The Effect of Time on Mycotoxins in Subsistence Farmed Maize from Zimbabwe

FACULTY OF PHARMACEUTICAL SCIENCES Department of Bio-Analysis Laboratory of Food Analysis

Academic year 2015-2016

THE EFFECT OF TIME ON MYCOTOXINS IN

SUBSISTENCE FARMED MAIZE FROM ZIMBABWE

Sarah GEHESQUIÈRE First Master of Pharmaceutical Care

Promoter Prof. Dr. Pharm. D. S. De Saeger

Supervisor Melody Hove

Commissioners Prof. Dr. Geert Haesaert Dr. Natalia Beloglazova

FACULTY OF PHARMACEUTICAL SCIENCES Department of Bio-Analysis Laboratory of Food Analysis

Academic year 2015-2016

THE EFFECT OF TIME ON MYCOTOXINS IN

SUBSISTENCE FARMED MAIZE FROM ZIMBABWE

Sarah GEHESQUIÈRE First Master of Pharmaceutical Care

Promoter Prof. Dr. Pharm. D. S. De Saeger

Supervisor Melody Hove

Commissioners Prof. Dr. Geert Haesaert Dr. Natalia Beloglazova COPYRIGHT

“The author and the promoters give the authorization to consult and to copy parts of this thesis for personal use only. Any other use is limited by the laws of copyright, especially concerning the obligation to refer to the source whenever results from this thesis are cited.”

August 16, 2016

Promoter Author

Prof. Dr. Pharm. D. S. De Saeger Sarah Gehesquière DANKWOORD

Ik had deze masterproef niet kunnen verwezenlijken zonder de hulp van bepaalde mensen.

Vooreerst wil ik graag Prof. Dr. Apr. Sarah De Saeger bedanken om mij de kans te geven om mijn masterproef op het Laboratorium voor Bromatologie te mogen uitvoeren.

Ook een oprechte dankjewel aan mijn begeleidsters Melody Hove en Marthe De Boevre, om mij steeds bij te staan met goeie raad, steeds een antwoord te geven op mijn vele vragen, mij te steunen, en om deze thesis na te lezen.

Verder wil ik nog mijn medestudenten en alle medewerkers van het laboratorium bedanken voor de gezellige sfeer, de leuke babbels, en de motiverende gesprekken toen ik het nodig had.

Ook wil ik mijn ouders, oma, zus en vrienden bedanken voor alle steun tijdens de voorbije maanden.

SAMENVATTING

Maïs is een basisvoedsel dat verbouwd wordt in Afrika ten zuiden van de Sahara, zoals Zimbabwe, maar het is ook graan dat vaak gekoloniseerd wordt door schimmels die mycotoxines kunnen produceren. Mycotoxines zijn de secundaire metabolieten van fungi en hebben een nadelig effect op de gezondheid van mens en dier, en kunnen gewassen aantasten. Dit kan resulteren in ziektes en economische verliezen. De belangrijkste toxigene schimmels en hun mycotoxines zijn de Aspergillus spp. (aflatoxines, ochratoxine A en sterigmatocystine), Fusarium spp. (trichothecenes, fumonisines en zearalenone), Alternaria spp. (alternariol en alternariol monomethylether), en Penicillium spp. (roquefortine C). Deze thesis maakt deel uit van een studie uitgevoerd door Melody Hove. Het doel van deze studie was tweevoudig. Ten eerste werden de aanwezigheid en quantiteit van mycotoxins bepaald in maïs afkomstig van kleine boerderijen in de provincies Mashonaland West en Manicaland. Het tweede doel van deze studie was om het effect van tijd op het mycotoxinegehalte te bestuderen. Hiertoe werden stalen die genomen werden op het moment van de oogst vergeleken met stalen van drie maanden na de oogst, en stalen van zes maanden na de oogst. De verzamelde stalen werden geanalyseerd gebruik makend van vloeistofchromatografie gekoppeld aan tandem massaspectrometrie. Van de 158 geteste maïsstalen was 46% positief voor tenminste één mycotoxine. De toxicologisch relevante mycotoxines FB 1, FB 2, FB 3, DON en ZEN kwamen voor in respectievelijk 40, 22, 11, 6 en 3% van de stalen, aan een gemiddelde van 243,30;

52,22; 13,67; 7,32 en 0,24 µg/kg. In één staal werd 14,80 µg/kg AFG 1 gedetecteerd. Dit was hoger dan de wettelijke limit van 10 µg/kg. Andere mycotoxines die in 1 tot 3% de stalen voorkwamen, waren: FX, 15-ADON, DAS, NIV en STERIG. Bij alle fumonisines werden dezelfde trend in de tijd gezien. Algemeen steeg het gemiddelde gehalte aan fumonisines significant tot minimum drie maanden na de oogst, waarna het gehalte terug significant daalde. Het gehalte aan fumonisines was significant hoger in Mashonaland West dan in Manicaland. Fumonisins zijn het grootste probleem in maïs verbouwd op kleine boerderijen in Zimbabwe. Goede praktijken vóór, tijdens en na het oogsten kunnen het voorkomen van mycotoxines in maïs, en bijgevolg ook de hieraan verbonden risico’s, reduceren.

