Cereal Mycotoxins Contamination and Associated Risk Factors: A Consideration of Lringa Municipality in Tanzania.

Background: Mycotoxins have various serious health effects when consumed by humans and animals including, acute aflatoxicosis when consumed in high levels. Chronic exposure to low levels of contamination in crops increases liver cancer risk and can suppress the immune system, particularly for populations that test positive for the hepatitis B virus (HBV). Also when contaminated foods are consumed by young children can lead to stunting and growth impairment. Methods: A cross-sectional study design was carried out at one-time point .A total of 296 participants were enrolled. Surveys were conducted to determine risk factors for mycotoxin contamination in cereal in Iringa municipality. The study involved 4 wards namely Kwakilosa, Mlandege, Kihesa and Ruaha, in which 2 streets were selected from each ward where a minimum of 25 respondents were selected in each street by using quota and convenience sampling techniques. A questionnaire was administered to assess community‟s level of knowledge on mycotoxin contamination in cereals. A checklist was used to explore the conditions and facilities used store cereal, Results: Majority of respondents 71(36.2%) their age ranged 29-39 years, few being above 61 making up 3.6%. Also 87.2% did not have knowledge on mycotoxin contamination in cereals, while only 12.8% had knowledge on mycotoxin contamination in cereals which showed that 56.6% of the storage rooms were not properly ventilated, with proper ventilation were 43.4% of the total storage rooms. The study also revealed that majority 67.1% placed cereals in rooms with no ceilings, while also 60.8% leaned their cereal bags directly against the wall without leaving space contributing to favorable conditions for fungal growth as it increases the likelihood of cereal bags to absorb moisture from the wall, 86.7% cleaned their packaging material before use while only 46.9% and 44.8% cleaned their rooms and milling machines before storage or processing respectively. Majority 80.4% used woven sacks while 22.4% and 46.9% had pallets and tarpaulin respectively. Conclusion: Majority of respondents 171 individuals equivalent to 87.2% did not have knowledge on mycotoxin contamination in cereals, while only 25 individuals equivalent to 12.8% had knowledge on mycotoxin contamination in cereals. While the storage conditions and facilities used, showed that majority of individuals 111(77.6%) piled up bags of cereals on bare floors (no pallets) and 87(60.8%) leaned them directly against the walls with majority of them 96(67.1%) placed under roofs with no ceiling.


Background
Mycotoxins are naturally occurring toxic secondary metabolites produced by filamentous fungi mainly Aspergillus flavus and Aspergillus parasiticus which can contaminate many kinds of agricultural products. Toxigenic fungi are capable of growing under a wide range of atmospheric conditions depending on the species and they can contaminate crops during pre-harvest, immediate post-harvest, storage, transport and processing (Bennett & Klich, 2003).
Aflatoxins and fumonisins are among the main types of mycotoxins of significant public health concern due to their widespread contamination in foodstuffs as well as chronic and acute health

MYCOTOXIN CONTAMINATION IN CEREALS AND THEIR ASSOCIATED EFFECTS Aflatoxin
Aflatoxins occur in nuts, cereals and rice under conditions of high humidity and temperature and present a risk to human health that is insufficiently recognized. Aflatoxins include both B and G aflatoxins. Aflatoxins M1 and M2 are oxidative metabolic products of aflatoxins B1 and B2 produced by animals following ingestion, and so appear in milk (both animal and human), urine and faeces. Aflatoxins are acutely toxic, immunosuppressive, mutagenic, teratogenic and carcinogenic compounds. The main target organ for toxicity and carcinogenicity is the liver [WHO, 1999].
Following the evaluation of epidemiological and laboratory results carried out in 1987 by the International Agency for Research on Cancer (IARC) naturally occurring mixtures of aflatoxins, were classified as Group 1 carcinogens, except for aflatoxin M1, which is possibly carcinogenic to humans. Aflatoxins have been suggested as an etiological factor in encephalopathy and fatty degeneration of viscera, similar to Reye syndrome, which is common in countries with a hot and humid climate. The clinical picture includes enlarged, pale, fatty liver and kidneys and severe cerebral oedema, accompanied by headache, nausea and jaundice (WHO, 1999).

