Microbial contamination and quantitative microbial risk assessment of high-density polyethylene (HDPE) film sachet drinking water in Ghana

Depending on an individual's age and gender, water may constitute between 50 and 60% of fat-free body mass (Jequier & Constant 2010). This body water is contributed largely by drinking water. However, contaminated water has been implicated in millions of deaths across the world over the years. From the infamous cholera pandemic of the industrial revolution that devastated Asia, Europe, and the Middle East in the mid-19th century (Tulchinsky 2018) to the more recent cholera outbreak in South America in the 1990s and the Walkerton water disaster in 2000 (Choffnes & Mack 2009), waterborne diseases have challenged and unsettled anthropogenic systems. In developing countries across the world, many children in rural communities and urban areas continue to die from exposure to water resources contaminated with pathogenic bacteria (Ntouda et al. 2013; Dos Santos et al. 2015; Ogbo et al. 2017). Several pockets of widespread morbidities and mortalities have also been reported in different parts of the world as a result of poor microbial loads in drinking water, prompting authorities to call for the provisioning of improved and secured drinking water resources (Mofidi 2016; Livingston 2021). Recent estimates by the United Nations Children's Fund revealed that more than 800 million people in Africa lack access to basic water, sanitation, and hygiene (WASH) services (UNICEF 2022). Within the Sub-Saharan Africa (SSA) area, only 30% of the population relies on improved sources of drinking water (United Nations 2020). It has been reported that more than half a million cases of diarrhoea were recorded in 2017 globally (Roth et al. 2018; Robert et al. 2021). In Ghana, diarrhoeal cases continue to headline reports on communicable diseases, especially among children under 5 years (Asamoah et al. 2016; Salifu 2020; Larbi et al. 2021; Asare et al. 2022) necessitating the need for improved water and sanitation infrastructure, and concerted data-reporting mechanisms.

Enterogastritis is a common condition in populations exposed to unsafe water and sanitation conditions (Ashbolt 2004; Ezzati et al. 2004). Infectious bacterial aetiologies are the leading cause of diarrhoeal gastroenteritis, especially among children in developing countries (Platts-Mills et al. 2015; Ugboko et al. 2020; Behera & Mishra 2022). Among the bacterial strains, an inviolable link has been established between pathogenic Escherichia coli (E. coli) O157:H7 and gastroenteritis since the 1980s when this strain was first identified and described (Mead & Griffin 1998; George et al. 2013; Zaychikova et al. 2020). Most E. coli are innocuous but E. coli O157:H7, a member of the verocytotoxic and enterohaemorrhagic group, causes shigella-like dysentery often characterised by blood-stained mucus and haemorrhagic colitis which can progress into renal failure in children and adults (Levine et al. 1987; Griffin & Tauxe 1991). The health and economic losses due to E. coli O157:H7 have been well established (Frenzen et al. 2005; Cabral 2010; Bivins et al. 2017; Wibisono et al. 2018; Islam & Islam 2020; MacKinnon et al. 2020; Powell et al. 2000; Barragán et al. 2021). According to the World Health Organization (WHO), the recommended level of risk due to pathogenic bacteria is a minimum of 1 × 10⁻⁶ attacks (World Health Organization 2017). E. coli O157:H7 has been isolated from drinking water samples across the world (Rompré et al. 2002; Thenmozhi 2010; Batabyal et al. 2013).

Water treatment and processing methods continue to evolve in an attempt to deliver safe and secure water supplies to populations across the world. In West Africa, the use of high-density polyethylene (HDPE) sachet water is a popular choice among water manufacturing and treatment companies due to its low cost and ease of access. Sachet water is used by all age groups in Ghana – babies, children, and adults, and across all income classes. Drinking water packaging businesses are considered lucrative due in large part to increasing demand, ease of production, ready access, and affordability. Sachet drinking water serves the primary drinking water needs for large sections of the community. This has led to the proliferation of mostly small and medium enterprises that also provide significant employment to the general population, and value chain services to suppliers and distributors. Some of these companies have evolved their processes to include bottled water and soft drinks. Previous studies have estimated that the minimum daily output for cottage manufacturers is 15,000 sachets while large-scale manufacturers may produce several million sachets (Quartey et al. 2015; Kaku 2019). The water is sourced from the public water system, underground water (borehole, well), or surface water (rivers, springs, streams, lakes). Manufacturers must register with the Ghana Standards Authority and the Ghana Food and Drugs Authority but most of them are unregulated and the safety of their supplies cannot be guaranteed. In some instances, the regulatory and compliance regimes have been questioned, raising doubts about institutional professionalism and technical competence (Aji et al. 2015; Vapnek & Williams 2016; Morinville 2017; Kusa & Joshua 2022). Records available at the Association of Sachet and Packaged Water Manufacturers Association (Ghana) suggest that about 100 million 500-mL sachet water packs are manufactured weekly in Greater Accra and distributed within the region and adjoining communities.

