|Roman Nega* and Samuel Sahile|
|Corresponding Author: Roman Nega, Microbial Biodiversity Directorate, Ethiopian Biodiversity Institute, P.O. Box 30726 Addis Ababa, Ethiopia|
|Received: December 04, 2018; Accepted: December 21, 2018; Published: December 30, 2018;|
|Citation: Nega R & Sahile S. (2018) The Effect of Irrigation Water the Microbial Quality and Safety of Vegetables: The Case of “Bisnet” River at Gondar Town. Int. J. Biopro. Biotechnol. Advance, 4(1): 127-143.|
|Copyrights: ©2018 Nega R & Sahile S. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.|
Vegetables are an extraordinary source of nutrients, micronutrients and vitamins for humans and are thus vital for health and wellbeing. Continued use of untreated waste water and manure as fertilizers for the production of vegetables is a major contributing factor to contamination. Microbial spoilage and contaminating pathogens pose a serious problem in food safety. Fresh produce can incorporate pathogenic microorganisms through the process of irrigation, harvesting, postharvest processing and distribution. The objective of this study was to assess the effect of irrigation water on the microbial quality and safety of in Gondar town. Fifty four vegetable yields and eighteen water (a total of seventy two) samples were collected from six farming sites for analysis. The water and vegetable quality was analyzed for total and fecal coliforms. Aerobic mesophilic, total coliforms, fecal coliforms counts were determined using standard methods. Additionally, 36 observation frequencies were used. Mean aerobic mesophilic count, total coliforms, fecal coliforms of vegetable yields were (4.93 to 4.90); (720.00 to 524.44) and (321.4 to 244.3) among the six sites, respectively. Escherichia coli, Enterobacter species, Proteous species, Klebsella species, Pseudomonas species and Penicillum species was isolated from vegetables. The water and vegetables samples showing high microbial load. The observational check list reveled that, the majority of growers did not practice sanitary cultivation and the farming environment also found unhygienic. One way ANOVA was used for comparing coliform counts among the six sites. There was statistically a significance difference in coliform counts of vegetable yields among sites (p<0.05). Most of the water samples were found to be contaminated. Prevention of contamination of fresh produce from both pre-harvest and post-harvest sources especially irrigation water still remains the only effective way to protect the public and to reduce the bacterial load.
Globally, over 70% of freshwater consumption is devoted to agricultural activities . The declining productivity of commercial farms has led international policy networks to recommend the promotion of urban and peri-urban (UPA) agriculture as an escape from food crisis situations . However, many households in poor areas lack access to fertilizers and have a limited supply of fresh water . Wastewater treatment and reuse at the individual level can provide a combined solution to these problems by supplying the water and nutrients needed for household food production . Indeed, this strategy is already in use by millions of farmers worldwide and it is estimated that 10% of the world’s population consumes foods irrigated with wastewater . Wastewater treatment and reuse for irrigation may well hold the key to easing demand on limited freshwater reserves while improving the food production capacity of households and farms . However, this practice is known to have adverse public health and environmental effects, especially because untreated wastewater or polluted water has high levels of pathogenic organisms .
Urbanization in the developing world is proving to be one of the greatest challenges of the 21st century with annual growth rate of 5.8% in Sub-Saharan Africa . The rapid rate of urbanization and the consequential rise in surface water pollution by wastewater discharge, combined with the scarcity of freshwater for irrigation in most cities, especially in arid areas, has led to a renewed interest in wastewater irrigation since the 1950’s . Numerous disease outbreaks are linked to contaminated fruit and vegetables . Urbanization in the developing world is proving to be one of the greatest challenges of the 21st century the urban population is growing at an estimated annual rate of about 2.3%, with only 4-5% of the population linked with sewage systems and sewerage treatment plants .
