SODIUM HYPOCHLORITE SUSCEPIBILITY OF COLIFORMS ISOLATED FROM 
CHLORINATED WATER SUPPLIES IN GWERU

By

EDISON MUPANDAGUTA

A dissertation submitted in partial fulfillment of the requirements for the Bachelor of Science Honours Degree in Biological Science

Department of Biological Sciences
Faculty of Science and Technology
Midlands State University

June 2014
Abstract
A study was carried out to determine the prevalence and evaluate the susceptibility to sodium hypochlorite of chlorinated water coliforms in Gweru municipal water supplies. Municipal water samples were collected from ten randomly selected suburbs between February and April 2014 from which the residual chlorine and presence of coliforms was determined. Evaluations of the susceptibility of isolated coliforms to sodium hypochlorite were done at concentrations ranging from 0.05 mg l-1 to 0.3 mg l-1  for 0, 5 10, 15, 20, 25 and 30 minute time intervals. The free residual chlorine concentration of municipal water obtained for the ten suburbs ranged from 0.05 mg l-1 to 0.4 mg l-1 depending on the proximity of the suburb to Gwenhoro water treatment plant. The furthest suburbs, Woodlands and Harben Park had the least mean residual chlorine levels (0.05 mg l-1) while the closest suburb, Southdowns, had the highest mean residual chlorine level (0.4 mg l-1). Chlorinated water coliform bacteria were isolated from 1 out of 30 samples collected, giving a prevalence of 3.33%. It took between 25 to 30 minutes for all the isolated chlorinated water coliforms to be killed by 0.2 mg l-1 of sodium hypochlorite, 10 minutes by 0.25 mg l-1 and 5 minutes by 0.3 mg l-1. The sodium hypochlorite concentration of 0.3 mg l-1 was most effective at killing chlorinated water coliforms as it does so within the minimum exposure time. This study serves as a baseline foundation on which further studies can be built in order to provide the most suitable and effective guidelines on sodium hypochlorite water disinfection in the homes that use municipal water supplies.

DEDICATION

To my late father. 

ACKNOWLEDGEMENTS

My sincere gratitude goes to Mrs Muteveri for her excellent supervision which made this project a success and the entire Biological sciences laboratory crew for their support during the course of the project session. I owe special thanks to my family, all my classmates and friends for providing support in all forms. Above all, it is the Lord whose loving kindness is channeling me towards a great destiny.

Table of contents
Contents                                                                                                                                                 page 

CHAPTER 1: INTRODUCTION
1.1 Background
1.2 Justification of study
1.3 Objectives of the study
1.3.1 Major objective
CHAPTER 2: LITERATURE REVIEW
2.1 Introduction
2.2 Coliforms
2.3 Occurrence of coliforms
2.4 Sodium hypochlorite
2.6 Municipal water treatment process
2.6.1 Preconditioning
2.6.2 Coagulation
2.6.3 Sedimentation and clarification
2.6.4 Filtration
CHAPTER 3: MATERIALS AND METHODS
3.1 Study area
3.2 Sampling
3.2.1 Sampling for microbial analysis
3.2.2 Sampling for determination of residual chlorine level
3.3 Media preparation
3.3.1 Kovac's reagent
3.4 Testing for residual chlorine
3.5 Presumptive coliform test
3.5.1 Confirmed test
3.5.2 Completed test
3.6 Estimation of bacterial densities from slants
3.7 Coliform susceptibility testing
3.8 Data analysis
CHAPTER 4: RESULTS
4.1 Prevalence of coliforms and residual free chlorine level
4.2 Coliform susceptibility to sodium hypochlorite
4.2.1 Coliform susceptibility to 0.05 mg l-1 sodium hypochlorite
4.2.2 Coliform susceptibility to 0.1 mg l-1 sodium hypochlorite
4.2.3 Coliform susceptibility to 0.15 mg l-1 sodium hypochlorite
4.2.4 Coliform susceptibility to 0.2 mg l-1 sodium hypochlorite
4.2.5 Coliform susceptibility to 0.25 mg l-1 sodium hypochlorite
4.2.6 Coliform susceptibility to 0.3 mg l-1 sodium hypochlorite
CHAPTER 5: DISCUSSION
5.1 Isolation and prevalence of coliforms
5.2 Residual chlorine level in water
5.3 Susceptibility of coliforms to sodium hypochlorite
5.4 Conclusion
5.5 Recommendations
REFERENCES
APPENDIX 1……………………………………………………………………………………………...37

LIST OF TABLES
Table                                                                                                                                          page 
Table 4.1: Mean residual chlorine, coliform prevalence and coliform density for the ten suburbs in Gweru……………………………………………………………………………………………………...20
Table 4.2: Initial counts for test coliform and the reference control…………………………………….. 21

LIST OF FIGURES
Figure                                                                                                                                    page 
Figure 4.1: Percentage coliform survival at 0.05 mg l-1 NaOCl concentration…………………………..22
Figure 4.2: Percentage coliform survival at 0.1 mg l-1 NaOCl concentration……………………………23
Figure 4.3: Percentage coliform bacteria at 0.15 mg l-1 NaOCl concentration…………………………..24
Figure 4.4: Percentage coliform survival at 0.2 mg l-1 NaOCl concentration……………………………25
Figure 4.5: Percentage coliform survival at 0.25 mg l-1 NaOCl concentration…………………………..25
Figure 4.6: Percentage coliform survival at 0.3 mg l-1 NaOCl concentration…………………………....26

CHAPTER 1: INTRODUCTION

1.1	Background
Disease-causing organisms are transmitted from host to host in many ways including through contaminated water supplies. Diseases caused by contaminated water consumption and poor hygiene practices are the leading causes of death among children worldwide (Tabor et al., 2011). Contaminated water is one of the oldest known public health concerns such that prevention of water-borne diseases has turned out to be one of the primary objectives of any drinking water supply system. The World Health Organisation declares in one of its primary goals that “all people have the right to have access to an adequate supply of safe drinking water, regardless of their stage of development and their social and economic conditions” (WHO, 2004). This idea has also been reinforced by the United Nations through the adoption of Millennium Development Goal 7 target 10 which  is aimed at halving the proportion of people without sustainable access to safe drinking water and basic sanitation by the year 2015 (U.N, 2006). Thus, water for home use should be free of pathogenic microbes that cause such illnesses as typhoid fever, dysentery, cholera, and gastroenteritis (Gleick, 2005).