While drinking water quality has been well documented in developed countries, very few reports are publicly available from developing countries, especially in Africa. Kangmennaang et al. (2020) postulated that the proliferation and use of packaged water in developing countries, although an improved source of drinking water, is a major health challenge due to poor handling and sanitation, and the influence of unsafe environmental conditions during storage. There has been perceived quality of plastic packaged drinking water (Page et al. 1993; Stoler et al. 2012; Wardrop et al. 2017), nevertheless, research has proven the presence of unacceptable levels of heterotrophic and pathogenic microorganisms (Al-Saleh et al. 2011; Osei et al. 2013; Fisher et al. 2015; Benjamin et al. 2017; Luo et al. 2018; Oßmann et al. 2018; Ali 2019; Jayaweera et al. 2020). Poor housekeeping and poor manufacturing practices at packaged drinking water facilities may become sources of microbial contamination for treated water.

The current literature relating to experimental microbiology of HDPE film water largely demonstrates plate counts and provides scanty information on dose–response. Kumar et al. (2021) have suggested that qualitative methods are suitable for monitoring purposes but may have limited applicability in estimating contamination and risk profiling. The lack of quantitative health risk assessments on the shelf stability of packaged drinking water may characterise inestimable acute and chronic health implications for the general population in many developing countries. The present research seeks to determine the impact of storage on microbial quality and the risk of morbidity due to the ingestion of sachet drinking water. It also inventoried secondary data on the microbial quality of sachet drinking water in Ghana where sachet water is a dominant source of primary drinking water (Wright et al. 2016; Vedachalam et al. 2017) using high-impact repositories between 2012 and 2022. To the best of the knowledge of the authors, the present work is the first quantitative microbial risk study dedicated entirely to HDPE film sachet drinking water in a developing country. On the premise that sachet water is perceptively considered better than tap water by the general population (Osei et al. 2013; Wardrop et al. 2017; Dzodzomenyo et al. 2018; Manjaya et al. 2019; Semey et al. 2020), the study hypothesises that the presence of microorganisms in sachet water should not be a concern to the general population.

Water samples were obtained in tropical Africa along the Greenwich Meridian. Two dominant sachet water manufacturing and distribution companies in Greater Accra (Ghana), named A and B, were selected for this study. There are currently more than 3,000 sachet water producers in Ghana and about 1,000 producers in the Greater Accra region alone (Mosi et al. 2019). The companies selected for this study are among the top 10 producers in the region, producing about 1,000 m³ of plastic packaged water daily between them. The companies source their water supplies from boreholes drilled over the Accra-Keta geological formation. The water is then filtered and softened through multiple layers to remove soluble salts, ions, and suspended solids using sand, carbon filters, reverse osmosis, and softening/deionizing chambers. Multiple disinfection processes using ultraviolet sterilisation, ozonation, and chlorination are also employed to remove microbial contamination. The processed water is then packed in 500-mL HDPE sachet films. The water packs are embossed with label and batch information including 6-month standard expiration date and a batch number. Apart from the Greater Accra region, produce from these two companies is also distributed in five other regions including the Volta, Eastern, Central, Western, Western North, and Asante Regions. The geographic spread of these supplies, therefore, makes their representation national in character.

The experimental sample size was estimated by computing the power for two independent sample proportions using the Statistical Analysis System of John's Macintosh Program (SAS JMP) version 16. A two-sided exact test was imputed at an error margin of 0.05 for 50% of the population that will be required to obtain 100% sampling power, resulting in 150 minimum samples for each company. 300 packets of 500-mL sachet water samples were thus collected from the two companies in the month of March. The samples were labelled A and B according to the company and stored over 4 weeks in three temperature regimes: 8 °C, refrigeration temperature; 30 °C, average room temperature (within the range of 21–37 °C); and 40 °C, average ambient air temperature (within the range of 25–55 °C). These temperatures were chosen to mimic the common storage environments of vendors and consumer sachet drinking water in Ghana. Sachet drinking water is generally sold ice cold (at ∼8 °C), or stored at room temperature and ambient air.