SUMMARY

Maize is a staple food that grows in sub-saharan Africa, like Zimbabwe. However, it is often colonized by mycotoxin-producing fungi. Mycotoxins are the secondary metabolites of fungi that have adverse effects on animals, humans and crops, and that result in economic losses and illnesses. Most important toxigenic fungi and their mycotoxins are the Aspergillus spp. (aflatoxins, ochratoxin A, sterigmatocystin), Fusarium spp. (trichothecenes, fumonisins, zearalenone), Alternaria spp. (alternariol, alternariol monomethyl ether), and Penicillium spp. (roquefortine C). This thesis is part of a larger study conducted by Melody Hove. The aim of this study was twofold. The first goal was to determine the presence and quantity of mycotoxins in maize obtained from subsistence farms in the provinces Mashonaland West and Manicaland, the two agricultural zones in Zimbabwe. The second goal was to study the effect of time on mycotoxin content by comparing contamination in samples taken at harvest, three months postharvest, and six months postharvest, to see whether there is a difference between the different stages or not. Samples were collected at harvest, three months postharvest, and six months postharvest, in two agricultural zones of Zimbabwe. Analysis was performed using liquid chromatography coupled to tandem mass spectrometry. Of the 158 maize samples tested, 46% were positive for at least one mycotoxin. Of the toxicologically relevant mycotoxins, FB 1, FB 2, FB 3, DON, and ZEN were detected in 40, 22, 11, 6, and 3% of the samples, at a mean level of 243.30, 52.22, 13.67, 7.32, and 0.24 µg/kg, respectively. AFG1 was detected in only one sample, at a level of 14.80 µg/kg, which exceeds the legal limit of 10 µg/kg. Other detected mycotoxins were FX, 15-ADON, DAS, NIV, and STERIG. The percentage of contamination for these mycotoxins ranged between 1 and 3% of the samples. The content of all fumonisins showed a significant change over time. In general, the average amount of fumonisins in the contaminated samples significantly increased until at least three months after harvest, and thereafter significantly decreased again. The fumonisin content in Mashonaland West was significantly higher than in Manicaland. Fumonisins are the major problem in subsistence farmed maize in Zimbabwe. Good pre- and postharvest practices can reduce the occurrence of mycotoxins and the associated risks.

TABLE OF CONTENTS

1. INTRODUCTION ...... 1 1.1. Aspergillus spp. Mycotoxins ...... 2 1.1.1. Aflatoxins ...... 2 1.1.2. Ochratoxin A (OTA) ...... 3 1.1.3. Sterigmatocystin ...... 4 1.2. Fusarium spp. Mycotoxins ...... 5 1.2.1. Trichothecenes ...... 6 1.2.2. Fumonisins ...... 9 1.2.3. Zearalenone (ZEN) ...... 11 1.3. Alternaria spp. Mycotoxins ...... 12 1.4. Penicillium spp. Mycotoxins ...... 13 1.5. Legislation ...... 13 2. OBJECTIVES ...... 16 3. MATERIALS AND METHODS ...... 17 3.1. Sampling ...... 17 3.2. Determination of mycotoxins in maize ...... 19 3.2.1. Standards ...... 19 3.2.2. Reagents and materials ...... 19 3.2.3. Sample preparation ...... 20 3.2.4. Apparatus ...... 21 3.2.5. Statistics and data analysis ...... 24 4. RESULTS AND DISCUSSION ...... 25 4.1. Investigation of the contamination level of mycotoxins in maize ...... 25 4.2. Determination of mycotoxin levels at three stages (harvest, three months postharvest and six months postharvest), and in two different agricultural zones (manicaland and mashonaland west) ...... 27 5. CONCLUSION ...... 36 REFERENCES ...... 37 APPENDIX A: Raw data ...... I 1. Raw data obtained from the samples from Mashonaland West in Zimbabwe ...... I 2. Raw data obtained from the samples from Manicaland in Zimbabwe ...... IX APPENDIX B: INTERNATIONALIZATION @ HOME ...... XIV

LIST OF ABBREVIATIONS

µg microgram µg/kg microgram per kilogram µL microliter µm micrometer 15-ADON 15-acetyl deoxynivalenol 3-ADON 3-acetyl deoxynivalenol

AFB 1 aflatoxin B 1

AFB 2 aflatoxin B 2

AFG 1 aflatoxin G 1

AFG 2 aflatoxin G 2 AME alternariol methylether AOH alternariol ATA alimentary toxic aleukia BEN Balkan Endemic Nephropathy CI95 95% confidence interval DAS Diacetoxyscirpenol DOM deepoxy-deoxynivalenol DON deoxynivalenol EC European Commission ELEM equine leukoencephalomalacia ESI electrospray ionization eV electron volt FAO Food and Agricultural Organization

FB 1 fumonisin B 1

FB 2 fumonisin B 2

FB 3 fumonisin B 3 FX fusarenon X g gram HPLC high-performance liquid chromatography HT-2 HT-2 toxin IARC International Agency for Research on Cancer JECFA Joint FAO/WHO Expert Committee on Food Additives kg kilogram kg bw kilogram bodyweight LC-MS liquid chromatography with mass spectrometry LC-MS/MS liquid chromatography with tandem mass spectrometry

LD 50 lethal dose 50%, median lethal dose m/z mass-to-charge ratio mg milligram min minute(s) mL milliliter mM millimolar mm millimeter NEO neosolaniol ng/kg bw/day nanogram per kilogram bodyweight per day NIV nivalenol NOEL No Observed Effect Level OTA ochratoxin A Phe phenylalanine PMTDI Provisional Maximum Tolerable Daily Intake ROQC roquefortine C SCF Scientific Committee on Food SPE solid phase extraction STERIG sterigmatocystin T-2 T-2 toxin TDI Tolerable Daily Intake UN United Nations UPLC ultra-performance liquid chromatography UPLC/MS ultra performance liquid chromatography with mass spectrometry V volt WHO World Health Organization ZAN zearalanone ZEN Zearalenone

1. INTRODUCTION

Maize ( Zea mays ) is a dietary staple crop that is grown widely throughout the world. It plays an important role in the diet of millions of African people, and it is the most important cereal crop in sub-Saharan Africa (Fandohan et al., 2003; IITA, 2016). Of its maize production, Eastern and Southern Africa uses 85% as food, while Africa as a whole uses 95% (IITA, 2016).

Unfortunately, maize is a cereal that is frequently colonized by filamentous fungi, or molds. Contamination with fungi not only makes maize unsuitable for consumption by discoloration and reduction of the nutritional value, but it can also result in the production of toxic secondary metabolites, also known as mycotoxins (Fandohan et al., 2003). The term is derived from the Greek word ‘mykes’ (meaning fungus) and the Latin word ‘toxicum’ (poison, toxin) (Moreno et al., 1999). Upon ingestion, inhalation, or skin contact, they may be toxic or have other debilitating effects on animals and humans (Dvorackova, 1989; Zain, 2011; Marin et al., 2013). The diseases they cause are known as mycotoxicoses (Zain, 2011; Marin et al., 2013). Characteristically, mycotoxicoses are not transmissible and drug treatments have little or no effect (Marin et al., 2013). Outbreaks are usually seasonal and associated with a specific foodstuff which, upon closer examination, often shows signs of fungal activity (Marin et al., 2013).

