactation (Heany,

1993 and Garzon and Eisenberg, 1998). It is essential in our body for teeth and

bones formation, blood coagulation, right functioning of our nervous system.

Drinking calcium poor water is considered dangerous for the risk of

coronary diseases. An excess in calcium can alter the water taste or cause

scaling problems in pipes and household appliances. The World Health

Organization (WHO, 2002) recommends a minimum calcium daily intake of

about 700 mg.

Also, magnesium plays an important role as a cofactor and activator of

more than 300 enzymatic reactions including glycolysis, ATP metabolism,

transport of elements such as sodium (Na), potassium (K) and calcium through

membranes, synthesis of proteins and nucleic acids, neuromuscular excitability

and muscle contraction. Magnesium deficiency increases risk to humans of

developing various pathological conditions such as vasoconstrictions,

hypertension, cardiac arrhythmia, atherosclerotic vascular disease, and acute

myocardial infarction, preeclampsia in pregnant women, possibly diabetes

mellitus of type II and osteoporosis. The daily recommended intake for Mg is

150-500 mg (WHO, 2002).

b. Sodium

The recommended daily intake of Sodium (Na) is 20 mg. numerous

studies have shown that a high Na+ intake is associated with hypertension

(Heany et al., 1982) and that dietary Na+ restriction, achieved by not adding salt

and avoiding Na+ rich foods, may effectively reduce blood pressure (Garzon, et

al., 1998). However, drinking certain waters may unnecessarily increase Na+

intake to a level may be detrimental for health, especially for individuals on a

Na+ restricted diet. (Garzon et al,. 1998 and Pomeranz, 2002).

c. Chloride

Chlorinated water in which the anion Cl- is dominant (hypertonic)

increases internal peristalsis. If water has low concentration (hypotonic) it

stimulates gastric secretion. It can have spasmodic, coleretic action and protect

liver from toxic compounds. The recommended daily intake is 7-15 g (Garzon,

1998 and Amato et al., 1999).

d. Fluoride

Fluorine (F) is useful for the good health of bones and teeth,

sometimes it is even essential. Fluorine values useful for our body are close to

toxic values, so a dispense not aimed and personalized can cause high risk of

overdosing and chronicle poisoning, with consequent skeleton deformation,

spots on tooth enamel, osteosclerosis, neurological disorders, damages on the

thyroids and even tumors. The maximum admissible content in water is 1.7 mg

L (Garzon, 1998 and Amato, 1999).

e. Toxic substances

Other inorganic toxic substances are found in water. The sulphate (SO4)

content of water should be lower than 200 mg L. Nitrates (NO4) are the main

source of nitrogen essential for nucleic acids and amino acids. Nitrate water

content of about 10 mg L is considered normal and natural. Excessive nitrate

presence can be transformed to nitrites (NO3) which are toxic to the human

body. Aluminum (Al) is not important for human nutrition. It can have toxic

effects even in small qualities. (Lopez et al., 2002 and Mazzeo-Farina, and

Cerulli, 1985)

The WHO (2005) recommends an Aluminum concentration in water lower

than 20 mg L. Bicarbonates (HCO3) in water should have a concentration lower

than 600 mg L. Mineral contents for both bottled and tap water samples were

determined by inductively coupled plasma-mass spectrometry (ICP-MS),

where variable and higher concentrations of different anions and cations were

observed in tap water rather than in bottled water samples. Water samples were

further subjected to solar disinfection system (SODIS) resulting in a notable

decrease in bacterial cell count present in water samples being tested.

f. Microbiological and physicochemical standards

The water sources from which the bottled water is produced must meet a

number of microbiological and physicochemical standards. In addition, the

water source should be free from parasites and pathogenic microorganism

(Warburton et al., 1998). The approved sources for bottled water may be

springs, wells, or other sources that have been analyzed and found to be safe

with or without treatment (Ramalho et al., 2001). The overall treatment of the

water source depends on the initial quality of water (Warburton and Austin,

1997). Bottled water is generally not sterile and may contain bacteria from

naturally occurring source as well as those introduced during manufacturing

and consumer handling (Warburton and Austin, 1997).

4. Effect of storage temperature on microbial bottled water content

Storage temperature has an effect on the bacterial count in bottled

mineral water. Viable counts, after incubation at 22°C, were 10 times higher in

water stored at 6°C compared with water stored at 20°C (Gonzalez et al.,

1987). Bottle size can also affect bacterial counts (Bischofberger et al., 1990).

