Aflatoxins are highly toxic and carcinogenic. The disease caused by aflatoxins is known as aflatoxicosis. It mainly targets the liver of humans and animals. Toxicity of aflatoxins depends upon the number of factors such as age, sex, species, and national factors [24]. The aflatoxin metabolic pathways are well studied in animals, but in human, data was very limited. In rats, the DNA/ RNA synthesis was inhibited by ingestion of aflatoxins (5 mg/kg). The B1 aflatoxin binds with the

 guanine by covalent bonding and formed AFB1-N7-guanine adducts. It results in the transversion of G to T which caused the DNA mutations and carcinogenic. In animals or in humans,

**Aflatoxin Molecular formula Molecular weight Melting point UV absorption max (e ), nm, methanol**

the pathway of aflatoxins is described as in **Figure 2**. While in humans (AFB1

AFB1 C17H12O6 312 268–269 12,400 21,800 AFB2 C17H14O6 314 286–289 12,100 24,000 AFG1 C17H12O7 328 244–246 9600 17,700 AFG2 C17H14O7 330 237–240 8200 17,100 AFM1 C17H12O7 328 299 14,150 21,250 (357) AFM2 C17H14O7 330 293 12,100 (264) 22,900 (357) AFd1 C16H14O5 286 250–300 11,200 20,120 AFRO C17H14O6 314 280–290 12,100 (310) 22,900 (320)

may also be formed in some other cases such as in aflatoxins RB1

Two pathways known for the formation of all these aflatoxins are:

.

**2.** Chemical reduction of sodium borhydride

**Table 4.** Chemical characteristic of different aflatoxins.

**Figure 2.** Aflatoxin metabolism in humans.

, and H. These aflatoxins have hydroxyl group in their ring's carbonyl group, D ring

and RB2

**265 360–362**

RB1 , RB2

N7

ring may be formed in B3

158 Aflatoxin-Control, Analysis, Detection and Health Risks

**1.** Microbial transformation

,

while an opened E

The human body could be exposed to mycotoxins through skin contact or by direct inhalation of spore-borne toxins, and these tend to accumulate within the body organs or tissues during their metabolism [24]. Infections caused by fungi such as *A. flavus* are known as *mycosis*, symptoms of high mycotoxin content in body cells is called *mycotoxicosis*, and diseases or symptoms of aflatoxin poison are referred to *aflatoxicosis*. Therefore, there is a high possibility that aflatoxins might play some important roles in mycotoxicosis. Mycotoxins are potent but silent killer endemic in most third world countries due to poor storage systems, leading to obnoxious levels of mycotoxins in food products [38]. These toxic compounds cause induction of jaundice, cancer, immune suppression, premature puberty in girls, reproductive dysfunction, birth defects, obstruction of liver metabolism, and liver cirrhosis by damaging DNA and sulfhydryl bonds in many enzymes [39, 40]; they also take part in gastrointestinal infection, kwashiorkor, Reye's syndrome, and hepatitis. Aflatoxin B1, for example, may cause chromosomal aberration in human. Generally, when aflatoxins B1, G1, and M1 are accumulated in human tissues, it results in epoxide formation at eight and nine positions in the terminal furan ring and then binds covalently with the nucleic acid.

Diagnostic features of mycotoxicosis:

#### **5.2. Aflatoxins and aflatoxicosis**

Many aflatoxins can cause acute mycotoxicosis (i.e., when the symptoms show within a short period after infection say like 7 days, it may lead to death if not treated on time) and/or chronic mycotoxicosis (i.e., when they appear and persist for a long period of time). In 1993 International Agency for Research on Cancer (IARC) has classified four types of naturally occurring aflatoxins AFs (AFB1 , AFB2 , AFG1 , AFG2 ) as the most active substances that cause mutagenicity and carcinogenicity [45]. Aflatoxin AFB1 is the most prevalent among all types of aflatoxins due to long-term chronic exposure to even very small amount of aflatoxin in food items, and it is an important concern for human health [10]. Its chronic exposure can lead to malnutrition, suppressed immune response, centrilobular necrosis, proliferation of bile duct, hepatic lesions and fatty infiltration of liver, and even hepatomas [41, 42] as shown in **Figure 2**.

Symptoms of mycotoxicosis depend on the quantity or concentration of the toxin, time of exposure, type of the mycotoxin involved, degree of toxin combination, host resistant capacity, physiological status of the host, and so on. Mycotoxin can cause heath effect in contact with the skin and alimentary canal, by inhalation or by other means. The toxins can enter into blood streams and the lymphatic system to inhibit the process of protein synthesis, damage macrophage system, or affect the lung's ability to clear particles or cause immune suppression.

### **5.3. Hepatocellular carcinoma (HCC)**

Carcinogenic chemicals like aflatoxin can bring different modifications in DNA sequence or protein structure which causes DNA adduct formation and finally cancer in effected people. AFB1 is a micro-component of nutrition that causes genetic alteration by inducing adduct formation, leading to DNA strand break. This DNA or oxidative damage can turn out to be hepatocellular carcinoma (HCC) or cancer [43–48].

Studies have demonstrated that HCC caused by AFB1 is due to *p53* gene mutation which occurs as a result of transversion at 249 codon (guanine to thymine). This kind of mutation ends up with the arginine to serine substitution that causes 50% HCC due to AFB1 [45, 46]. The liver is the target organ for AFB1 metabolism where mechanism of action starts with food ingestion. In the liver, cytochrome-P450 enzyme initiates metabolism of AFB1 by transformation of AFB1 to nucleophilic, reactive genotoxic intermediate (aflatoxin B1-8, 9-oxide, AFBO) or hydroxylation and demethylation (as shown in **Figure 3**). When AFBO binds to liver cells, it results in DNA adduct formation, termed as 8, 9, dihydro 8, (N7-guanyl) 9-hydroxy-AFB1 . If this phenomenon extends after DNA replication, adduct reacts with *p53* tumor suppressor gene and causes mutation in it, resulting in HCC. Expression of mutated protein (R249Sp53) may lead to inhibition of apoptosis, inhibition of *p53*-mediated transcription, and liver cell growth [45].

some important roles in mycotoxicosis. Mycotoxins are potent but silent killer endemic in most third world countries due to poor storage systems, leading to obnoxious levels of mycotoxins in food products [38]. These toxic compounds cause induction of jaundice, cancer, immune suppression, premature puberty in girls, reproductive dysfunction, birth defects, obstruction of liver metabolism, and liver cirrhosis by damaging DNA and sulfhydryl bonds in many enzymes [39, 40]; they also take part in gastrointestinal infection, kwashiorkor, Reye's syndrome, and hepatitis. Aflatoxin B1, for example, may cause chromosomal aberration in human. Generally, when aflatoxins B1, G1, and M1 are accumulated in human tissues, it results in epoxide formation at eight and nine positions in the terminal furan ring and then binds covalently with the nucleic acid.

Many aflatoxins can cause acute mycotoxicosis (i.e., when the symptoms show within a short period after infection say like 7 days, it may lead to death if not treated on time) and/or chronic mycotoxicosis (i.e., when they appear and persist for a long period of time). In 1993 International Agency for Research on Cancer (IARC) has classified four types of naturally

, AFG2

of aflatoxins due to long-term chronic exposure to even very small amount of aflatoxin in food items, and it is an important concern for human health [10]. Its chronic exposure can lead to malnutrition, suppressed immune response, centrilobular necrosis, proliferation of bile duct, hepatic lesions and fatty infiltration of liver, and even hepatomas [41, 42] as shown in **Figure 2**. Symptoms of mycotoxicosis depend on the quantity or concentration of the toxin, time of exposure, type of the mycotoxin involved, degree of toxin combination, host resistant capacity, physiological status of the host, and so on. Mycotoxin can cause heath effect in contact with the skin and alimentary canal, by inhalation or by other means. The toxins can enter into blood streams and the lymphatic system to inhibit the process of protein synthesis, damage macrophage system, or affect the lung's ability to clear particles or cause immune suppression.

Carcinogenic chemicals like aflatoxin can bring different modifications in DNA sequence or protein structure which causes DNA adduct formation and finally cancer in effected people. AFB1 is a micro-component of nutrition that causes genetic alteration by inducing adduct formation, leading to DNA strand break. This DNA or oxidative damage can turn out to be

) as the most active substances that cause

is the most prevalent among all types

, AFB2

mutagenicity and carcinogenicity [45]. Aflatoxin AFB1

, AFG1

Diagnostic features of mycotoxicosis:

160 Aflatoxin-Control, Analysis, Detection and Health Risks

**3.** Disruption associated with specific foodstuff.

**4.** Drug treatments have little or no effect.

**5.2. Aflatoxins and aflatoxicosis**

occurring aflatoxins AFs (AFB1

**5.3. Hepatocellular carcinoma (HCC)**

hepatocellular carcinoma (HCC) or cancer [43–48].

**1.** Non-transmissible.

**2.** Seasonal outbreak.

**Figure 3.** Biotransformation from AFB1 comprises CYP450-mediated reaction resulting in nucleophilic genotoxic reactive intermediate (AFBO), hydroxylation (to AFM1 and AFQ1 ), or demethylation (to AFP1 ). AFBO binds to the liver cell and causes mutation in *p53* which leads to HCC. AFBO when binds to protein amino acids causes aflatoxicosis.

#### **5.4. Immunosuppressive action of aflatoxins**

Aflatoxins are major factor causing suppression in the immune system which further affects humoral and cellular response. Animal exposure to aflatoxin showed dose-dependent response in percentage of splenic CD8+ T cells, CD3− CD8a+ cells, natural killer (NK) cells [51, 55, 56]. Reduced expression level of cytokine mRNA in the intestine was observed in a study that might be due to reduced percentage of T cells in broiler. This phenomenon directly affects the immune function of intestinal mucosa [49].

## **6. Factors affecting the aflatoxin production in street-vended foods**

Many fungal strains are genetically capable to produce mycotoxins, but they may not do so until certain conditions are met, and it is known that aflatoxin contents in food vary directly with the processing and storage methods and the associated producer microbe. Physical, chemical, and biological factors can affect the formation of aflatoxins in street-vended food including conditions such as temperature, moisture content, the location of vendors, the storage of utensils, personal hygiene, and method of reheating and storage of food. Availability of nutrients to enhance the fungal growth or energy sources such as sugar and vegetable oil will enhance the toxin production [26], for example, there was a higher aflatoxin produced by *A. flavus* on peanut and cottonseed than rice and sorghum; also, it was reported that some fungi like *Alternaria* and *Fusarium* can cause either preharvest or postharvest contamination of grains. In another study, *P. viridicatum* was reported to produce more stable citrinin and ochratoxin on grain compared to meat [50]. The most important climate factors favoring the aflatoxin production are temperature and moisture; optimum temperature for the growth of aflatoxins ranges from 25 to 37°C. *A. flavus* and *A. parasiticus* produced aflatoxin at 12–41°C, 14% moisture content, and humidity >62% followed by rapid drought [52, 53]; example is the 2004 drought-induced aflatoxicosis breakout in Kenya [49, 54].

The location and conditions set for preparing and storing street food may contribute to the production of fungal toxins. Jonathan et al. [31, 55, 56] reported variations in biodeteriorating fungi and aflatoxin contents of particular foodstuff collected from different locations in Nigeria. Most of the vendors are illiterate and do not have knowledge about the sanitary conditions [57]. A case reported in Africa showed that 85% of food stalls were located near garbage dumping sites [58]. Some storage molds such as *Aspergillus* and *penicillium* can survive environments with very low water content. In postharvest storage of food crops, aflatoxin production may be triggered by prolonged drought-associated elevated temperatures [58]. In some other cases, early harvest is also a good strategy to control mold contaminations [59]. Kaaya et al. [60] observed about 4 times increase in aflatoxin levels during the 3rd week and 7 times more in delay harvest after 4 weeks. However, following early harvest, crops have to be well dried to safe fungi contamination. Rachaputi et al. [61] observed least aflatoxin contents in early groundnut harvest and 27% increase in delayed harvest.

