An Assessment Of Food Safety Hazards Of Salad Vegetables Irrigated With Treated Municipal Effluent. A Case Study Of Umguza Irrigation Scheme, Bulawayo, Zimbabwe

Agricultural use of wastewater is a common practice and is increasing due to the escalation of water scarcity globally (Scott et al., 2004). Wastewater reuse in irrigation is largely considered an inevitable option to compensate water shortages in developing countries. Hence, crop irrigation with wastewater is a widespread practice in these countries (Sou et al., 2011). In the urban areas the use of wastewater in agriculture is a centuries-old practice that is receiving renewed attention with the increasing scarcity of freshwater resources in many arid and semi-arid regions (Ackerson et al., 2012). The growing wastewater volumes are driven by rapid urbanization (Scott et al., 2004). Economic and agronomic advantages are sometimes promoted in wastewater reuse but there are several studies warning about health risks and environmental impacts (Sou et al., 2011).
Uncooked (raw) vegetables constitute the essential element of a healthy diet. Vegetables can become contaminated with different pathogens such as enteric bacteria, viruses and parasites from primary production right across the supply chain. The degree of contamination depends on such factors as the use of untreated wastewater and water supplies contaminated with sewage for irrigation. Salad vegetables are rich in vitamins and minerals and consumed without any heat treatment, sometimes without washing and peeling and therefore the possibility of food borne diseases is more. Different egg parasites can adsorb to vegetables such as lettuce and cabbage and are not removed by basic disinfection and in some cases may cause serious illness. Furthermore, various bacterial infections such as Salmonella and Vibrio cholera are the main cause of diarrhoea especially in summer and detected in these vegetables (Amoah, 2006). In a study in Ghana, the salad vegetables were contaminated with, Streptococcus faecalis, Salmonella typhi, Shigella and Pseudomonas.
The irrigation of vegetables, which typically have high returns per volume of water invested in it, provides one of the most economically feasible agricultural uses of reclaimed water (Toze et al., 2006). However, the environmental and public health risks posed by wastewater irrigation are alarming, especially when untreated and/or partially treated wastewater is used for such purposes (Ackerson et al., 2012). Point sources of pollution to water bodies include industrial effluents and municipal/ domestic wastewater (Odjadjare et al., 2010). Health impacts are mainly due to pathogens (bacteria, viruses, protozoa, cysts and helminthic eggs) and toxic substances which are likely to exceed health protection standards (WHO, 2006). The pathogens are transmitted to the public through consumption of irrigated produce, especially crops eaten raw (Blumenthal et al., 2000).
Several studies throughout the world have demonstrated a very close relation between the consumption of fruits and vegetables irrigated with raw wastewater and many food borne diseases like gastroenteritis, cholera and chemical toxicity (Sou et al., 2011). Many of the sources that are thought to contribute to the epidemiology of diseases associated with raw fruits and vegetables are influenced by ecological conditions that affect survival or growth of pathogenic microorganisms (Steele et al., 2005). These sources include raw manure and irrigation water (Selma et al., 2007). The microbial quality of vegetables irrigated with wastewater is highly alarming (Ajayi et al., 2008). Microbial hazards continue to be one of the biggest threats to food safety (Al-Binali, 2006). The implications of the microbial contamination include spoilage, decreased sensory appeal and decreased shelf life.
Urban agriculture is practised in Ghana where vegetable farmers in search of irrigation water usually have no alternative but to resort to use of polluted waters to cultivate such vegetables as lettuce, spring onions, and cabbage which are usually consumed raw. However, the use of wastewater in producing these vegetables can expose farmers and produce consumers to a significant occupational and public health risk, respectively, (Blumenthal and Peasey, 2002; Smith et al., 2003; Minhas et al., 2006). The public health risks of using such contaminated waters for irrigation have been broadly elucidated (Blumenthal et al., 2000; Shuval et al., 1997; WHO, 2006).

Vegetables can become contaminated throughout the supply chain, that is: while growing, during harvesting, post-harvest handling or even during distribution and sale at the market (McMahon and Wilson, 2001). Several studies in West Africa have reported high levels of pathogen contamination in irrigation water; and on both farm and market vegetables, with a potential detriment to public health (Faruqui et al., 2004; Amoah et al., 2005; Niang, 1999). The risk of microbiological contamination of vegetables is greater on farms that make use of untreated wastewater and poorly composted manures (Beuchat, 2002).
The co-disposal of industrial and domestic waste also introduces heavy metal contamination into the aggregate effluent. Heavy metal pollution of agricultural soil and vegetables is one of the most severe ecological problems globally (Ahmad & Goni, 2010). Wastewater from industries or other sources carries an appreciable amount of toxic heavy metals which create a problem for safe rational utilization of agricultural soil (Yadav et al., 2002; Chenetal. 2005; Singh et al., 2004). Excessive accumulation of trace elements in agricultural soils through wastewater irrigation may not only result in soil contamination but also affect food quality and safety (Muchuweti et al., 2006; Sharma et al., 2007).
Some trace elements are essential in plant nutrition, but plants growing in the nearby zone of industrial areas display increased concentration of heavy metals, serving in many cases as bio-monitors of pollution loads (Mingorance et al., 2007). Long-term use of industrial or municipal wastewater in irrigation is known to have significant contribution to trace elements such as Cd, Cu, Zn, Cr, Ni, Pb, and Mn in surface soil (Mapanda et al., 2005). Studies conducted by Kisku et al., (2000) in Kalipur, Bangladesh, on the uptake of Cu, Pb, Ni and Cd by Brassica oleracea from fields irrigated with industrial effluent indicated widespread contamination from heavy metals despite plants showing a healthy and gigantic external morphology.
Use of wastewater for agricultural purposes in Zimbabwe is recommended for surface irrigation of grain crops, crops grown for industrial processing & pastures for slaughter stock. However, it is prohibited for the irrigation of leafy vegetable crops (NRB, 1974). Leafy vegetables have greater potential of accumulating heavy metals in their edible parts than grain or fruit crops. Studies on the uptake of heavy metals by plants have shown that heavy metals can be transported passively from roots to shoots through the xylem vessels (Krijger et al., 1999). In addition, plant organs such as fruit and seed that have low transpiration rates did not accumulate heavy metals because the storage organs are largely phloem-loaded and heavy metals are generally poorly mobile in the phloem. Zheljazkov and Neilsen (1996) found that the concentrations of heavy metals in vegetables per unit dry matter generally follow the order: leaves >fresh fruits >seeds.
In Zimbabwe, land disposal of sewage and industrial effluents has been implicated as the chief source of heavy metal enrichment of pasturelands and agricultural fields, particularly near Sewage Treatment Works in urban areas (Mangwayana, 1995; Nyamangara and Mzezewa, 1999; Mapanda et al., 2005). Furthermore, non-communicable diseases may result from consumption of crops which would have taken up heavy metals and other hazardous chemicals from wastewater (Gumbo, 2005). Research has shown that lead (Pb) and cadmium (Cd) are taken up by plants from the soil, thereby making plants potential sources of contamination for humans and animals (Madyiwa et al., 2004).
The need to reduce the potential health risks resulting from faecal contamination of vegetables required a more holistic approach. This approach involves applying various supply chain-wide multiple risk management practices, better known as the 'multiple barrier approach' where barriers (non'treatment interventions) are placed along the food chain to have aggregate or cumulative effects in reducing health risks as prescribed by the new WHO guidelines (WHO, 2006). WHO (2006) proposes measures such as wastewater treatment and other non-treatment interventions such as improvement on irrigation application techniques (that reduce direct contact of wastewater with the crop), crop restriction (which is useful and profitable to non-food crops) and cessation of irrigation before harvesting (which also allows natural pathogen die-off). Some of these measures like irrigation methods, cessation before harvesting and improved sanitary washing methods have been tested on lettuce under local conditions in Ghana (Keraita et al., 2007a,b,; Amoah et al., 2007b). The efficacies of these interventions on other vegetables which are also potentially consumed raw have not been assessed. In addition, these strategies have to be made locally relevant so that they can easily be adopted by farmers and other stakeholders in the food chain to mitigate potential health risks (Drechsel et al., 2006).