Historical evidence of the occurrence of mycotoxins reaches as far back as the time included in the writings of the Dead Sea Scrolls (408–318 B.C.), but it was only until quite recently that the true chemical nature of these entities was recognized, with the identification of the aflatoxins in the early 1960s (Miller, 1995; Richard, 2007). Currently, a lot of mycotoxins have been identified, and the search for new ones is still ongoing (Binder et al., 2007; Zain, 2011).

Maize is a cereal crop in which a range of mycotoxins have been found, the most important ones being aflatoxins, trichothecenes, fumonisins, ochratoxin A and zearalenone (Scudamore et al., 2000, Fandohan et al., 2003). Since it is a dietary staple food in a lot of countries over the world, including Zimbabwe, it can represent a source of human exposure, directly by its consumption, or indirectly through the

1 consumption of products derived from animals that were fed with contaminated feed (Placinta et al., 1999).

Mycotoxins not only affect the health of humans and animals. They also have an extensive economical impact, which includes increased costs for health care and veterinary care, reduction of livestock production and crop yield, and disposal of contaminated foods and feeds (Zain, 2011).

According to the Food and Agricultural Organization (FAO) of the United Nations (UN), estimations are that a large amount of the cereals produced in the world are contaminated by one or more mycotoxins (Mannon et al., 1985; Rice et al., 1994). Contamination can occur preharvest (on the field), during harvest and drying, or during storage (Marin et al., 2013). The degree of contamination depends on different factors such as temperature, water activity, humidity, physical damage (by insects and/or mechanical), and the presence of other mycobiota (Marin et al., 2013).

1.1. ASPERGILLUS SPP. MYCOTOXINS

1.1.1. Aflatoxins Among the known mycotoxins, aflatoxins are proven to be carcinogenic to both humans and animals. They are dangerous and widely distributed secondary fungal metabolites. They were first identified as the causal toxin of Turkey X disease in the early 1960s in England. This disease killed over 100,000 turkeys (and other poultry) and was due to contaminated groundnut meal (Moss, 2002; Zain, 2011).

Aflatoxins are produced by Aspergillus section flavi that grow in warm and humid conditions, mainly by Aspergillus flavus , Aspergillus parasiticus and the rare Aspergillus nomius (Sweeney et al., 1998). While there are a number of different types of aflatoxins produced in nature, the four main naturally produced ones are aflatoxin

B1 (AFB 1, Figure 1.1), aflatoxin B 2 (AFB 2, Figure 1.2), aflatoxin G 1 (AFG 1, Figure 1.3) and aflatoxin G 2 (AFG 2, Figure 1.4). While most A. parasiticus strains produce both aflatoxins B and G and are well adapted to a soil environment, A. flavus isolates are more adapted to the aerial parts of plants (leaves, flowers, and fruit) and produce only aflatoxins B (Dänicke et al., 2004). Aflatoxins may contaminate many agricultural products including groundnuts, tree nuts, maize, rice, dried fruits, spices and cocoa

2 beans, as a result of Aspergillus section Flavi infection before and/or after harvest (Dänicke et al., 2004).

O O O O

O O

OO O OO O

Figure 1.1. The structural formula of aflatoxin B 1 Figure 1.2. The structural formula of aflatoxin B 2 OO OO

OO OO

O O O O O O

Figure 1.3. The structural formula of aflatoxin G 1 Figure 1.4. The structural formula of aflatoxin G 2

AFB 1 and the mixtures of aflatoxins are known human carcinogens (IARC Group 1) and it was reported to cause liver cancer in African countries and Thailand (IARC, 1993; Pitt, 2000; Probst et al., 2007). In addition, aflatoxins can cause several toxic effects in human and animal health, including teratogenicity, mutagenicity, and immunotoxicity (Eaton et al., 1994). It is also considered that a number of diseases, including Reye’s syndrome, kwashiorkor and hepatitis may also be related to the intake of this group of mycotoxins (Hendrickse et al., 1982; Moss, 2002). The consumption of contaminated foods even with low levels of aflatoxins may result in chronic aflatoxicosis with stunting in children, suppression of immunity, cancer and reduced life expectancy (Shephard, 2008).

For most regulated mycotoxins the Tolerable Daily Intake (TDI) has been calculated except for aflatoxins, because of the known fact that AFB 1 is a genotoxic carcinogen, and therefore exposure at any level is considered unsafe (Yogendrarajah et al., 2014).

1.1.2. Ochratoxin A (OTA) Ochratoxin A (OTA) is the most toxic member of the ochratoxin mycotoxins (Marin et al., 2013). Its name is derived from Aspergillus ochraceus , the fungi from which it was first isolated (Van der Merwe et al., 1965). Apart from Aspergillus , OTA can also be produced by Penicillium (Marin et al., 2013). It can contaminate a large variety of food

3 products, such as grapes, dried fruits, cocoa beans, and coffee beans, especially when they are stored improperly (Bayman et al., 2002; Pfohl-Leszkowicz et al., 2007; Juan et al., 2008).

The structural formula of ochratoxin A is shown in Figure 1.5. It is structurally similar to phenylalanine (Phe), and has an inhibitory effect on a couple of enzymes that use phenylalanine as a substrate. This can result in the inhibition of protein synthesis (Marin et al., 2013).

O OH O OHO

N O H

CH3

Figure 1.5. The structural formula of ochratoxin A

The chronic toxicity of OTA in humans is associated with a kidney disease that was first described in the 1950s, and typically occurred in the Balkans (Pfohl-Leszkowicz et al., 2002; Marin et al., 2013). This disease, the so-called Balkan Endemic Nephropathy (BEN), consists of a bilateral chronic renal failure, and is often associated with urinary tract tumors (Pfohl-Leszkowicz et al., 2007; Marin et al., 2013). Toxicological studies on animals have reported OTA to be nephrotoxic, hepatotoxic, teratogenic, neurotoxic, carcinogenic, genotoxic and immunotoxic (Bayman et al., 2002; Juan et al., 2008; Marin et al, 2013). The IARC has classified OTA as a possible carcinogen to humans (Group 2B) (IARC, 1993).