Ascertaining the microbial quality of drinking water is a vital water quality test since a glass of water containing just a few disease causing organisms can cause illness. Infants, elderly and the immunocompromised individuals are the most vulnerable. Testing for presence of coliforms is therefore an ideal yardstick for determining a water supply's microbial quality (Stevens et al., 2003).

Coliform bacteria comprise of several bacterial genera belonging to the family Enterobacteriaceae. They are aerobic and facultative anaerobic, gram negative, non-spore forming, rod shaped bacteria that ferment lactose producing gas within 48hrs at 35ºC (Rompre et al., 2001). Most types of coliform bacteria are harmless to humans, but some can cause mild illnesses and a few can lead to serious waterborne diseases (Ignnnugbemi et al., 2009).

Coliforms are recognised as “indicator organisms” as they are indicative of the potential existence of pathogenic microbes in a water supply system (Campbell et al., 2011). Presence of coliforms in water may indicate the existence of a contamination pathway between their source and the water supply system (Stevens et al., 2003). A highly positive coliform test result shows considerable contamination requiring immediate action (Wohlsen et al., 2006).

Water disinfection is an important final step in the municipal water treatment process and is done in several ways (Bishankha et al., 2011). These include ozonation, chlorination, ultraviolet radiation, filtration and other physical means.  Ozone is produced by passing oxygen or dry air through a system of high voltage electrodes. Chlorine can be added in the form of chlorine gas, sodium hypochlorite or calcium hypochlorite and ultraviolet radiation disinfects water using light generated by special lamps (Pickard et al., 2006).  

Among all the disinfection methods used to get rid of water microbes, chlorination has been the most commonly adopted method due to its long term history of effectiveness as well as its low cost (Kumar et al., 2011). Chlorination reduces pathogenic microorganisms in water to levels that are tolerable from the public and health viewpoint thereby preventing the transmission of waterborne diseases (LeChevallier et al., 1988). 
Chlorination is a simple, reliable, effective, and safe way to protect drinking water supplies against contamination by microorganisms (Pickard et al., 2006). The methods and equipment needed to use chlorine safely are well designed and its effectiveness is well understood since it has been used for many years. One of the main advantages of using chlorine in drinking water is that once treated, a small residual amount of chlorine remains in the water distribution system to provide continuing protection from microbiological contamination while the water is delivered to taps at different distances. However chlorination can generate carcinogenic or other chemicals which are harmful to the environment (Kumar et al., 2011).

Chlorine has become one of the most widely used disinfectants found in homes, industry and health care facilities in the form of sodium hypochlorite. When used at levels that comply with the WHO Guidelines for drinking water-quality, chlorine is effective against almost all pathogenic microbes (WHO, 2004). Chlorine levels in water can fluctuate at or below the level at which it has been applied due to the flow rate of the water in the system, the proximity of the tap to the water treatment plant and also during maintenance periods. The concentration of the residual chlorine disinfectant in the distribution system can also be influenced by the assimilable organic carbon level, the type and concentration of disinfectant, water temperature, and system hydraulics (USEPA, 2004). Entry of foreign material into the distribution system from backflow may alter these factors and contribute to a loss of residual chlorine residual. The World Health Organization (WHO) has established a guideline value of not more than 5 mg l-1 residual chlorine in drinking water and such concentrations are considered acceptable for lifelong human consumption (WHO, 2004).

Occurrence of coliform bacteria in chlorinated water has become an issue since currently standardised chlorine levels are seemingly failing to get rid of these and other microbes (King, 1988). Occurrences of coliforms in chlorinated drinking water is associated with outbreaks of waterborne diseases. Researchers documented increased levels of antibiotic resistant bacteria as a result of water treatment processes including disinfection (LeChevallier et al., 1988). 

1.2	Justification of study
Chlorine is known to have very high antimicrobial properties that it is used in the treatment of water in cities and towns to reduce the microbial load of such water to safe levels for human consumption. However the occurrence of coliform bacteria and related diseases in chlorinated water is common, suggesting development of some resistance to disinfection by microorganisms. The Ministry of Health and Child Care Epidemic – Prone diseases weekly reports show that 55 deaths of common diarrhoea were recorded across Zimbabwe in January 2014.  Of the 55 deaths, 12 were recorded from the Midlands Province (UNOCHA, 2014).

The presence of coliform bacteria in municipal drinking water would be a significant reflection of how far Gweru water treatment plants are towards accomplishing the Millennium Development Goal 7 target 10 which is aimed at halving the proportion of people without sustainable access to safe drinking water and basic sanitation by 2015 (Gleick, 2005).

Municipal water in Gweru is treated with chlorine before being supplied to consumers. Currently, the public is being encouraged to use ‘Water guard’, a disinfectant containing 1.25% sodium hypochlorite as the active ingredient for further water disinfection. This study therefore is aimed at empirically determining the actual concentration of sodium hypochlorite that would be effective for municipal water disinfection before use by Gweru residents. To date, no such study has been conducted in Gweru and this study sought to generate baseline data on the susceptibility of chlorinated water coliforms to sodium hypochlorite.

1.3	Objectives of the study 
1.3.1	Major objective
The purpose of this study was to determine the concentration within the WHO guidelines at which sodium hypochlorite is effective (kills 99.9%) against chlorinated water coliforms under Gweru water treatment conditions.

1.3.2	Specific objectives
•	to determine the level of residual chlorine in Gweru chlorinated water supplies, 
•	to isolate chlorinated water coliforms from Gweru chlorinated water supplies,   
•	to determine the prevalence of coliforms in  chlorinated water supplies in Gweru, and
•	determine the most effective dose of sodium hypochlorite for further disinfection of Gweru municipal water.

CHAPTER 2: LITERATURE REVIEW

2.1 Introduction
Several waterborne diseases are caused by opportunistic and pathogenic bacteria that are found at lower levels than the traditional indicators of water quality (Martinez-Hernandez et al., 2013). The number of different types of pathogens that can be present in water as a result of pollution with human or animal faeces is very large and it is not possible to test water samples for each specific pathogen. The concept of coliforms as bacterial indicators of microbial water quality is based on the idea that because coliforms are present in high numbers in the faeces of humans and other warm-blooded animals and if faecal pollution has entered drinking water, it is likely that these bacteria will be present, even after significant dilution and thus pathogens might be present (Stevens et al., 2003).