1.2 Statement of the problem
There have been some outbreaks of diseases like typhoid and helminthic infections in parts of the world (e.g., Santiago, Egypt, Jerusalem, and Chile) that have been associated with crop contamination from wastewater irrigation (Blumenthal et al., 2000). Developing countries lack the capacity to effectively treat wastewater before disposal (Carr & Strauss 2001). Large volumes of untreated wastewater end up in urban water bodies, which farmers use for irrigation.
Vegetables are the crops most commonly irrigated with polluted water as they are the most demanded cash crops in urban areas (Scott et al., 2004; Obuobie et al., 2006). However, the use of contaminated irrigation water poses health risks to farmers and consumers (Blumenthal et al., 2000). In Ghana, water used by urban vegetable farmers has high levels of microbial contamination, and vegetables produced are equally contaminated (Amoah et al., 2005). This has been associated with the transmission of diarrhoea in the cities (Mensah et al., 2002).
Muchuweti et al., (2006), reported findings on their study of 'Heavy metal content of vegetables irrigated with mixtures of wastewater and sewage sludge in Zimbabwe: Implications for human health'. They noted the growing public concern in Zimbabwe over the illegal cultivation of vegetables on soils amended with sewage sludge or irrigated with admixtures of sewage and sewage sludge. Excessive accumulation of heavy metals in agricultural soils may not only result in environmental contamination, but also lead to elevated heavy metal uptake by crops, which may affect food quality and safety.
The work reported by Muchuweti et al., (2006) studied heavy metal concentrations in crops irrigated with sewage sludge and sewage/sewage sludge admixtures at Firle Municipal Farm in Harare and the crops analysed in the study were found to be heavily contaminated with the four regulated elements: Cd, Cu, Pb and Zn. This contamination is at its highest in two of the staple dietary crops maize and tsunga. The other plants (beans, maize, peppers and sugarcane) also contained concentrations of heavy metals above the permissible levels. Furthermore, the concentrations observed in this study were higher than those reported by other workers who have examined vegetation from other contaminated sites. This study highlighted the potential risks involved in the cultivation and consumption of vegetables on plots irrigated with sewage sludge, a practice which may place at risk the health of the urban population who consume these vegetables.
Umguza irrigation scheme farmers draw water from Umguza River which receives raw or partially treated sewage from Aisleby sewage treatment plant. Produce from these farms is supplied mainly to Bulawayo green market. Bulawayo population according to Census 2012 (ZimStat, 2013) is 304 446 males and 351 229 females giving a total of 676 650, all of whom are at potential risk of consuming contaminated vegetables produced at Umguza irrigation scheme. Aisleby sewage treatment plant is a conventional biological nutrient removal (BNR) plant not designed to remove heavy metals (Gumbo, 1995). The plant receives more sewage than its original design capacity and frequently experiences overload by faecal matter. Heavy metals and microbial pathogens find their way into Umguza River where 'treated effluent' is discharged and abstracted downstream for irrigation. Vegetables irrigated with this water are at risk of heavy metal and microbial contamination and the lack of heat or other form of treatment prior to consumption of salads made of these vegetables places consumers at enormous risk of food safety hazards. The vegetables selected for this study were Lactuca sativa (lettuce), Brassica oleracea var. capitata (cabbage) and Solanum lycopersicum (tomato)
1.3 Objectives of the study
The objectives of this study were as follows:

BROAD OBJECTIVE
The aim of the research is to characterise the physico-chemical and biological food safety hazards of vegetables that are common ingredients in salads and are produced using treated municipal effluent irrigation.
SPECIFIC OBJECTIVES
i. To determine the biological quality of effluent-irrigated vegetables (L.sativa, B.oleracea var. capitata, S.lycopersicum) in terms of coliforms, pathogenic Escherichia coli, Shigella, Salmonella & Staphylococcus aureus,
ii. To measure the physico-chemical properties of irrigation water and irrigated soils in terms of pH, electrical conductivity (EC) and total dissolved solids (TDS)
iii. To determine the levels of heavy metals (Pb, Cd, Cu, Ni and Zn) in irrigation water, irrigated soils and edible portions of effluent-irrigated vegetables (L.sativa, B.oleracea var. capitata, S.lycopersicum)
iv. To evaluate the food safety risks consumers of these vegetables are exposed to by comparing empirical measurements with Codex Alimentarius guidelines (FAO/WHO) as well as S.A.Z Food standards

1.4 Hypotheses

H01: There are no significant differences in the microbial loads of the effluent-irrigated vegetables (L.sativa, B.oleracea var. capitata, S.lycopersicum) in terms of coliforms, pathogenic Escherichia coli, Shigella, Salmonella & Staphylococcus aureus.
H11: There are significant differences in the microbial loads of the effluent-irrigated vegetables (L.sativa, B.oleracea var. capitata, S.lycopersicum) in terms of coliforms, pathogenic Escherichia coli, Shigella, Salmonella & Staphylococcus aureus.
H02: There are no significant differences in the levels of heavy metals (Pb, Cd, Cu, Ni, and Zn) in irrigation water, irrigated soils and edible portions of effluent-irrigated vegetables (L.sativa, B.oleracea var. capitata, S.lycopersicum)
H12: There are significant differences in the levels of heavy metals (Pb, Cd, Cu, Ni, and Zn) in irrigation water, irrigated soils and edible portions of effluent-irrigated vegetables (L.sativa, B.oleracea var. capitata, S.lycopersicum)

1.5 Justification of study
Salad can be defined as a food made primarily of a mixture of raw vegetables and/or fruits (Rajvanshi, 2010). Health benefits of salads are many, owing to the various vegetables present in them. Vegetables are a good source of antioxidants and phytonutrients. They are low in calories and are rich in complex carbohydrates, vitamins and minerals. Salads should be cleaned properly, as they are generally eaten raw or partially cooked. If these are not cleaned properly, these become source of food-borne illnesses. Pathogens on edible plants present a significant potential source of human illness. A significant portion of enteric pathogens can persist on the surface and proliferate. Proliferation of these dangerous pathogens can increase the likelihood of food-borne disease associated with fresh or minimally processed produce. Fresh vegetables and fruits become contaminated with microorganisms during production, harvest, packing, and distribution (Bartz & Wei, 2003).
Several outbreaks of gastroenteritis have been linked to the consumption of contaminated fresh vegetable. In 2011, cucumber E.coli outbreak left five dead in Germany and organic cucumbers from Spain were thought to be the source of bacteria. The Health Protection Agency of the United Kingdom went on to issue advice for travellers to Germany to avoid eating raw tomatoes, lettuce and cucumbers (www.telegraph.co.uk). Another outbreak was reported in Japan in 1996 in which 11,000 people were affected and about 6,000 cultures were confirmed. The outbreak involved the death of three children and was carried by Escherichia coli. These events contextualise the microbiological food hazard carried by vegetables which are normally eaten raw. The most common bacterial entero-pathogens associated with fruits and vegetables are Salmonella spp. (Thunberg et al., 2002). E. coli H1:O157 outbreaks were also associated with apple cider, lettuce, radish, and other mixed salads (Beuchat, 1996).
The World Health Organization (WHO) published guidelines for the safe use of wastewater in agriculture (WHO, 1989) to protect farmers' and consumers' health. These guidelines were to assist design engineers and planners in the choice of wastewater-treatment technologies and water management options. The guidelines encouraged specific measures and the adoption of a combination of other protective procedures, such as 'acceptable levels of microbiological contamination derived from the results of available epidemiological studies related to wastewater exposure, use and treatment' and 'health protection measures, especially crop selection, wastewater application measures (e.g., drip irrigation), and human exposure control through protective clothing to achieve its goal of protecting farmers and consumers'. However, the application of the guidelines has been found to be difficult in many field situations, especially in low income countries (Hyderabad Declaration, 2001) due to a number of reasons such as financial constraints and poor sewerage systems. Furthermore, crop restriction, one of WHO recommendations, is also unrealistic since the production of these crops provides the profit needed as livelihood support for farmers.
A meeting of experts convened by WHO (1973) concluded that primary treatment would be sufficient to permit re-use for the irrigation of crops that are not for direct human consumption. Secondary treatment and most probably disinfection and filtration are considered necessary if the effluent is to be used for irrigation of crops for direct human consumption. Most developing countries such as Zimbabwe unfortunately lack the capacity and resources required for secondary treatment. The lack of sanitation and hygiene at market level as observed by Amoah (2007) also results in significant post-harvest contamination of vegetables. Local authorities are committed to health and sanitation (BCC, 2010) but often lack the requisite resources for implementation.
Previous work done on microbial and heavy metal contamination of crops in Zimbabwe has concentrated on beans, maize, tsunga, peppers and sugarcane (Muchuweti et al., 2006); pasturelands and agricultural fields, particularly near Sewage Treatment Works in urban areas (Mangwayana, 1995; Nyamangara and Mzezewa, 1999; Mapanda et al., 2005); covo (Brassica oleracea variety, acephala); sugar beans (Glycene max) and maize (Zea mays) (Mutengu et al., 2007). From the literature reviewed, this author has not come across a study that looked specifically at both microbial and heavy metal contamination of effluent-irrigated salad vegetables whose food safety risk is exacerbated by lack of heat treatment prior to consumption. This is the reason for the selection of L.sativa, B.oleracea var. capitata and S.lycopersicum for this particular study. The study area selected by this author was also conspicuous by its absence from published literature. There was therefore a knowledge gap within the body of science that this research sought to fill. This study would thus expose the food safety hazards posed by contamination of soils and subsequent uptake of chemical and biological toxicants by crops irrigated with treated municipal effluent.
1.6 Delimitation
The study was conducted at the Umguza Irrigation scheme in Bulawayo, the second largest city in Zimbabwe. The study focused on bacterial and heavy metal contamination of effluent, soils and plants as a result of secondary discharge of partially treated municipal effluent from the city's Aisleby sewage treatment plants.