The Scientific Commission of the European Community established a maximum limit of 5 µg/kg in cereals, and 3 µg/kg for all products derived from cereals (Juan et al., 2008).

1.1.3. Sterigmatocystin Sterigmatocystin (STERIG, Figure 1.6) is a mycotoxin produced by many Aspergillus species, with A. versicolor being the most important (Rabie et al., 1977; Mallek et al., 1993; Lund et al., 1995; Mücke et al., 1998; Reijula et al., 2003). Optimal conditions for the production of sterigmatocystin by A. versicolor are temperatures between 23

4 and 29°C, water activity starting from 0.76 and a moisture content above 15% (Rabie et al., 1976; Abramson et al., 1999; Atalla et al., 2003).

O H O

O O O CH3 H

Figure 1.6. The structural formula of sterigmatocystin (STERIG)

Sterigmatocystin is a potential carcinogen, mutagen and teratogen (Sekijima et al., 1992; Sivakumar et al., 2001). This is probably due to the fact that sterigmatocystin is a biogenic precursor of AFB 1 (Hsieh et al., 1973; Betina, 1989; Wilkinson et al., 2004). This mycotoxin is associated with acute clinical symptoms of bloody diarrhea and death in dairy cattle fed with feed containing high levels of sterigmatocystin (Vesonder et al., 1985). It is also acutely toxic to the liver of the most animals tested (Veršilovskis et al., 2010). Despite its potent toxic and carcinogenic properties to animals, its importance as a human health hazard is unknown. (Purchase et al., 1969, 1973). However, it is assumed that sterigmatocystin may be involved in the etiology of chronic liver disease in humans, loss of muscle tone, generalized edema, and hemorrhage from the umbilical vessels (Lugauskas, 2005). Sterigmatocystin is classified by the IARC as ‘possibly carcinogenic to humans’ (Group 2B) (IARC, 1976, 1987, 1993).

1.2. FUSARIUM SPP. MYCOTOXINS The five most important toxigenic Fusarium species are F. sporotrichioides , F. graminearum , F. verticillioides ( syn. F. moniliforme ), F. poae and F. equiseti (Agag, 2004). Mycotoxin-producing strains of Fusarium are considered to be soil fungi that infect the crops while on the field (Robledo-Robledo, 1991; Eriksen, 1998; Pronk et al., 2002; Marin et al., 2013). However, when the environmental conditions are favorable, they can continue to grow on the crops held in storage (Pronk et al., 2002). Fusaria fungi are a worldwide problem, and have been isolated in both temperate and semitropical areas (Pronk et al, 2002; Marin et al., 2013). Especially for cereals, the Fusaria are major pathogens. They infect small grain cereals (such as wheat, oats,

5 barley, millet, sorghum, rice and rye) and maize, causing a number of frequently encountered plant diseases and therefore severe reductions in crops yield (Pronk et al., 2002). Head blight of small cereals and ear rot in maize are of the greatest concern, especially because maize, wheat and barley constitute almost two-thirds of the world production of cereals (Pronk et al., 2002). F. verticillioides is the most prevalent fungus infecting maize throughout the world, and reports of surveys showed that is also the case in some African countries (Marasas et al., 1988; Munkvold et al., 1997; Allah, 1998; Baba-Moussa, 1998; Kedera et al., 1999; Agag, 2004).

Fusaria are capable of producing a very diverse range of mycotoxins (Agag, 2004). The most important Fusarium toxins in maize are the trichothecenes, fumonisins and zearalenone (Marin et al., 2013).

1.2.1. Trichothecenes The trichothecenes (TCTs) are an important class of mycotoxins. They are known to be produced by various species of Fusaria , Trichoderma , Stachybotrys , Cephalosporium , Myrothecium , Trichothecium , Verticimonosporium , and Cylindrocarpon . The Fusaria are by far the most important genera, since they produce the greatest range of trichothecenes (Pronk et al., 2002). TCTs occur worldwide in corn, wheat, barley, oats, rice, rye, vegetables, and other crops (Zain, 2011). Natural occurrence of TCTs has been reported in Asia, Africa, South America, Europe, and North America (Scott, 1989).

The approximately 170 identified trichothecenes are subdivided in four groups (A–D), of which type A and type B are the most important ones (Grove 1988, 1993; Pronk et al., 2002; Marin et al., 2013). Type C and D trichothecenes are of lesser importance (Marin et al., 2013). Type A trichothecenes include T-2 toxin (T-2), HT-2 toxin (HT-2), neosolaniol (NEO) and diacetoxyscirpenol (DAS, also known as anguidine), while type B trichothecenes include deoxynivalenol (DON, also known as vomitoxin) and its acetyl derivatives 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15- ADON), nivalenol (NIV), and fusarenon X (FX) (Placinta et al., 1999; Pronk et al., 2002).

Trichothecenes are a family of closely-related sesquiterpenoid compounds. They have a tetraycyclic 12,13-epoxytrichothecene skeleton in common, which is responsible for their toxicological activity (WHO, 1990; Zain, 2011). Based on functional groups, they

6 can be divided into four categories (A–D) (WHO, 1990). Type A is characterized by a functional group other than a ketone at C-8, while type B is characterized by a carbonyl function at C-8 (Figure 1.7 and Figure 1.8, Table 1.1 and Table 1.2) (Placinta et al., 1999; Pronk et al., 2002).

HHH H3C O R1 O

R5 R2

R4 CH3 H

CH 2R3 Figure 1.7. The general structural formula of type A trichothecenes.

Table 1.1. The most important type A trichothecenes (Pronk et al., 2002). R1 R2 R3 R4 R5 DAS OH OAc OAc H H NEO OH OAc OAc H OH

T2 OH OAc OAc H OCOCH 2CH(CH 3)2

HT2 OH OH OAc H OCOCH 2CH(CH 3)2

HHH H3C O R1 O

O R2

R4 CH3 H

CH 2R3 Figure 1.8. The general structural formula of type B trichothecenes.