There have been some outbreaks of diseases like typhoid in Santiago, Chil and helminthes infections in Egypt and Jerusalem that have been associated with vegetable contamination from wastewater irrigation . The frequency of outbreaks epidemiologically associated with raw vegetables have increased in some industrialized countries as a result of change in dietary habits and increased import of food . In developing countries, foodborne illnesses caused by contaminated vegetables are frequent and in some areas they cause a large proportion of illness . However, due to lack of foodborne disease investigation and surveillance in most of these countries, most outbreaks go undetected and the scientific literature reports only on very few outbreaks . Untreated water is most likely to transmit several microorganisms, which may include pathogenic strains of Escherichia coli, Salmonella, Listeria, protozoa and viruses . Studies in different countries indicate that the use of untreated water for irrigation of vegetables is the practice most related to fresh produce safety issues . Contamination usually occurs before, during or after irrigation. In addition, the inhalation of aerosols from wastewater can affect farmers and surrounding communities .
The microbial safety of fresh vegetables is of global concern. Microorganisms that can cause illness or disease, collectively known as pathogens, are usually associated with human or animal fecal matter present in irrigation water and surface water sources . Irrigation water contaminated with pathogens has often been blamed for outbreaks of foodborne illness. Efforts to minimize the microbial contamination of fresh vegetables are essential and timely. However the current microbial contaminations of leafy vegetables at Gondar town are not known. So, the aim of this study is to assess the microbial quality and safety of leafy vegetables cultivated using irrigation water on “Bisnet” River in Gondar town.
Significance of the problem
It is expected that the result of this study will provide necessary information on the hygienic situation of a number of water samples and leafy vegetables taken from Bisnet River in Gondar town, and some potential risk factors associated with it. It will provide a framework to assess the on-going vulnerability of the supply to contamination and the major health problems that may make future contamination likely. It will also help for awareness creation in the health and sanitary workers of the town to minimize the microbial contamination of fresh vegetables.
· To assess the bacteriological water quality of irrigation water sources used for cultivation of cabbage, lettuce and spinach.
· To enumerate aerobic mesophilic count, fecal coliform and total coliform bacteria of leafy vegetables and irrigation water.
· To isolate and characterized the potential pathogens from the vegetables.
· To assess the safety practices around the farm sites and the irrigation water.
MATERIAL AND METHODS
Description of the study area
The study area is found in Gondar town, Amhara Regional State, Northern West Ethiopia. It is located 739 km far from Addis Ababa. Geographically Gondar is bounded by 12°36’ N and 37°28’ E longitude and it has a narrow range of altitude, i.e., 2133 m above sea level. Based on the Central Statistical Agency of Ethiopia (2012), Gondar has a total population of 308,257. The range of average monthly temperature is 19.5°C and the warmest average max/high temperature is 29°C (84°F) in March and May. The coolest average min/low temperature is 10°C (50°F) in December and January. The mean relative humidity for an average is recorded as 55.7% and on a monthly basis it ranges from 40% in January and February to 79% in July (http://www.gondar.climatemaps.com).
Design of the study
A cross sectional and experimental based study was conducted in Gondar town from October to December, 2013 to assess bacteriological quality of leafy vegetables like cabbage (Brassica oleracea L), spinach (Spinacea oleracea) and lettuce (Lactuca sativa L.) cultivated in six farming sites in connection with irrigating water. Additionally, sanitary practices around the farm and irrigation water were assessed using observational check list.
Vegetables and irrigation water sample collection
A total of 54 samples (9 from each farming site) comprising three types of fresh vegetables (cabbage, spinach and lettuce) and 18 water sample (3 from each farming sites), together a total of 72 samples were collected using simple random sampling technique from six farm lands on “Bisnet” vegetable farms around Gondar from October 2013-December 2013. Leafy vegetable samples in the six farming sites and the corresponding irrigation water (W) were coded as site 1 (Lettuce) (L1, L2, L3, L4, L5, L6,L7, L8, L9 and W1, W2, W3), site 2 (Lettuce) (L1, L2, L3, L4, L5, L6, L7, L8, L9 and W1, W2, W3), site 3 (Cabbage) (C1, C2, C3, C4, C5, C6, C7, C8, C9 and W1, W2, W3), site 4 (Cabbage) (C1, C2, C3, C4, C5, C6, C7, C8, C9 and W1, W2, W3), site 5 (Spinach) (S1, S2, S3, S4, S5, S6, S7, S8, S9 and W1, W2, W3) and site 6 (Spinach) (S1, S2, S3, S4, S5, S6, S7, S8, S9 and W1, W2, W3). All the collected vegetable samples were from cultivated and ready for harvest.