CHAPTER TWO
LITERATURE REVIEW
2.1 Introduction
The increase in urban populations in developing countries has resulted in larger amounts of freshwater being channelled to domestic, commercial, and industrial sectors, which consequently generate greater volumes of wastewater (Lazarova and Bahri, 2005; Qadir et al ., 2007a; Asano et al., 2007). Natural water bodies commonly receive wastewater discharged with little or no treatment high levels of pollution. Farmers in urban and peri-urban areas of most developing countries who are in need of irrigation water seldom have any other choice than using wastewater. They even deliberately use undiluted wastewater as it provides nutrients or is more reliable or cheaper than other water sources (Hussain et al., 2001; Keraita and Drechsel, 2004; Scott et al., 2004). Despite farmers' good reasoning, this practice can, however, severely harm human health and the environment (Qadir et al., 2007b) mainly due to the associated pathogens, but also heavy metals and other undesirable constituents depending on the source.
The lack of financial and technical resources in many developing countries makes comprehensive wastewater reticulation and treatment, a long term future strategy. Therefore, it becomes of necessity that in the short term, risk management and arbitrary solutions are implemented to prevent detrimental environmental and public health impacts that emanate from wastewater irrigation (IWMI, 2006; WHO, 2006). These include user and consumer health protection through interventions at farm level, post-harvest measures as well as public policies to motivate better management of wastewater.
There is an increasing trend to employ more efficient use of water resources, both in urban and rural environments. A major mechanism that can be utilised to achieve greater efficiencies is the reuse of water that previously would have been discarded into the environment after use (Toze, 2004). The reuse of water for agricultural irrigation is often viewed as a positive means of recycling water due to the potentially large volumes of water that can be used. Recycled water can have the advantage of being a constant, reliable water source and reduces the amount of freshwater extracted from the environment.

2.2 Salad and associated food safety hazards
Salad can be defined as a food made primarily of a mixture of raw vegetables and/or fruits (Rajvanshi, 2010). Salad is a term broadly applied to many food preparations that have mixture of chopped or sliced ingredients which may be mostly fruits or vegetables. Common vegetables used in salad include cucumber pepper, tomatoes, onions, red onions, carrots, spring fresh onions and radishes.
Health benefits of salads are many, owing to the various vegetables present in them. Vegetables are a good source of antioxidants and phytonutrients. They are low in calories and are rich in complex carbohydrates, vitamins and minerals. Salads should be cleaned properly, as they are generally eaten raw or partially cooked. If these are not cleaned properly, these become source of food-borne illnesses.
Pathogens on edible plants present a significant potential source of human illness. A significant portion of enteric pathogens can persist on the surface and proliferate. Proliferation of these dangerous pathogens can increase the likelihood of food-borne disease associated with fresh or minimally processed produce. Fresh vegetables and fruits become contaminated with microorganisms during production, harvest, packing, and distribution (Bartz, Wei, 2003). Several outbreaks of gastroenteritis have been linked to the consumption of contaminated fresh vegetable borne outbreak, occurred in Japan in 1996 in which 11,000 people affected and about 6,000 cultures were confirmed. The outbreak involved the death of three children and was carried by Escherichia coli.
The most common bacterial enteropathogens associated with fruits and vegetables are Salmonella spp. (Thunberg et al., 2002). E. coli O157:H7 outbreaks were associated with apple cider, lettuce, radish, alfalfa sprouts, and other mixed salads (Beuchat, 1996). There are reports of food borne illness associated with the consumption of fruit juices at several places in India and elsewhere (Bhaskar et al., 2004; Chumber et al., 2007; Ghosh et al., 2007). Such juices have shown to be potential sources of bacterial pathogens notably E. coli 0157:H7, species of Salmonella, Shigella, and S. aureus (Buchmann et al., 1999) In India the presence of coliforms and staphylococci in kinnow and mandarin juices in Patiala city could be reported (Ganguli et al. 2004). Similarly coliforms were observed, in fresh fruit and vegetable juices sold by the street vendors of Nagpur city (Titarmare, Dabholkar and Godbole, 2009). As the salads viz. carrot, coriander and cucumber have a very high consumer preference and eaten raw or partially cooked due to health effect throughout the country.
2.3 Wastewater generation, treatment, and current use
With urban water use, only 15% to 25% of water diverted or withdrawn is consumed, the rest being returned as wastewater to the urban hydrologic system (Bernstein, 2011). The wastewater is usually a mix of domestic and industrial wastewater as well as storm water. Industrial wastewater often contains elevated levels of metals and metalloids, while domestic wastewater is most harmful due to its pathogenic load.
In many Asian and African cities, population growth has outpaced improvements in sanitation and wastewater infrastructure, making management of urban wastewater a tremendous challenge (Bernstein, 2011). Some specific examples include India where only 24% of wastewater from households and industry is treated, and in Pakistan only 2% is treated (IWMI, 2003; Minhas and Samra, 2003). In West African cities, usually less than 10% of the generated wastewater is collected in piped sewage systems and receives primary or secondary treatment (Drechsel et al., 2006). In many developing countries, large centralized wastewater collection and treatment systems have proven difficult to sustain. Decentralized systems that are more flexible for long-term operation and financial sustainability and compatible with demands for local effluent use, have been promoted in many areas (Raschid-Sally and Parkinson, 2004), although not without challenges. In Ghana, for example, only 7 of 44 smaller treatment plants are functional and probably none meets the designed effluent standards (Obuobie et al., 2006).
Reliable estimates of projected wastewater use in agriculture are needed for better planning and managing risks, but limited information makes estimating future use difficult (Qadir et al ., 2007a). Data collection and comparison are challenging, due in part to the lack of a universally accepted typology (Van der Hoek, 2004). In some cases, information exists, but government policies make access difficult or the information is available only as grey literature. A further reason is that these farming activities remain informal and are not in official statistics (Drechsel et al., 2006). Jimenez and Asano (2004) and IWMI (2006) suggest that at least 3.5 million ha are irrigated globally with untreated, partly treated, diluted, or treated wastewater.
Globally, farmers in excess of 800 million farmers are engaged in urban agriculture. Of this group, about 200 million practise market-oriented farming on open spaces, often using poor-quality irrigation water when good-quality water is not available. Farmers enhance household income by producing perishable crops such as leafy vegetables for sale in local markets, providing a supply of vitamin-rich vegetables. For instance in most West African cities, 60% to 100% of the vegetables consumed are produced in urban and peri-urban areas (Drechsel et al., 2006). Economic benefits arising from wastewater-irrigated agriculture have so far been inadequately differentiated and quantified (Buechler and Devi, 2006; Obuobie et al., 2006; Drechsel et al., 2006). There is a growing interest in doing so to comprehend the importance of wastewater as a source of livelihoods.
Water availability is the most limiting factor for the necessary spread of extensive irrigated agriculture production in arid and semiarid regions of the world, particularly in view of the phenomenon of climate change. The utilization of marginal water for agricultural irrigation in these areas thus becomes an imperative for the maintenance and sustenance of productivity. The availability and relatively low cost of treated sewage effluents is providing the impetus for use of alternative marginal water for agricultural irrigation. Using reclaimed water in agriculture also minimizes the discharge of untreated wastewater directly into the environment. Therefore, the water resource planning policies of many countries are based on maximizing re-use of urban wastewater, and the use of wastewater for agricultural irrigation is increasing steadily world-wide (Scott et al., 2004).
2.4 Risks associated with wastewater reuse in agriculture
Utilization of treated wastewater for agriculture may carry risks to the environment and to public health. Bacteria, viruses, parasites, and other human pathogens are present in sewage water (Armon et al., 2002; Assadian et al., 2005) and, although their concentration decreases during the wastewater reclamation process (Van der Steen et al., 2000), the secondary treated effluents that are the effluents most commonly used for irrigation still contain pathogens that may pose a threat to public health (Kinde et al., 1997; Maynard et al., 1999; Armon et al., 2002). Health risks to farmers and consumers may result from direct contamination of crops by human pathogens present in the treated effluents used for irrigation, as well as indirect contamination of crops through contaminated soil at the agricultural site. Therefore a crucial issue in the reuse of treated wastewater for agricultural irrigation is its sanitation quality. Studies demonstrate that bacterial human pathogens can penetrate internal plant tissues via the root (Guo et al., 2002; Solomon et al., 2002; Bernstein et al., 2007a,c) and not only by direct contact with the above-ground parts of the plant. The root internalized bacteria were shown to translocate and survive in edible, aerial plant tissues (Guo et al., 2002; Bernstein et al., 2007a). Treated effluents contain higher levels of potentially plant-damaging substances such as heavy metals than the potable water from which they were derived (Feigin et al., 1991). Therefore, in addition to public health risks, treated effluents may also have detrimental effects on the irrigated crops (Feigin et al., 1991).
Developing countries lack the capacity to effectively treat wastewater before disposal (Carr & Strauss, 2001). Large volumes of untreated wastewater end up in urban water bodies, which farmers use for irrigation. Vegetables are the crops most commonly irrigated with polluted water as they are the most demanded cash crops in urban areas (Scott et al., 2004; Obuobie et al., 2006). However, the use of contaminated irrigation water poses health risks to farmers and consumers (Blumenthal et al., 2000). Waste water irrigation leads to accumulation of heavy metals in the soil (Singh et al., 2004, Mapanda et al., 2005, Sharma, Agarwal and Marshall 2007). Sewage waste has been implicated as a potential source of heavy metals such as Copper (Cu), Cadmium (Cd), Zinc (Zn), Lead (Pb), Nickel (Ni) and Iron (Fe) in the edible and non-edible parts of vegetables (Sharma, Agarwal and Marshall 2006). In Ghana, water used by urban vegetable farmers has high levels of microbial contamination, and vegetables produced are equally contaminated (Amoah et al., 2005). This has been associated with the transmission of diarrhoea in the cities (Mensah et al., 2002). Social attitudes/perceptions towards the use of crops that have been irrigated with recycled waters and the resulting impact on market value of crops are also a major consideration.
The increasing number of outbreaks of food poisoning has highlighted the importance of microbiological control in the food industry (Chaturvedi et al., 2013). Consumption of raw vegetables contaminated with harmful microorganisms may result in food poisoning. Contamination of vegetables may take place at all stages during pre and post-harvest techniques (De Roever, 1999). Raw fruits and vegetables are known potential for a wide range of microorganisms, including human pathogens (EC-SCF, 2002). Food- borne bacterial pathogens commonly detected in fresh vegetables are coliform bacteria, E. coli, Staphylococcus aureus and Salmonella species (Tambekar and Mundhada, 2006). These Microorganisms capable of causing human illness have been isolated in lettuce and salad vegetables (Francis et al., 1999).
Wastewater reuse in irrigation is largely considered an inevitable option to compensate water shortages in developing countries. Hence, crop irrigation with wastewater is a widespread practice in these countries (Sou et al., 2011). In the urban areas the use of wastewater in agriculture is a centuries-old practice that is receiving renewed attention with the increasing scarcity of freshwater resources in many arid and semi-arid regions (Ackerson et al., 2012). The growing wastewater volumes are driven by rapid urbanization (Scott et al., 2004). Economic and agronomic advantages are sometimes promoted in wastewater reuse but there are several studies warning about health risks and environmental impacts (Sou et al., 2011). One of the most economically feasible agricultural uses of reclaimed water is the irrigation of vegetables which typically have high returns per volume of water invested in it (Toze et al., 2006). However, the environmental and public health risks posed by wastewater irrigation are alarming, especially when untreated and/or partially treated wastewater is used for such purposes (Ackerson et al., 2012).
Point sources of contamination to water bodies include industrial effluents, municipal/ domestic wastewater, abattoir waste, while, non-point sources include wild animal defecation, storm water drainage and urban runoff (Odjadjare et al., 2010). Health impacts are mainly due to pathogens (bacteria, etc.) and other organic and inorganic toxic substances which are likely to exceed health protection standards (WHO, 2006). The pathogens are transmitted to the public through consumption of irrigated produce, especially crops eaten raw (Blumenthal et al., 2000). Several studies throughout the world have demonstrated a very close relation between the consumption of fruits and vegetables irrigated with raw wastewater and many food borne diseases like gastroenteritis, cholera and chemical toxicity (Sou et al., 2011). The World Health Organization estimates 200 000 deaths from food borne pathogens in Nigeria (WHO, 2009). Vibrio cholerae, the etiologic agent of cholera is consumed from contaminated water, foods including vegetables (Adesida et al., 2012) with water playing a central role in its transmission (Madoroba and Momba, 2012). Many of the sources that are thought to contribute to the epidemiology of diseases associated with raw fruits and vegetables are impacted by ecological conditions that affect survival or growth of pathogenic microorganisms (Steele et al., 2005). These sources include raw manure, inorganic amendments, irrigation water and dust (Selma et al., 2007).
There is evidence to indicate that agricultural soil also have increased levels of heavy metals as a results of increased in anthropogenic activities (Mc Laughlin and Singh 1999, Sharma Agrawal, and Marshall 2007). Wastewater carries appreciable amounts of trace toxic metals (Pescod 1992, Yadav et al., 2002) which often leads to degradation of soil health and contamination of food chain mainly through the vegetable grown on such soils (Rattan et al., 2002). The toxic elements accumulated in organic matter in soils are taken up by growing plants and lastly exposing humans to this contamination (Khan et al., 2008). Municipal solid waste applications in agricultural land and wastewater discharge from industries are the other major sources of the toxic heavy metals. Heavy metals contaminants can be found on the surface and in the tissues of fresh vegetables (Arif et al., 2011). Certain trace elements are essential in plant nutrition, but plants growing in a polluted environment can accumulate trace elements at high contaminations causing a serious risk to human health when they are consumed (Voutsa et al., 1996). Tricopoulos (1997) highlighted the carcinogenic effects of several heavy metals such as Cadmium (Cd), Iron (Fe), Lead (Pb), Mercury (Hg), Zinc (Zn) and Nickel (Ni).

