Longitudinal study of microbial load of drinking water and seasonal variation of water quality at the point of use in food establishments of Addis Ababa, Ethiopia

The study aimed to determine the status of microbial load of drinking water and seasonal variation of water quality. An institution-based longitudinal study was conducted. 1,141 food establishments were divided into slum and non-slum areas based on their location. Moreover, they were categorized as large and small food establishments. Then, 125 food outlets were selected using a simple random sampling technique. From the selected food outlets, 250 drinking water samples were collected directly from the drinking water storage in the rainy and the dry seasons. Data analysis was conducted using a repeated-measure ANOVA statistical model. The finding indicated that, 26.4% and 10.7% of the food establishments’ drinking water was positive for Escherichia coli in the wet and the dry season, respectively. Moreover, 3.2% and 1.6% of the food establishments’ drinking water had very high health risk to customers during the wet and the dry season, respectively. The drinking water at the point of use was found to be vulnerable to microbiological contamination and had a serious health risk. Therefore, good sanitation and proper handling of drinking water, and effective drinking water treatment, such as disinfection and filtration, should be practiced in all food establishments.


INTRODUCTION
Globally, drinking water quality is continuously deteriorating and becoming inappropriate for human use and wellbeing of society due to high population growth, expansion in industries, discharge of waste water and chemicals into canals and other water sources (Memon et al. ; Mohsin et al. ). As a result, access to potable water is one of the major challenges of the 21st century because 20% of the world population does not have access to pure drinking water (Saxena et al. ; Zameer et al. ).
Over the past several decades, many people in developing countries did not have a safe and sustainable water supply scheme (Hunter et al. ; World Health Organization ). Due to these and other human-made factors, the health burden of poor water quality and poor sanitation practice is considerable (Chauhan et al. ; Girmay et al. ). Worldwide, waterborne diseases have been estimated to cause more than two million deaths and four billion cases of diarrhea annually (El-Kowrany et al. ).
United States of America (USA)-based data indicated that of 9,040 food and waterborne disease outbreaks, approximately 4,675 (52%) of these were attributed to food establishments (Boro et al. ). Poor handling of drinking water, inadequate water supplies, and inadequate sanitation are responsible for a large proportion of disease transmission in developing countries (Mara ; Taylor et al. ). Moreover, poor environmental sanitation, poor personal hygiene practices, inappropriate storage of drinking water, and poor waste management practice of food establishments may cause water contamination and affect the health of customers (Ifeadike et al. ; Girmay et al. a).
Like many African countries, Ethiopia has a shortage of water, poor sanitation, and a lack of access to clean water sources (Hendrix ). Even though access to quality water and good sanitation are major determinants of preventable diseases in developing countries such as Ethiopia, a study conducted by Water.org stated that, only 42 and 11% of Ethiopians have access to a clean water supply and adequate sanitation services, respectively (Seyoum & Graham ).
This indicated that above half of the population did not have a chance to access clean and safe drinking water. Addis Ababa city gets water from Dire, Gefersa, and Legedadi reservoirs, as well as several boreholes concentrated around Akaki. Moreover, drinking water is supplied via water trucks for low pressure areas. However, nearly half of the drinking water demand of the city is not met. This supply deficit is causing frequent water supply interruptions. The food establishments of Addis Ababa suffer from a shortage of drinking water. As a result, they are suspected to be major sources of water-and foodborne disease which might arise from the inadequate drinking water, poor sanitation, and poor storage practices. Hence, this study aimed to assess the quality of drinking water at the point of use (drinking water storage) in Addis Ababa food establishments.

Description of the study area
The study was conducted in Addis Ababa, the capital city of the Federal Democratic Republic of Ethiopia and the seat for the African Union (Girmay et al. b). There are 1,141 licensed food establishments in the city. Of the total food establishments, 95 (8%) are high quality hotels with one or more stars, and the majority 1,046 (92%) are small food establishments, which include non-star rated hotels, bars, cafes, restaurants, etc. (Girmay et al. c). The Addis Ababa city administration government provides safe water supply to the food establishments. However, the safety of the supplied water handling practice varies from sub-cities to sub-cities. Moreover, the provided water also, in general, is inadequate and frequently interrupted. The location map of Addis Ababa city is depicted in Figure 1.