200 g of all the three leafy vegetable samples were collected in sterile polyethylene bags and each sample bags for each vegetable transported to Gondar University Department of Biology microbiology laboratory for analysis. The samples were cooled during transportation with ice box to keep the normal condition of the micro flora of vegetables. At the same time, two hundred ml of each water sample was collected from “Bisnet” vegetable farms around six vegetable farm stations in sterile plastic bottles. The samples were analyzed for total coliform count and fecal coliform count according to the procedure described by FSSAI . Each of irrigation water samples was taken from the source where the farmers drew. Samples were collected from 8:00 AM to 9:00 AM and analyzed within eight hours after collection.
25 g of each leafy vegetable sample was aseptically removed from using a sterile forceps and vigorously shaken in 225 ml of bacteriological peptone water (Oxoid, England). Serial dilutions were prepared using 9 ml sterile normal saline as diluents to enumerate, isolate and characterize bacteria groups from samples . Serial dilutions of samples were made up to 10-2 for total and fecal coliforms.
Similarly, from each eighteen (200 ml) water samples of plastic bottles 1 ml was taken and dropped to a test tube containing 9 ml sterile normal saline and thoroughly mixed to get 10-1 dilution. From 10-1 dilution, 1 ml was transferred to 9 ml of sterile normal saline in another test tube and thoroughly mixed to get 10-2 dilution. In such a way, serial dilutions of irrigation water samples were made up to 10-4 for total and fecal coliforms .
Bacteriological analysis of samples
Enumeration of aerobic mesophilic bacteria of vegetables (spinach, cabbage and lettuce): For aerobic mesophilic count (AMC), plate count agar (PCA) (Oxoid, England) was used as a culture medium . Zero point one ml of samples was taken aseptically from the most diluted one and spread plated in plates. The plates were then incubated at 37°C for 24 h. After growth of organisms, plates with colonies between 30-300 were counted using colony counter. The colonies were counted and reported as CFU/g of spinach, cabbage and lettuce.
Total and fecal coliform population estimations in irrigated vegetables: Total coliforms and fecal coliforms were determined using the most probable number (MPN) method as recommended by APHA-AWWA-WEF  and Downes and Ito . For the MPN method one ml of the sample was diluted up to a factor of 10-2 were made. 10 ml, 1 ml and 0.1 ml of the samples were inoculated in to triplicate tubes containing lactose broth (International diagnostics groups PIC, Lancashier, UK) and were incubated at 37℃ for total coliforms and 44℃ for fecal coliforms for 24-48 h. The tubes were examined for gas production. Tubes with gas formations at the end of the incubation periods were planted in to brilliant green bile broth (BGBB) and incubated at 44℃. Those tubes which formed a gas as a result of incubation process were evaluated according to the MPN table and results of a test were reported as MPN per ml of sample . The inoculums were transferred into a slant media and characterized by biochemical tests such as Indole test, catalase test, Simmone citrate test, methyl red test; VP test and TSI test .
Enumeration of total and fecal coliforms from irrigation water: Irrigation water samples were serially diluted before inoculation and incubated at 37°C for total coliform and 44°C for fecal coliforms for 24 to 48 h. Positive tubes (acid or gas production or both) were selected and the numbers of coliforms were obtained from MPN (Most Probable Number) index .