Samples were analysed for total heterotrophs using techniques for total plate count as specified in Method 9215 (Baird et al. 2017). Faecal coliforms and E. coli were tested with Method 9222 (Baird et al. 2017). Electrical conductivity (EC), turbidity, total chlorine, pH, and alkalinity were also determined using potentiometric methods for pH and EC, alkalinity by titrimetry, and total chlorine by colourimetry. The temperature of the samples was determined in situ. Temperature, pH, and EC were measured with pre-calibrated electrometric HANNA instruments using 100 mL of the water samples. Alkalinity was determined by titrating 0.1 N HCl against the water samples using methyl orange at an endpoint indicated by a purple colour. The alkalinity was reported as CaCO₃ in mg/L. During analysis of the samples for total chlorine, 10 mL of water samples were carefully transferred into N,N-diethyl-p-phenylenediamine (DPD) total chlorine test tubes and mixed uniformly, then analysed with a handheld HACH colourimetric instrument using standard methods (HACH Company 2012). The results were reported in mg/L Cl₂. Turbidity was measured with a Turbidimeter Model 2100P by reading the clarity of 25 mL of water samples in Nephelometric Turbidity Units (NTU) (APHA 1999). Between three and four samples were used for each parameter by mixing them thoroughly in a pre-sterilized stainless steel jug before drawing appropriate aliquots for the analysis.

All equipment for the micro lab was properly cleaned and sterilized in an ETHOS 900 Lab Station microwave set at 121 °C for 15 min. All laboratory procedures were performed under aseptic conditions to assure the high statistical power of the results. Surfaces were disinfected with 70% alcohol solution prepared from analytical grade ethanol (99.9%). All reagents used were of high analytical purity (≥99.9%) and aseptic grade. The results were reported in colony-forming units (cfu)/100 mL.

Among members of the E. coli, E. coli O157:H7 was chosen as the reference pathogen for the risk assessment because it is the commonest serotype of the Entorohaemorrhagic group and causes more widespread outbreaks than other strains (George et al. 2013; Wright et al. 2016). E. coli O157:H7 is also popular among waterborne pathogens (Howard et al. 2006).

Secondary data were curated from Scopus, Web of Science, and PubMed-indexed scholarly articles relating to experimental research on microbial assessment of sachet water in Ghana. These databases were chosen due to their reputation for indexing journals of high editorial rigour, scholastic quality, and adherence to best industry practices. The results were filtered for the period between 2012 and 2022 and inspected for countable total microbial loads and E. coli. Only maximum quantifiable contaminant values were used to reflect the maximum exposure loads possible. For studies reporting too numerous to count (TNTC) results, only the maximum numeric value was chosen.

Data were described with Statgraphics version 17 and regressed with SAS JMP version 16. Correlation coefficients were computed to determine the association between the different samples. Central composite designs using the fit least squares method, and response surface designs were modelled to evaluate the effect of time and temperature on the contamination of water samples. Stochastic models of exposure were simulated with the Markov Chain Monte Carlo package in Argo 4.1.3 software.

Deterministic risk assessment of exposure to sachet drinking water contaminated with E. coli O157:H7 was estimated by the following equations (Haas et al. 2014):

The daily and annual risks were estimated as beta-Poisson stochastic models using E. coli O157:H7 hazard indicators. This model proposes that the probability of an ingested organism surviving and infecting its host follows a beta distribution (Powell et al. 2000). This method has been validated for use in estimating the dose–response relationship for E. coli O157:H7 through ingestion exposure in human models (Haas et al. 2000). In the present study, the model is discriminated against children and adults using data from Ghana's (2021) Population and Housing Census (PHC) (Ghana Statistical Service 2021). According to the Ghana Statistical Service (GSS), 71% of the population in the Greater Accra Region depend on sachet water for their drinking water needs.

These physicochemical results are comparable to reports by Ankomah (2019), although Bowan (2022) reported lower turbidity values for borehole water in the Bole district of the Savannah region of Ghana. The high turbidity values reported in this study may be attributed to severe groundwater table disturbance due to the long periods of mechanical abstraction. Similarly, the high values of alkalinity and EC of the raw borehole water may be the result of dissolved ions while the lower values in the processed and packaged samples could be due to softening/deionizing and osmotic filtration processes that are designed to remove these ions. The pH for the raw water was near neutral due to the buffering capacity of bicarbonate, carbonate, and hydroxide ions, which also define its alkalinity. Treatment processes were also competent at maintaining an adequate level of alkalinity to stabilise the pH. The absence of chlorine in the raw water may be attributed to its groundwater origin. The near chlorine-free levels of the treated and packaged water samples may be the result of inadequate residual chlorination following purification. Total chlorine provides disinfection and anti-microbial activity in the water, and the lack thereof is a potential for microbial contamination during storage, transport, and distribution.