In experimental inoculation studies these authors found that, counts in 10 ml

bottles increased to over 106cfu ml-1, whilst counts only reached 10scfu ml-1 in

one liter glass flasks.

The reason for this is the increasing volume: surface area (V: S) ratio in

larger bottles. With low V: S ratios, a higher proportion of available nutrients

can become bound to the glass surface.

Burge and Hunter (1990) also found that E. coli was able to persist in

bottled mineral water for about 42 d. They reported, however, that Salmonella

typhimurium, Ps. aeruginosa and Aeromonas hydrophila persisted for at least

70 d in still mineral water and expressed concern about the use of E. coli as an

efficient indicator of potential faecal contamination with salmonellas. Burge

and Hunter (1990) also reported that, Campylobacter jejuni persisted for only

2-4 d but noted that Camp. jejuni can persist in a viable (and infectious) but

non-cultivable state in waters. When these same bacteria were inoculated in

carbonated waters, survival was reduced by about 50%, though only by about

25% for Salmonella typhimurium (Burge and Hunter, 1990).

5. Natural characteristics types' aquifer system

The groundwater in Egypt could be divided into 6 main aquifer

systems (Attia, 1999):

1. The Nile delta and the Nile Valley aquifer system (the aquifer system on

which the present study will conducted), assigned to the quaternary and

late Tertiary, occupies the Nile flood plain region from Aswan to delta.

The groundwater stored in aquifer is estimated to be in range of 200

billion m3. Groundwater in this system cannot be considered a separate

source of water, as the aquifer is mainly recharged as a result of

activities based on the Nile water. Including seepage from canals and

deep percolation from irrigation application (subsurface drainage). The

aquifer, however, can be utilized as a regulatory\storage reservoir.

2. The Nubian sandstone aquifer system, assigned to the Paleozoic-

Mesozoic, occupies mainly the Western Desert. It is the most potential

non-Nile aquifer system containing nonrenewable groundwater. The

total groundwater volume in storage in the Nubian sandstone is

estimated at 60,000 billion m3. The current total extraction amounts

about 0.5 billion m3 \year. However the economic annual maximum

extraction cannot exceed 5 billion m3\ years.

3. The moghra aquifer system assigned to the lower Miocene, occupies

mainly the western edge of the Delta.

4. The Coastal aquifer system assigned to the Quaternary and Late

Tertiary, located along the coastal zones of Egypt, Mediterranean Sea

coast and Red sea coast.

5. The Karstified Carbonate aquifer system, assigned to the Eocene and to

the Upper Cretaceous, outcrops in the northern part of the Western

desert and along the Nile system. About 200 natural springs are flowing

with total discharge of20, 000 m3 / day.

6. The Fissured and Weathered hard rock aquifer system, assigned to the

pre-Cambrian, outcrops in the Eastern Desert and the Sinai Peninsula.

6. Health risks associated with consumption of bad quality water

a. Physical and chemical contents

The presence of physical and chemical contamination in groundwater

consumed by human being performs great health risks. Dissolved substances

(TDS) may be organic or inorganic and many of them are undesirable in water

and produce displeasing color, taste and odors (Chapman, 1992). High contents

of nitrogen forms (nitrate, nitrite and ammonia) in water reduce the oxygen-

carrying capacity of the blood (David and Richared, 2000). Heavy metals

accumulated in human body tissues, for example arsenicis accumulated in hair

and nails, cadmium in kidney, mercury in hair and kidney and lead in bone and

teethe (Anspaugh et al., 1971; Hamilton et al., 1973; Snyder, 1975; and

Fergusson, 1990). Certain metals have been implicated carcinogenesis

including chromium, nickel, cobalt, lead, iron, and cadmium (Deligiannis et al.,

1986 and de Marteleare et al., 1988).

b. Pathogenic bacteria

On the other hand, the presence of pathogenic bacteria in groundwater

contributes a very dangerous health risks and diseases such as; cholera caused

by Vibrio chlera, Typhoid fever, diarrhea and Salmonillosis caused by S. typhi

and other Salmonella spp; Shigellosis and bacillary dysentery by Shigella, as

well as, gastroenteritis by E.coli and Campylobacter jejuni (Cheesbrough,

1984). About 70% of the waterborne microbial illness outbreaks in the United

States have been associated with groundwater (JIN and Flury, 2002).

Chryseobacterium meningosepticum is ubiquitous Gram- negative rod

historically associated with meningitis in premature neonates (Bloch et al.,

1997). The authors reported 15 positive cultures and 6 cases of infection among

immune-compromised adults.They found that there are 308 reports of positive

cultures, of which 59% were determined to represent true infection. Sixty-five

percent of those infected persons were younger than 3 months of age.