Ochratoxins
Ochratoxinsare secondary metabolites of Aspergillus and Penicillium strains, found on cereals (dry beans, maize, wheat, oats), coffee and bread, as well as on all kinds of food commodities of animal origin in many countries. The most frequent is ochratoxin A (OTA), which is also the most toxic. It has been shown to be nephrotoxic, immunosuppressive, carcinogenic and teratogenic in all experimental animals tested so far. This mycotoxin has been proposed as the causative agent of endemic nephropathy, the main features of endemic nephropathy are bilateral, primarily chronic lesions of the renal cortex (tubular degeneration, interstitial fibrosis and hyalinization of the glomeruli). In the advanced stage of the disease, the size and weight of kidneys are remarkably reduced, with diffuse cortical fibrosis, usually without signs of inflammation (WHO, 1999).

Fumonisins
Fumonisins are mycotoxins produced throughout the world by Fusarium moniliforme and related species when they grow in maize. Fumonisins B1 and B2 are of toxicological significance, while the others (B3, B4, A1 and A2) occur in very low concentrations and are less toxic (WHO, 1999).
They are found predominantly in maize and in a maize based animal feeds, FB(1) is a neurotoxic, hepatotoxic and nephrotoxic in animals, and it has been classified as possible carcinogen to humans. The cellular mechanisms behind FB (1) induced toxicity include the induction of oxidative stress, apoptosis and cytotoxicity as well as alterations in cytokine expression (Stockman & Savolainen, 2008).

Zearalenone
Zearalenone (previously known as F-2) is produced mainly by Fusarium graminearum and related species, principally in wheat and maize but also in sorghum, barley and compounded feeds (WHO, 1999). ZEN affects hematological and immunological parameters in humans and rodents.
The compound can cause cell death, lipid peroxidation, inhibit protein and exert genotoxic effects.
ZEN may cause increased phagolysosomal in the kidney, can induce liver carcinoma, pituitary adenoma and renal toxicity (Gao F, Jiang LP, et al., 2013).

Deoxynivalenol (DON)
DON is the most frequent myotoxin found in corn, barley, sunflower seeds, rye and wheat, exposure to elevated levels DON causes nausea, vomiting and diarrhea in farm animals and lower doses in cattle have been linked to weight loss and food refusal. Prolonged exposure to DON can cause reduced growth and damage organs such as spleen, thymus, liver and heart. Also known to be powerful inhibitors of protein synthesis by reacting with components of ribosomes (Lubinda Mbundi, et al., 2014).

Patulin
Patulin is produced by variety of molds especially Aspergillus and Penicillium species, which are commonly detected in variety of food stuffs such as cEreals, vegetables and fruits such as apples and pears. Patulin is generally a potent toxin but due to its demonstration of genotoxicity it shows potential signs of being carcinogen. WHO recommended maximum levels of patulin are 50ng/mL in apple juice and cider, 25ng/g in solid apple products and 10ng/g in products for infants and young children in the European Union, In addition the Joint FAO/WHO Expert Committee on Food Additives (JECFA) has established provisional maximum allowable daily intake of 0.4 μg/g of body weight per day for patulin (Lubinda Mbundi, et al., 2014).

Egort alkaloids
Ergot is the common name of the sclerotia of fungal species within the genus Claviceps, which produce ergot alkaloids. Ergot alkaloids are also secondary metabolites of some strains of Penicillium, Aspergillus and Rhizopus spp. The source of the ergot strongly influences the type of alkaloids present, as well as the clinical picture of ergotism. Claviceps purpurea produces ergotamine and ergocristine alkaloids, which cause the gangrenous form o fergotism because of their vasoconstrictive activity. The initial symptoms are oedema of the legs, with severe pains.
Paraesthesias are followed by gangrene at the tendons, with painless demarcation (WHO, 1999). Ergotism is extremely rare today, primarily because the normal grain cleaning and milling

CEREAL STORAGE
Toxigenic fungi are associated with both pre-harvest and postharvest mycotoxin contamination in cereals, The severity of post-harvest fungal infection and propagation during prolonged periods of grain storage can be managed more predictably through GAP and good manufacturing practices (GMP) that ensure that moisture levels in stored grain remain below levels. However, research has confirmed that spores of such species are ubiquitous in soils, equipment, and storage structures despite diligent cleaning. Therefore, it is important for the storage room to be designed properly such that it facilitates sanitary conditions to prevent mold growth, also storage equipments used Bins, silos, sheds and other buildings intended for grain storage are dry, well-vented structures that provide protection from rain, snow, ground water, moisture condensation, and the entry of rodents, birds and insects that cannot only contaminate grain, but damage grain kernels to render them more susceptible to mould infection. storage structures should be designed so as to minimize wide fluctuations in the temperature of the stored grain (CAC, 2003). Areas surrounding stores are well maintained. In particular, lawns, areas not covered with concrete and intake pits. Sites to be free-draining. Rodent traps are positioned in the areas surrounding food and feed materials and waste storage locations. Fencing is important for securing unauthorized entry, there should be enough lighting, gates and doors are fitted with good quality padlocks (WFP, 2012).

