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1 Public Health 123 (2009) Contents lists available at ScienceDirect Public Health journal homepage: Original Research Assessment of microbiological quality of sachet-packaged drinking water in Western Nigeria and its public health significance O.A. Olaoye a, *, A.A. Onilude b a Division of Food Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK b Microbial Physiology and Biochemistry Unit, Botany and Microbiology Department, University of Ibadan, Ibadan, Nigeria article info summary Article history: Received 17 June 2009 Received in revised form 1 September 2009 Accepted 23 September 2009 Available online 31 October 2009 Keywords: Public health Drinking water Regulatory agency Microbiological quality Nigeria Objective: To assess the microbiological quality of sachet-packaged drinking water in Western Nigeria and its impact on public health. Study design: Cross-sectional microbiological testing. Methods: Ninety-two sachet-packaged water samples were analysed for microbiological and metal qualities. Total bacterial and coliform counts were determined, and the presence of Escherichia coli, an important water quality indicator, was tested. The level of conformity of the water processors with the guidelines of Nigeria s quality regulatory agency was also determined. Results: Varying levels of microbial contamination were recorded in samples from the different sampling locations. The total bacteria count ranged between 2.86 and 3.45 log colony-forming units (cfu)/ml. The highest coliform count recorded was 1.62 log cfu/ml. Faecal coliform E. coli was detected in one sample from Oke-Iho and one sample from Okaka, representing 2.2% of total samples. Lead and manganese were not found in any of the samples. However, iron was detected and the highest iron concentration (0.10 mg/ l) was detected in samples from Ikorodu. The bacteria that were identified from the water samples included E. coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella sp., Proteus vulgaris, Alcaligenes faecalis, Bacillus cereus, Staphylococcus aureus, Streptococcus lactis, Aeromonas sp. and Micrococcus luteum. Many of the water processors did not comply with the guidelines of the quality regulatory agency. Conclusions: Some of the sachet-packaged samples of drinking water were of poor quality. The results indicate a need for Nigeria s quality regulatory agency to take appropriate measures in safeguarding public health. Ó 2009 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved. Introduction The microbiological quality of drinking water has attracted great attention worldwide because of implied public health impacts. Sachet-packaged drinking water is very common in Nigeria. It is usually called pure water, and is sold on the streets, highways and in market places. It is often found as a major source of water at food canteens, and sold by many food vendors in the country. It is consumed by the majority of the population, and hence there is a need to ascertain its quality in order to safeguard the health of consumers. Many common and widespread health risks have been found to be associated with drinking water in developing countries, a large percentage of which are of biological origin. 1 Unsafe water, poor * Corresponding author. address: olaayosegun@yahoo.com (O.A. Olaoye). sanitation and hygiene have been reported to rank third among the 20 leading risk factors for health burden in developing countries, including Nigeria. 2 Contamination of water, either directly or indirectly, by human or animal excreta and the micro-organisms contained in faeces is known to contribute to acquisition of disease by consumers. If the contamination is recent and those contributing to it include carriers of communicable enteric diseases, some of the micro-organisms responsible for causing these diseases may be present in the water, thereby questioning its safety. Drinking such contaminated water or using it in food preparation may lead to new cases of infection. 1 Various opportunistic pathogens that occur naturally in the environment may cause disease in humans. Those at greatest risk of infection are infants and young children, people whose immune system is suppressed, the sick and the elderly. In such individuals, drinking water containing large numbers of opportunistic pathogens can occasionally produce infections. Examples of such /$ see front matter Ó 2009 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved. doi: /j.puhe