Isolation and identification of microbes: The presence of microbes was confirmed by using the following standard method. 1 ml of the sample was diluted in nine ml of lactose broth and incubated at 37℃ for 24 h. The sample was examined for gas formation. One loop of culture from lactose positive broth was streak on L-EMB agar (HiMedia, India). The culture was incubated at 44.5℃ for 18-24 h. Dark center colonies with metallic sheen were considered as indicative of E. coli. The colonies were further confirmed by Indole test, MRVP test, TSI test, catalase test and Simmone citrate test.
The presence of other pathogens like Pseudomonas species, Klebsiealla species, Enterobacter species and Proteus species was characterized and identified by biochemical test. Biochemical test was done using Indole test, MRVP broth, triple sugar iron (TSI) agar (Oxoid), simmone citrate (SCA) agar (Oxoid). Colonies were picked from PCA (plate count agar), MacConkey Agar, Salmonella Shigella agar (Oxoid, England) and incubated in to tryptone soy agar slant. After incubation for about 24 h, a loop full of inoculums was taken for biochemical tests such as Indole test, MRVP test, TSI test, catalase test and simmone citrate test respectively. Thus, colonies were purified and tested biochemically .
Assessment of the sanitary condition of the farm land and the irrigation water: An Observation check list was used for assessment of the sanitary condition of the farm land and the irrigation water. The assessments were done on (a total of 36 observational frequencies) in the six farms three at the time of sample collection and other three in accidental time.
The data collected from all the experiments and field study were subjected to the analysis of SPSS-20 computer software. Average values were used and all the countable dilution were used to calculate the average number of colonies in terms of colony forming unit per gram (cfu/g) or (cfu/ml) for aerobic mesophilic plate count, most probable number per gram (MPN/g) or MPN/ml for the statistical estimation of coliform. P<0.05 was taken as statistically significant association.
RESULTS AND DISCUSSION
Aerobic mesophilic count of leafy vegetables
The finding of this result showed, higher mean counts of aerobic mesophilic count was recorded in site one (4.9 log 10 cfu/g) whereas lowest mean count was recorded in site 5 (4.5 log 10 cfu/g). Analysis of variance (ANOVA) showed that aerobic plate count was significantly different (p<0.05) among the various study sites.
Total coliforms count of leafy vegetables
Total coliform count and E. coli are used as indicator of the hygienic quality parameter of food. The presence of total coliform is an indicator for enteric pathogens . The present study of total mean total coliform levels of all the three vegetables was ranged from 79.4 to 720.0 in site 3 and site 5, respectively and it exceeds the International Commission on Microbiological Specifications for Food According to ICMSF the acceptable upper limit of total coliforms is 100 MPN/g. Analysis of variance (ANOVA) showed that total coliform count was significantly different (p<0.05) among the various study sites.
The World Health Organization  has recommended that vegetables to be eaten should be irrigated only with biologically treated effluent that has been disinfected to achieve a coliform level of not more than 100/100 ml in 80% of the samples. The result showed major content of bacterial burden; these results correlate with the probability of the analyzed vegetables to be in contact with the source of contamination during growth: soil, organic fertilizers and irrigation water .
Fecal coliform count of leafy vegetables
According to Nguz et al. , fecal coliform counts are efficient indicators of sanitization, but the presence of fecal coliforms does not necessarily indicate the presence of a pathogen. The total mean fecal coliform value of all the three vegetable samples was ranges from 321.4 to 39.8 in site 1 and site 3, respectively. Analysis of variance (ANOVA) showed that fecal coliform count was significantly different (p<0.05) among the various stations sampled.
Total coliforms count of irrigation water sample
The California (USA) State Health Department adopted a bacterial standard for unrestricted wastewater irrigation of <2.2 total coliforms/100 ml which was close to the existing drinking water standard. Many countries followed this lead and adopted the same criteria with little or no adaptation to local constraints or to the level of technology available to meet this standard . Duffy et al.  showed that irrigation water is the leading pre-harvest and post-harvest source of contamination of produce. Total coliform counts can be considered as a hygiene indicator, especially for fecal contamination. The presence indicates that pathogens might be present due to fecal contamination by human, animal or irrigation water .