Many traditional ways used for food preparation such as heating and roasting can reduce the aflatoxin contents in foods; ranging values in sausage rolls prepared from different locations in Nigeria were observed by Jonathan et al. [55] due to different methods adopted for its preparation; Fandohan et al. [62] determined the fates of aflatoxin and fumonisin in different traditional ways of preparing maize and maize foods based on washing, sorting, winnowing, and hulling combined with crushing of the grains; this is also supported by Park [63] and Lopez-Garcia and Park [64]. The presence of other microorganisms either bacteria or fungi may alter elaboration of mycotoxins on food materials, for example, there was reduced aflatoxin production when *A. parasiticus* was grown in the presence of some bacteria, *Streptococcus lactis* and *Lactobacillus casei* [65]. Meanwhile, fungal metabolites such as rubratoxins from *Penicillium purpurogenum*, cerulenin from *Cephalosporium caeruleus*, and *Acrocylindrium oryzae* enhance aflatoxin production even though they repress growth of aflatoxin-producing fungi [66–69].

Aflatoxin-producing fungi can contaminate food crops during cultivation, harvest, transport, storage, or food preparation [70–72]. Iaha et al. [70] reported that insect damage of maize enhances *Fusarium* mycotoxin through the inoculation of fungal spores into the wounds made on the kernels or stalks during feeding [73]. Higher aflatoxin contents were recorded in maize samples stored in high moisture content up till 10 [74–76]. Most of the people involved in street food vending are lower or middle class people who have more concerns for income or quantity than the quality of their products; many of them lack quality information on how to maintain good hygiene. Most street hawkers always believe in improvised cheap usage of materials during the food processing which may contravene the quality of the food product. However, these food products are sold to many people including the high class group of people. The lack of mycotoxin awareness among the street food vendors is one of the factor increasing mycotoxin incidences in foodstuffs. Many contaminated foods are still sold to avert loss of investment; many of the infected foodstuffs are sold at cheaper amount, and people tend to still buy them especially in countries with high poverty level.

## **7. Economic impact of mycotoxins**

**5.4. Immunosuppressive action of aflatoxins**

the immune function of intestinal mucosa [49].

2004 drought-induced aflatoxicosis breakout in Kenya [49, 54].

in early groundnut harvest and 27% increase in delayed harvest.

response in percentage of splenic CD8+

162 Aflatoxin-Control, Analysis, Detection and Health Risks

Aflatoxins are major factor causing suppression in the immune system which further affects humoral and cellular response. Animal exposure to aflatoxin showed dose-dependent

T cells, CD3−

**6. Factors affecting the aflatoxin production in street-vended foods**

55, 56]. Reduced expression level of cytokine mRNA in the intestine was observed in a study that might be due to reduced percentage of T cells in broiler. This phenomenon directly affects

Many fungal strains are genetically capable to produce mycotoxins, but they may not do so until certain conditions are met, and it is known that aflatoxin contents in food vary directly with the processing and storage methods and the associated producer microbe. Physical, chemical, and biological factors can affect the formation of aflatoxins in street-vended food including conditions such as temperature, moisture content, the location of vendors, the storage of utensils, personal hygiene, and method of reheating and storage of food. Availability of nutrients to enhance the fungal growth or energy sources such as sugar and vegetable oil will enhance the toxin production [26], for example, there was a higher aflatoxin produced by *A. flavus* on peanut and cottonseed than rice and sorghum; also, it was reported that some fungi like *Alternaria* and *Fusarium* can cause either preharvest or postharvest contamination of grains. In another study, *P. viridicatum* was reported to produce more stable citrinin and ochratoxin on grain compared to meat [50]. The most important climate factors favoring the aflatoxin production are temperature and moisture; optimum temperature for the growth of aflatoxins ranges from 25 to 37°C. *A. flavus* and *A. parasiticus* produced aflatoxin at 12–41°C, 14% moisture content, and humidity >62% followed by rapid drought [52, 53]; example is the

The location and conditions set for preparing and storing street food may contribute to the production of fungal toxins. Jonathan et al. [31, 55, 56] reported variations in biodeteriorating fungi and aflatoxin contents of particular foodstuff collected from different locations in Nigeria. Most of the vendors are illiterate and do not have knowledge about the sanitary conditions [57]. A case reported in Africa showed that 85% of food stalls were located near garbage dumping sites [58]. Some storage molds such as *Aspergillus* and *penicillium* can survive environments with very low water content. In postharvest storage of food crops, aflatoxin production may be triggered by prolonged drought-associated elevated temperatures [58]. In some other cases, early harvest is also a good strategy to control mold contaminations [59]. Kaaya et al. [60] observed about 4 times increase in aflatoxin levels during the 3rd week and 7 times more in delay harvest after 4 weeks. However, following early harvest, crops have to be well dried to safe fungi contamination. Rachaputi et al. [61] observed least aflatoxin contents

Many traditional ways used for food preparation such as heating and roasting can reduce the aflatoxin contents in foods; ranging values in sausage rolls prepared from different locations in

CD8a+

cells, natural killer (NK) cells [51,

Effect of aflatoxin on the economy require good imparts assessment model experts and data sets [80]; this may include income losses due to deaths of livestock, weight loss, reductions in productivity, and the yield of eggs, meat, and milk. It affects aves (poultry) such as ducks, chicken, and turkeys; mostly prime example is that of the 1960 outbreak of "Turkey-X disease" in the United Kingdom caused about 100,000 turkey deaths; it may also lead to loss in fishes, birds, rabbits, dogs, and other mammals. The effect of aflatoxin on economy, storage loss, and generally output loss in animal husbandry due to aflatoxin incidence would be threatened here. Aflatoxin lethal dose (LD-50) is generally between 0.5 and 10 mg/kg of livestock body weight; however, high dose of mycotoxin was responsible for, another case with more economic loss may still occur if mycotoxins are not controlled. There is a need for capacity building to enhance the analysis economic impact of mycotoxin and trade analysis.

Mycotoxin contaminations may also affect other sectors of food production and agriculture. Infected commodities can be rejected from shipments, and some prices may become reduced due to loss in quality; this can devastate export markets especially in developing countries. The consumers may be indirectly affected by increased price/costs of food materials as the sellers/producers levied extra cost due to maintenance against contamination or health risks if they are only able to afford contaminated products which are always cheaper. Farmers may suffer reduced income, feed loss, or low outputs due to aflatoxin/mycotoxin interference most especially on the stored products, and economy impact of mycotoxins stems from high mortality, immunity, weight loss, fertility, and quality of dairy products in livestock.

However, we need to checkmate the mycotoxin preventive cost over economic cost; preventive cost in most cases saves many products as the saying "prevention is better than cure" and yields better production and storage practices. Most African countries such as Gambia, Ghana, Mali, Niger, Nigeria, Senegal, as well as Sudan have reported about 7.5 million US\$ cost for mycotoxin control programs due to high-reported mycotoxin incidences on foodstuffs like peanuts and many cereals. In addition, fungi biodeterioration of foodstuffs has caused many countries economic loss due to rejection of exported foodstuffs and affected relationships with trading partners. Implementation of credible food safety controls by exporting countries is needed to foster their smooth exportation of food and agricultural produce. Quality assurance by importing countries has to be routine, and by so doing, the manufactured food will have insignificant mycotoxin levels. The exporting country must be able to comply with this requirement and demonstrate that compliance has been realized. Effectiveness of control measures requires important elements such as administrative structures, resource management, scientific and technical infrastructure, and financing and human capital. Lack of efficient management of resources in many countries has been verified to compromise the credibility of food safety controls. In 2010, foods worth more than US\$200,000 were destroyed by regulatory agencies in Nigeria as a result of contamination by mycotoxins.

Aflatoxin (B1 , B2 , G1 , and G2 ) level in product (ng/g) can give a mark of quality and can be used as a threshold for distinguishing low-, medium-, and high-quality product. Africa could have accrued an estimate of 67 million dollars annually, but this is lost due to export rejection of its food and agricultural produce contaminated with high levels of mycotoxins. Over the years, the European Union Rapid Alert System has notified Nigeria on its alarming export rejects. Contamination by mycotoxins is owed to hot and humid conditions, soil condition, storage method, crop variety, cultural practices, harvesting procedures, etc.

## **8. Prevention of aflatoxin contamination in street-vended foods**

Adequate practices such as pest and disease control management are needed for safe and healthy food production; this must be judiciously implemented to tackle the menace of aflatoxin spoilage of storage and dairy agricultural produce. Early harvest of agricultural produce may avert aflatoxin/mycotoxin contaminations as the fungi that produce these toxins are usually associated with deterioration of the organic compounds. More also, mycotoxin prevention starts right from the cultivation and rearing of livestock. The use of contaminant-free tools, sterile water for washing, and some other hygienic measures needs to be taken seriously either the products are to be consumed or for sale. Some scientists suggest harvesting at dry period, and storage of agricultural produce in dry environment as wet condition improves the growth of molds [77]. Proper cleaning of harvested products, removal of spoilt products (most especially grains), and proper storage methods are good for mycotoxin control. Good transport and storage mechanisms and frequent check for daily temperature and humidity are good for mycotoxin control.

Many scientists have suggested the use of living organisms to nullify or reduce mycotoxin contamination [78]; this is called *biological control measures*. In this method, the mycotoxinproducing fungi or the pest that can act as vector are targeted. The organisms that kill, feed, or attack the toxin production organisms or vector are introduced. In the United States, biological control measures for mycotoxin have been encouraged for various crops like cotton, peanuts, and maize. In Nigeria, the International Institute for Tropical Agriculture (IITA) has reported much success in biocontrol measures on aflatoxins reported as *Aflasave* measures; this has been tried on many crops [79].

Physical methods such as sorting out bad or infected products from healthy ones, hygienic handling, and maintenance of clean environment can reduce aflatoxin/mycotoxin incidence in street-vended foods or industrial food products [67]. A center control point for testing all streetvended food may be set up by the government to reduce mycotoxin incidence. Regulatory measure on mycotoxin should be strengthened in every country. Food management system point will save a lot from food poisoning [80]. Conduction of test at different steps in food production is also important in big food industries; this may be coupled with the use of supplier's schemes. Hazard and analysis critical control point (HACCP) for pre- and postharvest stage plan has save many commodities from contaminants; this has been used on coconuts and corn in South East Asia and on nuts in South and West Africa; it has also been used on apple juice and pistachio nut in South America [79]. Detailed information on HACCP was reported by FAO [79] and suggested the implementation of HACCP for adequate fungal toxin management.

## **9. Regulations on fungal toxin (mycotoxin)**

sellers/producers levied extra cost due to maintenance against contamination or health risks if they are only able to afford contaminated products which are always cheaper. Farmers may suffer reduced income, feed loss, or low outputs due to aflatoxin/mycotoxin interference most especially on the stored products, and economy impact of mycotoxins stems from high mor-

However, we need to checkmate the mycotoxin preventive cost over economic cost; preventive cost in most cases saves many products as the saying "prevention is better than cure" and yields better production and storage practices. Most African countries such as Gambia, Ghana, Mali, Niger, Nigeria, Senegal, as well as Sudan have reported about 7.5 million US\$ cost for mycotoxin control programs due to high-reported mycotoxin incidences on foodstuffs like peanuts and many cereals. In addition, fungi biodeterioration of foodstuffs has caused many countries economic loss due to rejection of exported foodstuffs and affected relationships with trading partners. Implementation of credible food safety controls by exporting countries is needed to foster their smooth exportation of food and agricultural produce. Quality assurance by importing countries has to be routine, and by so doing, the manufactured food will have insignificant mycotoxin levels. The exporting country must be able to comply with this requirement and demonstrate that compliance has been realized. Effectiveness of control measures requires important elements such as administrative structures, resource management, scientific and technical infrastructure, and financing and human capital. Lack of efficient management of resources in many countries has been verified to compromise the credibility of food safety controls. In 2010, foods worth more than US\$200,000 were destroyed

tality, immunity, weight loss, fertility, and quality of dairy products in livestock.

by regulatory agencies in Nigeria as a result of contamination by mycotoxins.