Open weave sacks
For keeping grain on farm for periods of three months or less, open weave sacks are the most convenient option. Sacks may be made of polypropylene, jute or sisal. If second-hand bags are to be used then they must be thoroughly cleaned before before filling with grain. The bags should not be overfilled with grain, after filling they should be closed by hand stitching or by using a stitching machine (WFP, 2012).

Metal silos
Metal silos are insect-proof but can be made hermetic by tying rubber from a bicycle inner tube very tightly around the grain input and output ports. In order to have a quick change in gas composition, a lighted candle may be placed on the grain surface at the time the inlet and outlet ports are sealed (do not do this with plastic grain stores as they may catch fire).
The candle will burn the oxygen and in so doing create carbon dioxide, this will extinguish the candle and within two weeks will kill any insects that are present. Do not open the silos until after two weeks as this will let in fresh air and the insects will survive (WFP, 2012).

Pallets
The sacks must be prevented from making contact with the floor or walls of the house, from which they might absorb moisture, causing the grain to rot. To do this the bags are placed on pallets made of sticks and/or stones so they are suspended at least 12 cm above the floor (if no pallets can be constructed then a plastic sheet could be used) and away from contact with the wall (WFP, 2012).

Tarpaulins
Tarpaulins that can be used to cover bag stacks to prevent insect infestation moving from one stack to another and as a base on which to do grain conditioning operations. Also can be used to cover bag stacks when store is cleaned to prevent dust that may contain fungal spores to contaminate cereals (WFP, 2012)..

SANITATION OF THE STORE ROOM AND ITS FACILITIES
Before the new harvest arrives in store it is best to ensure that all the old harvest has been discharged. If it cannot be discharged then it would be helpful to isolate it from the new harvest by covering with a tarpaulin and weighting the edges of the tarpaulin with lengths of wood or stones.
This will help reduce the movement of insects from old infested grain to the new grain (WFP, 2012).
The store should then be thoroughly swept, to remove all grain residues from the floor, any cracks and crevices, or sliding door runners. The grain residues should be taken out of the store and burnt or fed to animals. The floor should be thoroughly inspected for cracks and if any are found they should be filled with cement. Cracks in walls should also be filled (WFP, 2012).
Once the store is clean the pallets should be laid out to receive the incoming crop, with a gap of 1m from the store walls. The pallets should be brushed clean to remove any old grain or grain dust. They should be checked for any protruding nails, which might tear grain sacks, any nails should be removed or hammered in (WFP, 2012).
Storage facilities should be cleaned prior to receiving grain to remove dust, fungal spores, grain, crop residues, animal and insect excreta, soil, insects, foreign material such as stones, metal and broken glass, and other source of contamination (CAC, 2003).

Sieving cleaning
Removing kernels with extensive mold growth, broken kernels, and fine materials such as dirt and debris can be achieved by sieve cleaning, which significantly lowers total mycotoxin contamination. After sieving off corn screenings, it was determined that intact kernels contained about 10 times less fumonisins than broken corn kernels or smaller parts. Removing broken kernels and smaller parts from maize reduced DON and ZEN contamination by around 70-80 % however, up to 69 % of the total maize was rejected as well (Petr Karlovsky, et al., 2016).

Flotation and density segregation
The different physical properties of mold-damaged kernels compared to non-damaged kernels can be exploited to separate them by density segregation or by fractionation on gravity tables. Ergot

Heat treatment
The time/temperature combination undoubtedly remains one of the most important interventions by which industrial processing can affect the mycotoxin content in a finished food product.

Study Area
The study was conducted in Iringa municipal which is one among the five districts of Iringa  Figure 1 below.