Meningitis was the most common infectious syndrome among neonates, seen

in 84% of cases and associated with a 57% mortality rate.

Less commonly reported infection among infants included sepsis (13%)

and pneumonia (3%). Pneumonia was the most frequent infection among the

post- neonatal group, accounting for 40% of cases, followed by sepsis (24%),

meningitis (18%), endocarditic (3%), cellulites (3%), abdominal infections

(3%), eye infections (3%) and single case reports of sinusitis, bronchitis as well

as epididymitis. The 6 cases in their report were all adults with a mean age of

58.7 years. Sites of C. meningosepticum infection were limited to the lungs,

bloodstream and gall bladder. Infection was associated with prolonged

hospitalization, prior exposure to multiple antibiotics and host immune

compromise, particularly neutropenia.

In an outbreak of gastroenteritis affecting 730 students in Taiwan in

September 1993. Shigella sonnei and Entamoeba histolytica were isolated from

the fecal specimens of patients. Environmental investigations reveal the source

of infection to be contamination of underground well water by sewage from

toilet (Chen et al., 2001).

Miettinen et al. (2001) reported 14 waterborne epidemics occurred in

Finland during 1998-1999 and about 7300 illness cases were registered in these

outbreaks; all were associated with nomadic infected groundwater.

The main reasons of groundwater outbreaks were floods and surface

runoffs which contaminated the water. In most cases the outbreaks ceased by

boiling the drinking water before use and starting chlorination.

The world health organization (WHO, 1987) has estimated that up to

80% of all sickness and diseases in the world is caused by inadequate sanitation

polluted water or unavailable of water. Concerning the epidemics caused by

waterborne pathogens in Egypt, Ezzat (2002) reported some of them; in 1947

cholera epidemic affected the whole country, about 50% of the total infected

patients were died. In 1973 typhoid epidemic was localized in a small village in

Damietta Governorate, where about 400 students and villagers fell ill. In 1986

diarrhea outbreak occurred in many villages in Hosainia district, Sharqiya

Goverorate. Jousilahti et al. (1997) estimated the incidence of diarrhea in

Egypt, Geographically the survey was limited to two Governorates, Daqahliya

and Gharbiya, which have the largest population (7.12 million) and where

thought to be representative of Delta Egypt. The total sample size was 11032

people. Seasonally adjusted diarrhea incidence was 3.6 episodes per child

under five years of age per year. This means a minimum estimate of 30 million

cases annually in Egypt.

Recently, three more epidemics of typhoid were recorded; one of them

was at Desouq district, Kafr El-Shaykh Governorate in 2005 where about 400

patients fell ill, the second was at Al-Mansoura city. Daqahliya Governorate in

2006 and the third were at El-Baradaa village, Kalubiya Governorate in July

2009. In most cases the investigations revealed that the groundwater was the

route of infection.

c. Protozoa

The fresh-water free-living protozoa have a cosmopolitan distribution as

has been pointed out by Bovee (1957), Cairns (1962) and Corliss (1973). It is

also well known that, the diversity, density, and kind of protozoa present in

waters may be used as indicators of their quality and portability (Cairns &

Dickson, 1972). A widely used method to determine pollution is one employed

in Europe named "Saprobic System" (Kolwitz & Marsson, 1908 and Cairns et

al., 1972).

Therefore, since protozoa can pollute the drinkable waters which flow

through the distribution duct system of a city, a study on this matter is well

justified, especially in countries such as Mexico where the knowledge on

ecology of free-living protozoa is scant and mostly superficial.

d. Fungi

Fungi are widely distributed in nature and can occur as unicellular yeast

or filamentous and, multi cellular molds. There are over 70,000 species of

fungi. Fewer than 300 have been implicated in human diseases, and fewer than

a dozen cause about 90% of all fungus infections. They are involved in

different forms of diseases, including allergies to fungal antigens, production of

toxins, or direct invasion of hosts (McGinnis, 1996).

A one year fungal survey of a water bottling plant was conducted in order

to evaluate the incidence and fluctuations of the biota. The dominant fungal

genera in order of highest number were order Penicillium, Cladosporium and

Trichoderma followed by Aspergillus and Paecilomyces. As expected, highest

number of isolates collected was during the summer months, particularly May

and June. Indeed during these two months there were more fungi present in the

water after it had passed through the filtration system (0.4^m filter), indicating

that during those times of the year when fungal contamination is high, filters

should be changed on a more regular basis.