**8. Prevention of aflatoxin contamination in street-vended foods**

method, crop variety, cultural practices, harvesting procedures, etc.

as a threshold for distinguishing low-, medium-, and high-quality product. Africa could have accrued an estimate of 67 million dollars annually, but this is lost due to export rejection of its food and agricultural produce contaminated with high levels of mycotoxins. Over the years, the European Union Rapid Alert System has notified Nigeria on its alarming export rejects. Contamination by mycotoxins is owed to hot and humid conditions, soil condition, storage

Adequate practices such as pest and disease control management are needed for safe and healthy food production; this must be judiciously implemented to tackle the menace of aflatoxin spoilage of storage and dairy agricultural produce. Early harvest of agricultural produce may avert aflatoxin/mycotoxin contaminations as the fungi that produce these toxins are usually associated with deterioration of the organic compounds. More also, mycotoxin prevention starts right from the cultivation and rearing of livestock. The use of contaminant-free tools, sterile water for washing, and some other hygienic measures needs to be taken seriously either the products are to be consumed or for sale. Some scientists suggest harvesting at dry period, and storage of agricultural produce in dry environment as wet condition improves the growth of molds [77]. Proper cleaning of harvested products, removal of spoilt products (most especially grains), and

) level in product (ng/g) can give a mark of quality and can be used

Aflatoxin (B1

, B2 , G1

, and G2

164 Aflatoxin-Control, Analysis, Detection and Health Risks

Legislation and food inspections are very important for food security. For thousands of years back, food has always been subjected to legislation and inspection as the need was felt for some control on the quality of food materials. This control was intended to safeguard the health of consumers and as well prevent cheating in terms of the composition of food; the principle that food contaminated with a hazardous substance is unfit for human consumption and shall not be sold or offered for sale was not always applied as intended. Before, local and municipal affair/ordinances were used for regulated food quality as there were no advanced scientific methods and tools, but today, advances in science of bacteriology, microscopy, and chemistry have aided official legislations on food. Today, we have legislations that prohibit adulterated or misbranded foodstuffs and incidence of contaminants in food particularly incidence of these contaminants above the tolerance limit in either humans or animals.

Mycotoxins as a food toxic substance have recently been considered in food regulation (**Table 5**). It started shortly after the aflatoxin discovery in the 1960s, and some other specific mycotoxins such as ochratoxin, zearalenone, deoxynivalenol, patulin, and phomopsin were later considered. An attempt to study the whole world's legislation on mycotoxins was made by the International Union of Pure and Applied Chemistry (IUPAC) and the Food and Agriculture Organization which led to an updated paper that was published by Schuller et al. [80] in the

**Country Food material Tolerance level (μg/kg) Authority**

Peanuts, peanut products,

Infant foods based on cereals

except milling and shelled

Milling and shelled products and derived products children

foodstuffs for children from 0 to 2 years and for school meals Imported foodstuffs

maize products

166 Aflatoxin-Control, Analysis, Detection and Health Risks

and AH foods

Australia All foods except peanut products

Australia Peanut (products), all foods,

products

foods

Brazil Industrially prepared

China Rice, peanuts, maize, sorghum,

millet)

Czechoslovakia Cereals, grains, peanuts

children foods

beans, wheat barley, oats

Cereals, grains (sorghum,

All foods except infant and

Infant foods on milk basis (calculated on basis of reconstituted product) Other infant foods and children foods

**B1 B2 + G1 + G2**

Agriculture

5 20 Ministry of Public Health, Ministry of Agriculture

2 Ministry of Public Health, Ministry of Agriculture

Proposed

Canada (official)

536, edicion 1981

30 Ministry of Public Health (official)

Ministry of Health (official)

5 10 Ministry of Health (official)

50 Ministry of Public Health, Council

(official)

Research Council, 1982 (recommendation)

Ministry of Public Health

Ministry of Public Health Ministry of Public Health

Ministry of Agriculture (not official)

of Agriculture and Local Authorities

Colombiana de normas technicas "Icontec." Norma Colombiana nr.

3 – National Health and Medical

5 Not official

Argentine Sugar-coated nuts – 5 Ministry of Public Health, Ministry of

15 1

2 0.02

Belgium All foods 5 Ministry of Public Health and

Other foodstuffs 15 30 Proposed

Colombia Sesame seed 20 Publication official del Instituto

Cuba Cereals, grains, peanuts 0 Ministry of Agriculture

0.1 1

Canada Nuts and nut products 15 Health and Welfare

5 3

5 0.02

10

Oil seeds (peanuts) 10 Ministry of Public Health (official)

0.2 2

Denmark Peanuts 10 Ministry of the Environment (official)

**Table 5.** Legal aflatoxin tolerance levels in foodstuffs for humans [80].

**Country Food material Tolerance level (μg/kg) Authority**

Kenya Peanuts and other vegetable oils, peanut products

168 Aflatoxin-Control, Analysis, Detection and Health Risks

Mauritius Groundnuts

The Netherlands Others

New Zealand Peanut butter, shelled nuts

Infant foods

Norway Nuts, buck wheat, other foodstuffs

Philippines Coconut, peanut products (export)

> Infant foods Other foods

Nigeria All foods

Portugal Peanuts

Peanuts and peanut products All foods and food ingredient

and nut portion of products containing nuts, other foods

**B1 B2 + G1 + G2**

Luxembourg Peanuts and peanut products 5 Règlement Grand-Ducal of 22.09.1978

Malawi Peanuts (export) 5 Letter of Malawi Bureau of Standards

Mexico All foods 20 Ministry of Official Public Health,

15 10

Malaysia All foods 35 Food Regulations 1985

5 5

5 5

20 0

Peru Maize and peanuts 5 Code of practice

Poland All foods 0 Ministry of Public Health

25 5 20

Romania All foods 0 Joint papers of veterinary specialists

20 Food, Drugs, and Chemical

Public Health

19.9.1979

BS/1/1 of 24.06.1976

Food and Drug (Control of Aflatoxins) Regulations, 1979. Government 1: Notice No. 222 of

Ministry of Agriculture

Koninkrijk der

and Cultural Affairs

15 Food Regulations 1984, section 257

(official)

(official)

(official)

5 Rundskriv IK-1/85 of 08.02.1985

20 Ministry of Health

Algemeen Besluit (Warenwet) art.3 quinquies. Staatsblad van het

Nederlanden 46, artikel 1, 19.01.1974, Ministry of Welfare, Public Health

Ministry of Welfare, Public Health and Cultural Affairs Official

Food and Drug Administration

Food and Drug Administration

Decreto-Lei no.6/83 nr. Diärio da Républica of 14.01.1983, Ministry of Public Health, Ministry of Agriculture, Ministry of Commerce

and doctors of medicine 1978,

Ministry of Health Ministry of Agriculture

Health

Substances Regulations Kenya Gazette, 01.07.1978, Ministry of

Loi 25.09.1953, art.7f.ll and 12 Benelux Arrêté M(77) 5-03.05.1977 Ministry of

1987 (second) mycotoxin joint International annual Conference of FAO/WHO/UNEP. The mycotoxin legislation has since then grow exponentially in various countries. Many countries are known to enforce or propose certain aflatoxin regulations for foodstuffs to continue to increase, and many countries expanded their regulations to specify more types of foodstuffs. As in 1981, the maximum limits for aflatoxins in food (aflatoxin B1 or the sum of aflatoxins B1, B2, G1, and G2) vary from zero detectable to 50 jtg/kg. The strategies which may be employed to limit the establishment of mycotoxins in food should include both on-field and post-field measures. The regulation of a country is to ensure that any food contaminated in an amount that is intolerable from a public health point of view, particularly, at a toxicological level, is not tradable in that country. Contaminant levels are required to be kept as low as possible by proper measures. Regulations are established in many countries to control food contamination so as to protect human health; these regulations may include specific maximum limits for several contaminants for different foods and a reference to the sampling methods and methods of analysis used [81, 82]. Report by FAO in 2003 shows that about 100 countries have existing regulations on mycotoxins in specific foods and feeds; this was about 30% increase over 1995 report.

However, studies have shown that most African countries have adequate mycotoxin regulations due to the fact that many countries in Africa account for highest countries with massive aflatoxin incidence. Morocco had the most detailed regulations on mycotoxins with about 15 nations out of about 99 countries with known mycotoxin regulations in 1995. Nigeria, for example, adopted its regulatory system from the European commission used primarily on export commodities. However, mycotoxin regulatory status quo in African countries still needs to be improved upon especially for effective implementation.

## **10. Conclusion**

In this chapter, it would be understood how historical events should gear enforcement of regulatory limits for foods produced locally in order to maintain a high standard especially in foreign trading. This chapter elucidated the need to devise principal methods of mycotoxin control in food and human. It would be understood that mycotoxins in food commodities are a result of fungi infection. It would reiterate the need for proper cultural practice, sanitation, and good storage procedures, among others, as possible measures.

## **Author details**

Michael Dare Asemoloye1 \*, Segun Gbolagade Jonathan1 , Rashid Saddaf2 , Zaffar Habiba2 , Edward Ehidiabhen Okoawo1 and Tunde Sheriffdeen Bello1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Mycology/Biotechnology Unit, Department of Botany, University of Ibadan, Ibadan, Nigeria

2 Department of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan

## **References**

1987 (second) mycotoxin joint International annual Conference of FAO/WHO/UNEP. The mycotoxin legislation has since then grow exponentially in various countries. Many countries are known to enforce or propose certain aflatoxin regulations for foodstuffs to continue to increase, and many countries expanded their regulations to specify more types of foodstuffs. As in 1981, the maximum limits for aflatoxins in food (aflatoxin B1 or the sum of aflatoxins B1, B2, G1, and G2) vary from zero detectable to 50 jtg/kg. The strategies which may be employed to limit the establishment of mycotoxins in food should include both on-field and post-field measures. The regulation of a country is to ensure that any food contaminated in an amount that is intolerable from a public health point of view, particularly, at a toxicological level, is not tradable in that country. Contaminant levels are required to be kept as low as possible by proper measures. Regulations are established in many countries to control food contamination so as to protect human health; these regulations may include specific maximum limits for several contaminants for different foods and a reference to the sampling methods and methods of analysis used [81, 82]. Report by FAO in 2003 shows that about 100 countries have existing regulations on mycotoxins in specific foods and feeds; this was about 30% increase over 1995 report.

However, studies have shown that most African countries have adequate mycotoxin regulations due to the fact that many countries in Africa account for highest countries with massive aflatoxin incidence. Morocco had the most detailed regulations on mycotoxins with about 15 nations out of about 99 countries with known mycotoxin regulations in 1995. Nigeria, for example, adopted its regulatory system from the European commission used primarily on export commodities. However, mycotoxin regulatory status quo in African countries still

In this chapter, it would be understood how historical events should gear enforcement of regulatory limits for foods produced locally in order to maintain a high standard especially in foreign trading. This chapter elucidated the need to devise principal methods of mycotoxin control in food and human. It would be understood that mycotoxins in food commodities are a result of fungi infection. It would reiterate the need for proper cultural practice, sanitation,

\*, Segun Gbolagade Jonathan1

and Tunde Sheriffdeen Bello1

1 Mycology/Biotechnology Unit, Department of Botany, University of Ibadan, Ibadan, Nigeria 2 Department of Environmental Sciences, COMSATS Institute of Information Technology,

, Rashid Saddaf2

, Zaffar Habiba2

,

needs to be improved upon especially for effective implementation.

and good storage procedures, among others, as possible measures.