The wastewater treatment is a combination of the water and carried wastes removed from residential, institutional and commercial establishments together with infiltration of water, surface water and runoff water (Al- Enezi et al., 2004). The methods of wastewater treatment were first developed in response to the concern for public health and the adverse conditions caused by the discharge of wastewater to the environment (Jamrah, 1999). The characteristics of treated wastewater and sludge that affect its suitability for land irrigation and beneficial use include the presence the heavy metals, toxic organics, pathogens nutrient and organic content. The use of wastewater treated in irrigation has agronomic and economic benefits (Pescod, 1992; Shatanawi and Fayyad, 1996). It causes some problems such as; salinity, which causes soil problems and adversely affects yield quality beside, the ionic toxicities, which also causes problems to both land and crops.

Trace metals are sometimes found in wastewater. The use of water leads to accumulation of these metals in crops (Wang and Keturi, 1990; Pescod, 1992; Wong et al., 2001; Karvelas et al., 2003; Mireles et al., 2004; Al- Enezi et al., 2004; Wang et al., 2005). Urban effluents of wastewater always contain trace metals, while the concentration in the water is related to the source of the water and activities in the urban environment. Trace metals are widely used in industrial activities (Page and Chang., 1985; Smith et al., 1996). Major urban inputs to sewage water include household effluents drainage water, industrial effluents, atmospheric deposition and traffic related emissions (vehicle exhaust, brake lining, tires, and asphalt wear, gasoline and oil leakage). These are transported with storm water into the sewerage system (Wang et al, 2005; Karvelas et al., 2003; Sorme and Lagerkvist, 2002).

2.5 Wastewater and foodborne infections and intoxications
Bacteria are the most common of the microbial pathogens found in recycled waters (Toze 1999). There are a wide range of bacterial pathogens and opportunistic pathogens which can be detected in wastewaters. Many of the bacterial pathogens are enteric in origin; however, bacterial pathogens which cause non-enteric illnesses (e.g., Legionella spp., Mycobacterium spp., and Leptospira) have also been detected in wastewaters (Fliermans 1996, Neuman et al. 1997, Wilson and Fujioka 1995). Bacterial pathogens are metabolically active microorganisms that are capable of self-replication and are therefore, theoretically capable of replicating in the environment. In reality, however, these introduced pathogens are prevented from doing so by environmental pressures (Toze and Hanna 2002). Like other enteric pathogens, a common mode of transmission is via contaminated water and food and by direct person to person contact (Haas et al. 1999). A number of these bacterial pathogens can also infect, or be carried by wild and domestic animals.

Public and commercial concern does exist regarding pathogens through the use of recycled water and biosolids on cereal crops (Crute et al. 2004). It should be expected, however, that if there is a reduction of risk for the consumption of raw vegetables irrigated with partially treated effluent, then it can be surmised that grain crops irrigated with treated recycled waters would have even less risk from microbial pathogens. Even more specifically, grains are commonly processed further before they are consumed by humans which decreases the human health risk even further. Little is known, however, about the risk to domestic grazing animals that may be fed this grain unprocessed as a stock feed source. It is acknowledged that more scientific research is needed to confirm this lack of confirmatory information and funding is being provided to undertake this research (Crute et al. 2004).

In many countries, excreted infections, including faeco-oral diseases are common and excreta and wastewater contain correspondingly high concentrations of excreted pathogens. Scott et al. (2000) reported that wastewater can contain a wide spectrum of enteric pathogens which may have negative impact on the environment and human health. For example, Jimenez et al. (2001) reported concentrations in wastewater of faecal coliforms, Salmonella spp. and helminthic ova of 107'109 MPN/100 ml, 106'109 MPN/100 mL, and 6'93 ova/L, respectively. It is important we understand the transmission routes of these diseases and the health risk factors involved, in order to design and implement or modify wastewater use schemes to avoid any increased transmission of these diseases.
2.5.1 Salmonellosis (Salmonella)
Bacteria from the genus Salmonella are responsible for enterocolitis and the enteric fevers (typhoid and paratyphoid fevers) in man. Salmonellosis is in fact any infection with bacteria of genus Salmonella. Salmonella enteritidis and Salmonella typhimurium are the most frequently reported non-typhodal serotypes in many countries and outbreaks have been associated with a diverse range of food vehicles (SCF, 2002). Human salmonellosis is generally associated with enterocolitis, diarrhea, abdominal pain, and vomiting usually lasting 4-7 days (Anon, 2001). Septicaemia is rarely associated with Salmonella.

In developing nations where raw sewage is used to irrigate salad crops and where the lack of adequate sanitation and hygiene is a reality, typhoid and paratyphoid fevers are still endemic. The endemic nature of typhoid fever in Santiago, Chile is thought to result from irrigation of vegetables with raw sewage (Sears et al., 1984). These infections are rare in the developed world.