The practice of disinfection of water using sodium hypochlorite has become a widely adopted technique for controlling microbial contaminants in homes. The development of resistance to chemical compounds emerges from the selective pressure bacterial populations undergo when fronted by these chemicals. Bacterial resistance poses a considerable concern due to the genetic variability and adaptive ability shown by these microorganisms when fronted by disinfectants (Silveira et al., 2006).

The regrowth of bacteria within any water distribution system is of concern to water authorities for a variety of reasons. High numbers of environmental coliforms undermine the validity of routine sampling programs and often lower annual water quality results into the non-compliance categories. Many microorganisms found in distribution systems also have the capacity to behave as opportunistic pathogens (Stevens et al., 2003).
2.2 Coliforms
The coliform group is comprised of Gram-negative, nonspore-forming, aerobic to facultative anaerobic rods, which ferment lactose to acid and gas. Two organisms in this group include Escherichia coli and Enterobacter aerogenes. However, the only true faecal coliform is E. coli, which is found only in faecal material of warm-blooded animals. Escherichia coli is the only thermotolerant member of this group. The presence of this organism in a water supply is evidence of faecal contamination and is sufficient to order the water supply closed until tests no longer detect E. coli. The outer membrane of gram-negative bacteria acts as a barrier that limits the entry of many chemically unrelated types of antibacterial agents (Stevens et al., 2003). 

2.3 Occurrence of coliforms 
Water may be contaminated with pathogens at the source but contamination may also occur during distribution, transportation, or handling in households or other working places. Regrowth of thermotolerant coliforms in the distribution system are unlikely unless sufficient bacterial nutrients are present or the water temperature is above 15°C, and there is no residual free chlorine. Such warm conditions can favour the regrowth of organisms like thermotolerant coliforms in the distribution systems (WHO, 2004)

Studies to determine the factors that contribute to the occurrence of coliform bacteria in chlorinated water have shown that the occurrence of coliform bacteria can be related to the following factors: filtration, temperature, disinfectant type and residual, assimilable organic carbon (AOC) level, corrosion control and pipe material selection ( LeChevallier, 2003). Good quality drinking water can suffer serious contamination in distribution systems because of breaches in the integrity of pipelines and storage reservoirs (Bishankha et al., 2011). 
Occurrence of coliforms maybe attributed to failure to maintain adequate residual disinfectant within the distribution system. Low pipeline water pressure, intermittent service, excessive network leakages, corrosion of parts, and inadequate sewage disposal also have a contributory effect (Lee and Schwab, 2005). Coliform occurrences in a distribution system may also be attributed to bacterial regrowth in the mains and interrupted water supply which allows pathogens to grow in the distribution lines. Other causes are attributed to unskilled persons tampering with lines to connect water into homes or to repair broken lines. Underground leakages can also be a significant contributory factor (LeChevallier, 2003). 

Resistance to stress factors like chlorine can be induced by environmental conditions resulting in expression of genes that increase bacterial resistance. Suspended solid particles and organic matter can provide protection to microorganisms by generating a demand for free residual chlorine, which decreases the availability of chlorine and weakens the disinfection process. Clumping of microbes within the distribution system is another factor that confers resistance to chlorine disinfection (Martinez-Hernandez et al., 2013). 

The way through which physiological adaptation can play a role in conferring intrinsic resistance to disinfection in bacteria is demonstrated by biofilms. These are microbial populations that grow on the inside of pipes and other surfaces. Biofilms in pipework can also provide ecological niches that are suited to the potential survival of pathogens. They can be responsible for a wide range of water quality and operational problems (LeChevallier, 2003). 

Biofilms can also contribute to loss of distribution system disinfectant residuals, increased bacterial levels, reduction of dissolved oxygen, taste and odour changes, red or black water problems due to iron or sulphate-reducing bacteria, microbial influenced corrosion, hydraulic roughness and reduced material life (Stevens et al., 2003). Coliform bacteria have traditionally been used to indicate the adequacy of municipal water treatment. An interpretation of this indicator concept implies that municipal water is not adequately treated if coliform bacteria can proliferate in distribution systems (LeChevallier, 2004). 

Chlorinated water coliforms may develop resistance to chlorine within the distribution systems. This usually results from the alteration of cell surface structures which may lead to increased aggregation and clumping of cells, adherence of these microbes to pipework, extrusion of protective extracellular capsular or slime layers and formation of resistant spores. Some of these mechanisms may also influence the survival and persistence of various opportunistic pathogenic microbes within chlorinated water distribution systems (Bishankha et al., 2011).

The fact that relatively high numbers of microorganism could be isolated from chlorinated supply systems suggests that certain bacteria may possess mechanisms enabling them to survive in chlorinated environments. Survival of opportunistic pathogenic microbes and their persistence in the water supply systems may be due to their capability to resist chlorine (Bishankha et al., 2011). The fact that disinfection itself can select for a variety of bacteria has been demonstrated by the results of work of several researchers who have indicated that chlorination of water supplies select for survivors which are antibiotic resistant (LeChevallier et al., 1988). 

2.4 Sodium hypochlorite
Sodium hypochlorite (NaOCl) is a compound that can be effectively used for water purification. It is used on a large scale for surface purification, bleaching, odour removal and water disinfection. Every day, millions of households throughout the world rely on sodium hypochlorite bleach for their disinfection, deodorizing and cleaning needs (Sarbatly et al., 2007).

Sodium hypochlorite (NaOCl) is made up of the sodium salts of hypochlorous acid and is a chlorine-containing compound that can be used as a disinfectant. It is produced when chlorine gas is dissolved into a sodium hydroxide solution. It is supplied in liquid form, clear with a light yellow colour and has a strong chlorine smell. Of all the different types of chlorine available for use, this is the easiest to handle (WHO, 2004). 

Sodium hypochlorite provides a higher concentration of available chlorine and is less susceptible to inactivation by organic matter. Sodium  hypochlorite  (NaClO)  is  a  widely  used  disinfectant  due  to  its  strong  oxidizing  capacity. When  it  comes  into  contact  with  water,  this  molecule  produces  both  HClO  (hypochlorous  acid, the  more  active  fraction  of  chlorine)  and  ClO− (hypochlorite  ion).  These fractions constitute the free available chlorine.  Sodium hypochlorite  affects  the  plasmatic  membranes  of  bacterial  cells  and  disables enzymatic  active  sites.  It  also  diminishes  the  biological  functions  of  proteins,  and  it  produces deleterious  effects  on  DNA. This is attributed to a higher penetration of the disinfectant through the bacterial cell envelope (Martinez-Hernandez et al., 2013).