\*Address all correspondence to<EMAIL_ADDRESS>
**10. Conclusion**

170 Aflatoxin-Control, Analysis, Detection and Health Risks

**Author details**

Michael Dare Asemoloye1

Abbottabad, Pakistan

Edward Ehidiabhen Okoawo1

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## **Leaching of Cyanogens and Mycotoxins from Cultivated Cassava into Agricultural Soil: Effects on Groundwater Quality**

Elie F. Itoba-Tombo, Seteno K.O. Ntwampe and John B.N. Mudumbi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68715

#### **Abstract**

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Cyanogens and mycotoxins are vital in protecting flora against predation. Nevertheless, their increased concentrations and by-products in agricultural soil could result in produce contamination and decreased crop yield and soil productivity. When exposed to unsuitable weather conditions, agricultural produce such as cassava is susceptible to bacterial and fungal attack, culminating in spoilage, particularly in arid and semi-arid regions, and contributing to cyanogen and mycotoxins loading of the arable land. The movement of cyanogen including mycotoxins in such soil can result in sub-surface and/ or groundwater contamination, thus deteriorating the soil's environmental health and negatively affecting wildlife and humans. Persistent cyanogen and mycotoxins loading into agricultural soil changes its physico-chemical characteristics and biotic parameters. These contaminants and their biodegradation by-products can be dispersed from soil's surface and sub-surface to groundwater systems by permeation and percolation through the upper soil layer into underground water reservoirs, which can result in their exposure to humans and wildlife. Thus, an assessment and monitoring of cyanogen and mycotoxins loading impacts on arable land and groundwater in communities with minimal resources should be done. Overall, these toxicants impacts on agricultural soil's biotic community, affect soil's aggregates, functionality and lead to the soil's low productivity, cross-contamination of fresh agricultural produce.

**Keywords:** agricultural soil, cassava, cyanogen, groundwater, mycotoxin

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Cyanogens have been widely demonstrated to be an important component within the earth's system. These compounds have been reported to have an influential role in the lives of several organisms on earth [1]. Cyanogens are characterised by the presence of two elements: a carbon: nitrogen functional group held together by a triple bond (─C≡N). The simplest form, which is predominant in the environment, is hydrogen cyanide (HCN), with nitriles and cyanogenic glycosides (CGs) being other forms of these compounds [2–5]. Generally, free cyanide originates from both anthropogenic and natural processes [6]. The anthropogenic sources of cyanide range from effluents discharged from municipal wastewater treatment plants, agricultural run-off, mining activities and electroplating industries [7, 8], including the application of some cyanide containing insecticides in the agricultural industry, which culminates in environmental contamination [9]. Cyanides and CGs have also been generated in plants and agricultural produce such as *Manihot esculenta* (cassava), with the waste generated through processing of such produce contributing to the cyanide load into the environment. During cassava harvesting and processing, plant-borne hydrolases result in CGs' conversion into by-products which are released into the soil, although sometimes this is due to rot produce, a consequence of microbial contamination of the produce and wastewater generated for processing of such produce [10, 11].

As a result of produce-facilitated microbial decay due to the availability of pathogenic organisms in soil where the produce grows, mycotoxins are produced. Mycotoxins are fungal secondary metabolites that also have a negative impact on human and animal wellbeing [12–14]. They co-occur with other bacterial toxins in spoiled agricultural produce such as cassava. Previous studies on mycotoxins revealed that these compounds are hazardous to animals and humans. Generally, it has been reported that CGs as well as mycotoxins occur naturally in flora and organisms (fungi) as a result of biosynthesis, with their prevalence being quantifiable in many agricultural products, such as cassava, apples, spinach, apricots, cherries, peaches, plums, quinces, almonds, sorghum, lima beans, corn, yams, chickpeas, cashews and kirsch [15, 16]. Although some microorganisms and plants synthesise these compounds for their survival when exposed to harsh environmental conditions, their cumulative production can contribute to ecological disturbances. Furthermore, various arthropods and invertebrates were also determined to produce cyanogens as a defence mechanism and for a control of mating behaviour [17, 18], although on a minute scale, with research by Jones [19] indicating that plants including microorganisms are known to be major producers of these compounds owing to their physiology. Thus, the presence and loading of these cyanogens and mycotoxins into terrestrial ecosystems are largely overlooked, although they have some negative effects on the physico-chemical and biological properties of soil, particularly arable land as well as the environment in general [10, 20].

Previous studies have stated that cyanogen and mycotoxin loading in agricultural soil can have a serious impact, disturbing the terrestrial ecosystem functionality [10]. Current evidence suggests that most studies on agricultural produce such as cassava, known for its high cyanogen content, have predominantly focused on the production of the crop for nutritional and industrial purposes, with its effects on soil (including the surrounding environment) overlooked [2, 10] Accordingly, minimal research has been completed on cyanogen and mycotoxin loading, including their behaviour and movement in soil that can culminate in groundwater contamination. This is because a large amount of agricultural produce, such as cassava tubers, perishes prior to harvesting for a variety of reasons. Although free cyanide and mycotoxin toxicity is widely reported, their level of toxicity is also influenced by cumulative exposure and the continuation of their release from produce into the environment. Cyanogen and mycotoxin loads and their movement in soil, including their potential to contaminate groundwater, which is used in impoverished communities where cassava is cultivated mostly as a source of protein and starch, are largely under-reported.

The highlights of this review are:

**1. Introduction**

178 Aflatoxin-Control, Analysis, Detection and Health Risks

processing of such produce [10, 11].

well as the environment in general [10, 20].

Cyanogens have been widely demonstrated to be an important component within the earth's system. These compounds have been reported to have an influential role in the lives of several organisms on earth [1]. Cyanogens are characterised by the presence of two elements: a carbon: nitrogen functional group held together by a triple bond (─C≡N). The simplest form, which is predominant in the environment, is hydrogen cyanide (HCN), with nitriles and cyanogenic glycosides (CGs) being other forms of these compounds [2–5]. Generally, free cyanide originates from both anthropogenic and natural processes [6]. The anthropogenic sources of cyanide range from effluents discharged from municipal wastewater treatment plants, agricultural run-off, mining activities and electroplating industries [7, 8], including the application of some cyanide containing insecticides in the agricultural industry, which culminates in environmental contamination [9]. Cyanides and CGs have also been generated in plants and agricultural produce such as *Manihot esculenta* (cassava), with the waste generated through processing of such produce contributing to the cyanide load into the environment. During cassava harvesting and processing, plant-borne hydrolases result in CGs' conversion into by-products which are released into the soil, although sometimes this is due to rot produce, a consequence of microbial contamination of the produce and wastewater generated for

As a result of produce-facilitated microbial decay due to the availability of pathogenic organisms in soil where the produce grows, mycotoxins are produced. Mycotoxins are fungal secondary metabolites that also have a negative impact on human and animal wellbeing [12–14]. They co-occur with other bacterial toxins in spoiled agricultural produce such as cassava. Previous studies on mycotoxins revealed that these compounds are hazardous to animals and humans. Generally, it has been reported that CGs as well as mycotoxins occur naturally in flora and organisms (fungi) as a result of biosynthesis, with their prevalence being quantifiable in many agricultural products, such as cassava, apples, spinach, apricots, cherries, peaches, plums, quinces, almonds, sorghum, lima beans, corn, yams, chickpeas, cashews and kirsch [15, 16]. Although some microorganisms and plants synthesise these compounds for their survival when exposed to harsh environmental conditions, their cumulative production can contribute to ecological disturbances. Furthermore, various arthropods and invertebrates were also determined to produce cyanogens as a defence mechanism and for a control of mating behaviour [17, 18], although on a minute scale, with research by Jones [19] indicating that plants including microorganisms are known to be major producers of these compounds owing to their physiology. Thus, the presence and loading of these cyanogens and mycotoxins into terrestrial ecosystems are largely overlooked, although they have some negative effects on the physico-chemical and biological properties of soil, particularly arable land as

Previous studies have stated that cyanogen and mycotoxin loading in agricultural soil can have a serious impact, disturbing the terrestrial ecosystem functionality [10]. Current evidence suggests that most studies on agricultural produce such as cassava, known for its high cyanogen content, have predominantly focused on the production of the crop for nutritional and

## **2. Cyanogen and mycotoxin reduction**

Several methods of cyanogen reduction have been proposed and include physical, chemical and biological methods [6, 21]. However, it has been reported that some of these methods require high input costs and sophisticated knowledge and/or training to implement successful strategies for their reduction [4]. Meanwhile, scientists have embarked on intense research and simplify reduction methods for these toxicants in the environment by using techniques which are considered environmentally benign, as such novel ways of reducing both cyanogen and mycotoxin levels in the environment, including in agricultural produce destined for consumption, are generally considered cost effective when compared with long-term outcomes of none implementation of control measures [22–24].

### **2.1. Biological reduction of cyanogens**

The biological reduction of CGs as a source of cyanide, as well as mycotoxins, has gained popularity and has been a huge research focus area [17, 22, 23]. As such, genetically modified cassava cultivars, with a suppressed cytochrome P450 gene (producers of enzymes CYP79D1 and CYP79D2) functionality, may inhibit the infiltration of linamarin as it can be converted to free cyanide from valine [25].

Furthermore, other biological treatments for free cyanide involve microorganisms; these organisms are known to be toxin producers and are organisms, such as *Pseudomonas* sp., *Nocardia* sp., *Flavobacterium* sp., *Bdellovibrio* sp., as well as nitrifiers, such as *Nitrosomonas* sp., *Nitrobacter* sp., *Sphingomonas* sp., *Exophiala* sp., *Bacillus* sp., and fungi such as *Aspergillus* sp. and *Penicillium* sp. [4, 8, 22, 26–28]. Among these microorganisms, *Aspergillus* sp. and *Penicillium* sp. are the most prevalent species able to grow successfully in stringent weather conditions, with some, including *Cunninghamella* sp. being common in soil [29], with the ability to grow on a variety of agricultural produce such as maize, peanuts and tubers [30, 31].

In soil consisting of fungal biocatalysts of different origins, scientific evidence seems to indicate that agricultural produce appears to be susceptible to spoilage due to substrate availability, which results in the proliferation of microbial spoilage organisms [32, 33]. It has also been reported that fruit or produce has trace elements, such as Ca, Na, K and Zn, and low relative molecular weight hydrocarbons, including proteins and moisture, providing conditions which facilitate microbial growth and thus spoilage [34, 35]. Owing to this, some microorganisms produce hydrolases, reducing primary compounds in produce to by-products, furthering physico-chemical changes in the environment in which they are leached [30]. These seem to be the ideal conditions in which cyanide reduction biocatalysts proliferate, i.e. conditions that are nutrient rich as a result of nutrient availability from decaying produce.

Some of the cyanogens are reduced to by-products such as bicarbonate and ammonia. The ammonia formed during the process is further utilised by the microorganisms as a source of nitrogen, supporting increased microbial growth [36, 37]. In the agricultural industry, the reduction of both cyanogens and related compounds is complex, as *in-situ* quantification of such processes is minimally reported. The development of processes and strategies that are environmentally benign; i.e. of biological origin, is gaining popularity due to their simplicity and advantages, as they are considered less harmful, and can be beneficial in the economical management urged for, in the improvement of commercial agro-produce manufacturers [28, 38, 39]. Owing to the exposure to primary and by-products of cyanogen conversion/transformation, some species became tolerant, thus biologically evolve.