Study design and setting.
A cross-sectional study design was carried out at one-time point ohe study was descriptive in form of survey. Usually there is no hypothesis as such, but the aim is to describe a population or subgroup within the population with respect to an outcome and set of risk factors, the purpose of the study was to find prevalence of the outcome of interest, for the population or subgroups within the population at a given time point (Kate Ann Levin, 2006).
A quantitative research approach was used, which emphasized objective measurements and the statistical, mathematical or numerical analysis of data collected through questionnaires and checklist. The approach focused on gathering numerical data and generalizing it across groups of people or to explain a particular phenomenon (Babbie, Earl R., 2010).

Sampling.
Multistage sampling method was used to select participants. Sample frame of 18 wards was used to select 4 wards randomly. From each ward that was selected a sampling frame consisting of all streets was used to select 2 streets from each selected ward, in which a total of 8 streets was obtained.
From the 8 streets selected, a minimum of 25 respondents was obtained from each street by using both quota and convenience sampling techniques. Quota sampling technique was used to select respondents of a sample characteristic (age), in which the study focused on respondents who are 18 years and above while convenience sampling technique was used to obtain readily available respondents, who were found buying, processing (milling and grinding) or selling cereals at that period of time, until a total of 196 respondents was reached.

Data collection.
Objectives were met using two questionnaires and a checklist administered in Iringa Municipality particularly in 4 wards namely Kwakilosa, Mlandege, Kihesa and Ruaha. The first questionnaire assessed general knowledge that respondents have on mycotoxin contamination in cereals and the second questionnaire was used to assess respondents" awareness on food processing methods used to reduce mycotoxin contamination in cereals.
Lastly, a checklist explored storage facilities and conditions used to store cereals, in which the researcher was able to determine if storage conditions used favor mold growth facilitating mycotoxin contamination in cereals.

Data analysis
Data collected from respondents were analysed using Statistical Packages for Social Sciences (SPSS) version 22.0 for quantitative data, which were then presented in graphs, charts and tables.

RESULTS.
Social-demographic characteristics of respondents. Table 1 shows that majority of respondents (76.5%) were males while (23.5%) being female. Iringa one among the region that engage highly in agricultural activities, the occupation distribution showed Majority (29.6%) were owners of cereal processing stations and cereal retailers (25%) with the least (16.3%) and (10.7%) being entrepreneur and unemployed.

KNOWLEDGE ON MYCOTOXIN CONTAMINATION IN CEREALS
The study conducted showed that majority of the respondents that is 171 individuals equivalent to The study (with respect to Table 2) showed that level of education had negative correlation with mycotoxin knowledge where by (r ꞊ -0.161, p< 0.05). Correlation is significant at the 0.05 level (2-tailed).  Table 3, 13 (52%) Respondents out of 25(100%) who had knowledge on mycotoxin contamination, did not understand corresponding factors causing the contamination.

Table 3. Knowledge on factors for mycotoxin contamination in cereals
Majority 13 (52%) of the total respondents with mycotoxin knowledge i.e. 25(100%) in figure   2 understood that blood vomiting is the acute health effects associated with consumption of    Table 4 notes that majority 81% stored their cereals in rooms with improper ventilation while 67.1% stored in rooms with no ceiling where 60.8% did not place their cereal bags away from the walls. The sanitation condition of cereal storage rooms as indicated in table 5 shows that Majority 53.1% do not clean their rooms before storage but 86.7% clean their packaging materials before storing cereals in them while milling machines for those processing cereals majority are not cleaned prior to processing.   Sorting of damaged cereals is one among food processing methods that can be used to reduce the risk of fungi contamination in cereals, Table 7 indicayes Majority 50.5% do not sort cereals while among 49.5% who sort 24.5% uses the damaged cereals as feed.

RECOMMENDATIONS
1. Intervention should be taken to raise awareness regarding mycotoxin contamination in cereals, as majority 87.2% do not have knowledge concerning mycotoxin contamination.
2. Use of pallets to place cereal bags away from the ground, as majority 77.6% do not have pallets. Cereal packaging materials, such as woven sack can be made hermetic by tightly folding and stitching the sack to prevent attraction of fungi and other insects, should be placed under roofs with ceiling to be able to control temperature of the storage room and prevent entry of water during rain.
3. Facilities should be cleaned before storage or usage, as majority 55.2% do not clean their milling machines before processing, leads to feedstuffs building up on the inside walls of machines and facilities, but also packages scattered all over the ground, potentially encouraging fungal growth and cross-contamination as majority 53.1% do not clean their storages. Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.