Fecal coliform count of irrigation water sample
The values of fecal coliform count for water ranges from 23 MPN/ml to 460 MPN/ml. The highest FCC (460 MPN/ml) was recorded in farming site 1, 2, 3 and 4. The lowest FCC (23 MPN/ml) was recorded in farming site 6. Analysis of variance (ANOVA) showed that fecal coliform count was significantly different (p<0.05) among the various study sites.
The presence of fecal coliform is an index of the bacteriological quality of water . The US-Environmental Protection Agency (US-EPA) in their 1992 guidelines has recommended the use of much stricter standards for wastewater use in the USA, than those recommended by the WHO. The main guideline is that fecal coliforms should not exceed 14 MPN/100 ml in any sample, which in practice means not detectable (Table 7).
Isolated pathogens in the growing field
A total of eighteen samples were tested to isolate pathogens from the three leafy vegetables among the six sites are shown in Table 8. Five bacteria and one fungus were isolated: Escherichia coli, Klebsiella species, Proteus species, Enterobacter species, Pseudomonas species and fungus (Penicillum species).
Assessments of sanitary practices around the farm and irrigation water
Growers farming practices, cares being taken during growing and the farm land, irrigation water and the surrounding environment have significant role for contamination of the product. In the present study in Bisnet vegetable farm 24 (66.6%) of farming sites in the production field stored manure near the vegetable farm (Table 9). Animal manure is a well-known source of food borne pathogenic bacteria and its inappropriate use in vegetable crops contributes a risk to consumer health . In developing countries, continued use of manure as fertilizers for the production of vegetables is a major contributing factor to contamination that causes numerous foodborne disease outbreaks . For this problem, the use of poultry droppings can be converted to compost and vegetable farmers must be educated on the proper way of applying it to the soil .
From the farm assessment 18 (50%) of the farming sites removed the house hold wastes/trashes around the growing fields (Table 9). The common sources of irrigation water used in South Africa are large reservoirs, farm dams, rivers, ground water, municipal supplies and industrial effluent .
Observations result shows that (0%) none of the farming sites fecal waste is not well-aged or properly treated (Table 9). Properly treated fecal waste (manure and human excreta) is an effective and safe fertilizer . Dangerous microorganisms in human and animal fecal waste can survive for long period of time and contaminate fruits and vegetables. Fecal waste must be treated to kill the microorganisms .
In the present day, 12 (33.3%) of the irrigation water of the farming sites, has fences to keep poultry or cattle from defecating in water sources. Dirty storage facilities and the presence of rodents, birds and insects may increase the risk of contamination with food borne pathogens . Possible corrective actions may include fencing to prevent large animal contact, appropriate well casing and head maintenance and placement of wells, filtering water, not stirring the sediment when drawing water, building settling or holding ponds and water treatment facilities. Settling or holding ponds that are used for subsequent irrigation may be microbiologically safe but may attract animals or in other ways increase the microbial risks associated with water for irrigating crops .
The sanitary survey also revealed that 12 (33.3%) of the irrigation water latrine was located downhill or away from water sources. Locating latrine downhill or away from the growing field prevent contamination during heavy rain fall or natural flooding (FAO (CAC/RPC, 2003).
Result from the observation check list showed that (0%) or none of the family was applying control measures when using contaminated water. Animal and human fecal waste can contaminate water with dangerous microorganisms, with surface waters especially prone to contamination. Measures need to be put in place to prevent such contamination passing to fruits and vegetables and impacting on human health . If the quality of water is poor, unknown or cannot be controlled, crop contamination is minimized by applying control measures. Properly treated fecal waste is a good source of nutrients for fruits and vegetables. However, fecal waste must be properly treated to kill dangerous microorganisms. Helping participants understand the control measures needed to treat fecal waste is important for maintaining the safety of the fruits and vegetables (FAO (CAC/RPC, 2003).