For example, Sing et al. [30] successfully isolated a fungus*, Cunninghamella* sp. UMAS SD12 from sawdust, with an ability to biodegrade 51.7% pentachlorophenol (PCP) within 15 days in a controlled static environment. However, more research needs to be conducted to assess direct evolvement of the microbial ecosystem, as other microorganisms that constitute a community, for the betterment of soil, can reduce such soils' viability, and/or result in some organisms producing extracellular secondary metabolites such as mycotoxins.

### **2.2. Biological reduction of mycotoxins**

There are numerous mycotoxins known to contaminate agricultural produce such as cassava. Among these mycotoxins, fumonisin B1 and deoxynivalenol (DON) are common. The biodegradation of fumonisin B1 and deoxynivalenol (DON) can be achieved through their direct conversion using detoxification processes with different pathways [22]. For example, fumonisin biodegradation was observed through the elimination of the tricarballylate side chains and amino groups. The enzymatic hydrolysis of such mycotoxins might involve carboxylesterases and aminotransferases from bacteria such as *Sphingomonas* and *Sphingopyxis*

normally found in soil, which have the ability to detoxify recalcitrant persistent organic pollutants (PoPs) such as polycyclic aromatic hydrocarbons (PAHs) [40–43]. Other researchers have reported degradation or detoxification of fumonisin, including by-products, by oxidative deaminase from *Exophiala* sp., a common soil organism [42–44]. *Bacillus* sp., including non-Saccharomyces yeast commonly found in soil, were also suggested to destabilise these mycotoxins' structure, and thus reduce their amino acid functional groups albeit at elevated pH [45].

In most instances, the biodegradation process of most mycotoxins involves a consortium of organisms, which utilises a variety of degradation pathways [42, 44]. Overall, the initial biodegradation stage starts at extracellular level by deamination or facilitation by esterase with the last biodegradation step involving microbial/enzymatic decoupling of the aliphatic chain within the mycotoxin molecule [22]. For example, the first biodegradation steps of DON using *Curtobacterium* sp. and *Eubacterium* sp. were determined to be initiated by the de-epoxidation step which subsequently followed oxidation [22, 46].

## **3. Toxicity of cyanide as a cyanogen from cassava**

### **3.1. Toxicity of** *Manihot esculenta*

*Nocardia* sp., *Flavobacterium* sp., *Bdellovibrio* sp., as well as nitrifiers, such as *Nitrosomonas* sp., *Nitrobacter* sp., *Sphingomonas* sp., *Exophiala* sp., *Bacillus* sp., and fungi such as *Aspergillus* sp. and *Penicillium* sp. [4, 8, 22, 26–28]. Among these microorganisms, *Aspergillus* sp. and *Penicillium* sp. are the most prevalent species able to grow successfully in stringent weather conditions, with some, including *Cunninghamella* sp. being common in soil [29], with the ability to grow on a variety of agricultural produce such as maize, peanuts and tubers [30, 31].

In soil consisting of fungal biocatalysts of different origins, scientific evidence seems to indicate that agricultural produce appears to be susceptible to spoilage due to substrate availability, which results in the proliferation of microbial spoilage organisms [32, 33]. It has also been reported that fruit or produce has trace elements, such as Ca, Na, K and Zn, and low relative molecular weight hydrocarbons, including proteins and moisture, providing conditions which facilitate microbial growth and thus spoilage [34, 35]. Owing to this, some microorganisms produce hydrolases, reducing primary compounds in produce to by-products, furthering physico-chemical changes in the environment in which they are leached [30]. These seem to be the ideal conditions in which cyanide reduction biocatalysts proliferate, i.e. conditions that are nutrient rich as a result of nutri-

Some of the cyanogens are reduced to by-products such as bicarbonate and ammonia. The ammonia formed during the process is further utilised by the microorganisms as a source of nitrogen, supporting increased microbial growth [36, 37]. In the agricultural industry, the reduction of both cyanogens and related compounds is complex, as *in-situ* quantification of such processes is minimally reported. The development of processes and strategies that are environmentally benign; i.e. of biological origin, is gaining popularity due to their simplicity and advantages, as they are considered less harmful, and can be beneficial in the economical management urged for, in the improvement of commercial agro-produce manufacturers [28, 38, 39]. Owing to the exposure to primary and by-products of cyanogen conversion/transformation, some species

For example, Sing et al. [30] successfully isolated a fungus*, Cunninghamella* sp. UMAS SD12 from sawdust, with an ability to biodegrade 51.7% pentachlorophenol (PCP) within 15 days in a controlled static environment. However, more research needs to be conducted to assess direct evolvement of the microbial ecosystem, as other microorganisms that constitute a community, for the betterment of soil, can reduce such soils' viability, and/or result in some

There are numerous mycotoxins known to contaminate agricultural produce such as cassava. Among these mycotoxins, fumonisin B1 and deoxynivalenol (DON) are common. The biodegradation of fumonisin B1 and deoxynivalenol (DON) can be achieved through their direct conversion using detoxification processes with different pathways [22]. For example, fumonisin biodegradation was observed through the elimination of the tricarballylate side chains and amino groups. The enzymatic hydrolysis of such mycotoxins might involve carboxylesterases and aminotransferases from bacteria such as *Sphingomonas* and *Sphingopyxis*

organisms producing extracellular secondary metabolites such as mycotoxins.

ent availability from decaying produce.

180 Aflatoxin-Control, Analysis, Detection and Health Risks

became tolerant, thus biologically evolve.

**2.2. Biological reduction of mycotoxins**

Worldwide, cassava is utilised as a primary foodstuff for disadvantaged and needy rural communities of Africa, Asia and South America [23, 47, 48]. Cassava's toxicity is due to cyanogens such as linamarin, lotaustralin and 2-((6-O-(b-d-apiofuranosyl)-b-d-glucopyranosyl) oxy)-2-methylbutanenitrile) that are biologically transformed into hydrogen cyanide [25, 49]. As a result of enzymatic hydrolysis, for which the linamarin from the plant tissue is transformed into acetone cyanohydrin through linamarases [3]. At an increased temperature and pH of >30°C and 5, respectively, conditions associated with arid regions which are suitable for microbial proliferation and thus agricultural produce contamination or spoilage, acetone cyanohydrin is released, resulting in its decomposition into acetone and hydrogen cyanide [3, 25] (**Figure 1**). Several studies have been done on the impact of the cultivar on humans as a result of direct ingestion [24, 48, 50], as cyanide concentration in the tuber is estimated to reach 50 mg/kg [51].

**Figure 1.** Enzymatic hydrolysis of linamarin to hydrogen cyanide.

Thus, its prolonged consumption may be toxic. However, there is minimal information on hydrogen cyanide loading into irrigable land in which cassava is cultivated. Free metal ions in such soil exposed to hydrogen cyanide can form metallic cyanide complexes under suitable conditions, further prolonging cyanide-based compounds' prevalence in the soil, which might leach into groundwater.

#### **3.2. Production of mycotoxins**

Terrestrial ecosystems are populated by a diversity of microorganisms that contribute to and maintain the ecological and biological balance. These organisms contribute to the characteristics of the soil that directly influence soil productivity and crop yield in the agricultural sector [52–54], although some have been shown to exhibit pathogenicity toward mature produce. For example, during the growth and up to the harvest stage of cassava tubers, several pathogenic organisms with mycotoxin-production potential can dominate several other types of bacteria and fungi on the tuber and in cassava-cultivated soils [30]. Some of these organisms are resistant even to the free cyanide in cassava, and with their inherent characteristics, such as their predisposition for survival, they produce mycotoxins such as ochratoxin A, aflatoxins, fumonisin B, pyranonigrin A, tensidol B, funalenone, naphtho-y-pyrones, deoxynivalenol (DON) and malformins [55–57]. Research revealed that exposure to mycotoxins pre/post-harvest and their presence in soil can render the cassava tubers inedible [58, 59], leading to their cumulative and increased levels due to sustained use of pre-recovery land for cultivation to produce an essential food source—a method that will affect the soil's ecology.

### **3.3. Mycotoxins' effects on soil ecology**

Soil ecology is influenced by the biochemical including biotic relationships and physical conditions paramount for its good health [52, 54]. The biochemical aspect of soil used for cultivation is related to its microbial diversity as well as its chemical/pollutant content [53], with the soil's microbial community playing a transformative role with regard to the soil nutrient availability, health and fertility, which enhance the soil's quality, including its productivity [52]. The microbial ecology of any soil facilitates nutrient flow through immobilisation processes, which may result in its bioaugmentation [54, 60, 61], contributing to suitable soil structure that assists in the formation of nutrient-rich aggregates. According to Knudsen [53], soil aggregates are created by microbial activity, albeit at a microscopic level, linking soil particles, while the external polysaccharide tissues of bacterial cells play a role in holding soil aggregates together [52], with subsequent structuring and compaction, parameters influencing the quality of the soil's texture, porosity, aeration, moisture permeability, water circulation and organic matter content [52]. Soil grain cohesion, porosity, permeability and organic matter content are vital for soil quality and fertility, particularly for soil demarcated for sustaining the production of agricultural produce.

All these parameters are indispensable for sustainable use of arable soil for food production and productivity for crop yield [52, 53]. Additionally, soil health can also be affected by surface, subsurface and groundwater supply, including quality.

Soil moisture content is vital for soil functionality as it serves as a water reservoir for the terrestrial ecosystem, playing a major role in the water cycle between surface and subsurface water, thus affecting the quality of groundwater [62, 63]. High mycotoxin loading into the soil may impact its functionality. Thus, the interaction of microorganisms, invertebrates, vertebrates, and planted crops, which eventually leads to the depletion of groundwater quality, leads to sustained leaching or periodic contamination of the water, which can easily lead to human exposure. The disturbance in a terrestrial hydrological movement may have longterm disastrous consequences for surface, subsurface and groundwater supplies [62, 64].

Thus, its prolonged consumption may be toxic. However, there is minimal information on hydrogen cyanide loading into irrigable land in which cassava is cultivated. Free metal ions in such soil exposed to hydrogen cyanide can form metallic cyanide complexes under suitable conditions, further prolonging cyanide-based compounds' prevalence in the soil, which

Terrestrial ecosystems are populated by a diversity of microorganisms that contribute to and maintain the ecological and biological balance. These organisms contribute to the characteristics of the soil that directly influence soil productivity and crop yield in the agricultural sector [52–54], although some have been shown to exhibit pathogenicity toward mature produce. For example, during the growth and up to the harvest stage of cassava tubers, several pathogenic organisms with mycotoxin-production potential can dominate several other types of bacteria and fungi on the tuber and in cassava-cultivated soils [30]. Some of these organisms are resistant even to the free cyanide in cassava, and with their inherent characteristics, such as their predisposition for survival, they produce mycotoxins such as ochratoxin A, aflatoxins, fumonisin B, pyranonigrin A, tensidol B, funalenone, naphtho-y-pyrones, deoxynivalenol (DON) and malformins [55–57]. Research revealed that exposure to mycotoxins pre/post-harvest and their presence in soil can render the cassava tubers inedible [58, 59], leading to their cumulative and increased levels due to sustained use of pre-recovery land for cultivation to

produce an essential food source—a method that will affect the soil's ecology.

Soil ecology is influenced by the biochemical including biotic relationships and physical conditions paramount for its good health [52, 54]. The biochemical aspect of soil used for cultivation is related to its microbial diversity as well as its chemical/pollutant content [53], with the soil's microbial community playing a transformative role with regard to the soil nutrient availability, health and fertility, which enhance the soil's quality, including its productivity [52]. The microbial ecology of any soil facilitates nutrient flow through immobilisation processes, which may result in its bioaugmentation [54, 60, 61], contributing to suitable soil structure that assists in the formation of nutrient-rich aggregates. According to Knudsen [53], soil aggregates are created by microbial activity, albeit at a microscopic level, linking soil particles, while the external polysaccharide tissues of bacterial cells play a role in holding soil aggregates together [52], with subsequent structuring and compaction, parameters influencing the quality of the soil's texture, porosity, aeration, moisture permeability, water circulation and organic matter content [52]. Soil grain cohesion, porosity, permeability and organic matter content are vital for soil quality and fertility, particularly for soil demarcated for sus-

All these parameters are indispensable for sustainable use of arable soil for food production and productivity for crop yield [52, 53]. Additionally, soil health can also be affected by sur-

might leach into groundwater.