In order to assess whether contamination was single or multi-cellular,

molecular methods based on PCR were used. Overall fungal contamination

arose from multiple sources. Some fungal strains were very "alike" and were

detected during different sampling times, indicating that some strains were

endemic to the plant. There was little evidence to suggest that fungi detected in

the source water passed through to other parts of the plant. However, there was

evidence that fungal strains isolated from the water filter were detected

elsewhere in the factory, confirming the need to change filters more regularly

during periods of high fungal contamination. In order to improve quality

control, a HACCP programmer was implemented and Best Practice Guidelines

introduced (Ribeiro et al., 2006).

Several species of fungi are capable of infecting healthy hosts and

causing diseases ranging from mucosal to life-threatening disseminated

infections. In addition, there is an increasing number of severe fungal diseases

by commensally or fully saprophytic species in immunocompromised hosts.

These diseases are frequently associated with abrogated host immunity as a

result of viral infections, mainly the human immunodeficiency virus,

hematological and hormonal disorders, organ transplants, antibiotic usage, and

more intensive and aggressive medicals practices (van Burik and Magee,

2001). Fungal infections were difficult to treat since the agents were

eukaryotes, as human cells. Despite their wide occurrence, little attention has

been given to their presence and significance in aquatic environments (Souza et

al., 2003). Drinking water distribution systems are colonized by saprophytic

heterotrophic microorganisms (such as bacteria, fungi, yeast) that grow on

biodegradable organic matter (Servais et al., 1992). However, potentially

pathogenic microorganism and microorganisms of fecal origin can also find

favorable condition and proliferate in these systems (Petrucio et al., 2005).

Zoosporic fungi from different water habitats have been studied in many

parts of the world by numerous researchers (Ziegler, 1958; Suzuki, 1960;

Roberts, 1963; Alabi, 1971 a and b, 1973 and 1974; Hunter, 1975; Hasija and

Batra, 1978 and EI-Hissy, 1994). In Egypt, zoosporic fungi have been isolated

and studied from waters of the River Nile, Ibrahimia canal, in some ponds and

irrigation canals (EI-Hissy, 1974; EI-Hissy et aI., 1982; EI-Nagdy and Abdel-

Hafez, 1990; EI-Nagdy and Khallil, 1991 and Khallil et al., 1991). The

terrestrial fungal flora of Aswan High Dam Lake (AHDL) have been studied by

EI-Hissy et al. (1990) and Moharram et al. (1990).

e. Bacteria

The bacterial flora of bottled mineral water increases in numbers after

bottling, reaching a peak by the end of 1 week (Yurdusev and Ducluzeau 1985;

Gonzalez et al., 1987; Bischofberger et al., 1990 and Morais and da Costa,

1990). After this time the bacterial count remains fairly constant for at least 6

months. The bacterial counts in still waters reach higher levels than in

carbonated waters. There is some debate about whether bacterial counts reach

higher levels in plastic compared with glass bottles. Several authors have found

higher counts in waters stored in plastic or PVC bottles when compared with

glass (Del Vecchio and Fischetti, 1972; De Felip et al., 1976 and Yurdusev and

Ducluzeau, 1985). The reason put forward by these authors was that the inner

surface of plastic bottles was rougher, so promoting adhesion and colonization.

By contrast Morais and da Costa (1990) found no real difference.

The quantity of bacteria in commercial mineral water is generally

dependent of good manufacturing practices and autochthonous flora of the

spring. It is well known that natural mineral water is characterized by its

bacterial flora, chemical and physical composition. In addition to natural

contamination, the product can also be deteriorated before it reaches the

consumer (Nsanze and Babarinde, 1999).

The Brazilian directives (ANVISA, 2000 and 2004) regulate water from

municipal water supplies on the basis of coliform content and heterotrophic

plate count, whereas more stringent bottled mineral water regulations prohibit

the presence of a group of potentially pathogenic bacteria (Pseudomonas, fecal

streptococci and clostridia).

Although microbiological standards exist for bottled water, the same

product once installed on a dispenser is generally not regulated and is rarely

controlled.

f. Microbial diversity in aquifer system

The FDA as well as International Bottled Water Association (IBWA)

classified bottled water according to its origin. Some of the more common

types of bottled water are: artesian water which is derived from a well tapping a

confined aquifer (a water bearing rock, rock formation, or group of rocks) in

which the water level stands above the water table; mineral water that contains

not less than 500 ppm total dissolved solids (TDS); sparkling water or

carbonated that contains carbon dioxide; spring water which is derived from an

underground formation from which water flows naturally to the earth's surface;

well water which is from a hole bored, drilled, or otherwise constructed in the

ground which taps the water of an aquifer and purified water which has been

produced by a process of distillation, deionization, reverse osmosis, or other

suitable process that contains not more than 10 mg/L TDS and it has an

electrical conductivity (EC) of not more than 10 AS/cm (Bullers, 2002).