A correlation among bacterial count of vegetables, water (AMC, TCV, FCV, TCW and FCW) and sanitary survey of farming sites (ASSVEG and ASSWA) is shown on Table 11. A positive relationship existed between AMC and TCV (r=0.370, p<0.001) and AMC and FCV (r=0.408, p<0.001). TCV exhibited significant positive correlation with FCV (r=0.991, p<0.001), TCW (r=0.616, p<0.001) and FCW (r=0.517, p<0.0001). FCV exhibited significant positive correlation with TCW (r=0.637, p<0.001) and FCW (r=0.557, p<0.0001). TCW also exhibited significant positive correlation with FCW(r=0.946, p<0.001). Positive correlation means that high scores on one are associated with high scores on the other, and that low scores on one are associated with low scores on the other [63-66].
CONCLUSION AND RECOMMENDATION
The study revealed that there was bacterial contamination of fresh leafy vegetables (lettuce, cabbage and spinach) grown in Gondar Bisnet river vegetable farms. Spinach farm site 1 and 2 (4.93, 4.90); (720.00, 524.44) and (321.4, 244.3) was found to be the most contaminated vegetable by aerobic mesophilic bacterial count, total coliform count and fecal coliform count respectively. This might be due to the fact that spinach have wider leaf surface for contact with wastewater, soil and dust. In contrast lettuce (farm site 5 and 6) was the least in aerobic mesophilic bacterial count, total coliform count and fecal coliform count. Therefore, great attention should be paid in using contaminated water for production of vegetables for the public health perspective. The presence of coliforms is an indication of fecal contamination that comes from river water used for irrigation. We tested the vegetables samples for the presence of Salmonella and they were not detected [67-73].
The presence of low level coliforms may be because of pathogens survived on leaf surfaces for a shorter time than in the soil as they are less well protected from the harsh effects of sunlight and desiccation and also the temperature of the environment. The results obtained have demonstrated that the microbiological quality of fresh leafy salad vegetables grow in Bisnet River is acceptable [74,75]. Escherichia coli, Klebsiella species, Proteus species, Enterobacter species, Pseudomonas species and fungi (Penicillum species) were isolated from leafy vegetables. The safety measures also shows high burden of water and vegetable farm contamination using observational check lists and concluded that manure, animal and human feces, from house hold wastes and factories, clinical wastes etc. might be the sources of contaminations. Analysis of variance has shown there was significant difference (p<0.05) between bacteriological quality and safety of the samples [76-82].
There is a need for improved surveillance systems on food-borne pathogens, on food products and on outbreaks.
Water used in the irrigation vegetables should be of a quality that does not introduce microorganisms at a level that might cause harm to the consumer [83-88].
Further studies are required on the microbiological status and survival of various pathogens in/on raw vegetables and the most efficient decontamination procedures [89-96].
There should be public food hygiene and safety education on the consumption of leafy vegetables [97-101].
Local authorities should provide alternative means facilities for growers for safer cultivation of fresh produces [102-111].
Firstly, my special gratitude goes to my heavenly Father for giving me the capabilities, courage and perseverance for this study.
My special gratitude and appreciation goes to my advisor Dr. Samuel Sahile for his expert guidance and support during the course of my study and unreserved encouragements and advice in conduction of this thesis. Following him, I thank Miss. Atsede Muleta who shared and supported me her expertise and professional guidance in laboratory activity.
I would like to express my heartfelt gratitude for the peoples behind my success, my parents, my mother Wudinesh Sintayehu, my father Nega Mengistu and my brother Biniam Nega. My thanks also goes to my friend Askal Molla, Tigist Malede, her mother and also all her families for their endless encouragement, love, support and motivation throughout my studies.
I would like to express my gratitude to University of Gondar for giving me this opportunity.
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