Table 1.2. The most important type B trichothecenes (Pronk et al., 2002). R1 R2 R3 R4 NIV OH OH OH OH F-X OH OAc OH OH DON OH H OH OH 3-ADON OAc H OH OH 15 -ADON OH H OAc OH

As can be seen in Table 1.3, a certain TCT can be produced by different Fusarium species. Also, within one Fusarium species, more than one TCT can be produced (depending on the strain and/or the environmental conditions). Fusarium graminearum and F. sporotrichioides are considered the two most important species in the

7 production of these mycotoxins. Fusarium sporotrichioides is generally associated with the production of type A trichothecenes, while F. graminearum is the major producer of type B trichothecenes (Pronk et al., 2002).

Table 1.3. Trichothecene production by Fusarium species (Pronk et al., 2002).

Type A T-2, HT-2 F. sporotrichioides, F. acuminatum, F. poae DAS F. poae, F. equiseti, F. sambucinum, F. sporotrichioides, F. acuminatum NEO F. sporotrichioides, F. poae, F. acuminatum, F. equiseti

Type B DON F. graminearum, F. culmorum NIV F. crookwellense, F. poae, F. graminearum, F. culmorum, F. nivale *, F. equiseti F-X F. crookwellense, F. poae, F. graminearum, F. culmorum, F. nivale *, F. equiseti 3-ADON F. graminearum, F. culmorum 5-ADON F. graminearum, F. culmorum * atypical strain of F. sporotrichioides

The main species associated with these diseases are Fusarium graminearum and F. culmorum , two major producers of type B trichothecenes (Table 1.3) (Pronk et al., 2002).

Trichothecene compounds produce a variety of toxic symptoms in humans and animals, including mortality; skin and gastrointestinal irritation or necrosis; haematological disorders (initial leukocytosis followed by leucopenia); diarrhea; vomiting and feed refusal; decreased body weight gain; damage to the haematopoietic system in bone marrow, spleen, thymus and lymph nodes; and immunological alterations. Trichothecenes are very cytotoxic, causing cell lysis and inhibition of mitosis. They can also interact with the cell membrane. Furthermore, they are very potent inhibitors of protein, DNA, and RNA synthesis. The inhibition of protein synthesis can be of two types. The first one is inhibition of the initial step of protein synthesis. Examples of this type of inhibition are T-2, HT-2, DAS, NIV, and FX. The second type of protein inhibition happens through inhibition of the elongation-termination step. DON works through this type of inhibition. Since TCTs are potent inhibitors of DNA and RNA, they are very toxic to tissues with a high cell division rate, such as thymus, spleen, bone marrow, ovary, testes, lymph nodes and intestinal mucosa. Because of this action being very similar to the actions induced by radiation, TCTs are classed as radiomimetic substances (Pronk et al., 2002).

8 Trichothecene-producing Fusarium species have been known to be associated with a number of human and animal mycotoxicoses (Pronk et al., 2002). Animal mycotoxicoses include the haemorrhagic syndrome; feed refusal and emetic syndromes; and oral and gastrointestinal lesions. Outbreaks have been reported to occur all over the world, thereby severely compromising livestock health, welfare, and productivity (Nelson et al., 1994; D’Mello et al., 1999).

Mycotoxin outbreaks affecting humans have mainly been reported to occur in the former Soviet Union and Asia (Japan, China, Korea) (Pronk et al., 2002). One of those outbreaks associated with TCT-producing Fusarium species is alimentary toxic aleukia (ATA) (Joffe, 1971; Pronk et al., 2002). This disease is closely related to the haemorrhagic syndrome in animals (Pronk et al., 2002). Symptoms include nausea, vomiting, abdominal pain, diarrhea, dizziness and headache (IARC, 1993). Although there is a strong suspicion of the involvement of TCTs in this disease, none has positively been identified (Pronk et al., 2002).

1.2.2. Fumonisins Fumonisins are mainly produced by Fusarium verticillioides (formerly known as Fusarium monoliforme ) and Fusarium proliferatum (Shephard et al., 1995; Agag, 2004; Marin et al., 2013). Of the twelve known fumonisin analogues known, the B series

(Figure 1.9) are the ones most abundantly found in foods: fumonisin B 1 (FB 1), fumonisin B 2 (FB 2), and fumonisin B 3 (FB 3), with fumonisin B 1 usually being found at the highest levels (Shephard et al., 1995; Rheeder et al., 2002; Marin et al., 2013). These mycotoxins are very commonly found in maize in the United States of America, South- America Europe, China, and Africa (Sydenham et al., 1991; Thiel et al., 1992; Visconti et al., 1993; Shephard et al., 1995). As a result of invasion with Fusarium , fumonisins can contaminate maize before harvest and during the early stages of storage (Doko et al., 1995; Marin et al., 2013).

O OH O O

OH O R1 OH

CH3 H3C

CH3 CH3 R2 NH2

OH O R1 R2 FB OH OH O O 1 OH O FB 2 OH H

FB3 H OH Figure 1.9. The general structural formula of fumonisins B

Animal studies have shown that fumonisins do not have a high acute toxicity (Marin et al., 2013). Fumonisins have been reported to cause equine leukoencephalomalacia (ELEM) in horses and related species (Prelusky et al., 1994; Marasas, 1996; Placinta et al., 1999; Agag, 2004; Zain, 2011; Marin et al., 2013). ELEM is an acutely fatal neurological disorder with symptoms such as paresis, ataxia, locomotor derangements, and hypersensitivity (Placinta et al., 1999). In pigs, fumonisins cause pulmonary edema syndrome (PES), as well as damage of the pancreas and liver (Harrison et al., 1990; Colvin et al., 1992; Prelusky et al., 1994; Placinta et al., 1999; Zain, 2011). Additionally, it has been demonstrated that prolonged exposition of fumonisin B 1 is hepatocarcinogenic to rodents (Gelderblom et al., 1991, 2001; Howard et al., 2001a, 2001b; Zain, 2011). The effects of fumonisins on humans are not yet well understood, but studies on the prevalence of esophageal cancer revealed an association between this disease and the consumption of maize contaminated with fumonisins (Franceschi et al., 1990; Rheeder et al., 1992; Chu et al., 1994; Marasas, 1996; Ueno et al., 1997; Shephard et al., 2000; Wang et al., 2000; Fandohan et al.,

2003). Based on the thus far obtained research results, FB 1 has been classified as ‘possibly carcinogenic to humans’ (Group 2B) by the International Agency for Research on Cancer (IARC, 1993, 2002).