2 730 O.A. Olaoye, A.A. Onilude / Public Health 123 (2009) opportunistic agents are Pseudomonas aeruginosa, Klebsiella sp., Aeromonas sp. and certain slow-growing mycobacteria. 3 Microbiological quality is the most important aspect of drinking water in relation to waterborne diseases. Detection of bacterial indicators in drinking water implies the presence of pathogenic organisms that are the source of waterborne diseases which could be fatal. 4 These enteropathogenic bacteria are responsible for a variety of diseases such as cholera, typhoid, dysentery and bacillary dysentery in humans and livestock. 5 They belong to the Enterobacteriaceae family of organisms which comprise members of the genera Escherichia, Enterobacter, Salmonella and Klebsiella, among others. 6 Generally, their occurrence in water is due to faecal contamination. 3 The faecal indicator bacterium (Escherichia coli), excreted in the faeces of all warmblooded animals and some reptiles, 7 has been considered as a bioindicator of faecal contamination of drinking water. The major pathogenic bacteria responsible for waterborne diseases are spread by the faecal oral route, in which water may play an intermediate role. In Nigeria, the National Agency for Food Administration and Control (NAFDAC) has responsibility for regulating the standards of drinking water. The agency has published guidelines for the production of sachet-packaged drinking water. Unfortunately, most producers do not adhere to these guidelines. 9 Nigeria is one of the developing nations in the world where water has contributed to infections and disease outbreaks, mainly due to lack of good manufacturing and hygienic practices by the water handlers. 10 There have been many reported cases (mostly unpublished) of epidemic outbreaks caused by consumption of sachet-packaged drinking water in the country. 11 Therefore, this study was undertaken to assess the microbiological quality of sachet-packaged drinking water samples from Western Nigeria, with a view to advising the agencies concerned in order to safeguard public health. Methods Study area Twenty areas in the major states of Western Nigeria were strategically selected for this study (Fig. 1). The areas comprised of towns and villages where sachet-packaged drinking water is sold by hawkers. Sampling of sachet-packaged drinking water Fig. 1 shows the towns and villages where water samples were collected. The sites are all located in Western Nigeria. The collected samples were taken to the laboratory on ice in clean containers, and stored at 4 C for min before commencement of analyses. Hygienic and aseptic practices were observed during sampling of the sachet-packaged drinking water samples. Microbiological analysis Aerobic plate count. Plate count agar (Oxoid, Hampshire, UK) was used for enumeration of total bacterial count. This was performed using the serial dilution agar plating method at suitable dilution levels. 12 One millilitre each of appropriate diluted suspensions was transferred into sterile Petri dishes, and molten agar medium (w45 C) was carefully poured while swirling gently. The plates were then incubated at 37 C for 24 h, after which visible colonies were counted and results were expressed in cfu/ml. Plating of samples was performed in triplicate. Bacterial isolation. This was accomplished following the methods described by Aneja. 12 Spread and pour-plate techniques were adopted. Isolation of pure cultures was completed by the streak plate method on various agar media including plate count, Mac- Conkey and eosin methylene blue agars (Oxoid). Gram staining Figure 1. (A) Sites where water samples were obtained in Western Nigeria. (B) Locations of towns and villages where sachet-packaged drinking water samples were taken.

3 O.A. Olaoye, A.A. Onilude / Public Health 123 (2009) was carried out on respective isolates to confirm purity and cell shapes. The bacterial isolates were subcultured on solid agar of their respective media at regular intervals to maintain viability. Pure colonies were preserved in liquid media containing 20% (v/v) glycerol, and stored at -80 C until further analysis. Characterization of isolates. Identification of the isolates was carried out on the basis of their cultural characteristics on agar plates and microscopic examinations. Further identification of the isolates was performed based on their biochemical characteristics, which helped in distinguishing the isolates sharing similar morphological characteristics. 13,14 Occurrence and detection of E. coli as an indicator of faecal coliform. The media used contained o-nitrophenyl-b-d-galactopyranoside and 4-methylumbelliferyl-b-d-galacuronidase (MUG), both useful in detecting the faecal coliform E. coli in water samples. 