The Centers for Disease Control and Prevention (CDC) reported a 1994

outbreak of cholera in the United States associated with bottled water. The

brand of water involved was not named. The bottled water plants producing the

water involved in this outbreak reportedly obtain their water mainly from

municipal water and some of the wells used tested positive for fecal coliform

bacteria. In Portugal, although cholera outbreak occurred in the mid-1970s due

to the use of bottled water from a contaminated limestone aquifer by broken

sewers from a nearby village but it demonstrates the continuing potential for

contaminated bottled water to spread waterborne disease (Natural Resources

Defense Council, NRDC, 1999).

American legislation does not distinguish between bottled waters and

bottled mineral waters, classing them all as bottled waters (Rosenberg. 1990).

SUMMARY

Water is the most important resource for humans. It is an essential

element to life on the earth. Everyone needs water to survive, especially when

up to 60% of the human body is composed of it. Approximately 75% of the

earth’s surface is covered by water, but only 1% of that is drinkable. Therefore,

clean drinking water is not as abundant as it may seem. With water as a limited

available resource and not as plentiful in some regions as it is in others, it

recently become common for water to be bottled and sold.

Twelve bottled water brands {9 domestic i.e. A1, B2, A3, D4, H5, M6,

N7, S8 and S9 and 3 imported i.e. E10, H11 and Y12} were collected.

Number of samples were collected once time then a different analysis

was performed immediately at zero time for twelve samples then repeated after

3 months passed for another twelve samples from the same collected samples

and so on for five times subsequently to complete a one year of analysis to

assure the guarantee of the bottled water as the manufactures are ensuring and

to study the effect of storing conditions on the microbial community of each

bottled water brand.

Total bacterial count at 22oC and 35oC for all the tested brands of

bottled water are laying under the limits of the Egyptian low for bottled water

for year 2007 which mentioned that they should not exceed 100 and 50 CFU/

ml, respectively. Baraka brand was the highest counts (98 and 49 CFU/ ml for

22oC and 35oC , followed by S8 brand (30 and 20 CFU/ ml), M6 brand (28 and

16 CFU/ml), D4 brand (26 and 14 CFU/ ml), H5 brand (21 and 10 CFU/ml),

N7 brand (14 and 8 CFU/ml), A3 brand (12 and 5 CFU/ml) and in the end S9

brand (9 and 5 CFU/ ml, respectively). The spore forming bacteria presented in

few counts ranged from 2 to 7 CFU/ ml in D4, H5, M6, N7 and S8 brands,

respectively. It is worth to mention that, A1, E10, H11, and Y12 brand were

found free from bacteria.

The tested brands of bottled water were almost free from indicator

bacteria (coliform and fecal coliform) and some other pathogenic bacteria

where the total count of coliform bacteria, fecal coliform bacteria and fecal

streptococcoi were zero, in addition, the Aerumonas hydrophilla and

Staphylococcus aureus did not detected. Unfortunately two brands (D4 and

M6) were found to be contaminated with Pseudomonas aeruginosea, with

counts of 285 and 400 CFU/ 100 ml respectively.

Protozoa detected in five tested bottled water brands B2, A3, D4, N7 and

S8.

The physical characteristics showed that all samples were under the

recommended limits, where the pH values ranged from 7.20 to 7.92 and the

TDS values ranged from 137 to 485 mg /l.

Anions and cations were also under the recommended limits except for

potassium whereas two local samples had 16 and 15 ppm.

On the other hand, heavy metals content of all bottled water samples

showed results under the guidelines mandated by Egyptian law, WHO and

USEPA which indicates that it cannot be a reason for health hazard.

Effect of the refrigerator on the samples showed that local brands

containing bacteria at the zero time and slight elevation in total count of

bacteria was shown after 3 and 6 months, but the counts start to decline again at

the 9th month and continued to decrease until the end of the year, while the

foreign brands were proved to be free of bacteria during the study.

The storage at room temperature showed that the water brands contained

bacteria at zero time, and total count of bacteria increased during the twelve

months of the study. The bad storing conditions (Sunny ambient) have a great

threat since the bacterial counts recorded in the selected bottled water brands as

it is highly increased during the study period.