182 Aflatoxin-Control, Analysis, Detection and Health Risks

**3.2. Production of mycotoxins**

**3.3. Mycotoxins' effects on soil ecology**

taining the production of agricultural produce.

face, subsurface and groundwater supply, including quality.

Previous studies on mycotoxin mobility in soil revealed that the movement of these contaminants is influenced by processes such as deposition, decomposition, distribution and accumulation [2, 65], while the compounds' concentration increases with depth. A soil with a high moisture content creates conditions that lead to the furtherance of the contaminants' ability to be transferred, based on processes such as infiltration, percolation and leaching into groundwater [62, 63]. The detoxification bioprocesses and strategies may involve extended periods during which the land is unusable. Furthermore, several studies on the effects of cassava effluents on soil, including microbiota, stated that a high mycotoxin concentration in soil is harmful to these soil microorganisms. Some of these mycotoxins are produced because of inhibitive competition, i.e. organisms will produce these mycotoxins to limit the proliferation of others, particularly under nutrient-depleted conditions.

A study by Knudsen [53] revealed that mycotoxins from cassava are mobile in soil and destroy the resident soil's organisms. Additionally, Okechi et al. [10] showed that the effects of cassava effluents on soil microbial populations revealed a discrepancy in bacterial and fungal populations at different pH levels and soil depths. This indicates that the bacterial populations from the upper layers of soil counts revealed an increase in comparison with those recorded in the lower soil layers, with high concentrations of the mycotoxins observed on the lower soil layers, a process furthered by leaching. Similar total fungal population counts revealed a similar phenomenon with surface, subsurface and deeper soil layers.

## **3.4. Impacts of hydrovgen cyanide on biochemical and physical properties of agricultural soil**

Although the conditions and diversity of habitats contribute to and thus influence the biochemical and physical properties of arable soil [62, 64], a high cyanogen load in soil can have a negative impact on soil microbial populations, with sustained exposure and an increased concentration of cyanogens hindering the microbial activity, and thus the functionality of soil microorganisms, leading to the deformation of the biochemical and physical properties of the soil. A high hydrogen cyanide concentration load in such soil was determined to contribute to an increase in the total oxygen carbon (TOC) and chemical oxygen demand (COD), reducing the ability of *Nitrobacter* sp. to sustain nitration processes [29, 64]. Therefore, an increase in the hydrogen cyanide loading could lead to an imbalance between *Nitrospira* and *Nitrobacter* sp., resulting in a higher count of species with a hydrogen cyanide-resistant ability. The change in the microbial population balance could lead to stunted growth and/ or variations in the growth of a cultivar. This can easily culminate in the dominance of the species, which can be a spoilage organism with free cyanide-resistant characteristics contributing to spoilage patterns/microbial contamination of the produce of interest.

For mitigation strategies in the post-harvesting period, preparation of soil for re-cultivation could lead to inadequate organic matter (OM), variation in total nitrogen (TN) content and availability, which could interfere with soil biochemical and physical properties [64]. Research on the physico-chemical characteristics of cassava-cultivated soil has shown a correlation between continuous cassava cultivation and a decline in the soil's physico-chemical properties (Haplic Acrisols) [64]. Therefore, continuous cultivation of cassava, which normally happens in impoverished communities, could result in a decrease in soil quality, bulk density, organic carbon (OC), OM, trace elements, moisture, infiltration rate, including holding capacity, and aggregate stability. Howeler [66] further reported that the average nutrient removal rate per ton of cassava tuber harvested is equivalent to: N=2.53 (38%), P=0.37 (49%), K=2.75 (56%), Ca=0.44 (16%) and Mg=0.26 (30%). Thus, cyanogen loading indirectly has an impact on C:N ratio, which will result in a pH increase with depth, while OC, nitrogen (N) and OM distortions will be entrenched.

Similarly, Boadi et al. [67] examined the relationship between cyanogen concentration, pH and soil moisture, determining that with an increase in cyanogen values, soil pH increases with moisture content, further supporting the retention of cyanogens at a lower pH. The concentration of cyanogenic compounds was shown to be varied from soil to groundwater and from one site to another [61–63, 68], which suggested that the discrepancies in distribution could be due to the mobility of the contaminant [67].

### **3.5. Behaviour of cyanogen and mycotoxin in soil**

Cyanogen and mycotoxin behaviour in soil, groundwater and the environment is largely controlled by a multitude of chemical reactions and processes. There are similarities and differences between the processes involved for the behaviour of each contaminant, which is largely controlled by conditions the contaminant undergoes when in soil and groundwater. These processes are primarily influenced by numerous biochemical processes and by the compounds' structures, properties and behaviour in the environment. According to Kjeldsen [65], the behaviour of cyanogen and mycotoxins from soil into groundwater is largely influenced by processes such as deposition, dissolution, infiltration, leaching, degradation, transformation and complexation (see **Figure 2**).

Furthermore, human, wildlife exposure and environmental contamination are directly associated with other pathways, such as volatilisation, dermal contact and ingestion of other degradation by-products from the transformation of the primary source due to the transportation pathways facilitated by leaching mechanisms into groundwater [62, 65]. Thus, when not monitored, the environmental prevalence and exposure of these contaminants can be harmful to human health/ wildlife. For example, the concentration of leached iron-cyanide complexes in groundwater ranged between 2 and 12 mg/L [59, 69]. The prevalence of such complexes is influenced by the reactivity of free metal ions and free hydrogen cyanide from cassava. These compounds may be transformed (through decomposition) to free cyanide at a later stage, although most are stable with longer half-life, thus they enter an aquifer through processes such as infiltration and leaching.

Leaching of Cyanogens and Mycotoxins from Cultivated Cassava into Agricultural Soil: Effects on Groundwater Quality http://dx.doi.org/10.5772/intechopen.68715 185

species, which can be a spoilage organism with free cyanide-resistant characteristics contrib-

For mitigation strategies in the post-harvesting period, preparation of soil for re-cultivation could lead to inadequate organic matter (OM), variation in total nitrogen (TN) content and availability, which could interfere with soil biochemical and physical properties [64]. Research on the physico-chemical characteristics of cassava-cultivated soil has shown a correlation between continuous cassava cultivation and a decline in the soil's physico-chemical properties (Haplic Acrisols) [64]. Therefore, continuous cultivation of cassava, which normally happens in impoverished communities, could result in a decrease in soil quality, bulk density, organic carbon (OC), OM, trace elements, moisture, infiltration rate, including holding capacity, and aggregate stability. Howeler [66] further reported that the average nutrient removal rate per ton of cassava tuber harvested is equivalent to: N=2.53 (38%), P=0.37 (49%), K=2.75 (56%), Ca=0.44 (16%) and Mg=0.26 (30%). Thus, cyanogen loading indirectly has an impact on C:N ratio, which will result in a pH increase with depth, while OC, nitrogen (N) and OM distor-

Similarly, Boadi et al. [67] examined the relationship between cyanogen concentration, pH and soil moisture, determining that with an increase in cyanogen values, soil pH increases with moisture content, further supporting the retention of cyanogens at a lower pH. The concentration of cyanogenic compounds was shown to be varied from soil to groundwater and from one site to another [61–63, 68], which suggested that the discrepancies in distribution could be due

Cyanogen and mycotoxin behaviour in soil, groundwater and the environment is largely controlled by a multitude of chemical reactions and processes. There are similarities and differences between the processes involved for the behaviour of each contaminant, which is largely controlled by conditions the contaminant undergoes when in soil and groundwater. These processes are primarily influenced by numerous biochemical processes and by the compounds' structures, properties and behaviour in the environment. According to Kjeldsen [65], the behaviour of cyanogen and mycotoxins from soil into groundwater is largely influenced by processes such as deposition, dissolution, infiltration, leaching, degradation, transforma-

Furthermore, human, wildlife exposure and environmental contamination are directly associated with other pathways, such as volatilisation, dermal contact and ingestion of other degradation by-products from the transformation of the primary source due to the transportation pathways facilitated by leaching mechanisms into groundwater [62, 65]. Thus, when not monitored, the environmental prevalence and exposure of these contaminants can be harmful to human health/ wildlife. For example, the concentration of leached iron-cyanide complexes in groundwater ranged between 2 and 12 mg/L [59, 69]. The prevalence of such complexes is influenced by the reactivity of free metal ions and free hydrogen cyanide from cassava. These compounds may be transformed (through decomposition) to free cyanide at a later stage, although most are stable with longer half-life, thus they enter an aquifer through processes such as infiltration and

uting to spoilage patterns/microbial contamination of the produce of interest.

tions will be entrenched.

to the mobility of the contaminant [67].

184 Aflatoxin-Control, Analysis, Detection and Health Risks

tion and complexation (see **Figure 2**).

leaching.

**3.5. Behaviour of cyanogen and mycotoxin in soil**

**Figure 2.** Cassava cyanogen and mycotoxin movement in agricultural soil. Key: NO3 − (nitrate), NO2 − (nitrite), Fe(II)(CN)63− (ferrocyanide), Fe(III)(CN)64− (ferricyanide), SCN (thiocyanide), NH4 -N (ammonium nitrogen), CN- (cyanide ion), -OCN (cyanate), N2 O (nitrous oxide) and Ny Ox (nitrogen oxides).

It is also important to point out that the behaviour of contaminant movement in terrestrial or/ and aquatic ecosystems and the environment in general is also influenced by parameters such as wash-off, periodic moisture saturation and time. Based on the stability of each individual contaminant, including its by-products, the mobility can also be spontaneously influenced by the rate of conversion, thus degradation, and can even become volatilised under suitable conditions [65, 70], depending on vapour pressure. Time or length of exposure is a very important aspect, particularly where human exposure is assessed, which is generally neglected or unclear in many recent studies. Similarly, contamination gradients must be established because of groundwater variations in the water flow, as well as the influence of the insolation surrounding the water body that might contribute to acute exposure levels. Furthermore, from produce itself, volatilised compounds can undergo photodecomposition due to UV effects contributing to pseudohalogen accumulation in the troposphere/stratosphere.

#### **3.6. Cyanogen and mycotoxin effects on humans and animals**

The focus of this review is that cassava can be toxic when consumed in large quantities owing to its cyanogen content [49]. The prolonged consumption of cassava in different forms can be harmful for humans in particular, owing to inadequacies in post-harvest treatment techniques [8, 21]. For instance, studies on cassava-cyanide effects in humans revealed that a permanent consumption of low-level concentrations of cyanide from poorly processed cassava could result in goitres and Tropical Ataxic Neuropathy (TAN) [24, 59], whereas a high consumption of the produce could result in neurological disorders, such as konzo [23, 50]. Most post-harvest cyanogen removal techniques focus on free-cyanide removal techniques, without accounting for transformed varieties of the cyanogens (see **Figure 2**), such as thiocyanate, etc.

A team of researchers conducting studies on the thiocyanate concentration in urine samples of pupils, who consumed cassava in Mozambique, revealed that a mean concentration of urinary thiocyanate in school children ranged from 225 to 384 mol/L, whereas mean total cyanogen concentrations in processed cassava flour varied with seasons and years from 26 to 186 mg/L [71]. Similarly, a study by Shifrin et al. [59] revealed that mycotoxin can easily be absorbed through dermal contact, ingestion and inhalation. Some mycotoxins are hazardous and are proposed to be carcinogens facilitating mutation in human cells—an effect that can be postulated to suggest their facilitation of cell mutation in humans.