15 One hundred millilitres of sample was enriched overnight in 50 ml of triple-strength lauryl tryptose broth at 37 C, and streaked inoculated on sorbitol MacConkey (SMaC) agar containing potassium tellurite. The sorbitol-negative strains were tested for enzyme b-d-galacuronidase using MUG as the substrate. A colony of test isolate was applied to MUG-impregnated filter paper, moistened with a drop of saline and incubated at 37 C for 20 min. The absence of enzyme was indicated by the lack of fluorescence under ultraviolet light. The water samples which showed the presence of coliforms were further analysed using the Most Probable Number (MPN) test. Enumeration of total coliforms. The MPN test was used to enumerate the total number of coliforms in the packaged water samples. 16 This test was performed sequentially in three stages: presumptive, confirmed and completed tests. Double- and singlestrength lauryl tryptose (lactose) broths in tubes were incubated with 10 ml, 1.0 ml and 0.1 ml of water in presumptive tests. Tubes that were positive for gas production after 24-h incubation at 35 C were inoculated into brilliant green lactose bile broth for confirmed tests. Tubes showing positive reactions were used to calculate the MPN of coliforms in the water samples with reference to the statistical table described by Tillett. 17 For completed tests, inoculations were made on eosin methylene blue agar plates, nutrient agar slants and brilliant green lactose broths, and Gram staining was used to establish the presence of coliforms in the samples. Metal analysis. Metal concentrations were analysed in all brands of sachet-packaged drinking water samples using standard atomic absorption spectrophotometric methods (Type Perkin Elmer 7100AV, Branchburg, USA). 18 The metals analysed included iron, lead and manganese. Results Ninety-two sachet-packaged drinking water samples were analysed for microbiological and metal qualities. Table 1 shows the bacterial profiles and iron levels in the water samples. Results indicated that the samples showed varying levels of contamination at different sampling locations. The total bacterial count ranged between 2.86 and 3.45 log cfu/ml, with the lowest figure being recorded in samples from Ife and the highest figure being recorded in samples from Iseyin. E. coli was detected in one sample from Oke-Iho and one sample from Okaka, representing 2.2% of the total samples. Lead and manganese were not found in any of the water samples; however, iron was detected in low concentrations. The highest iron concentration (0.10 mg/l) was recorded in samples from Ikorodu. Table 1 Bacterial profiles and iron concentrations of sachet-packaged drinking water samples in Western Nigeria. Sampling sites The coliform count ranged from 1.40 to 1.62 log cfu/ml among the sachet-packaged drinking water samples in Western Nigeria. The distribution and presence of bacterial isolates in sachetpackaged drinking water samples in Western Nigeria (Table 2) indicated undesirable contamination of some of the samples. The bacteria that were identified from the water samples included E. coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella sp., Proteus vulgaris, Alcaligenes faecalis, Bacillus cereus, Staphylococcus aureus, Streptococcus lactis, Aeromonas sp. and Micrococcus luteum (Table 2). In the 92 water samples analysed, E. coli was least prevalent and M. luteum was most prevalent. Generally, the types of bacteria detected varied widely among the different samples in the respective sampling locations. M. luteum, Aeromonas sp., S. lactis and P. vulgaris were detected most frequently in the samples (Fig. 2). Generally, the frequency of occurrences and distribution were noted to vary within the sites of water collection. Results testing the compliance of the water processors with the NAFDAC regulatory requirements of printing production and expiry dates on the water packages indicated 100% non-compliance (Table 3). It was also observed that many of the processors did not comply with the requirement to print their plant locations and NAFDAC registration numbers on the packaged water sample. Discussion Samples Iron (mg/l) Log cfu/ml TBC Coliform count E. coli Iseyin (0.001) 3.45 (0.06) 1.50 (0.00) Oke-Iho (0.001) 3.29 (0.07) 1.58 (0.01) þ (1) a Ibadan (0.001) 3.31 (0.07) 1.52 (0.01) Iwo (0.001) 3.29 (0.08) 1.50 (0.00) Abeokuta (0.001) 3.33 (0.09) 1.49 (0.00) Ado-Ekiti (0.001) 3.28 (0.13) 1.40 (0.02) Ikorodu (0.001) 3.31 (0.23) 1.55 (0.01) Lagos (0.001) 3.45 (0.16) 1.48 (0.01) Ijebu Ode (0.001) 3.11 (0.09) 1.45 (0.00) Shagamu (0.001) 3.39 (0.17) 1.62 (0.00) Epe (0.001) 3.18 (0.12) 1.49 (0.01) Ibadan (0.001) 3.36 (0.25) 1.50 (0.02) Oyo (0.001) 3.29 (0.11) 1.50 (0.02) Ede (0.001) 3.15 (0.14) 1.49 (0.01) Ilobu (0.001) 3.34 (0.16) 1.45 (0.00) Ogbomoso (0.001) 3.33 (0.22) 1.51 (0.02) Ilorin (0.001) 3.07 (0.20) 1.49 (0.01) Okaka (0.001) 3.97 (0.18) 1.42 (0.01) þ (1) a Ife (0.001) 2.86 (0.15) 1.50 (0.00) Osogbo (0.001) 3.35 (0.15) 1.58 (0.02) Total samples taken 92 TBC, total bacteria count; cfu, colony-forming units. Values are means of three replicates. a No. of sample(s) testing positive. Poor personal hygiene of handlers and environmental hygiene have been reported to contribute significantly to the level of contamination in packaged drinking water in developing countries. 5 The poor microbiological quality of the drinking water samples recorded in this study has also been observed in other developing countries. 19,20 In a study carried out by Obiri-Danso et al. 19 on the microbiological evaluation of drinking water in Ghana, the authors reported that the water samples were of poor quality. According to them, the occurrence of indicator organisms in the water constitutes a serious threat to the community, and they called for strict observance of good manufacturing practices

4 732 O.A. Olaoye, A.A. Onilude / Public Health 123 (2009) Table 2 Prevalence of bacterial isolates in sachet-packaged drinking water samples in Western Nigeria. Sampling sites No of Escherichia samples coli Pseudomonas aeruginosa Enterobacter aerogenes. Klebsiella sp Proteus vulgaris Alcaligenes faecalis Bacillus cereus Staphylococcus aureus Streptococcus lactis Aeromonas sp. Iseyin 4 þ þ þ þ þ þ þ þ þ Oke-Iho 4 þ þ þ þ þ þ þ þ Ibadan 01 6 þ þ þ þ þ þ þ Iwo 4 þ þ þ Abeokuta 6 þ þ þ þ þ þ þ Ado-Ekiti 6 þ þ þ þ þ þ þ þ Ikorodu 4 þ þ þ þ þ Lagos 8 þ þ þ Ijebu Ode 4 þ þ þ þ þ þ þ þ Shagamu 4 þ þ þ þ þ Epe 4 þ þ þ þ þ þ þ Ibadan 02 6 þ þ þ þ þ þ Oyo 4 þ þ þ þ Ede 4 þ þ þ þ þ Ilobu 4 þ þ þ þ þ Ogbomoso 4 þ þ þ þ þ þ Ilorin 4 þ þ þ þ þ þ þ þ Okaka 4 þ þ þ þ þ þ þ Ife 4 þ þ þ þ Osogbo 4 þ þ þ þ þ þ Positive incidences % in total samples Micrococcus luteum by the processors and handlers. In a related study, Yassin et al. 4 reported poor microbiological quality of water in Gaza, which deviated from the recommended limits of the World Health Organization (WHO). The detection of E. coli, E. aerogenes and Klebsiella sp. implies that the water samples were potentially contaminated from faecal sources and, as such, were not safe for consumption. 3 WHO has reported that the occurrence of pathogens or indicator organisms in water sources mainly depends on the intrinsic physical and chemical characteristics of the catchment area, and the magnitude and range of human activities and animal sources that release pathogens to the environment. 21 Hence the bacteria found in the drinking water samples in Western Nigeria could be due to the sources of water used as the raw material by the processors. NAFDAC has published guidelines for small-scale production of sachet-packaged drinking water 8 with which processors are expected to comply. Unfortunately, many of the processors fail to do so, as this study has shown. Moreover, poor practices of the water handlers, during processing as well as distribution from factory to consumer, may contribute to contamination with unwanted organisms. It has been observed that sanitation and hygiene practices by many food handlers in Nigeria is poor. 22 It is the authors candid opinion that public enlightenment through reinforced and consistent health education on hygiene could contribute significantly towards improving microbiological safety in food and water processing plants in the country. The coliform count recorded in this study falls within the slightly polluted limits as published by WHO on the pollution of drinking water. 21 Detection of coliforms shows the danger of faecal pollution and the consequent hazard of contracting disease through pathogenic organisms. Disease-causing organisms transmitted via drinking water are predominantly of faecal origin, but the coliform count would not constitute much concern without the detection of E. coli in the water samples. It has been reported that typical enteropathogenic E. coli is a leading cause of infantile diarrhoea in developing countries, whereas this is rare in industrialized countries. 23 Among the microbial isolates identified from the drinking water samples and which are of public health concern is S. aureus. The micro-organism is a pathogenic bacterium responsible for severe health problems such as food spoilage, chronic infections and vomiting in humans. 24 S. aureus generally occurs in water that Micrococcus luteum Pseudomonas aeruginosa Aeromonas sp. Streptococcus lactis Staphylococcus aureus Bacillus cereus Alcaligenes faecalis Proteus vulgaris Klebsiella sp. Enterobacter aerogenes Pseudomonas aeruginosa Escherichia coli Frequency of occurrence in total samples Figure 2. Number of isolates in sachet-packaged drinking water samples in Western Nigeria