In 2003, the Scientific Committee on Food concluded a TDI of 2 µg/kg bw for the total of fumonisins B 1, B 2, and B 3 (alone or in combination) (SCF, 2003).

10 1.2.3. Zearalenone (ZEN) Zearalenone (ZEN, Figure 1.10) is mainly produced by Fusarium graminearum , and also by F. equiseti, F. culmorum, F. cerealis, F. incarnatum and F. verticillioides (Marin et al., 2013).

OH O CH3

OH O Figure 1.10. The structural formula of zearalenone (ZEN)

The toxicity of ZEN is due to its ability to competitively bind estrogen receptors, thus causing reproductive problems in specific animals, and possibly also in humans (Wood, 1992; Gromadzka et al., 2009). It has been shown that ZEN potentially can stimulate the growth of cells with estrogenic receptors in human mammary glands, thereby supporting the hypothesis that ZEN may have a role in the development of breast cancer (Minervini et al., 2005; Yu et al., 2005). However, the International Agency for Research on Cancer (IARC) concluded that there is insufficient evidence for ZEN to be carcinogenic, and therefore that it is not classifiable as to its carcinogenicity to humans (Group 3) (IARC, 1993).

There is little data available on the acute toxicity of ZEN, but it appears to be relatively low, with oral LD 50 doses varying from 2,000 to 20,000 mg/kg bw (depending on the tested species) (Flannigan, 1991). The toxic effects of chronic dietary exposure in animals have been well-documented, and include genotoxicity, carcinogenicity, immunotoxicity, reproductive toxicity and endocrine effects (Marin et al., 2013). Pigs are one of the most susceptible animals, with NOEL values of 40 mg/kg bw/day (compared to 100 mg/kg bw/day in rats). Chronic exposure with ZEN has severe effects on their production (Marin et al., 2013). In females pigs, ZEN gives retention or complete absence of milk, vulvar dilatation and redness, vulvovaginitis, and rectal prolapsed, and, during pregnancy, reduces embryo survival and fetal weight (Zinedine et al., 2007). In boars, ZEN can reduce testosterone levels, spermatogenesis, weight of the testes, the libido, and induce feminization (Zinedine et al., 2007).

In general, Fusarium species grow and invade crops in moist cool field conditions during blooming, but growth and toxin production can also occur after harvest when storage conditions are poor (EFSA, 2011)

11 1.3. ALTERNARIA SPP. MYCOTOXINS Several Alternaria species are responsible for the production of toxic secondary metabolites (Ostry, 2008). They infest numerous foodstuffs, such as apples, mandarins, olives, pepper, tomatoes, sunflower seeds, sorghum, and wheat (Scott, 2001). Apart from foodstuffs, Alternaria species also grow on other materials like soil, wall papers, textiles, decaying wood, wood pulp and compost (Gravesen et al., 1994). The most important Alternaria species is A. alternata , which produces a number of mycotoxins, including altenuene, alternariol (AOH, Figure 1.11), and alternariol monomethyl ether (AME, Figure 1.12) (King et al., 1984; Visconti et al., 1986; Logrieco et al., 1990; Ozcelik et al., 1990; Ansari et al., 1990; Sanchis et al., 1993; Müller, 1992; Bilgrami et al., 1994; Andersen et al., 1996; Bottalico et al., 1998).

H3C OH H3C OH

OH O H3C O O

OH O OH O Figure 1.11. The structural formula of alternariol Figure 1.12. The structural formula of alternariol (AOH) monomethyl ether (AME)

Alternariol and alternariol monomethyl ether are not very acutely toxic (Pero et al., 1973; Hilblink, 1982; Olsen et al., 1988). There are several reports on the mutagenicity and genotoxicity of these mycotoxins (McCann et al., 1975; Scott et al., 1980; DiCosmo et al., 1985; An et al., 1989; Davis et al., 1994; Schrader et al., 2001, 2006; Brugger et al., 2006). The possibility that A. alternata may be a factor in the etiology of esophageal cancer has been suggested by Dong et al. (1987), Zhen et al. (1991), and Liu et al. (1990, 1992). Liu et al. (1992) studied the mutagenicity and carcinogenicity of AME and AOH and their relevance to the etiology of esophageal cancer in Linxian (China), an area with high incidence of esophageal cancer (Liu et al., 1992; Ostry, 2008). Based on the results, it was concluded that A. alternata plays an important role in the etiology of esophageal cancer (Liu et al., 1992). Despite this potential mutagenicity and carcinogenicity, there are no legislative norms for Alternaria toxins.

12 1.4. PENICILLIUM SPP. MYCOTOXINS The most widespread Penicillium spp. mycotoxin is roquefortine C (Figure 1.13), which is produced by 25 different species (Lugauskas, 2005). Among the producing fungi are Penicillium roqueforti , P. griseofulvum, P. chrysogenum, P. claviforme, P. hirsutum, P. expansum, P. crustosum, P. italicum, P. verrucosum, P. carneum, P. hordei, P. melanoconidium, and P. paneum (Bräse et al., 2009). Penicillium roqueforti is the principal species used in the production of blue-veined cheese (Roquefort, Gorgonzola) (Nelson, 1970; Scott et al., 1976).

Roquefortine C is a neurotoxin (Scott et al., 1976; Wagener et al., 1980). In cows, it causes extensive paralysis that is unresponsive to treatment with calcium, and in mice it provokes convulsive seizures (Scott et al., 1976; Häggblom, 1990). The estimated

LD 50 in mice after intraperitoneal injection is 189 mg/kg (Arnold et al., 1978). Little is known about the toxicity of ROQC for humans.