There was no statistical difference between water samples from the two sites (p-value > 0.05) but the results showed a weak positive relationship between HPC and E. coli (Pearson correlation coefficient = 0.14).

These results are consistent with prior works by Angnunavuri et al. (2022a), Aslan et al. (2020), and Mosi et al. (2019) who investigated the quality of packaged water in Ghana. Temporal microbial quality has been determined to deteriorate in bottled water (Herath et al. 2012) and pasteurised milk (Petrus et al. 2010) although E. coli O157:H7 contamination was not reported. Combined ultraviolet and chlorination disinfection, and ozonation processes may have contributed significantly to the absence of microbial contamination in the initial water samples. Packaged water has become very popular in Ghana, serving as the most convenient form of potable water and a key index for quality health and well-being among the populace. As such, it is expected that such water should have the highest level of quality and must be shelf-stable. Water producers may bypass safety protocols and apply uncertified procedures and equipment in water production with attendant negative public health consequences (Dzodzomenyo et al. 2018). Sachet water continues to attract research attention due to its tendency to contribute to enteric epidemic outbreaks (Moore et al. 2015; Banu et al. 2018; Mbala-Kingebeni et al. 2021). Microbial quality in plastic packaged water has been observed to decrease in the order of PET < HDPE (Angnunavuri et al. 2022a) < LDPE (Herath et al. 2012), assuming standard manufacturing guidelines and hygiene practices are adhered to.

A major criterion for assessing the safety of potable water is its level of microbial contamination (Thompson et al. 2007; World Health Organization 2017). Generally, food-product spoilage is caused by an aggregation of microorganisms of varying incubation periods but they become a health concern when they or their by-products like toxins reach their infective doses for both immune-competent and immune-compromised individuals. Despite many interventions for improved microbial water quality, inadequate treatment systems, poor housekeeping and manufacturing/processing practices, and compromised or inappropriate distribution systems contribute to poor microbial profiles of drinking water. Studies by Osei et al. (2013), Awuah et al. (2014), Mosi et al. (2019), and Aslan et al. (2020) have revealed significant exceedances of various toxic microorganisms, including faecal and pathogenic subtypes, in sachet water sampled from different parts of Ghana, urging the need for strict monitoring by regulatory agencies. Packed sachet water in Niger and Nigeria has also been determined to have significant levels of microorganisms, and questionable safety (Dada 2009; Fisher et al. 2015). Human exposure to pathogenic bacterial strains of E. coli, Cryptosporidium, Klebsiella, Salmonella, Shigella, Vibrio, and Giargia in low- and medium-income countries has been majorly attributed to drinking water (Brouwer et al. 2018; Khabo-Mmekoa et al. 2022).

The growth profiles shown above agree with Addo et al. (2016) and Chinenye & Amos (2017) when they examined sachet water under storage in Ghana and Nigeria. It is not uncommon to find distribution outlets that store water under direct sunlight that is then sold to consumers in these countries (Ajekunle et al. 2015; Vedachalam et al. 2017). According to the Ghana Multiple Indicator Cluster (MIC) Report, 41.6% of primary drinking water sources are contaminated by E. coli at moderate to very high levels (between 1 and 100 cfu/100 mL) in the Greater Accra Region where the prevalence of sachet drinking water was estimated at 74% (Ghana Statistical Service 2019). Poor housekeeping and lack of compliance with good manufacturing practices at production facilities may introduce dormant or culturable bacteria into processed water. In the case of E. coli O157:H7, the duration of incubation has been estimated between 2 and 8 days under ambient conditions (Soller et al. 2010). High bacterial counts have also been blamed on poor handling and environmental conditions, especially outdoor ambient air storage (Adewoye et al. 2013; Addo et al. 2016). The early emergence of bacteria in water samples has been associated with low levels of chemical disinfectants (Soller et al. 2010; World Health Organization 2017; Udoh et al. 2021; Angnunavuri et al. 2022a), and microbial contamination in sachet water has also been determined to be inversely related to residual chlorine levels (Karikari & Ampofo 2013). Rice et al. (1999) reported a significant reduction of E. coli O157:H7 up to four orders of magnitude in water samples dosed with 1.1 mg/L of free chlorine. Apart from poor microbial concentrations, high concentrations of organic chemicals at unfavourable environmental conditions during storage may be a source of public health concern, considering the large number of people that depend on sachet water (Machdar et al. 2013; Momtaz et al. 2013; Harkin 2019; Kangmennaang et al. 2020; Angnunavuri et al. 2022a). Endocrine-disrupting chemicals have been variously found in plastic packaged water with indications that they may have leached from the primary package during storage (Amiridou & Voutsa 2011; Bach et al. 2012; Guart et al. 2014; Zaki 2015; Khaustov et al. 2020; Pourzamani et al. 2020; Razali et al. 2021; Ibeto et al. 2022; Kumawat et al. 2022). Co-contamination of plastic packaged drinking water due to matrix components and pathogenic microorganisms may pose severe adverse health effects to consumers. In our previous works on HDPE sachet packaged water, we determined a positive correlation between storage temperature and the concentration of phthalates (Angnunavuri et al. 2022b). Ibeto et al. (2022) also found a similar relationship between plasticisers in sachet water and incubation duration.

