1. Introduction

Water is indispensible for life considering the fact that it is an essential part of human nutrition. Water of good drinking quality is of basic important for man’s continuous existence. It is also require for maintenance of personal hygiene and prevention of diseases. Lack of safe drinking water is a serious threat to national development and may have a negative impact on the health status of citizens. In many developing countries, availability of water has a critical and urgent problem and it is a matter of great concern to families and communities depending on non-public water supply system (Okonko et al., 2008).

The challenges associated with drinking water have encouraged the production of packaged drinking water by private enterprises that have little knowledge about manufacturing practices (Edema et al., 2011). The introduction of sachet water in Nigeria was a laudable idea, but studies suggested that this innovative idea is not risk free (Alli et al., 2011). Today, the easy accessibility to drinking water in packaged forms has resulted in big thriving water industries with several millions of litres of these products consumed every year by Nigerians (Ogundipe, 2008). Most people in the cities and rural communities depend on sachet water as their ultimate source of drinking water. The integrity of sachet water is doubtful in fact, it has been reported that most of the vendors do not treat their sachet water before selling to the public and many engaged in the production, do not follow strictly the standard set by Federal Environmental Protection Agency and World Health Organisation (Oladipo et al., 2009; Okpako et al., 2009). The consumption of sachet water in Nigeria is on alarming rate and people are not mind full of the source, quality and possible consequences associated with sachet water consumption. Despite its popularity, studies conducted on the microbial quality of sachet water in some African cities have catalogued various levels of contamination (Adenkunle et al., 2004; Ifenyi et al., 2006; WHO, 2011).

Fungi are widely distributed in nature and can occur as unicellular yeast or filamentous and, multicellular molds. Despite their wide occurrence, little attention has been given to their presence and significance in aquatic environments (Kirk et al., 2001). In 1980s and 1990s, more cases of health problems caused by fungal contaminated drinking water were reported from Finland and Sweden (Muittari et al., 1980; Aslund, 1984). Fungi have been reported from all types of water which included bottled and sachet drinking water (Mirian et al., 2007; Okpako et al., 2009). The most commonly isolated genera from drinking water are Penicillium, Cladosporium, Aspergillus, Phialophora and Acremonium (DEFRA, 2011). They are involved in different forms of diseases, including allergies to fungal antigens, production of toxins, or direct invasion of hosts (McGinnis et al., 1996). Several species of fungi are capable of infecting healthy hosts and cause diseases ranging from mucosal to life threatening disseminated infections (Mirian et al., 2007). The study aimed at assessing fungi which are capable of contaminating sachet water, Zaria, Nigeria.

2. Materials and Methods

Sample collections

A total of three hundred and sixty sachets from six brands of sachet water sold within Ahmadu Bello University, Zaria, Kaduna State, Nigeria. These were randomly collected from the production companies for six months. From each brand of sachet water, ten sachets were collected each month which were divided into two, halves of five sachet each. One half was stored at ambient temperature while the other half was refrigerated. Within 24 hours after collection, sample were analysed to assess the rate of contamination by fungi. Subsequently, each week single sachet was randomly picked from the stored samples for analysis within the month.

Preparation of Culture Medium for the Isolation of Fungi

Potato Dextrose Agar was used for the isolation, was prepared according to manufacturer’s instruction. The molten medium was poured into conical flasks, plugged with aluminum foil. The medium was sterilized by autoclaving at 121°C at a pressure of 15 pound for 15 minutes. After sterilization, 15 ml of medium was aseptically dispensed into sterile petri dishes and allowed to solidify. The petri dishes were labeled accordingly.

Method for Isolation Fungi

Many methods for isolation of fungi have been mentioned in literatures; however, there is no standard method for isolation of fungi in water (DEFRA, 2011). Based on result obtained from pilot, Cheesbrough (2004) method was adopted by replacing the membrane filter with filter paper of 0.45μm pores. From each sample 100 ml was dispensed into two sterilized test tubes each and centrifuged. The centrifuged samples were filtered through a small disc of sterilized filter paper of 0.45μm pores and the membrane filter was placed on Potato Dextrose Agar plates in duplicates and labeled accordingly. All isolates from the medium were sub cultured into Potato Dextrose Agar (PDA) slant labeled appropriately and refrigerated for further assay.

Identification of Fungi

Isolated fungi were identified by examining both microscopic and macroscopic characters. The identification was aided by using identification keys of Barnett and Hunter (1972), Larone (2002), Klich (2002) and Samson et al, (2004).

Plate 1. Pure Culture of 1. Aspergillus flavus 2. Aspergillus fumigates 3. Aspergillus niger 4. Penicillium glabrum 5. Fusarium oxysporum

Plate 2. Pure Culture of 1. Cephalosporium curtipes 2. Oidiodendron griseum 3. Rhizoctonia solani 4. Rhizoctonia solani

3. Results

A total 9 of fungal species were isolated from five sachet water samples while one was devoid of any fungus even thought their distribution was not even (Table 1). The isolates were identified by studying their macroscopic and microscopic characters and were compared with already described species using identification keys by Barnnet and Hunter, (1972), Larone, (2002), Klich, (2002) and Samson et al., (2004) (Table 2). It is evident from Table 3 that Aspergillus niger was more prevalence out of the three Aspergillus species as it was isolated in three different samples with higher percentage of 12.50% followed by Pencillium glabrum and Fusarium oxysporum with 8-33% each while Aspergillus flavus, Aspergillus fumigates, Cephalosporium curtipes, Odiodendron griseuum, Rhizoctonia solani and Trichoderma viride had the same percentage of 4.17% each. There was no significant difference in the mean of the fungal colony count at (P˃0.05) between after collection and when stored at ambient temperature and when refrigerated except OJT which was significantly different (P˂0.05). It is evident that ambient temperature fovours the multiplication of fungi as the higher colony count was recorded at ambient temperature (Table 3).