2.5 Free residual chlorine
When chlorine is added to water, some of the chlorine reacts first with organic materials and metals in the water and is not available for disinfection, this is called the chlorine demand of the water. The remaining chlorine concentration after the chlorine demand is accounted for is called total chlorine. Total chlorine is further divided into:  the amount of chlorine that has reacted with nitrates and is unavailable for disinfection which is called combined chlorine and, the free chlorine, which is the chlorine available to inactivate disease-causing organisms, and thus a measure to determine the potability of water (Wohlsen et al., 2006). The World Health Organisation suggests that free residual chlorine levels of 0.2-0.5mg l-1 to be maintained in water as it is supplied to taps of various distances away from the treatment plant (WHO, 2004).

Maintenance of a residual disinfectant throughout the distribution system is intended to produce conditions unfavourable for bacterial survival in drinking water. However, in some cases, improper application of a disinfectant can actually enhance bacterial growth conditions (LeChevallier et al., 1996). At low chlorine levels, microorganisms that survive the treatment may be injured rather than inactivated. Under suitable conditions injured cells might repair cellular damage and recover. An appreciation of the nature of sublethal injury and repair is therefore important in devising chlorination strategies and in developing combination treatments with synergistic actions against the target microorganisms (McDonnell and Russell, 1999). 

Chlorine residuals of drinking water have long been recognized as an excellent indicator for studying water quality in the distribution network. In the absence of a residual disinfectant, microorganisms in the distribution network will be recovered at high levels. 
Keeping residual chlorine at a certain level in tap water is effective not only in improving sanitary conditions but also in suppressing the regrowth of microorganisms and preventing the formation of biofilms on the internal surface of distribution pipelines (Karikari and Ampofo 2013).

2.6 Municipal water treatment process
The Municipal water treatment process involves several steps which are preconditioning, coagulation, sedimentation and clarification, filtration and disinfection (ZINWA, 2011). Disinfection involves the application of chlorine, which can be added in the form of chlorine gas, sodium hypochlorite or calcium hypochlorite.   

2.6.1 Preconditioning
This stage involves the correction of pH through the addition of either soda ash or sulphuric acid depending on the pH of water so that water so that disinfectants and other chemicals used in the course of the treatment process can be added at a neutral pH. Activated carbon is added at this stage so as to get rid of the odorous smell produced by algae and other microorganisms that may be present within the water reservoir (ZINWA, 2011).

2.6.2 Coagulation
The coagulation stage is aimed at removing the colour or even turbidity of water from through the addition of aluminium sulphate as the coagulant. This process coagulate suspended particles in water forming flocs in a process known as flocculation. Tests are carried out to determine the appropriate dose of the coagulant needed during this stage (ZINWA, 2011).
2.6.3 Sedimentation and clarification
Here, the heavy flocs formed during flocculation settle down the sedimentation tank forming a sludge at the bottom. The supernatant flows from the sedimentation tank to the filter beds. The settled sludge is removed by scouring the sedimentation tank with scour valves (ZINWA, 2011).

2.6.4 Filtration
The supernatant is filtered by a three layered channel of different soil particle sizes so as to trap some suspended solids which passed the coagulation. Microbes like protozoans are also entrapped here (ZINWA, 2011). 

2.6.5 Disinfection  
This is the final stage in water treatment process and chlorine is added to the filtrate as a disinfectant. Other disinfection methods like ozonation and ultraviolet radiation may also be used. After chlorination water is given appropriate contact time for the distribution and disinfection of chlorine after which quality tests are then carried out to ascertain compliance with both physiochemical and microbiological parameters. Water is then pumped to a treated water reservoir from where it is pumped into different distribution pipelines (ZINWA, 2011). 

CHAPTER 3: MATERIALS AND METHODS

3.1 Study area
Chlorinated municipal water was sampled from 10 randomly selected suburbs which were supplied by Gwenhoro water treatment plant in Gweru, Zimbabwe. The samples were collected within a period of three months from February to April 2014. Laboratory analysis were conducted at Midlands State University, Biological Sciences Laboratories.  Water samples were collected from Harben Park, Woodlands, Dalesford, Mkoba 11, Irvine, Lundi Park, Kopje, Windsor Park, Senga and Southdowns.

3.2 Sampling
Samples adding up to 30 were collected within a period of three months. Two samples of water were collected per every identified tap, one for microbial analysis and the other one for determination of residual chlorine level. Municipal water samples were collected from the suburbs which were supplied by the same treatment plant. Aseptic techniques were followed during sampling to ensure that results will be reliable. 

3.2.1 Sampling for microbial analysis
Municipal water was sampled aseptically into sterile 500 ml bottles containing 0.50 ml of 1.8% sodium thiosulphate per every identified tape. The tape was left to run for a period of two minutes in order to collect water from the main line. Sodium thiosulphate neutralises the effect of chlorine in municipal water. The bottles were labelled for easy of identification, packed into a cooler box, carried to MSU laboratory and tested within a period of 4 hours.

3.2.2 Sampling for determination of residual chlorine level 
Municipal water was sampled into one litre clean plastic containers per every identified tap. Residual chlorine determination tests were carried out on site. 

3.3 Media preparation
Double Strength MacConkey Broth (McB) was prepared according to manufacturer’s instructions and 50 ml volumes were dispensed into 100ml capped Duran bottles. Additionally, 10 ml aliquots were dispensed into capped test tubes and inverted Durham’s tubes were placed into each test tube.

Peptone water and Brilliant Green Bile Broth (BGBB) were prepared according to manufacturer’s instructions and 10 ml aliquots of each were dispensed into capped test tubes. Durham’s tubes were also placed into tubes containing BGBB. Tryptone Soya Agar (TSA) was prepared following manufacturer’s instructions and 10 ml aliquots were dispensed into caped McCartney bottles. Normal saline was prepared as 0.9% sodium chloride (w/v) and 9 ml aliquots were dispensed into capped McCartney bottles. MacConkey agar (McA) was prepared following manufacturer’s instructions in a capped 1000ml Duran Scott bottle. All the culture media was sterilised at 121oC for 15 minutes and then cooled to 45 ºC in a water bath. TSA bottles were cooled in a slanting position till the agar solidified making sure it does not reach the inside of the cap.
3.3.1 Kovac's reagent
4-dimethylaminobenzaldehyde (2.5 g) was dissolved in 37.5 ml of amyl alcohol followed by the slow addition of 12.5 ml of concentrated hydrochloric acid (HCL) to make a final volume of 50ml. The reagent was labelled and stored in a dark bottle away from direct light.