CH2 O CH3 H C 3 NH N

N H H O NH

N Figure 1.13. The structural formula of roquefortine C (ROQC)

1.5. LEGISLATION Zimbabwe has no legislation on maximum levels for mycotoxins, but generally adopts the European legislation. This European legislation was conducted to keep contaminants at levels which are toxicologically acceptable in order to protect public health. An important one is Commission Regulation (EC) No 1881/2006 of 19 December 2006, setting maximum levels for certain contaminants in foodstuffs, including mycotoxins. It states that maximum levels of mycotoxins should be set at a strict level which is reasonably achievable by following good agricultural practices and taking into account the risk related to the consumption of the food. In order to protect the public health, products exceeding the maximum levels should not be placed on the market.

13 Aflatoxins are genotoxic carcinogens (SCF, 1994). Therefore, it is appropriate to limit the total aflatoxin content in food, as well as the content of aflatoxin B 1 alone, since

AFB 1 is by far the most toxic compound (EC 1881/2006, 2006).

Maximum levels have not yet been established for every mycotoxin. The ones that have been established are shown in Table 1.4.

Zimbabwe uses European legislation, but the intake of maize in Zimbabwe is much higher than in Europe, since in the former maize is a staple food. So, eventually, Zimbabwe needs to establish its own legislation, customized to the intake of foodstuffs by the Zimbabwean people.

Table 1.4. Maximum levels for mycotoxins in foodstuffs according to EC No 1881/2006 in Europe.

Mycotoxin Foodstuffs Maximum levels (µg/kg)

Aflatoxins AFB 1 Sum of AFs Maize to be subjected to sorting or other physical treatment before 5.0 10.0 human consumption or use as an ingredient in foodstuffs Ochratoxin A Unprocessed cereals 5.0 Deoxynivalenol Unprocessed maize, with the exception of unprocessed maize 1,750 intended to be processed by wet milling Milling fractions of maize with particle size > 500 micron falling 750 within CN code 1103 13 or 1103 20 40 and other maize milling products with particle size > 500 micron not used for direct human consumption falling within CN code 1904 10 10 Milling fractions of maize with particle size ≤ 500 micron falling 1,250 within CN code 1102 20 and other maize milling products with particle size ≤ 500 micron not used for direct human consumption falling within CN code 1904 10 10 Zearalenone Unprocessed maize with the exception of unprocessed maize 350 intended to be processed by wet milling Maize intended for direct human consumption, maize-based snacks 100 and maize-based breakfast cereals Milling fractions of maize with particle size > 500 micron falling 200 within CN code 1103 13 or 1103 20 40 and other maize milling products with particle size > 500 micron not used for direct human consumption falling within CN code 1904 10 10 Milling fractions of maize with particle size ≤ 500 micron falling 300 within CN code 1102 20 and other maize milling products with particle size ≤ 500 micron not used for direct human consumption falling within CN code 190 10 10

14 Table 1.4 (continued)

Fumonisins Sum of FB 1 and FB 2 Unprocessed maize, with the exception of unprocessed maize 4,000 intended to be processed by wet milling Maize intended for direct human consumption, maize-based foods 1,000 for direct human consumption, with the exception of maize-based breakfast cereals, maize-based snacks and processed maize- based foods and baby foods for infants and young children Milling fractions of maize with particle size > 500 micron falling 1,400 within CN code 1103 13 or 1103 20 40 and other maize milling products with particle size > 500 micron not used for direct human consumption falling within CN code 1904 10 10 Milling fractions of maize with particle size ≤ 500 micron falling 2,000 within CN code 1102 20 and other maize milling products with particle size ≤ 500 micron not used for direct human consumption falling within CN code 1904 10 10 T-2 and HT-2 Unprocessed cereals 100 Maize for direct human consumption 100

15 2. OBJECTIVES

Maize is an important staple food in Zimbabwe, but mycotoxin contamination is a big problem. Since the major part of the maize production happens through small stakeholders and subsistence farming, and since there is no monitoring by the government, nor a legislation, people have no notion of the level of mycotoxin contamination or the danger that comes with it. Since maize is so important in Zimbabwe, it is important to evaluate whether mycotoxin contamination is occurring, and, if so, whether it is a potential threat to human safety.

This thesis is part of a larger study conducted by Melody Hove. The aim of this study was twofold. The first goal was to determine the presence and quantity of mycotoxins in maize obtained from subsistence farms in the provinces Mashonaland West and Manicaland, the two agricultural zones in Zimbabwe. The second goal was to study the effect of time on mycotoxin content by comparing contamination in samples taken at harvest, three months postharvest and six months postharvest, to see whether there is a difference between the different stages or not.

16 3. MATERIALS AND METHODS

3.1. SAMPLING Sampling was executed according to the method developed by Hove et al. (2016). According to rainfall characteristics, Zimbabwe is divided into six agro-ecological zones (Table 3.1). The two provinces of Manicaland and Mashonaland contain all these agro- ecological zones, except for Region I, which does not occur in Mashonaland West (Figure 3.1) (Hove et al., 2016). The sampling protocol used can be seen in Figure 3.2. Basically, only wards with subsistence farming activities located within a range of ten kilometers of an agrometeorology station were selected in each agro-ecological zone in each province. In every ward, a number of households were randomly selected (Hove et al., 2016).

Table 3.1. The six agro-ecological zones in Zimbabwe (Hove et al., 2016).

Agro -ecological zone Surface area (km²) Altitude (m) Annual rainfall (mm) Region I 7000 1100–2600 > 1000 Region IIa 41000 1100–1800 700–1000 Region IIb 18000 1100–1600 700–1000 Region III 72900 1100–1200 500–800 Region IV 147800 600–1200 450–650 Region V 104400 300–900 < 450

Figure 3.1. Geographical locations of the sampled households in accordance to the different agro-ecological zones (Hove et al., 2016).

Figure 3.2. Sampling protocol for the selection of households from different provinces and the five agro-ecological zones of Zimbabwe (BYO – Bulawayo, HRE – Harare, MCL – Manicaland, MBN – Matebeleland North, MBS – Matebeleland South, MSC – Mashonaland Central, MSE – Mashonaland East, MWS – Mashonaland West, MSV – Masvingo, MID – Midlands) (Hove et al., 2016).

For the present study, samples of homegrown maize were collected in 2015 at three different times: at harvest, three months postharvest and six months postharvest. The number of collected samples is shown in Table 3.2. In total, 158 samples were collected.