Study design
An institutional-based longitudinal study was conducted in Addis Ababa city.

Source population
All food establishments were located within Addis Ababa city administration.

Study population
All selected food establishments were located within Addis Ababa city administration.

Inclusion criteria
All food establishments that provided services in the city were included during the first selection.

Exclusion criteria
Establishments that provide only packed drinking water were excluded from the study, because they are less likely to be contaminated.

Sample size determination
The sample size was calculated using an unmatched cohort and cross-sectional study formula (

Sampling procedure
At the first instance, a listing of the 1,141 licensed food establishments was obtained from Addis Ababa Food, Medicine Health Care Administration and Control Authority (AAFMHACA). These food establishments were divided into slum and non-slum areas based on their location and into large and small food establishments, based on their size. Sample allocation was conducted. Then, the required food establishments were selected using simple random sampling technique. From the non-slum area, 7 large and 44 small food establishments were included. From the slum area, 3 large and 71 small food establishments were selected. In total, 10 samples from the large and 115 samples from the small food establishments (total of 125 samples of food establishments) were included. Addresses of these selected food establishments were registered, to make the next visit simpler. To assess seasonal variation of drinking water quality, 125 samples of drinking water were collected during the dry season (December-March) from water storage facilities and repeated during the wet season (June-September). In summary, the sampling procedure for this study is depicted in Figure 2.

Data collection procedures
Drinking water samples were collected at both seasons of the year from the point of use (POU) in food establishments and were bacteriologically tested in the laboratory. To collect the drinking water samples from the food establishments, heat-sterilized bottles of 100 ml capacity were used and the methods of sampling were adapted from the WHO guidelines for drinking water quality. The bottles were delivered to the laboratory within 6 hours and kept refrigerated at 4 C until the time of analysis.

Data analysis procedures
All laboratory results were recorded and coded appropriately. Then, data were entered to the SPSS (Statistical Package for the Social Sciences) software version 20. Data analysis was conducted using a repeated-measure ANOVA statistical model.

Microbial load assay
Membrane filtration method was used for microbiological analysis of the drinking water. In this method, a measured volume of the water sample (100 ml) was filtered through a membrane with a pore size small enough to retain the indicator bacteria to be counted. In the presumptive test, the membrane was placed on Eosine Methylene Blue (EMB) agar and incubated at 44.5 C for 24 hours, so that the indicator bacteria grew into colonies on its surface. According to Osman et al. () and Cowan et al. (), these colonies, which are recognized by their color, morphology and ability to grow on the selective EMB medium, were counted as separate colonies. Moreover, in the confirmatory test, to confirm whether positive samples were fecal coliform or not, they were re-inoculated into peptone broth test tubes for 24 hours at 44.5 C. Then, drops of Kovac's reagent were added to the re-incubated peptone broth test tubes. Finally, test tubes which indicated reddish color at the top were identified as positive for fecal coliforms and/or E. coli.

Operational definitions
According to WHO drinking water standard 2004, water samples with <1 CFU/100 ml were considered to be uncontaminated and samples with !1 CFU/100 ml to be contaminated (World Health Organization ). Moreover, based on previous studies, the fecal coliform count contamination levels in the drinking water samples were categorized into 0 (no health risk), 1-10 (low health risk), 11-100 (high health risk) and >100 CFU per 100 ml (very high health risk) (Lloyd & Bartram ).
Food establishments: Institutions that provide food and drinks for selling to customers.
Food: A material consisting of nutritious substances that people eat or drink in order to maintain life and growth.
Large food establishment: Hotels ranked with one or more stars.
Small food establishment: Small vendors, non-star ranked hotels, bars, restaurants, cafes.
Slum area: Area with poorer sanitation infrastructure.
Non-slum area: Area with better sanitation infrastructure.