3.4 Testing for residual chlorine 
Residual chlorine was measured using a calorimetric method which employed the N, N-diethyl-p-phenylenediamine (DPD) technique. Two cuvettes were filled with 10ml of water sample each. A DPD 1 tablet was crushed and added to one of the cuvettes containing 10ml of water sample. The sample was shocked for 30 seconds and left to stand for 2 minutes to allow the tablet to dissolve completely in water. Both cuvettes were placed into a lovibond comparator. Colour comparisons were used to estimate the amount of free residual chlorine in milligrams per liter (mg l-1).

3.5 Presumptive coliform test
Water samples to be tested were mixed by inverting the sample bottle and 50 ml aliquots were aseptically transferred into capped Duran bottles with 50 ml double Strength McB that had been incubated for 24 hours. Water aliquots measuring 10 ml were aseptically added into five test tubes containing 10 ml double strength McB that has been incubated for 24 hours. Untreated samples were incubated along with test sample and these were regarded as controls. All The bottles were incubated at 35oC and inspected for colour change and gas production after 24 hours and 48 hours. Gas detection within the Durham’s tubes and the broth colour change from purple to yellow after an incubation period of 48 hours was regarded as presumptive evidence for presence of coliform bacteria in water.
The positive tubes were counted and the Most Probable Number (MPN) of coliforms was determined from the standard table (Appendix 1).

3.5.1 Confirmed test
From each tube containing acid and gas, a tube of BGBB and a tube of peptone water were inoculated. Slants of TSA were also prepared from positive tubes. Tubes containing peptone water and BGBB were incubated at 44oC for 24 hours in a water bath.  TSA slants were incubated at 35oC for 24 hours. Gas formation and turbidity were observed in the tubes containing BGBB as positive of coliforms. The presence of a rose pink colour on adding kovac’s reagent on peptone water was an additional confirmation for presence of coliform bacteria in water.

3.5.2 Completed test
Gram staining was performed for pink colonies with precipitation zones on McA plates. Coliform bacteria were confirmed as gram negative rods. Presumptive coliforms were streaked on McA plates and incubated at 37˚C for 24 hours. 
 
3.6 Estimation of bacterial densities from slants 
From each dilution, 0.1ml aliquots were cultured in triplicate plates on TSA and incubated at 35oC for enumeration of viable coliforms. The number of colonies on each plate were counted on a colony counter and averaged by multiplying the number of colonies on plates to which 0.1ml of sample was added by 10 to give the average count per ml. 
The equation:	 

3.7 Coliform susceptibility testing
Intermittent time intervals of 5 minutes were chosen so as to monitor the changes resulting from the response given by chlorinated water coliforms to each dose of sodium hypochlorite. All tests were given maximum exposure time of 30 minutes. The 0.3mg/l chlorine concentration was used as the maximum after preliminary tests proved that none of the coliforms were able to survive this concentration for a period of five minutes.  

Commercial stock (5.25 mg l-1) of sodium hypochlorite was diluted using distilled water to give a variety of percentage concentrations ranging from 0.05 mg l-1 up to 0.3 mg l-1. Known concentrations of sodium hypochlorite were inoculated with known concentration of the bacterial suspension. The samples were mixed thoroughly to ensure homogeneous distribution. The change in concentrations of the coliform resulting from the treatment with sodium hypochlorite were monitored using the serial dilutions and standard plate count method. Percentage of viable coliforms after treatment with sodium hypochlorite concentrations of 0.05 mg l-1, 0.1 mg l-1, 0.15 mg l-1, 0.2 mg l-1, 25 mg l-1 and 0.3 mg l-1 for 0, 5, 10, 15, 20, 25 and 30 minutes exposure times were determined. At the end of each exposure time, sodium thiosulphate was added to neutralise the effect of sodium hypochlorite. A suspension of coliform bacteria isolated from sewage water was used as a standard reference. 

3.8 Data analysis 
The residual chlorine level and total coliform counts were analysed for means. The decrease in coliform densities resulting from treatment with sodium hypochlorite was expressed as percentages and graphically presented as a function of time.
CHAPTER 4: RESULTS

4.1 Prevalence of coliforms and residual free chlorine level
Coliform bacteria were identified in one of the samples obtained from Harben Park. No coliforms were isolated from samples collected from all the other nine suburbs. Coliforms were isolated from a water sample collected from Harben Park which had a mean residual chlorine level of 0.05 mg l-1. The species found was E. coli. No coliforms were isolated from samples obtained from Woodlands although residual chlorine levels were the same (0.05 mg l-1). The prevalence of coliforms in Gweru municipal water was found to be 3.33%. The mean residual chlorine level for the 10 suburbs ranged between 0.05 - 0.4 mg l-1 (Table 4.1). Harben Park and Woodlands had the lowest mean residual chlorine (0.05 mg l-1). Southdowns had the highest mean residual chlorine level (0.4 mg l-1). Dalesford, Mkoba 11 and Irvine had equal mean chlorine residual levels (0.1 mg l-1). Lundi Park, Kopje, Windsor Park and Senga also had the same mean residual chlorine levels (0.2 mg l-1). Five of the suburbs had mean chlorine residuals below the WHO guidelines (0.2- 0.5 mg l-1). These included Harben Park (0.05 mg l-1), woodlands (0.05 mg l-1), Dalesford (0.1 mg l-1), Mkoba 11 (0.1 mg l-1) and Irvine (0.1 mg l-1). The other five had mean residual chlorine within the WHO guideline. Four of these had a mean residual chlorine of 0.2 mg l-1. These were Lundi Park, Kopje, Windsor Park and Senga. The fifth suburb, Southdowns had a chlorine residual of 0.4 mg l-1.coliforms from Harben park had an MPN value of 6/100 ml. The prevalence of coliforms, mean estimated coliform densities (MPN /100ml) and the mean residual chlorine for the ten suburbs are shown in Table 4.1. 