2.5.2 Diarrhoea (Pathogenic Escherichia coli)
Pathogenic E. coli is the most common cause of infantile diarrhoea in several countries, particularly in the developing world. There is currently evidence that pathogenic E. coli is one of the most common pathogens associated with the endemic life-threatening diarrhoea in many countries, e.g. Brazil (Guerrant et al., 1983), Bangladesh (Black et al., 1981), and Guatemala, El Salvador and Indonesia (Feachem et al., 1983) among others. The clinical symptoms of the diarrhoea produced by pathogenic E. coli resemble those of similar diseases caused by other enteric pathogens. It may be the severe diarrhoea similar to the cholera syndrome, a dysentery-like syndrome or a milder form of diarrhoea.

2.5.3 Shigellosis (Shigella)
The natural habitat of shigellae is limited to the intestinal tracts of humans and other primates, and a number of species are known to cause bacillary dysentery. In South America, Asia, and Africa, S. dysenteriae is commonly the cause of severe illness. The usual route of transmission is faeco-oral where food handling, faeces and flies transmit shigellae from person to person. Shigella infection usually presents itself as abdominal cramps, fever and diarrhoea, which may contain blood and mucus. The duration of illness is 4-7 days (Anon,
2001).

2.6 Survival of organisms in the environment
The viabilities of most pathogens in the environment decrease over time. Survival of bacteria, like many other organisms, depends greatly on the environment. This includes other microorganisms in the water that might provide competition or predation. As occurs in the environment, cooler temperatures promote survival of pathogenic microorganisms on fruits and vegetables. E. coli O157:H7 survived on the surface of harvested lettuce for up to 15 days when it was stored at 4??C (Beuchat, 1999) and on the surfaces of harvested fresh and frozen strawberries for at least 1 month (Knudsen et al., 2001; Yu et al., 2001).

Pathogen survival depends upon a number of factors. Parameters influencing the persistence of pathogenic microorganisms in water, crops, and soil are temperature, pH, moisture, antagonism from the soil microflora, and exposure to sunlight (Feachem et al., 1983; Bitton and Harvey, 1992 in Oron et al., 2001). Generally, high temperatures lead to rapid die-off and low temperatures prolong pathogen survival in the environment. Pathogen inactivation is much more rapid in hot sunny weather than in cool, cloudy, or rainy conditions. This is particularly relevant for post-harvest storage. It has been observed that when plants are harvested, transported and stored in refrigerated conditions (e.g., 4 ??C), pathogens may be able to survive long enough to infect product consumers.

Enteric bacteria have a shorter survival period in soils possessing a low pH (Gerba et al., 1975; Ellis and McCalla, 1976) with pH of 6 to 7 being optimum for bacterial survival (Cuthbert et al., 1955; Reddy et al., 1981). Sjogren (1994) found E. coli survived longer at a neutral to alkaline pH than at an acidic pH in soils of similar texture and organic matter content.

2.7 Trace metals
The mean concentrations of trace metals of effluent wastewater can be low possibly due to enhancing precipitation of metals under high pH value ( Karvelas et al, 2003; Wang et al, 2005). Similar findings were reported in Cairo/Egypt (El-Nennah and El-Kobbia, 1983) in Greece (Karvelas et al, 2003), in Kuwait (Al-Enezi et al, 2004) and in Mexico City (Mireles et al, 2004) and in China (Wang et al, 2005). On the other hand, if the concentrations of most trace metals are found to be high and outside guidelines for irrigation water, a possible explanation would be the high level of industrialization activities in the area under investigation. However, a possible long-term problem with wastewater irrigation is that toxic materials may accumulate in the soil. As the unsaturated zone removes chemical pollutants, particularly trace metals, their concentration in the soil will increase with time and, after many years of irrigation, it is possible that levels will reach toxic level to human.
2.7.1 Natural Occurrence in Water
Trace elements occur in almost all water supplies but at very low concentrations, usually less than a few mg/l with most less than 100 micrograms per litre (??g/1). They are not often included in a routine analysis. Surface water normally contains lower concentrations than groundwater, but this is variable and no general guidelines can be given. As a rule of thumb, irrigation water supplies do not need to be checked for trace elements unless there is some reason to suspect toxicity. In almost all cases where trace elements are at high levels, they are the result of man's activities, particularly wastewater disposal. Any project using wastewater should check for trace elements.
2.7.2 Toxicities
Not all trace elements are toxic and in small quantities many are essential for plant growth. However, excessive quantities will cause undesirable accumulations in plant tissue and growth reductions. There have been few field experiments from which toxic limits could be established, especially for irrigation water. However, research dealing with disposal of wastewater has gained sufficient experience to prove useful in defining limitations. It is now recognized that most trace elements are readily fixed and accumulate in soils, and because this process is largely irreversible, repeated applications of amounts in excess of plant needs eventually contaminate a soil and may either render it non-productive or the product unusable. Recent surveys of wastewater use have shown that more than 85 percent of the applied trace element accumulates in the soil and most accumulates in the surface few centimetres. Although plants do take up the trace elements, the uptake is normally so small that this alone cannot be expected to reduce appreciably the trace element in the soil in any reasonable period of time. The recommended maximum concentrations of some trace elements in irrigation water are given in Table 2.7 below.

Table 2.7 Recommended Maximum Concentrations of Trace Elements in Irrigation Water
Element Recommended Maximum Concentration
(mg/l) Remarks
Cd (cadmium) 0.01 Toxic to beans, beets and turnips at concentrations as low as 0.1 mg/l in nutrient solutions. Conservative limits recommended due to its potential for accumulation in plants and soils to concentrations that may be harmful to humans.
Cu (copper) 0.20 Toxic to a number of plants at 0.1 to 1.0 mg/l in nutrient solutions.
Pb (lead) 5.0 Can inhibit plant cell growth at very high concentrations.
1 Adapted from National Academy of Sciences (1972) and Pratt (1972).
2 The maximum concentration is based on a water application rate which is consistent with good irrigation practices (10 000 m3 per hectare per year). If the water application rate greatly exceeds this, the maximum concentrations should be adjusted downward accordingly. No adjustment should be made for application rates less than 10 000 m3 per hectare per year. The values given are for water used on a continuous basis at one site.

2.7.3 Human Health Effects of Heavy Metals
Heavy metals are individual metals and metal com- pounds that can impact human health (Martin and Griswold, 2009). Common heavy metals such as cadmium, chromium, lead and mercury are all naturally occurring substances which are often present in the environment at low levels. In larger amounts, they can be dangerous. Generally, humans are exposed to these metals by ingestion (drinking or eating) or inhalation (breathing).
Metals are notable for their wide environmental dispersion from anthropogenic activity; their tendency to accumulate in select tissues of the human body; and their overall potential to be toxic even at relatively minor levels of exposure. Some metals, such as copper and iron, are essential to life and play irreplaceable roles in, for example, the functioning of critical enzyme systems. Other metals are xenobiotic, i.e., they have no useful role in human physiology (and most other living organisms) and, even worse, as in the case of lead and mercury, may be toxic even at trace levels of exposure. Even those metals that are essential, however, have the potential to turn harmful at very high levels of exposure, a reflection of a very basic tenet of toxicology--'the dose makes the poison.'
One reflection of the importance of metals relative to other potential hazards is their ranking by the U.S. Agency for Toxic Substances and Disease Registry (ATSDR), which lists all hazards present in toxic waste sites according to their prevalence and the severity of their toxicity. The first, second, and sixth hazards on the list are heavy metals: lead, mercury and cadmium, respectively. Exposure to metals can occur through a variety of routes that include involuntary ingestion through food and drink. The amount that is actually absorbed from the digestive tract can vary widely, depending on the chemical form of the metal and the age and nutritional status of the individual. Once a metal is absorbed, it distributes in tissues and organs. Excretion typically occurs primarily through the kidneys and digestive tract, but metals tend to persist in some storage sites, like the liver, bones, and kidneys, for years or decades.
The toxicity of metals most commonly involves the brain and the kidney, but other manifestations occur, and some metals, such as arsenic, are clearly capable of causing cancer. An individual with metals toxicity, even if high dose and acute, typically has very general symptoms, such as weakness or headache. This makes the diagnosis of metals toxicity in a clinical setting very difficult unless a clinician has the knowledge and training to suspect the diagnosis and is able to order the correct diagnostic test. Chronic exposure to metals at a high enough level to cause chronic toxicity effects (such as hypertension in individuals exposed to lead and renal toxicity in individuals exposed to cadmium) can also occur in individuals who have no symptoms. Much about metals toxicity, such as the genetic factors that may render some individuals especially vulnerable to metals toxicity, remains a subject of intense investigation. It is possible that low-level metals exposure contributes much more towards the causation of chronic disease and impaired functioning than previously thought.
2.7.4 Bioaccumulation
Bioaccumulation is the accumulation of substances or chemicals in an organism. A few plants are capable of easily absorbing high levels of metals from the surrounding soil and these are called hyper-accumulators. Harvesting of such plants, for human use, results in exposure to harmful levels of metals. Ordinarily, this is a concern only if plants are collected from areas with high concentrations of metals in the soil. Metals uptake by plants is dependent on soil acidity (pH). The higher the acidity, the more soluble and mobile the metals become, and the more likely they are to be taken up and accumulated in plants. Root crops (like potatoes and carrots), leafy vegetables (like spinach and lettuce), and parts of plants that grow near the soil (like strawberries) are a higher risk for exposure to metal contamination than the higher portions of plants, like fruits or berries.