Table 1. Fungi Species Isolated and their Distribution from Six Different Brands of Sachet Water

Table 2. Description of the Macroscopic and Microscopic Characteristics Fungi Species Isolated

Table 3. Fungi Species Isolated from Six Brands of Sachet Water and their Percentage Occurrence in Parenthesis

Table 4. Mean (±S.E.) Value of Fungi Colony Count of Six Brands of Sachet Water

4. Discussion

Five out of the six brands of sachet water were to be contaminated with varied number of fungi species (Table 1). The occurrence of varied number of fungal species in different sachet water indicates the status of the treatment rendered to the water during production. The presence of fungi species in any sample indicates inadequate treatment while the absences of fungus in BRT imply the treatment was probably effective (Table 1). Aspergillus niger had the higher percentage of occurrence of 3 (12.50%) followed by Pencillium glabrum and Fusarium oxysporum which recorded 2 (8.33%) each (Table 2). The higher percentage of occurrence by Aspergillus niger, Pencillium glabrum and Fusarium oxysporum could be attributed to its ability to secrete pigment called melanin which provides protection against a range of stresses making them resistant to water treatment coupled with inadequate treatment (Waipara, 1998; Langfelder et al., 2003). Aspergillus flavus, Aspergillus fumigatus, Cephalosporium, Odiodendron griseuum, Rhizoctonia solani and Trichoderma viride had the low percentage of occurrence of 1 (4.17%) each (Table 2). The low percentage of occurrence could be adduced to effective water treatment methods such as ozone and chlorine dioxide against the fungal spores as reported by Kelley et al., (2003).

Mean of the colony count also varied even though it was not significantly different (P˃0.05) except OJT which was significantly different (P˂0.05). OJT had the highest mean of colony counts than other samples which indicated high level of contamination This varied percentage of occurrence and mean of colony count could be attributed to variation in level of treatment, poor hygiene practices, poor storage of treated water and poor management of storage facilities, unhygienic working environment, use of unsterilized materials, difference in sources of water used for production coupled with inadequate treatment. Similar observation was made by Hageskal (2007); Okpako et al., (2009); Tanveer et al., (2011).

The occurrence of these fungi may cause diverse effects on human health as they have the potential of producing mycotoxins. The concentrations of these substances may increase during storage of water due increase in the population of the fungi species, hence, daily intake of such water containing mycotoxin could result in bioaccumulation in the body which could be hazardous to human health. Aspergillus flavus, Aspergillus fumigatus and Aspergillus niger are known to produce aflatoxins, ochratoxins and fumitremoigin which are carcinogenic and are capable of causing kidney and liver disorders, invasive and non invasive aspergillosis, allergic and sinusitis (Bryce, 1999; Bennett and Klich, 2003 and Samson et al., 2004). Penicillium glabrum produce citromycetin which is known to cause allergy, asthma and some respiratory problems (Cooley et al., 1998; Frisvad et al., 1998; Samson et al., 2004 and Gunhild et al., 2006). Fusarium oxysporum is capable of producing fusaric acid, monilifornun, gibepyrones and naphthoquinone pigment which have been recognized as agents of superficial infections (Guarro and Gene, 1995 and Samson et al., 2004). Trichoderma virides produces trichothecenes, alamethicins, emodin, trichotoxin and suzukacillin and have been reported to cause mycosis and allergy in humans (Jaakkola et al., 2002; Tang et al., 2003 and Samson et al., 2004).

5. Conclusions

The findings revealed the presence of pathogenic fungi species in the samples which indicate risk involved in consumption of such products and are therefore could be hazardous to human health. Based on the results obtained in this study, it could be concluded that sachet water are contaminated with fungi. The predominant fungi genera associated with the sachet water are Aspergillus, Penicllium and Fusarium. The presence of those microbes in sachet water has been traced to inadequate treatment and poor hygiene practices during production which has significantly affected the quality of the sachet water making the water unfit for human consumption. It also revealed that ambient temperature favours multiplication of fungi. It revealed that storage has significant effect on the microbial quality of sachet water if the treatment is inadequate during production. It also revealed that the quality of sachet water can be deteriorated within the shelf life if the water produced is not adequately treated.

6. Recommendations

It is recommended that all manufacturing industries must adhere to National Agency for Food and Drug Administration and Control (NAFDAC) guidelines and all the existing laws should be enforced considering the high patronage of sachet water in the area of study. In addition, there is need for awareness programs in order to educate the general public on the potential health implication associated with consumption of such products. Furthermore, to safeguard the health of the people there is need for regular monitoring of the quality of the water and the environment by National Agency for Food and Drug Administration and Control (NAFDAC).

ACKNOWLEDGEMENTS

We are grateful to the Department of Biological Sciences, Ahmadu Bello University, Zaria for providing the basic materials that was used for the analysis and the staffs of Microbiology Laboratory at the Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria for their support in the laboratory.