Table 4.1: Mean residual Chlorine, coliform prevalence and coliform density for ten suburbs in Gweru 
	WHO guideline	A	B	C	D	E	F	G	H	I	J	
residual chlorine	0.2-0.5 mg l-1	0.05	0.05	0.1	0.1	0.1	0.2	0.2	0.2	0.2	0.4	
												
coliform+/-	-	+	-	-	-	-	-	-	-	-	-
	
MPN/100 ml	0/100ml	6	<1	<1	<1	<1	<1	<1	<1	<1	<1	

A - Harben park, B - Woodlands, C - Dalesford, D - Mkoba 11, E - Irvine, F - Lundi Park, G - Kopje, H - Windsor Park, I - Senga and J - Southdowns.
+; present, -; absent. 
The MPN of value of <1/100ml represents that the samples were negative of coliform bacteria.

4.2 Coliform susceptibility to sodium hypochlorite 
The initial density of coliform bacteria from a positive sample (Harben Park) as determined by serial dilution was 4×105 colony forming units per milliliter (cfu ml-1). The reference coliforms isolated from sewage had a cfu count of 1×108 ml-1. The first and second dilutions for chlorinated water coliforms had the greatest colony counts exceeding 300 cfu in a single plate. These were recorded as too numerous to count (TNTC). The reference coliform had too numerous colonies in the first four dilutions.  Results of serial dilution tests to determine the initial concentration of the coliforms harvested from TSA slants are summarised in Table 4.2. 
Table 4.2: Initial counts for test coliform and the reference control 
Dilution factor	10-1	10-2	10-3	10-4	10-5	10-6	10-7	10-8	
Test
Coliform (cfu ml-1)
	TNTC	TNTC	8.8×102	4.3×103	4×105	0	0	0	
Ref control (cfu ml-1)
	TNTC	TNTC	TNTC	TNTC	1.1×103	7.1×105	2.3×106	1×108	
Too numerous to count (TNTC) applied to colony forming units exceeding 300 in a single plate. 

4.2.1 Coliform susceptibility to 0.05 mg l-1 sodium hypochlorite
About 67% of chlorinated water coliforms survived treatment with 0.05 mg l-1 of sodium hypochlorite for 30 minutes. About 61% of the reference coliforms survived treatment with 0.05 mg l-1 of sodium hypochlorite. Chlorinated water coliforms were less susceptible to 0.05% sodium hypochlorite than those isolated from sewage water. A 100% coliform density was recorded on the control sample throughout 30 minutes (Figure 4.1). Sodium hypochlorite at a concentration of 0.05 mg l-1 was not effective against chlorinated water coliforms within 30 minutes exposure time. 

Figure 4.1: Percentage coliform survival at 0.05 mg l-1 NaOCl concentration.

4.2.2 Coliform susceptibility to 0.1 mg l-1 sodium hypochlorite
About 47% of the test coliforms survived treatment with 0.1 mg l-1 of sodium hypochlorite for 30 minutes. About 40% of reference coliforms survived treatment with 0.1 mg l-1 of sodium hypochlorite for 30 minutes. A 100% coliform density was recorded for the control sample throughout 30 minutes (Figure 4.2). The sodium hypochlorite disinfectant was more effective within the first 10 minutes killing almost 40% of the coliforms. Sodium hypochlorite at a concentration of 0.1 mg l-1 was not effective against chlorinated water coliforms within 30 minutes exposure time.

Figure 4.2: Percentage coliform survival at 0.1 mg l-1 NaOCl concentration.

4.2.3 Coliform susceptibility to 0.15 mg l-1 sodium hypochlorite
About 33% of chlorinated water coliforms survived treatment with 0.15 mg l-1 of sodium hypochlorite. About 26% of the reference coliform survived treatment with 0.15mg l-1 sodium hypochlorite. A 100% coliform density was recorded on the control sample throughout the 30 minutes. The highest reduction in coliform density was observed during the first 15 minutes (Figure 4.3). Sodium hypochlorite at a concentration of 0.15 mg l-1 was not effective against chlorinated water coliforms. 

Figure 4.3: Percentage coliform bacteria at 0.15 mg l-1 NaOCl concentration.

4.2.4 Coliform susceptibility to 0.2 mg l-1 sodium hypochlorite
None of the chlorinated water coliforms survived treatment with 0.2 mg l-1 sodium hypochlorite for 30 minutes. A 100% coliform density was recorded for the control sample throughout the 30 minutes (Figure 4.4). The sodium hypochlorite at a concentration of 0.2 mg l-1 was effective against coliforms requiring 30 minute exposure time. 

Figure 4.4: Percentage coliform survival at 0.2 mg l-1 NaOCl concentration.

4.2.5 Coliform susceptibility to 0.25 mg l-1 sodium hypochlorite
There were no coliform bacteria enumerated after treatment with 0.25 mg l-1 sodium hypochlorite for a period of 10 minutes. A 100% coliform density was still maintained by the control sample for the 30 minutes (Figure 4.5). Sodium hypochlorite at a concentration of 0.25 mg l-1 was effective against chlorinated water coliforms.

Figure 4.5: Percentage coliform survival at 0.25 mg l-1 NaOCl concentration.
4.2.6 Coliform susceptibility to 0.3 mg l-1 sodium hypochlorite
None of the coliforms survived treatment with 0.3 mg l-1 sodium hypochlorite within the first five minutes. A 100% coliform density was maintained by the control sample throughout 30 minutes. There was no pronounced difference in response to sodium hypochlorite between chlorinated water coliforms and sewage water coliforms at higher concentrations of the hypochlorite disinfectant as indicated by the line graphs which are close to each other (Figure 4.6). Collectively, the minimum effective concentration of sodium hypochlorite against chlorinated coliforms from Harben Park was 0.2 mg l-1 requiring 30 minutes exposure time. The most effective dose was 0.3 mg l-1 requiring 5 minutes. However a concentration of 0.25 mg l-1 was as well effective requiring 10 minutes of exposure time.

Figure 4.6: Percentage coliform survival at 0.3 mg l-1 NaOCl concentration.

CHAPTER 5: DISCUSSION

5.1 Isolation and prevalence of coliforms
Gweru municipal water had a relatively low prevalence of coliform bacteria (3.33%). This means that the water treatment method employed by Gwenhoro water treatment plant in Gweru is effective against coliform bacteria and is thus microbiologically safe for human consumption. The isolation of coliforms from a sample from Harben Park where the mean residual chlorine was (0.05 mg l-1) could be related to the relatively low residual chlorine level. 