In animals, on the other hand, an increased consumption of tuber debris and waste by-products of produce processing could lead to neuronal disturbances, weight loss and dysfunctional thyroid [23, 50, 72]. Observations reported by Wade et al. [73] on cassava waste in fish, i.e., in the Nile tilapia (*Oreochromis niloticus*), revealed that some cyanogens caused oedema, gill lamellae telangiectasia, gill enlargement, formation of vacuoles and liver cell deterioration. Similar health outcomes for humans and animals observed in acute mycotoxin exposure, including ingestion, were inter alia, weight loss, internal organ bleeding, respiratory diseases (asthma, pneumonia), diarrhoea, liver and kidney cancer and skin irritation [74–76]. Therefore, a large-scale propagation of agricultural produce with cyanogens, which is susceptible to a high concentration of spoilage organisms, particularly mycotoxin producers, requires continuous monitoring to ascertain its quality. Such produce should be free of both cyanogen and mycotoxins, primarily if it is destined for human and animal consumption and/or exposure. In this case, required strategies for the reduction of exposure must be implemented.

## **4. Conclusion**

Cassava, in general, and cassava tubers, in particular, are indispensable for daily self-nourishment of several poor communities worldwide owing to their nutritional value. However, when exposed to environmental processes and bacterial and fungal attacks that can occur prior to harvesting, the produce is susceptible to release cyanogen and mycotoxin compounds that are hazardous to humans, animals and the environment. These contaminants and their by-product mobility into the terrestrial ecosystem are similar and are facilitated by environmental processes such as transformation, complexation, percolation and volatilisation as they can travel from surface and subsurface to groundwater level, which can result in exposure to both animals and humans. The presence of these compounds in arable land can lead to their accumulation, which can negatively affect soil properties, groundwater quality and the environment, thus contributing to a decline in the production of useful produce, such as cassava. Monitoring, particularly in communities that use such arable soil on a continuous basis, can mitigate intoxication of humans and animals, by effectively implementing suitable reduction strategies thus prevent environmental pollution. Therefore, continuous monitoring, quality assurance and a novel *in-situ* biological method (for treatment of the contaminants) are paramount to ensure a healthier agricultural soil, clean surface and groundwater quality.

## **Acknowledgements**

of the produce could result in neurological disorders, such as konzo [23, 50]. Most post-harvest cyanogen removal techniques focus on free-cyanide removal techniques, without accounting

A team of researchers conducting studies on the thiocyanate concentration in urine samples of pupils, who consumed cassava in Mozambique, revealed that a mean concentration of urinary thiocyanate in school children ranged from 225 to 384 mol/L, whereas mean total cyanogen concentrations in processed cassava flour varied with seasons and years from 26 to 186 mg/L [71]. Similarly, a study by Shifrin et al. [59] revealed that mycotoxin can easily be absorbed through dermal contact, ingestion and inhalation. Some mycotoxins are hazardous and are proposed to be carcinogens facilitating mutation in human cells—an effect that can be

In animals, on the other hand, an increased consumption of tuber debris and waste by-products of produce processing could lead to neuronal disturbances, weight loss and dysfunctional thyroid [23, 50, 72]. Observations reported by Wade et al. [73] on cassava waste in fish, i.e., in the Nile tilapia (*Oreochromis niloticus*), revealed that some cyanogens caused oedema, gill lamellae telangiectasia, gill enlargement, formation of vacuoles and liver cell deterioration. Similar health outcomes for humans and animals observed in acute mycotoxin exposure, including ingestion, were inter alia, weight loss, internal organ bleeding, respiratory diseases (asthma, pneumonia), diarrhoea, liver and kidney cancer and skin irritation [74–76]. Therefore, a large-scale propagation of agricultural produce with cyanogens, which is susceptible to a high concentration of spoilage organisms, particularly mycotoxin producers, requires continuous monitoring to ascertain its quality. Such produce should be free of both cyanogen and mycotoxins, primarily if it is destined for human and animal consumption and/or exposure.

In this case, required strategies for the reduction of exposure must be implemented.

mount to ensure a healthier agricultural soil, clean surface and groundwater quality.

Cassava, in general, and cassava tubers, in particular, are indispensable for daily self-nourishment of several poor communities worldwide owing to their nutritional value. However, when exposed to environmental processes and bacterial and fungal attacks that can occur prior to harvesting, the produce is susceptible to release cyanogen and mycotoxin compounds that are hazardous to humans, animals and the environment. These contaminants and their by-product mobility into the terrestrial ecosystem are similar and are facilitated by environmental processes such as transformation, complexation, percolation and volatilisation as they can travel from surface and subsurface to groundwater level, which can result in exposure to both animals and humans. The presence of these compounds in arable land can lead to their accumulation, which can negatively affect soil properties, groundwater quality and the environment, thus contributing to a decline in the production of useful produce, such as cassava. Monitoring, particularly in communities that use such arable soil on a continuous basis, can mitigate intoxication of humans and animals, by effectively implementing suitable reduction strategies thus prevent environmental pollution. Therefore, continuous monitoring, quality assurance and a novel *in-situ* biological method (for treatment of the contaminants) are para-

for transformed varieties of the cyanogens (see **Figure 2**), such as thiocyanate, etc.

postulated to suggest their facilitation of cell mutation in humans.

186 Aflatoxin-Control, Analysis, Detection and Health Risks

**4. Conclusion**

This research was funded by the Cape Peninsula University of Technology (CPUT), through the University Research Fund (URF - RK 16). Members of the Bioresource Engineering Research Group (BioERG), Dr Richard Mundembe, Department of Biotechnology (CPUT) and Dr Arnelia N. Paulse, Department of Environmental and Occupational Studies (CPUT) are acknowledged for their support.

## **Declaration of conflicting interests**

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this manuscript.

## **Author details**

Elie F. Itoba-Tombo\*, Seteno K.O. Ntwampe and John B.N. Mudumbi

\*Address all correspondence to<EMAIL_ADDRESS>
Bioresource Engineering Research Group (BioERG), Department of Biotechnology, Cape Peninsula University of Technology, Cape Town, , South Africa

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## **First Report of Mycotoxins in Second Peanuts Crop in Adana and Osmaniye at Harvest, Drying, Prestorage and Storage Periods**

Isilay Lavkor, Isil Var, Sevcan Oztemiz and Manaf AlMatar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68210

#### **Abstract**

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Aflatoxin (AF) and cyclopiazonic acid (CPA) contaminations are very important problems for peanuts and its products. The aim of the study was to detect aflatoxin (types B and G) and cyclopiazonic acid (CPA) occurrence and critical periods of toxin production in peanuts collected from different research areas of Osmaniye and Adana, Turkey, in 2015. Peanut kernels toxin analysis was performed in four different periods during the harvest, drying, prestorage, and storage. Total aflatoxin occurrence in peanut kernels was analyzed by immunoaffinity chromatography‐reversed‐phase high‐performance liquid chromatography (IAC‐HPLC) analysis and cyclopiazonic acid occurrence in peanut kernels was analyzed by thin layer chromatography (TLC). Aflatoxin levels in 76 out of 102 contaminated samples were from 0.3 to 1333.42 μg/kg. Cyclopiazonic acid levels in 18 out of 102 peanut samples were from 16.6 to 44.44 μg/kg. An unusual pattern of mycotoxin production (aflatoxin types B and G simultaneously with CPA) was seen in 11 of 102 peanuts samples. Six of nine samples were from the storage period. Aflatoxin contamination during harvesting (64%) and drying (75%) were higher than prestorage (53%). Aflatoxin (93%) and cyclopiazonic acid (30%) were the most produced during storage. The results showed that storage period was significantly important for the presence of two mycotoxins according to the statistical analysis.

**Keywords:** aflatoxin, cyclopiazonic acid, peanut, drying, harvest, prestorage, storage

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Peanut (*Arachis hypogaea* L., Family: *Fabaceae*) is a rich source of fat, proteins, and vitamins. Peanuts are grown on a large scale in almost all the tropical and subtropical countries, especially in India, China, the USA, and West Africa [1]. Also, peanut is a member of the legume family, an important food and oil crop. It is currently grown on approximately 42 million acres worldwide. It is the third major oilseed of the world after soybean and cotton [2]. Peanut is used for human consumption, oil production, food industries, and animal feeding [3]. It is grown in China (37%), India (20%), Nigeria (6.5%), and the USA (4.1%). Turkey supplies about 0.3% of the world production of peanut [4]. The total production of peanut was 147,537 tons harvested from 377,729 da, with an average yield of 391 kg/da in Turkey in 2015 [5].

Poor agricultural practices and postharvest treatments of peanuts can lead to an infection by mold fungus *Aspergillus flavus* and *Aspergillus parasiticus* releasing the toxic substance aflatoxins (AFs). Contamination may occur when either the grown crops [6] or more badly stored harvests are infested by molds [7, 8]. *A. flavus* also produces other mycotoxins such as cyclopiazonic acid (CPA) and indole‐tetramic acid [9]. CPA occurs naturally in peanuts [10–12] and corn [12, 13].

The occurrence of aflatoxins in foods has been also recognized as a potential threat for human health. Aflatoxin, naturally occurring secondary metabolites, are potent hepatotoxic, mutagenic, and carcinogenic toxins, causing serious health hazards in humans and in animals. Aflatoxin B1 , B2 , G1 , and G2 are found predominantly as the hydroxylated metabolic products of aflatoxins B1 (AFB1 ) and B2 (AFB2 ), respectively [14]. The most toxic aflatoxin known, AFB1 , is cited as a group I carcinogen by the International Agency for Research on Cancer [15, 16].

Another mycotoxin is cyclopiazonic acid (CPA), which causes necrotic foci in internal organs such as the liver and exerts neurotoxic effects [17]. Natural occurrence of CPA has been reported in peanuts, corn, cheese, tomato products, and also meat, eggs, and milk of animals that are fed by contaminated feeds [18]. Incidence of aflatoxigenic *A. flavus* strains was higher in peanuts (69%) than in wheat (13%) or soybeans (5%), while the ratio of CPA producers [12]. Risk of aflatoxin contamination hits top values in such commodities as nuts [19]. The maximum levels of AFB1 and total aflatoxins allowed in peanut as determined by Commission of the European Communities are 2 and 4 μg/kg, respectively.

Fungal infection of seeds before and after harvest remains a major problem of food safety in most parts of Turkey. Problems associated with this infection include loss of germination, mustiness, moldy smell [20–22], and aflatoxin contamination [23–25]. These problems are, however, dealt with most developed world where a careful commodity screening and improved storage conditions are provided [7, 23, 26]. Though aflatoxins producing fungus are a natural contaminant of peanut and other agricultural commodities, it is aggravated due to poor agricultural practices, harvesting practice, postharvest handling, and storage methods. Some studies undertaken in Turkey in different foods show that aflatoxins levels are substantially higher. One important aspect is traditional harvesting and storage practices [22].

In this study, aflatoxin and cyclopiazonic acid contamination were determined in the second peanut crops at harvest, drying, pre‐storage, and the storage periods in Adana and Osmaniye provinces of Turkey in 2015. Aflatoxin is always the most important toxin for peanut because of its toxicity, and the CPA presence in peanuts is also important as it causes necrotic foci in internal organs such as the liver and exerts neurotoxic effects, therefore, in peanuts, aflatoxins and besides, CPA should be investigated.

## **2. Materials and methods**

## **2.1. Location of the study**

**1. Introduction**

194 Aflatoxin-Control, Analysis, Detection and Health Risks

corn [12, 13].

Aflatoxin B1

of aflatoxins B1

mum levels of AFB1

practices [22].

, B2 , G1

(AFB1

, and G2

) and B2

(AFB2

the European Communities are 2 and 4 μg/kg, respectively.