2.7.5 Cadmium
Certain compounds of cadmium (Cd) are highly toxic to humans. Cadmium is employed in several industrial processes such as: protective coatings (electroplating) for metals like iron; and preparation of Cd-Ni batteries, control rods and shields within nuclear reactors and television phosphors. Some compounds are used as stabilizers for PVC. For non-smoking population the major exposure pathway is through food. Cadmium is readily taken up by plants. Potential source of cadmium toxicity is the use of commercial sludge for fertilizing agricultural fields. Some root crops (carrots and parsnip) and some leafy crops (lettuce and spinach) are able to accumulate more cadmium compared to other plant foods. Grain crops like rice and wheat can accumulate relatively high amounts of cadmium. Its absorption is increased by calcium, protein and vitamin D. Internal organs of mammals such as liver and kidneys may also contain high amounts of cadmium (Tallkvist et al., 2001).
It has been documented that Itai-itai disease was caused by large amounts of cadmium in the village's water supply of Toyama city, Japan, from 1939 to 1954. Multiple fractures and severe pain in the legs and lower back affected mainly post-menopausal women with abnormal levels of glucose, calcium, and amino acids in their urine also the inhabitants of the community had for years been consuming rice contaminated by the effluent of a lead- zinc mine upstream from their rice paddies (Oudeh et al., 2002). Cadmium is a cumulative toxicant and carcinogenic that affects kidneys, generates various toxic effects in the body, disturbs bone metabolism and deforms reproductive tract as well as endocrine system. There are several morpho-pathological changes in the kidneys due to long-term exposure to cadmium. Increasing intakes of zinc can reduce the renal toxicity of cadmium. An exposure to cadmium in- creases calcium excretion thus causes skeletal demineralization, probably leading to increases in bone fragility and risk of fractures (Wu et al., 2001). Cadmium and its compounds are currently classified by IARC as a Group 1 carcinogen for humans. Occupational human exposure has been correlated with lung cancer. Cadmium exposure, during human pregnancy, leads to reduced birth weights and premature birth (Henson and Chedrese, 2004).
2.7.6 Lead
Lead (Pb) is used in storage batteries, cable coverings, plumbing, ammunition, manufacture of tetraethyl Pb, sound absorbers, radiation shields around X-ray equipment and nuclear reactors, paints, while the oxide is used in producing fine "crystal glass" and "flint glass" with a high refractive index for achromatic lenses, solder and insecticides. Lead enters the human body in many ways. It can be inhaled in dust from lead paints, or waste gases from leaded gasoline. It is found in trace amounts in various foods which are heavily subjected to industrial pollution. Pb can contaminate water and consequently enter the aquatic food chains (Kaste et al., 2003). Pb is a toxic metal and most people and animals receive the largest portion of their daily Pb intake via food.
Children under 6 years are especially susceptible to the adverse effects of Pb, as the blood-brain barrier is not yet fully developed in young children, haematological and neurological adverse effects of Pb occur at lower threshold levels than in adults. Pb has effects on erythropoiesis and haem biosynthesis. Chronic Pb intoxication in adults resulted in to anaemia, some types of cancer, reproductive harm in males while in young children hormonal imbalance of metabolite of vitamin D, namely 1, 25-dihydroxy-vitamin D, drop in IQ, (Tandon et al., 2001; Siddiqui et al., 2002; Lindbohm et al., 1991).
EPA has determined that lead is a probable human carcinogen. Lead can affect every organ and system in the body. Long-term exposure of adults can result in decreased performance in some tests that measure functions of the nervous system; weakness in 'ngers, wrists, or ankles; small increases in blood pressure; and anaemia. Exposure to high lead levels can severely damage the brain and kidneys and ultimately cause death. In pregnant women, high levels of exposure to lead may cause miscarriage. High level exposure in men can damage the organs responsible for sperm production.

2.7.7 Copper
Copper is a reddish metal that occurs naturally in rock, soil, water, sediment, and, at low levels, air. Copper also occurs naturally in all plants and animals. It is an essential element for all known living organisms including humans and animals at low levels of intake. At much higher levels, toxic effects can occur (ATSDR, 2010). Copper is primarily used as the metal or alloy in the manufacture of wire, sheet metal, pipe, and other metal products. Copper compounds are most commonly used in agriculture to treat plant diseases, like mildew, or for water treatment and as preservatives for wood, leather, and fabrics. Ingestion of copper may result in nausea, vomiting, stomach cramps and diarrhoea. However, the EPA does not classify Copper as a human carcinogen due to lack of adequate scientific evidence.
2.7.7 Nickel
Pure nickel is a hard, silvery-white metal, which has properties that make it very desirable for combining with other metals to form mixtures called alloys. Some of the metals that nickel can be alloyed with are iron, copper, chromium, and zinc. These alloys are used in making metal coins and jewellery and in industry for making items such as valves and heat exchangers. Most nickel is used to make stainless steel. There are also compounds consisting of nickel combined with many other elements, including chlorine, sulphur, and oxygen. Many of these nickel compounds are water soluble (dissolve fairly easily in water) and have a characteristic green colour. Nickel and its compounds have no characteristic odour or taste. Nickel compounds are used for nickel plating, to colour ceramics, to make some batteries, and as substances known as catalysts that increase the rate of chemical reactions.
Nickel is released into the atmosphere during nickel mining and by industries that make or use nickel, nickel alloys, or nickel compounds. These industries also might discharge nickel in waste water. Nickel is also released into the atmosphere by oil-burning power plants, coal-burning power plants, and trash incinerators.
The most common harmful health effect of nickel in humans is an allergic reaction. The most common reaction is a skin rash at the site of contact. In some sensitized people, dermatitis (a type of skin rash) may develop in an area of the skin that is away from the site of contact. For example, hand eczema (another type of skin rash) is fairly common among people sensitized to nickel. Some sensitized individuals react when they eat nickel in food or water or breathe dust containing nickel. The U.S. Department of Health and Human Services (DHHS) has determined that nickel metal may reasonably be anticipated to be a carcinogen and nickel compounds are known human carcinogens. The International Agency for Research on Cancer (IARC) has determined that some nickel compounds are carcinogenic to humans and that metallic nickel may possibly be carcinogenic to humans.
2.7.8 Zinc
Zinc is one of the most common elements in the Earth's crust. Zinc is found in the air, soil, and water and is present in all foods. In its pure elemental (or metallic) form, zinc is a bluish-white, shiny metal. Powdered zinc is explosive and may burst into flames if stored in damp places. Metallic zinc has many uses in industry. A common use for zinc is to coat steel and iron as well as other metals to prevent rust and corrosion; this process is called galvanization. Metallic zinc is also mixed with other metals to form alloys such as brass and bronze. Metallic zinc is also used to make dry cell batteries.
Zinc can also combine with other elements, such as chlorine, oxygen, and sulphur, to form zinc compounds. Zinc compounds that may be found at hazardous waste sites are zinc chloride, zinc oxide, zinc sulphate, and zinc sulphide. Most zinc ore found naturally in the environment is in the form of zinc sulphide. Zinc compounds are widely used in industry. Zinc sulphide and zinc oxide are used to make white paints, ceramics, and other products. Zinc oxide is also used in producing rubber. Zinc compounds, such as zinc acetate, zinc chloride, and zinc sulphate, are used in preserving wood and in manufacturing and dyeing fabrics. Zinc compounds are used by the drug industry as ingredients in some common products, such as vitamin supplements, sun blocks, diaper rash ointments, deodorants, athlete's foot preparations, acne and poison ivy preparations, and antidandruff shampoos.
Taking too much zinc into the body through food, water, or dietary supplements can affect health. The levels of zinc that produce adverse health effects are much higher than the Recommended Dietary Allowances (RDAs) for zinc of 11 mg/day for men and 8 mg/day for women. If large doses of zinc (10-15 times higher than the RDA) are taken by mouth even for a short time, stomach cramps, nausea, and vomiting may occur. Ingesting high levels of zinc for several months may cause anaemia, damage the pancreas, and decrease levels of high-density lipoprotein (HDL) cholesterol. The EPA has determined that because of lack of information, zinc is not classifiable as to its human carcinogenicity.
Consuming too little zinc is at least as important a health problem as consuming too much zinc. Without enough zinc in the diet, people may experience loss of appetite, decreased sense of taste and smell, decreased immune function, slow wound healing, and skin sores. Too little zinc in the diet may also cause poorly developed sex organs and retarded growth in young men. If a pregnant woman does not get enough zinc, her babies may have birth defects.

2.8 Abnormal pH
The pH of water is an indicator of its acidity or basicity, but is seldom a problem by itself. The main use of pH in a water analysis is for detecting abnormal water. The normal pH range for irrigation water is from 6.5 to 8.4. An abnormal value is a warning that the water needs further evaluation. Irrigation water with a pH outside the normal range may cause a nutritional imbalance or may contain a toxic ion.
Water sometimes has a pH outside the normal range. Such water normally causes few problems for soils or crops but is very corrosive and may rapidly corrode pipelines, sprinklers and control equipment. Any change in the soil pH caused by the water will take place slowly since the soil is strongly buffered and resists change. An adverse pH may need to be corrected, if possible, by the introduction of an amendment into the water, but this will only be practical in a few instances. It may be easier to correct the soil pH problem that may develop rather than try to treat the water. Lime is commonly applied to the soil to correct a low pH and sulphur or other acid material may be used to correct a high pH. Gypsum has little or no effect in controlling an acid soil problem apart from supplying a nutritional source of calcium, but it is effective in reducing a high soil pH (pH greater than 8.5) caused by high exchangeable sodium. The greatest direct hazard of an abnormal pH in water is the impact on irrigation equipment. Equipment will need to be chosen carefully for unusual water.
2.9 Health risk reduction methods
2.9.1 Crop restriction
Crop restriction can be used to protect the health of consumers when water of sufficient quality is not available for unrestricted irrigation. For example, water of poorer quality can be used to irrigate non-vegetable crops such as cotton or crops that will be cooked before consumption (e.g., potatoes). In Chile, the use of crop restriction when implemented with a general hygiene education program reduced the transmission of cholera from the consumption of raw vegetables by 90% (Monreal, 1993).