The absence of coliform bacteria in samples collected from the other nine suburbs might be attributed to the effectiveness of the treatment process. The treatment process adopted by Gwenhoro water treatment plant might be effective at removing the probable food sources that necessitates coliform growth. This makes treated water a poor medium for coliform growth and is thus safe for human use. At low chlorine levels, microorganisms that survive the treatment may be injured rather than inactivated. Under suitable conditions injured cells might repair cellular damage and recover (McDonell and Russell, 1999). 
 
Samples from Woodlands, where the mean residual chlorine level was the same as that of Harben Park (0.05 mg l-1) were however free of coliforms.  The regrowth of coliform bacteria within the water distribution system can only occur when sufficient bacterial nutrients are available, at a temperature exceeding 15°C, and when there is no residual free chlorine. Survival of the opportunistic pathogenic microorganisms in the water distribution systems may be due to their capability to resist chlorine (Bishanka et al., 2011). 

5.2 Residual chlorine level in water
Chlorination of municipal water is intended at ensuring that water is microbiologically safe for human consumption. Chlorine residuals of municipal water are intended at ensuring continual disinfection, should contamination occur within the distribution system (Karikari and Ampofo, 2013). Gweru water supplies maintains free residual chlorine between 0.05 and 0.40 mg l-1. The residual chlorine levels in Gweru municipal water were correlated to the distance between the suburb and the treatment plant. The suburbs which were distal to treatment plant, Harben Park and Woodlands had the least mean residual chlorine levels (0.05 mg l-1). The suburb proximal to the treatment plant (Southdowns), had the highest mean residual chlorine level (0.4 mg l-1).  

Studies have shown that most of the chlorine in water is lost when water flows through the pipework satisfying the chlorine demand (LeChevallier, 2003). The suburb which was proximal to the treatment plant had the greatest mean residual chlorine (0.4 mg l-1). Only a small fraction of chlorine will be directed towards satisfying the chlorine demands of water. Halve of the sampled suburbs had residual chlorine levels below the WHO guideline (0.2- 0.5 mg l-1) whilst the other halve had their chlorine residual within the WHO guideline. This is attributed to the distances of these suburbs from the treatment plant. The shorter the distance between the user point and the treatment plant, the higher the residual chlorine level, the further the user point the lower the residual chlorine. The ability of treated water to maintain a residual chlorine facilitate continual disinfection of water and thus keeping it free of pathogenic microbes (Karikari and Ampofo, 2013).

5.3 Susceptibility of coliforms to sodium hypochlorite
The sodium hypochlorite concentrations used in this study were similar to residual chlorine levels that were found in Gweru municipal water distribution system. However, they as well fall within the WHO guidelines. A minimum concentration of 0.05 mg l-1 was chosen since it was the least residual chlorine level detected in Gweru municipal water (Table 4.1). The zero minute counts serve to indicate the effectiveness of sodium thiosulphate at neutralising the effect of sodium hypochlorite. In this study sewage water coliforms were used to represent the wild type coliform bacteria that may not be adapted to chlorine environments.

Sodium hypochlorite at concentrations of 0.15mg l-1 and below were not effective against coliform bacteria as they could not kill 99.9% of coliform bacteria within 30 minutes of exposure. However, these concentrations were also below the WHO guideline (0.2-0.3 mg l-1). The differences in susceptibility between chlorinated water coliforms and sewage water coliforms at these sodium hypochlorite concentrations might be related to development of resistance mechanisms by chlorinated water coliforms. The effectiveness of sodium hypochlorite within the first 10 minutes could be related to the delayed dissociation of sodium hypochlorite into salt and water (Figure 4.5). This suggests that sodium hypochlorite works best before it dissociates into salt and water. The dissociation of sodium hypochlorite into its constituents lowers the disinfectant concentration as well as the disinfectant capabilities (LeChevallier, 2003).

There was a small difference in susceptibility to sodium hypochlorite between chlorinated water coliforms and the reference coliforms from sewage water at high concentrations of sodium hypochlorite (0.2- 0.3 mg l-1) (Figure 4.4, 4.5 and 4.6). This could be related to the disinfectant effectiveness of sodium hypochlorite at higher concentrations. 
Sodium hypochlorite concentrations between 0.2 - 0.3 mg l-1 were effective against coliform bacteria. The effectiveness increased with increase in sodium hypochlorite concentration thereby reducing the exposure time required.  

Water guard for home use disinfection recommends a 5 ml stork concentration of 1.25% sodium hypochlorite in 20 litres gives a final concentration of 0.00025 mg l-1. However when this is combined with 0.05 mg l-1 residual chlorine in municipal water in which coliforms were isolated, it makes a final concentration of 0.05025 mg l-1. This concentration is below the 0.3 mg l-1 recommended by this study. Therefore, the set of conditions prescribed on such ‘Water guards’ may not be effective against chlorinated water coliforms under Gweru water treatment conditions.  

5.4 Conclusion
In this study it was found that a sodium hypochlorite concentration of 0.3 mg l-1 would be more effective for further disinfection of Gweru municipal water. Sodium hypochlorite works best within the first 10 minutes before its concentration is reduced by its dissociation into salt and water. A relatively low prevalence (3.33%) of coliform obtained in this study shows that the treatment process at Gwenhoro plant is effective and municipal water is not a good medium for coliform growth. Suburbs which are distal to the water treatment plant have a likelihood of receiving water that is contaminated with coliform bacteria. This is mainly due to the reduced residual chlorine as distance increases from the treatment plant.

5.5 Recommendations
Based on this study findings a sodium hypochlorite concentration of 0.3 mg l-1 is recommended when conducting further disinfection of water in homes under Gweru water treatment conditions. Since this was a baseline study, there is still need for more research using a large sample size, to isolate and evaluate the susceptibility of different species coliforms to sodium hypochlorite under Gweru water treatment conditions in different seasons in order to consolidate the findings in this study.

REFERENCES

Bishankha, S., Bhatta, D. R., Josh, D. R. and Joshi, T. P. (2011). Assessment of microbial quality of chlorinated drinking tap water and susceptibility of gram negative bacterial isolates towards chlorine. Nepal Journal of Science and Technology. 13:173-178.

Campbell, J. W., Watson, A., Watson, C., Ball, H. and Pirkle, R. (2011). Escherichia coli, other coliform, and environmental chemoheterotrophic bacteria in isolated water pools from six caves in northern Alabama and northwestern Georgia. Journal of Cave and Karst Studies. 73: 75-82.