Peanut (*Arachis hypogaea* L., Family: *Fabaceae*) is a rich source of fat, proteins, and vitamins. Peanuts are grown on a large scale in almost all the tropical and subtropical countries, especially in India, China, the USA, and West Africa [1]. Also, peanut is a member of the legume family, an important food and oil crop. It is currently grown on approximately 42 million acres worldwide. It is the third major oilseed of the world after soybean and cotton [2]. Peanut is used for human consumption, oil production, food industries, and animal feeding [3]. It is grown in China (37%), India (20%), Nigeria (6.5%), and the USA (4.1%). Turkey supplies about 0.3% of the world production of peanut [4]. The total production of peanut was 147,537 tons

harvested from 377,729 da, with an average yield of 391 kg/da in Turkey in 2015 [5].

Poor agricultural practices and postharvest treatments of peanuts can lead to an infection by mold fungus *Aspergillus flavus* and *Aspergillus parasiticus* releasing the toxic substance aflatoxins (AFs). Contamination may occur when either the grown crops [6] or more badly stored harvests are infested by molds [7, 8]. *A. flavus* also produces other mycotoxins such as cyclopiazonic acid (CPA) and indole‐tetramic acid [9]. CPA occurs naturally in peanuts [10–12] and

The occurrence of aflatoxins in foods has been also recognized as a potential threat for human health. Aflatoxin, naturally occurring secondary metabolites, are potent hepatotoxic, mutagenic, and carcinogenic toxins, causing serious health hazards in humans and in animals.

is cited as a group I carcinogen by the International Agency for Research on Cancer [15, 16]. Another mycotoxin is cyclopiazonic acid (CPA), which causes necrotic foci in internal organs such as the liver and exerts neurotoxic effects [17]. Natural occurrence of CPA has been reported in peanuts, corn, cheese, tomato products, and also meat, eggs, and milk of animals that are fed by contaminated feeds [18]. Incidence of aflatoxigenic *A. flavus* strains was higher in peanuts (69%) than in wheat (13%) or soybeans (5%), while the ratio of CPA producers [12]. Risk of aflatoxin contamination hits top values in such commodities as nuts [19]. The maxi-

Fungal infection of seeds before and after harvest remains a major problem of food safety in most parts of Turkey. Problems associated with this infection include loss of germination, mustiness, moldy smell [20–22], and aflatoxin contamination [23–25]. These problems are, however, dealt with most developed world where a careful commodity screening and improved storage conditions are provided [7, 23, 26]. Though aflatoxins producing fungus are a natural contaminant of peanut and other agricultural commodities, it is aggravated due to poor agricultural practices, harvesting practice, postharvest handling, and storage methods. Some studies undertaken in Turkey in different foods show that aflatoxins levels are substantially higher. One important aspect is traditional harvesting and storage

are found predominantly as the hydroxylated metabolic products

and total aflatoxins allowed in peanut as determined by Commission of

), respectively [14]. The most toxic aflatoxin known, AFB1

,

In the second crop of peanuts, 102 samples were collected randomly throughout the peanut fields, that is, about 5949.5 da from Adana and Osmaniye provinces during October and November in 2015. Peanut samples were collected from 72 representative fields of 16 different districts of Adana and Osmaniye. Sampling was done according to Bora and Karaca [27] and have been followed in 1% of the survey areas. Also, 30 peanut samples were collected from storage in Adana and Osmaniye provinces. The samples were collected during the following periods of production: preharvest, drying, and prestorage. Also, 30 unshelled peanut samples were collected from the storage.

## **2.2. Collection of peanut samples**

Second crop peanut samples were collected during harvest, drying, prestorage of eliminated soil, and storage periods. Seventy‐two crusted peanuts samples were collected at harvest, dried for 7 days, and eliminated soil to prestorage. Each sample has been obtained from different farmer fields. Samples of pods (about 5 kg each) were divided manually and homogeneously to obtain working samples (about 1 kg each) for mycotoxins analyzes. The shells were removed manually. Additionally, 30 unshelled peanut samples were collected from storage. The samples (about 1 kg each) were collected in paper bags for analyzing mycotoxins. All the samples were kept at +4°C [28].

### **2.3. Analysis of mycotoxins**

## *2.3.1. Determination of AFs with immunoaffinity chromatography*‐*reversed phase high*‐*performance liquid chromatography (IAC*‐*HPLC) analysis*

Analysis of aflatoxins was performed using immunoaffinity columns, as described below. Identification and deperiodination of aflatoxins B1 , B2 , G1 , and G2 in peanut product samples were carried out by high‐performance liquid chromatography (HPLC) according to Arzandeh and Jinap [29]. To 50 g of tested sample, 5 g of NaCl and 125 ml of methanol and water (70:30)was mixed in a blender for 2–3 min at high speed. The mixture was filtered through Whatman no. 4 filter paper. Then 30 ml of water was added to a 15 ml of filtrate. About 10 ml of the second filtrate was quantitatively passed through the immunoaffinity column at flow rate of 1 ml/min. The column was washed with 10 ml of water. Aflatoxins were eluted with 1 ml of methanol in an amber vial at flow rate of 1–2 ml/min. The elution step was repeated with 1 ml of water. Thus, Agilent 1100 HPLC was ready for injection. Fluorescence detector (excitation at 360 nm and emission above 440 nm). Mobile phase consisted of methanol/water/acetonitrile (300:600:200, v/v/v) with a flow rate of 1.0 ml/min followed by derivatization with bromine (132 mg/l KBr, 385 μL nitric acid) in C‐18 (R‐Biopharm Rhône). HPLC column was maintained at a constant temperature (*T* = 25°C). The results were expressed as a μg/kg.

## *2.3.2. Determination of CPA with thin*‐*layer chromatography (TLC) analysis*

The peanut samples were tested for cyclopiazonic acid (CPA) production following the method modified by Somuncuoglu [18]. On each samples, 45 g of the contents of each test plate was macerated in a waring blender with 150 ml of methanol and 2% sodium bicarbonate (7:3). The slurry was twice filtered through a Büchner funnel with Whatman no. 4 filter paper and then concentrated to dryness with a rotary evaporator. The residue was partitioned between 200 ml of dichloromethane‐distilled H2 O (1:1), and the dichloromethane layer was extracted three times with a saturated NaHCO3 solution (100 ml). The dichloromethane layer, containing AFs, was rotary evaporated and concentrated under a gentle stream of nitrogen. The aqueous layer, containing CPA, was acidified to pH 2.0 with 0.5 N HCl and extracted two times with 25 ml kloroform (500 ml). The extract was evaporated and concentrated as for CPA.

A total of 45 g of peanuts was used for extraction of CPA. After adding 150 ml methanol and sodium bicarbonate (2%) (7:3), it was stirred in a high‐speed mixer for 5 min. The mixture was filtred using Whatman no. 4 filter paper, and then 80 ml was taken from the fitrate obtained. To the filtrate, 30 ml of 0.05 M solution of lead acetate was added and stirred and the precipitate was removed by filtration. 0.5 N HCl reduced the pH to 2 with 50 ml of the filtrate that was extracted twice with 25 ml of chloroform, and the bottom phase was collected. The water in the chloroform phase (lower phase) was removed with 10 g anhydrous sodium sulfate by filtrating through a paper filter. The extract was collected in the flask after extraction and dried at 40°C by rotary vacuum evaporator. The extract in the flask was taken to the tubes with 3‐4 ml of chloroform and then the content is dried under nitrogen gas.

The qualitative presence of CPA was determined by thin‐layer chromatography (TLC) separation on silica gel plates 60 EM‐5721 (20 × 20 cm; Merck). The plates were first dipped in a 2% (wt/wt) solution of oxalic acid in methanol for 10 min, after being heated at 100°C for 1 h and cooled. The plates were spotted with 60–80 μl of the respective extract and developed in the solvent ethyl acetate/2 propanol/sodium hydroxide (50:15:10, v/v/v) for 35–40 min. After this, the plates were being heated at 35–40°C for 1 h and cooled. CPA was viewed after spraying with Ehrlich's reagent (1.0 g of 4‐dimethylaminobenzaldehyde in 25 ml of HCl and 75 ml etanol) with subsequent development of a purple color in daylight [30].

The results were calculated applying the formula, and a concentration of CPA in μ/kg = (*S* × *Y* × *V*)/(*X* × *W*)

where *S* is the μl aflatoxin CPA standard equal to unknown; *Y* is the concentration of CPA standard μg/ml; *V* is the μl of final dilution of sample extract; *X* is the μl of sample extract spotted to give fluorescent intensity equal to *S* (CPA standard); and *W* is the weight of sample in gram of original sample contained in the final extract [18].

#### **2.4. Statistical analysis**

column at flow rate of 1 ml/min. The column was washed with 10 ml of water. Aflatoxins were eluted with 1 ml of methanol in an amber vial at flow rate of 1–2 ml/min. The elution step was repeated with 1 ml of water. Thus, Agilent 1100 HPLC was ready for injection. Fluorescence detector (excitation at 360 nm and emission above 440 nm). Mobile phase consisted of methanol/water/acetonitrile (300:600:200, v/v/v) with a flow rate of 1.0 ml/min followed by derivatization with bromine (132 mg/l KBr, 385 μL nitric acid) in C‐18 (R‐Biopharm Rhône). HPLC column was maintained at a constant temperature (*T* = 25°C). The results were

The peanut samples were tested for cyclopiazonic acid (CPA) production following the method modified by Somuncuoglu [18]. On each samples, 45 g of the contents of each test plate was macerated in a waring blender with 150 ml of methanol and 2% sodium bicarbonate (7:3). The slurry was twice filtered through a Büchner funnel with Whatman no. 4 filter paper and then concentrated to dryness with a rotary evaporator. The residue was partitioned

containing AFs, was rotary evaporated and concentrated under a gentle stream of nitrogen. The aqueous layer, containing CPA, was acidified to pH 2.0 with 0.5 N HCl and extracted two times with 25 ml kloroform (500 ml). The extract was evaporated and concentrated as

A total of 45 g of peanuts was used for extraction of CPA. After adding 150 ml methanol and sodium bicarbonate (2%) (7:3), it was stirred in a high‐speed mixer for 5 min. The mixture was filtred using Whatman no. 4 filter paper, and then 80 ml was taken from the fitrate obtained. To the filtrate, 30 ml of 0.05 M solution of lead acetate was added and stirred and the precipitate was removed by filtration. 0.5 N HCl reduced the pH to 2 with 50 ml of the filtrate that was extracted twice with 25 ml of chloroform, and the bottom phase was collected. The water in the chloroform phase (lower phase) was removed with 10 g anhydrous sodium sulfate by filtrating through a paper filter. The extract was collected in the flask after extraction and dried at 40°C by rotary vacuum evaporator. The extract in the flask was taken to the

tubes with 3‐4 ml of chloroform and then the content is dried under nitrogen gas.

etanol) with subsequent development of a purple color in daylight [30].

The qualitative presence of CPA was determined by thin‐layer chromatography (TLC) separation on silica gel plates 60 EM‐5721 (20 × 20 cm; Merck). The plates were first dipped in a 2% (wt/wt) solution of oxalic acid in methanol for 10 min, after being heated at 100°C for 1 h and cooled. The plates were spotted with 60–80 μl of the respective extract and developed in the solvent ethyl acetate/2 propanol/sodium hydroxide (50:15:10, v/v/v) for 35–40 min. After this, the plates were being heated at 35–40°C for 1 h and cooled. CPA was viewed after spraying with Ehrlich's reagent (1.0 g of 4‐dimethylaminobenzaldehyde in 25 ml of HCl and 75 ml

The results were calculated applying the formula, and a concentration of CPA in μ/kg = (*S* ×

O (1:1), and the dichloromethane layer was

solution (100 ml). The dichloromethane layer,