2.9.2 Wastewater application methods
Choosing a wastewater application method can impact on health protection of farm workers, consumers, and nearby communities. Because of the formation of aerosols, spray/sprinkler irrigation has the highest potential to spread contamination on crop surfaces and affect nearby communities. Where spray/sprinkler irrigation is used with wastewater it may be necessary to set up a buffer zone to prevent health impacts on local communities (Mara and Cairncross, 1989). Localized irrigation techniques (e.g., drip) offer farm workers the most health protection because the wastewater is applied directly to the plants.

2.10 Overview
It is clear from the literature reviewed in this thesis that urban effluent is generally contaminated with heavy metals, bacteria and other potentially toxic materials. The scarcity of freshwater resources and the disproportionate increase in demand for food has resulted in the exploration of wastewater reuse as an alternative to freshwater reuse in urban agriculture. Some of the crops irrigated with urban effluent are consumed raw in salads and thus the lack of heat treatment which would perhaps make the foods safe for human consumption therefore poses a risk to public health. The associated disease burden on populations of developing countries as a result of foodborne infections is of particular fundament. There is therefore an observable link between wastewater reuse, bacterial and heavy metal toxicants, effect of lack of heat treatment as well as disease burden. This link is illustrated in the conceptual framework below.

Fig. 2.10 Conceptual framework for this research
CHAPTER THREE
RESEARCH METHODOLOGY
3.1 Research Design
This study was a quantitative exploratory study whereby samples were collected from the identified study sites and then analyzed in a laboratory for the specified chemical constituents.
3.2 Study area
Bulawayo is located in the drought prone, semi-arid Matabeleland region and receives about 590 mm rainfall per annum. It has a hot wet summer from October to March with average temperatures of 25oC and a cool dry season with an average temperature of 15oC for the rest of the year (Taigbenu and Ncube, 2005). Due to its relatively high altitude, the city has a subtropical climate despite lying within the tropics. The city experiences three broad seasons: a dry, cool winter season from May to August; a hot dry period in early summer from late August to early November; and a warm wet period in the rest of the summer, early November to April. The city's average annual rainfall supports a natural vegetation of open woodland, dominated by Combretum and Terminalia trees. Due to proximity to the Kalahari Desert, Bulawayo is vulnerable to droughts and rainfall tends to vary sharply from one year to another.

The study was conducted at Umguza Irrigation Scheme on the outskirts of Bulawayo, Zimbabwe. The site was in a low-lying area where farmers had access to irrigation water from the perennial Umguza River, and soils are predominantly loamy. The polluted river is a common source of irrigation water, and canals, pipes connected to sprinklers as well as watering cans are used for irrigation. The four main vegetables cultivated are lettuce, spring onion, tomato and cabbage. These vegetables are mostly eaten raw (no prior heat treatment) in salads hence they were chosen for investigation (Amoah et al., 2005). The cultivar of lettuce grown in the study area is the bunching type, which takes a month to mature after transplanting.


The map of the study area is given in Figure 3.2 below.



3.3 Population
The project involved the collection of quantitative data (amounts of Cadmium, Lead and Copper in the samples) as well as extrapolation of qualitative environmental data (known effects of observed levels of heavy metals). Quantitative data was obtained from heavy metal assay of treated effluent; irrigated soil and vegetables using standard analytical methods. Literature review was extensively conducted to draw parallels between the outcomes of this research and those done before.

Various methodologists (e.g. Neuman, 1994:317) have tabulated the differences between qualitative and quantitative research as shown in Table 3.3 below. These differences are the reason this researcher settled for quantitative research.

Table 3.3 Differences between qualitative and quantitative research


An exploratory research design was also selected for this project. An exploratory design is conducted about a research problem when there are few or no earlier studies to refer to. The focus is on gaining insights and familiarity for later investigation or undertaken when problems are in a preliminary stage of investigation.
The goals of exploratory research are intended to produce the following possible insights:
i. Familiarity with basic details, settings and concerns.
ii. Well-grounded picture of the situation being developed.
iii. Generation of new ideas and assumption, development of tentative theories or hypotheses.
iv. Determination about whether a study is feasible in the future.
v. Issues get refined for more systematic investigation and formulation of new research questions.
vi. Direction for future research and techniques get developed.

Exploratory research is useful in that it can provide the following:
1. Design is a useful approach for gaining background information on a particular topic.
2. Exploratory research is flexible and can address research questions of all types (what, why, how).
3. Provides an opportunity to define new terms and clarify existing concepts.
4. Exploratory research is often used to generate formal hypotheses and develop more precise research problems.
5. Exploratory studies help establish research priorities.

3.4 Sampling
3.4.1 Sampling sites
Multi-stage sampling was performed whereby the study site was purposively sampled since it represents the hallmark of the project which is effluent reuse for vegetable production. cluster sampling of soil, effluent and plant samples was then conducted and the individual samples from each cluster were collected on the basis of simple random sampling. The vegetables that were used in the study were those that the researcher found on the study site at the time of sampling which translates to convenient sampling. Sampling sites selection was based on; the availability of vegetables on the farms, the cooperation of the farmers, the type of vegetables grown, the source of irrigation water and its point source of contamination. Five designated irrigation sites were selected for the study.

3.4.2 Sample size
Fifty samples consisting of thirty vegetables as per the recommendation of FAO (1992), ten irrigation water samples and ten soil samples were collected and analyzed during the study. Samples were collected in the wet (January - March, 2014) season.
3.5 Sample preparation
3.5.1 Water Sample preparation
Sample bottles were soaked overnight in dilute hydrochloric acid before use and were rinsed two times with the sample to be collected before filling with sample as recommended by APHA (1985). Water samples were collected according to the procedure recommended by American Public Health Association (APHA, 1992). Water samples were collected in sterile wide mouth, screw capped 250 ml bottles.

3.5.2 Vegetable Sample preparation
Using 90% ethanol sterilized scissors; vegetables were cut into factory sterile polythene bags. All samples were packed on ice during the transportation to the laboratory. To obtain an accurate assessment in this situation, it is necessary to produce composite samples by collecting individual samples at known time intervals throughout the period and then carrying out the appropriate analytical procedures (Tebbutt, 1998).

3.5.3 Soil Sample preparation
Soil samples were collected at depths ranging from 0'0.10 m using a soil Dutch auger and put in polythene bags. Surface litter was first scraped away at each sampling spot to remove plant debris. Samples were collected from nine sites, three sites inside each of the effluent-irrigated vegetable fields to make one composite sample from each field to make a composite sample at each depth. The soil samples were analysed following recommendations by Page et al. (1982).

3.5.4 Sterilization of equipment and material
The best means of avoiding microbial contamination is to sterilize the materials used for microbiological analysis under laboratory conditions. Hence all the materials used were sterilized using standard procedures. Petri dishes and test tubes were washed with soap and rinsed with water after which they were air-dried. The working benches were also cleaned or disinfected with 70% ethanol before and after use.
3.6 Specific analytical methods
3.6.1 Chemical Analytical Methods
3.6.1.1 Determination of soil pH, total dissolved solids (TDS) and electrical conductivity (EC)

The method used was extracted from ISO 10390:1994(E).
Scope
The afore-stated international standard specifies an instrumental method for the routine determination of pH using a gas electrode in a 1:5 (v/v) suspension of soil in a solution of 0.01mol/L Calcium chloride (pH_CaCl2).
Principle
A suspension of soil is made up in five times its volume of a 0.01mol/L solution of Calcium chloride in distilled water. The pH, EC and TDS of the suspension was measured using a portable Hannah multi-meter H300 equipped with a combination gas electrode.

3.6.1.2 Determination of effluent pH, total dissolved solids (TDS) and electrical conductivity (EC)
The pH, total dissolved solids (TDS) and electrical conductivity (EC) of effluent were measure in situ using a portable Hannah multi-meter H300 equipped with a combination gas electrode.

3.6.2 Heavy metal Analytical Methods
Heavy metals were analysed using a Perkin Elmer AAnalyst 100 Atomic Absorption Spectrophotometer using an air-acetylene flame. The wavelengths at which sample metal concentrations were read were as follows:
Copper 324.8 nM
Nickel 352.5 nM
Zinc 213.9 nM
Lead 217.0 nM
Cadmium 228.8 nM
3.6.2.1 Analysis of heavy metals in effluent samples
The reagents needed include Lanthanum solution, 5% (w/v) as well as concentrated Hydrochloric acid. All standard solutions of the heavy metals to be analysed were also prepared by suitable dilutions of the stock solutions for standardization of the Atomic Absorption Spectrometric process. Sample preparation involved filtering each sample through a Whatman *42 filter paper to avoid clogging of the burner capillary. Each sample was aspirated directly into the AA Spectrometer (Perkin Elmer AAnalyst 100) and a routine determination of the concentration of the heavy metal of interest was performed.