Independent or predictor or explanatory variables
In this study, the independent study variables are defined as a factor or phenomenon that causes or influences the dependent variable. The predictor variables of the study are: • time, • type of food establishment, • location of food establishments, either in slum area and non-slum area.

Dependent or outcome or response variables
In this study, dependent variables are defined as phenomena that are changed by the effect of independent variables. The outcome variable of this study is: • microbiological quality of drinking water (fecal coliforms, E. coli).

Ethical consideration
The ethical procedure followed a series of three stage-based validation processes that incorporate (1) research approval, (2) technical clearance, and (3)

RESULTS
Microbial load of drinking water and seasonal variation of water quality The majority (98.4%) of the food establishments' drinking water source was municipal. In this study, the drinking water in 73.6% and 89.3% of the food establishments was found to be clean, safe, and free from E. coli in the rainy and dry season, respectively. In the presumptive test, 32.8% and 16.4% of the samples had thermo-tolerant/fecal coliforms during the wet and the dry season, respectively.
However, in the confirmatory test, 26.4% and 10.7% of the food establishments' drinking water was positive for E. coli in the wet and the dry season, respectively. Out of the total of 247 drinking water samples, 46 (18.6%) had fecal coliforms and/or E. coli. The mean scores of thermotolerant/fecal coliforms count per 100 ml were found to be 7.59 and 3.12 in the rainy and dry season, respectively (Table 1).

Microbial load, seasonal variation, and impact of health risk implications
The finding of the study revealed that 3.2% of the food establishments had drinking water with very high health risk to customers during the wet season and 1.6% of them during the dry season. Moreover, 12% and 2.5% of the food establishments had high health risk to customers due to their drinking water in the wet and the dry season, respectively ( Table 2).

Tests of within-subjects effects of time on the number of fecal coliforms' count
Having fulfilled the assumption of the 'Mauchly's test of sphericity' (non-significant P-value), 'tests of within-subjects effects' were conducted. Then, as observed in Table 3, the value of F is 5.631, which reached significance with a P-value of 0.019 (less than the 0.05 alpha levels). This indicated that there was a statistically significant difference between the means of the number of fecal coliforms' count per 100 ml between the dry and the rainy seasons (Table 3).  Table 4).

Tests of within-subjects effects of time on E. coli in drinking water
Similar to the above stated results, after fulfilling the assumption of the 'Mauchly's test of sphericity' (non-significant P-value), 'tests of within-subjects effects' was conducted. As indicated in Table 5, the value of F was 16.244, which researched significance with a P-value of 0.000. This indicated that there is a statistically significant difference between the means of E. coli per 100 ml between the dry and rainy seasons (Table 5).
Pairwise comparisons to observe effect of time on the occurrence of E. coli As seen in Table 6, there was a statistical significance in the occurrence of E. coli and season of the year although it was not yet known in which season the mean difference was significant. A repeated-measure ANOVA determined that the mean of occurrence of E. coli had the same statistical

CONCLUSION AND RECOMMENDATION
The majority of the food establishments' drinking water was safe, clean, and free from pathogenic microorganisms. However, a large number of food establishments have drinking water with E. coli. The levels of selected indicator bacteria (fecal coliforms and/or E. coli) exceeded the WHO recommended guidelines for drinking water. The microbial load of the drinking water at the POU in food establishments greatly differs between the dry and the rainy seasons. The occurrence of fecal coliforms and/or E. coli was higher during the rainy season. There was a time effect for the presence of fecal coliforms and/or E. coli in the drinking water. The findings of the study revealed that a large number of food establishments' drinking water had health risks. Therefore, there must be creation of awareness in food establishments for the appropriate management of drinking water to curb outbreaks of waterborne/foodborne diseases. This includes the practice of effective drinking water treatment such as disinfection, filtration, good sanitation and hygiene at the food establishment level. In addition, all concerned decision-making bodies, in particular, the government, should conduct regular and continuous microbial drinking water monitoring, evaluation and learning (MEL) practices to improve drinking water quality in food establishments.