Gleick, P. H. (2005). The Millennium Development Goals for Water: Crucial Objectives, Inadequate Commitments. Washington, D.C. Island Press.

Ignnnugbemi, O. O., Babalola, B. O., Olayemi, A. B. and Kolawole, O. M. (2009). Survival of coliforms in chlorinated and dechlorinated water under storage. Journal of applied sciences in environmental sanitation.  119-126.

Karikari, A. Y. and Ampofo, J. A. (2013). Chlorine treatment effectiveness and physico-chemical and bacteriological characteristics of treated water supplies in distribution networks of Accra-Tema Metropolis, Ghana. Journal of applied water sciences. 3: 535-543.

King, C.H., Shotts, E.B., Wooley, R.E. and Porter, K. G. (1988). Survival of Coliforms and Bacterial Pathogens within Protozoa during Chlorination. Journal of Applied and Environmental Microbiology. 54: 3023-3033.

Kumar, D., Tyagi, N. and Gupta, A. B. (2011). Selective Action of Chlorine Disinfection on Different Coliforms and Pathogens Present in Secondary Treated Effluent of STP. IACSIT Press, Singapore 4: 179-184.

LeChevallier, M. W. (2003). Conditions favouring coliform and HPC bacterial growth in drinking water and on water contact surfaces. IWA Publishing, London, UK.

LeChevallier, M. W. (2004). Water Treatment and Pathogen Control: Process Efficiency in Achieving Safe Drinking Water. IWA Publishing, London, UK.

LeChevallier, W. M., Cawthon, C. D. and Lee, R. M. (1988). Factors Promoting Survival of Bacteria in Chlorinated Water Supplies. Journal of environmental microbiology. 54: 649-654.

LeChevallier, M. W., Welch, N. J. and Smith, A. B (1996). Full-Scale Studies of Factors Related to Coliform Regrowth in Drinking Water. Journal of applied and environmental microbiology. 62: 2201-2211.

Lee, E. L., Schwab, K. J. (2005). Deficiencies in drinking water distribution systems in developing countries. Journal of Water Health. 3:109-127.

Martinez-Hernandez, S., Vazquez-Rodriguez, G. A., Beltran-Hernandez, R. I., Prieto-Garcia, F., Miranda-Lopez, J. M., Franco-Abuin, C. M., Alvarez-Hernandez, A., Ulises Iturbe, U. and Coronel-Olivares, C. (2013). Resistance and inactivation kinetics of bacterial strains isolated from the non-chlorinated and chlorinated effluents of a WWTP. International Journal of Environmental Research and Public Health. 10: 3363-3383.

McDonnell, D. and Russell, A. D. (1999). Antiseptics and Disinfectants: Activity, Action, and Resistance. Clinial microbiology review. 12: 1147-1179.

Pickard, C. B., Clarke, S. and Bettin, W. (2006). Chlorine disinfection in the use of individual water purification devices report.  Deatrick and Associates Inc. Alexandria.

Ridgway, H.F. and Olson. B. H. (1982). Chlorine resistance patterns of bacteria from two drinking water distribution systems. Applied and Environmental Microbiology. 44: 972-987.

Rompre, A., Servais, P., Baudart, J., Marie-Renee de-Roubin, Laurent, P. (2001). Detection and enumeration of coliforms in drinking water: current methods and emerging approaches. Journal of Microbiological Methods. 49: 31-54.

Sarbatly, R. H. J. and Krishnaiah. D. (2007). Free chlorine residual content within the drinking water distribution system. International Journal of Physical Sciences. 2: 196-201.

Silveira, A. B, Bechtlufft, M. P., Van Der Sand, S. T. and Corcao, G. (2006). Evaluation of the activity of disinfectants against coliform bacteria group strains isolated from a sewage treatment plant. 34: 71-76.

Stevens, M., Ashbolt, N. and Cunliffe, D. (2003). Review of Coliforms as Microbial Indicators of Drinking Water Quality. Biotext Pty Ltd, Canberra. 

Tabor. M., Kibret. M. and Abera. B. (2011). Bacteriological and Physicochemical Quality of Drinking Water and Hygiene-Sanitation Practices of the Consumers in Bahir Dar City, Ethiopia. Ethiopian Journal of Health Sciences. 21: 19-26.

United Nation (U.N). (2006). Millennium Development Goals Report. Geneva.

United Nations Environment Programme and the World Health Organization (UNEP/WHO). (1996). Water quality monitoring - A practical guide to the design and implementation of freshwater quality studies and monitoring programmes. Geneva.

United Nations Office for the Coordination of Humanitarian Affairs (UNOCHA). (2014). Zimbabwe Humanitarian Bulletin. Harare.

U.S. Environmental Protection Agency (USEPA). (2004). The effectiveness of disinfectant residuals in the distribution system.  [Online]. Available at
http://www.epa.gov/safewater/disinfection/tcr/regulation revisions.html. Accessed 22 february 2013.

Wagenet, L., Heidekamp, A. and Lemley, A. (2005). Chlorination of drinking water. Cornell cooperative Extension, College of Human Ecology.

Wohlsen, T., Bates, J., Vesey, G., Robinson, W. A. and. Katouli, M. (2006). Evaluation of the methods for enumerating coliform bacteria from water samples using precise reference standards. Journal of Applied Microbiology. 42: 350-356.

World Health Organization (2004). Guidlinines for drinking water quality. Volume I, II and III. IWA Publishing, London, UK.

World Health Organization (2004). Water Treatment and Pathogen Control: Process efficiency in Achieving Safe Drinking Water. Edited by Mark W LeChevallier and Kwok-Keung Au. IWA Publishing, London, UK.

ZINWA. (2011). Annual water reports. Government printers. Harare.

APPENDIX 1

MPN index and 95% confidence for various combinations of positive results for a set of one 50 ml and five 10ml portions of sample.

Number of tubes giving a positive reaction	MPN index per 100 ml	        95% confidence limits	
   1 × 50 ml	  5 × 10 ml		        Lower	      Upper	
0	0	          <1			
0	1	1	0.5	4	
0	2	2	0.5	6	
0	3	4	0.5	11	
0	4	5	1	13	
0	5	7	2	17	
1	0	2	0.5	6	
1	1	3	0.5	9	
1	2	6	1	15	
1	3	9	2	21	
1	4	16	4	40	
1	4	         >18			
Source; UNEP/WHO, 1996.