3.6.2.2 Analysis of heavy metals in soil samples
The extracting solution used to determine extractable cations in the soil samples was a 0.005N HCl in 0.025N H2SO4 solution (Double Acid). An air-dried ground soil was weighed out (5g) and placed in a flask, and extracting solution added followed by agitation of the mixture. The samples were then filtered through Whatman *42 filter paper into a 50ml volumetric flask and diluted to 50ml with extracting solution. The extract was analyzed directly as serial dilutions of 1:10, 1:20 and 1:50 so as to come up with the concentration that could be picked within the range of the AA Spectrometer.

3.6.2.3 Analysis of heavy metals in vegetable samples
About 1.0 g portion of the ground sample was weighed into a 100cm3 Kjeldhal digestion flask; 5ml of concentrated nitric acid (HNO3) was added followed by 1ml each of concentrated sulphuric acid (H2SO4) and perchloric acid (HClO4). The flask was heated in the fume cupboard until dense white fumes appeared at the end of the digestion. The flask was cooled and the content was diluted with distilled water and filtered through Whatman #42 ashless filter paper into 100cm3 volumetric flasks (USEPA, 2002). The content was made up to the mark with distilled water and transferred to 120cm3 plastic bottles and stored for heavy metal determination using Atomic absorption Spectrophotometer (Perkin Elmer AAnalyst).

3.6.3 Bacteriological Analytical Methods
All bacteriological analytical methods are based on FAO (1992).

3.6.3.1 Determination of E.coli from vegetable samples
Presumptive test
About 50g of vegetable sample was weighed into a sterile high-speed blender jar. This was followed by addition of 450ml of Butterfield's phosphate buffer and blended for two minutes at 10000 ' 12000 rpm. Decimal dilutions were prepared with 90ml Butterfield's phosphate buffer plus 10ml from previous dilution, and these were shaken 25 times in 30cm arc for 7s. Portions of 1ml each were transferred to 3 Lauryl tryptose tubes for each dilution. The tubes were incubated for 48hrs at 350C. The tubes were examined at 24hrs for gas, i.e., displacement of medium in fermentation vial or effervescence when tubes are gently agitated. Negative tubes were re-incubated for additional 24hrs and examined a second time for gas. A confirmed test for E.coli was then performed on all presumptive positive (gassing) tubes.
Confirmed test
A loopful of suspension from each gassing medium tube was streaked to Levine's eosin-methylene blue (L-EMB) agar and the plates were incubated at 350C for 24hrs. The plates were examined for typical E.coli colonies, i.e., dark centered with or without metallic sheen. The most probable number (MPN) was subsequently computed. This test is illustrated in Appendix 1.

3.6.3.2 Determination of Salmonella from vegetable samples
About 25g of sample was aseptically weighed into a sterile blending container followed by the addition of 225ml of sterile lactose broth. This mixture was blended for 2minutes and homogenate was aseptically transferred to a sterile, wide-mouth screw-cap jar (500ml) and allowed to stand for 60minutes at room temperature with jar securely capped. Swirling was done to mix well and pH was determined and adjusted, if necessary, to 6.8 ?? 0.2. The mixture was incubated for 24hrs at 350C. The incubated sample was gently shaken and 1ml of mixture transferred to 10ml selenite cysteine broth followed by incubation at 350C for 24hrs. The tube was shaken and 3mm loopful of broth was streaked and incubated on xylose lysine desoxycholate (XLD) agar and the mixture was incubated for 24hrs at 350C. The plates were examined for presence of Salmonella colonies which appeared as pink colonies with or without black centers. The most probable number (MPN) was subsequently computed. This test is illustrated in Appendix 2.

3.6.3.3 Determination of Shigella from vegetable samples
Enrichment
About 25g of sample was aseptically weighed into 225ml Shigella broth to which novobiocin had been added to a level of 3??g/ml. the suspension was held for 10minutes at room temperature whilst being shaken periodically. The eluent was poured into sterile 500ml Erlenmeyer flask and incubated anaerobically in 440C water bath for 20hrs. The suspension was agitated and streaked on MacConkey agar plates and then incubated for 20hrs at 350C. The plates were then examined for Shigella colonies which appeared slightly pink and transluscent. The most probable number (MPN) was subsequently computed. This test is illustrated in Appendix 3.
3.6.3.4 Determination of Staphylococcus aureus
For each dilution to be plated, 1ml sample suspension was aseptically transferred to 3 plates of Baird-Parker medium, distributing 1ml of inoculum equitably to 3plates. The inoculum was spread over surface of agar plate using a sterile bent glass streaking rod. The plates were inverted and incubated for 45-48hrs at 350C. plates were examined for S. aureus whose colonies are tyoically circular, smooth, convex, moist, 2-3mm in diameter on uncrowded plates, gray to jet-black, frequently with light-coloured (off-white) margin, surrounded by opaque zone and frequently with an outer clear zone; colonies have buttery to gummy consistency when touched with inoculating needle. The most probable number (MPN) was subsequently computed. This test is illustrated in Appendix 4.

3.5 Statistical analysis
Using the statistical software SPSS for windows 17.0, analysis of variance (ONE WAY ANOVA) was used to evaluate statistical significance of the data and results were quoted at P < 0.05 levels of significance. Faecal coliform populations (MPN) were normalised by log transformation before analysis of variance (ANOVA). ANOVA was used to compare faecal coliform levels on different crops. Unless otherwise stated, results of analysis are quoted at p < 0.05 level of significance.

'

CHAPTER FOUR
RESULTS AND DISCUSSION

4.1 Results of heavy metal analysis

4.2 Copper results One-way Analysis of Variance (ANOVA)

The P value is < 0.0001, considered extremely significant.
Variation among column means is significantly greater than expected by chance.
Tukey-Kramer Multiple Comparisons Test
If the value of q is greater than 4.087 then the P value is less than 0.05.
Mean
Comparison Difference q P value
================================== ========== ======= ===========
Lettuce vs Lettuce soil 0.01300 5.035 ** P<0.01
Lettuce vs Cabbage 0.2790 108.06 *** P<0.001
Lettuce vs Tomato 0.1780 68.939 *** P<0.001
Cabbage vs Cabbage soil -0.5510 213.40 *** P<0.001
Cabbage vs Tomato -0.1010 39.117 *** P<0.001
Tomato vs Tomato soil -0.8990 348.18 *** P<0.001

4.3 Cadmium results One-way Analysis of Variance (ANOVA)

The P value is > 0.9999, considered not significant.
Variation among column means is not significantly greater than expected by chance.

Post tests
Post tests were not calculated because the P value was greater than 0.05.

4.4 Zinc results One-way Analysis of Variance (ANOVA)

The P value is < 0.0001, considered extremely significant.
Variation among column means is significantly greater than expected by chance.

Tukey-Kramer Multiple Comparisons Test
If the value of q is greater than 3.913 then the P value is less than 0.05.

Mean
Comparison Difference q P value
================================== ========== ======= ===========
Lettuce soil vs Cabbage 0.9020 333.09 *** P<0.001
Lettuce soil vs Cabbage soil 0.3140 115.95 *** P<0.001
Lettuce soil vs Tomato 0.4880 180.21 *** P<0.001
Lettuce soil vs Tomato soil 0.05700 21.049 *** P<0.001
Cabbage vs Cabbage soil -0.5880 217.13 *** P<0.001
Cabbage vs Tomato -0.4140 152.88 *** P<0.001
Tomato vs Tomato soil -0.4310 159.16 *** P<0.001


4.5 Lead results One-way Analysis of Variance (ANOVA)

The P value is < 0.0001, considered extremely significant.
Variation among column means is significantly greater than expected by chance.

Tukey-Kramer Multiple Comparisons Test
If the value of q is greater than 3.913 then the P value is less than 0.05.

Mean
Comparison Difference q P value
================================== ========== ======= ===========
Cabbage vs Cabbage soil -0.2080 76.809 *** P<0.001
Cabbage vs Tomato 0.1040 38.405 *** P<0.001
Tomato vs Tomato soil -0.3800 140.32 *** P<0.001

4.6 Nickel results One-way Analysis of Variance (ANOVA)

The P value is < 0.0001, considered extremely significant.
Variation among column means is significantly greater than expected by chance.
Tukey-Kramer Multiple Comparisons Test
If the value of q is greater than 3.913 then the P value is less than 0.05.

Mean
Comparison Difference q P value
================================== ========== ======= ===========
Cabbage vs Cabbage soil -1.091 402.88 *** P<0.001
Cabbage vs Tomato -0.1200 44.313 *** P<0.001
Tomato vs Tomato soil -0.3810 140.69 *** P<0.001

Appendix 1

Conventional method for enumeration of Escherichia coli (FAO, 1992)
Appendix 2

Conventional method for detection of Salmonella (FAO, 1992)
Appendix 3

Conventional method for detection of Shigella (FAO, 1992)
Appendix 4

Conventional method for enumeration of Staphylococcus aureus (FAO, 1992)
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