5 O.A. Olaoye, A.A. Onilude / Public Health 123 (2009) Table 3 Sachet-packaged drinking water samples conforming to some NAFDAC regulations. Sample no. Location No of samples Confirmation on sachet package P/E date Address NAFDAC no. 1 Iseyin Oke-Iho Ibadan Iwo Abeokuta Ado-Ekiti Ikorodu Lagos Ijebu Ode Shagamu Epe Ibadan Oyo Ede Ilobu Ogbomoso Ilorin Okaka Ife Osogbo Values are means of three replicates. P/E date, production/expiry date. contains organic pollutants, i.e. mineral ions and organic matter. 15 The organic matter content provides a better environment for the development of this bacterium in water sources. The occurrence of this bacterium in drinking water samples indicates contamination with organic pollutants. The risk of water contamination resulting in waterborne disease has been noted to be high in developing countries, 5 with the reasons being identified as: inadequate availability of water; poor quality of water at source; ill-maintained water pipelines and sewer lines; open air defaecation; lack of disposal of human, animal and household wastes; and lack of awareness of good sanitation and personal hygienic practices. Lead, iron and manganese are the greatest sources of consumer complaints in the water industry. 25 However, no lead and manganese were detected in the water samples in this study. Water distribution pipes in some processing facilities in Nigeria and other developing countries are mainly made of iron. As such, they may have undergone considerable corrosion over time and could readily contaminate the water supply with metal particulates. Although high iron concentrations may not constitute a direct health risk, this could have a negative impact on odour and taste. 26 It is interesting that the levels of iron found in this study were within the recommended limits. 3 The non-compliance of the water processors with NAFDAC regulations, as noted in this study, could pose serious concerns to public health. Many of the defaulting processors may not have been licensed for their operations, and this could be the reason why their plant location details were not printed on the water samples. They also do this to avoid their unlicensed plants from been sealed off by the regulatory agency. Such practices may have led to outbreaks of waterborne diseases in many parts of Nigeria. 10,11 Conclusion The overall results suggest the need for processors of sachetpackaged drinking water in Nigeria to improve their processing operations, especially in terms of hygiene, and to ensure strict compliance with guidelines as set by Nigeria s quality regulatory body. This study also revealed that the consumers of sachet-packaged drinking water could be at serious risk if necessary measures are not taken to improve quality. In addressing the public health concerns that are being generated by the sachet-packaged drinking water phenomenon in developing countries, especially Nigeria, intensive efforts are required in educating the processors, distributors and handlers of the product. Training should focus on the need for good personal hygiene and strict compliance with good manufacturing practices. Possession of hygiene certificates by staff of organizations seeking a licence for the production of sachet-packaged drinking water could be a useful criterion before approval is given. These measures will greatly enhance quality in operational procedures with subsequent promotion of public health safety among consumers of the product. The Government should give NAFDAC the necessary support to enforce their guidelines. Public enlightenment will also help consumers to be aware of what to look for on the package label before purchasing water for consumption. Ethical approval None declared. Funding The Gen Foundation, London, UK. Competing interests None declared. References 1. Suthar S, Chhimpa V, Singh S. Bacterial contamination in drinking water: a case study in rural areas of northern Rajasthan, India. Environ Monit Assess; 2008;. doi: /s World Health Organization. Emerging issues in water and infectious disease. Geneva: WHO; World Health Organization. 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