The annual probability of infection was strongly associated with increasing storage duration and increasing temperature. The estimated population level risks in terms of the number of cases based on the annual probability of exposure were between 8,910 and 12,914 for children under 5 years; 22,700 and 30,189 for children in general; and 44,778 and 332,542 for adults in general. As reported by Dos Santos et al. (2015), the dependence on an improved drinking water source is not foolproof against enteric infections. Due to their low level of immunity and smaller body sizes, children are more prone to developing waterborne infections from pathogenic bacteria than adults. One in 10,000 morbidity ratios of E. coli O157:H7 risk levels in household drinking water samples has been estimated in rural and urban areas in KwaZulu Natal (Khabo-Mmekoa et al. 2022), instigating the need for continuous monitoring of water resources and adequate drinking water treatment and WASH services to protect public health. Generally, unsafe drinking water has been complicit in the high morbidity and mortality of underaged children in developing countries (Adane et al. 2017).

Ten (10) historical studies were reported from the renowned repositories out of which 90% contained information relevant to this study. About 56% of these studies were dedicated to the Ashanti Region of Ghana and the remainder to other geographic locations as presented in Table 2. All the listed studies were mostly cross-sectional, using random samples across different brands. The maximum E. coli concentration was 4.0 × 10⁴ and the minimum was 0.2 × 10¹. Although the present study is longitudinal, the results of the HPC and E. coli counts were within the range of the historical set.

The dose–response model of the historical data did not indicate any safe level for sachet water, as all reports presented higher than the WHO recommended level of 1 × 10⁻⁵ annual risk of infection. The mean daily rate of infection was 2.36 × 10⁻² attacks per person per day within a sensitivity of 1.60 × 10⁻⁴ and 2.26 × 10⁻², given the ingestion of 3 L of water per day.

The historical exposure to E. coli O157:H7 from HDPE sachet water also depicted a high probability of infection in the general population. Extrapolation of the annual infection rates to the target population of 3,866,828 persons indicated that about 200,000 persons were at risk of E. coli O157:H7 complications annually. A 5-year retrospective study of national MIC data by Apanga & Kumbeni (2021) estimated a 17% prevalence of diarrhoea among 8,879 children in Ghana. Diarrhoeal cases among children are widespread in developing countries and are mostly associated with maternal age, maternal education, poverty, poor sanitation and unsafe drinking water (Mohammed & Tamiru 2014; Amugsi et al. 2015; Tesfamariam et al. 2019; Manandhar & Kakchapati 2021; Tareke et al. 2022). Children's age has also been significantly associated with diarrhoea by Mulatya & Mutuku (2020). Piped drinking water alternatives such as water jars and water tankers in Nepal (Kobayashi et al. 2022) and water jerry cans in Ethiopia have been found to contribute significantly to diarrhoeal cases (Soboksa et al. 2020). A weak association between the ingestion of sachet water and diarrhoeal morbidity in Nigeria has also been established by Ugboko et al. (2021). Pathogenic E. coli has been reported in sachet water and found to be resistant to antibiotics (Umar et al. 2019; Umar et al. 2021).

This study was confined to regulated water producers which are expected to produce under hygienic and standard good manufacturing practices as specified by regulations. The results determined higher microbial loads and pathogenic microbes of faecal origin in sachet water supplies in Ghana. The probability of daily and annual infection due to E. coli O157:H7 was significantly higher than WHO threshold recommended exposure limits. Results from this study may engender scientific discussions on the need to enforce regulations during sachet drinking water manufacturing. Further risk assessment models that conscript larger sample cohorts are also needed to understand the full extent of exposure to sachet drinking water. Enteric diarrhoea contributes to adult and child morbidity, and interventions are necessary to avoid preventable deaths and improve the general well-being of the population.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.