Table 3.2. Number of samples collected in Mashonaland West and Manicaland (Zimbabwe) at harvest, 3 months postharvest and 6 months postharvest.

Province Zone Number of Number of samples collected households Harvest 3 months 6 months postharvest postharvest Mashonaland West Karoi 10 8 9 8 Chinhoyi 8 8 8 6 Mhondoro 10 9 7 4 Kadoma 10 10 9 7 Total 38 35 33 25 Manicaland Chipinge 8 8 7 2 Chisengu 6 5 5 4 Nyanga 7 7 6 6 Rusape 8 7 7 1 Total 29 27 25 13

18 3.2. DETERMINATION OF MYCOTOXINS IN MAIZE The multi-mycotoxin LC-MS/MS method developed by Ediage et al. (2015) for the detection and quantification of mycotoxins was used to analyze and quantify the mycotoxin contamination in the maize samples.

3.2.1. Standards Nivalenol (NIV) in acetonitrile (100 µg/mL), fusarenon X (FX) in acetonitrile (100 µg/mL), and HT-2-toxin (HT-2) were obtained from Fermentek Ltd (Jerusalem,

Israel). Deoxynivalenol (DON), aflatoxin G 2 (AFG 2), aflatoxin G 1 (AFG 1), aflatoxin B 2

(AFB 2), aflatoxin B 1 (AFB 1), fumonisin B 1 (FB 1), fumonisin B2 (FB 2), altenariol (AOH), T-2 toxin (T-2), ochratoxin A (OTA), zearalenone (ZEN), and sterigmatocystin (STERIG) were purchased from Cfm Oskar Tropitzsch GmbH (Marktredwitz, Germany). Neosolaniol (NEO) in acetonitrile (100 µg/mL), 3-acetyldeoxynivalenol (3- ADON) in acetonitrile (100 µg/mL), 15-acetyldeoxynivalenol (15-ADON) in acetonitrile (100 µg/mL), roquefortine C (ROQC) in acetonitrile (100 µg/mL), alternariol monoethylether (AME) in acetonitrile (100 µg/mL) and the internal standard deepoxy- deoxynivalenol (DOM) were obtained from Biopure, RomerLabs. Diacetoxyscirpenol (DAS) in acetonitrile (100 µg/mL), and the internal standard zearalanone (ZAN) were purchased from Sigma (Diegem, Belgium), and fumonisin B 3 (FB 3) was obtained from South African Medical Research Council (Tygerberg, South Africa).

From the individual standards, a standard mixture was prepared with the following concentrations: AFG1, AFB2, DAS, OTA, and STERIG (1 ng/µL); T-2, AFG2, and AFB1 (2 ng/µL); NEO, ROQ C, HT-2, ZEN, and AME (2.5 ng/µL); ALT (5 ng/µL); DON (10 ng/µL); 3ADON (12.5 ng/µL); AOH (20 ng/µL); FX, and 15ADON (25 ng/µL); and NIV (40 ng/µL). This standard mixture was prepared in methanol, stored at –18°C, and renewed every 3 months. A separate mixture of fumonisins was prepared at the following concentrations: FB2, and FB3 (30 ng/µL); and FB1 (15 ng/µL). This fumonisin mixture was prepared in methanol and stored at 4°C.

3.2.2. Reagents and materials All reagents used were of analytical grade, unless specified otherwise. Ethyl acetate and dichloromethane were purchased from Acros Organics (Geel, Belgium). HPLC grade methanol and n-hexane were supplied by VWR International S.A.S. (Fontenay-

19 sous-Bois, France). Formic acid and ammonium acetate were obtained from Merck (Darmstadt, Germany), and LC-MS grade methanol and acetic acid (UPLC/MS grade) were purchased from Biosolve BV (Valkenswaard, the Netherlands).

GracePure TM Amino solid phase extraction (SPE) cartridges (1000 mg/6 mL) were used from Grace Discovery Sciences (Lokeren, Belgium). Ultrafree-MC GV centrifugal filter devices (0.22 µm) of Millipore (Merck Millipore, Tullagreen, Ireland). Water was purified with a Milli-Q plus apparatus (Merck Millipore, Brussels, Belgium).

3.2.3. Sample preparation Samples were homogenized, accurately weighed (3.000 g ± 0.003 g), spiked with internal standards ZAN (60 µL) and DOM (20 µL) and left to equilibrate in the dark for 15 minutes. Extraction was performed with 20 mL of methanol/ethyl acetate/water (70/20/10, v/v/v) on an end-over-end shaker (Agitelec, France) for 40 minutes. This was followed by centrifugation for 15 minutes at 4,000 g, and transfer of the supernatant to clean extraction tubes. Defatting was carried out by adding 10 mL of hexane to the supernatant, and shaking it on the end-over-end shaker for 15 minutes. The hexane layer (upper layer) was discarded. The lower layer, that contained the mycotoxins, was subjected to different clean-up procedures. Because fumonisins and ochratoxin A strongly bind the resin of the amino SPE cartridges, the defatted extract was split. A 5 mL portion was transferred to a fresh glass tube. From this portion, 2.5 mL was subjected to a liquid-liquid extraction with 10 mL of a dichloromethane/formic acid (90/10, v/v) solution, vortexed and centrifuged for 10 minutes at 4,000 g. The remaining fraction of the defatted extract (approximately 12.5 mL) was applied on an amino SPE column, mounted on a vacuum SPE manifold and pre-equilibrated with 12 mL (two times 6 mL) of the extraction solvent (methanol/ethyl acetate/water, 70/20/10, v/v/v). The eluate from the column was collected in a fresh glass test tube. Both phases (from the amino SPE and the dichloromethane/formic acid clean-up step) from the extract were pooled and evaporated to dryness under a gentle nitrogen flow at 60°C. This residue then was reconstituted in 300 µL of injection solvent composed of methanol/water/acetic acid (57.2/41.8/1, v/v/v) and 5 mM ammonium acetate. To this redissolved extract, 200 µL of n-hexane was added, vortexed and centrifuged for 10 minutes at 3,300 g. Thereafter, the extract was transferred in a centrifuge filter, and centrifuged for 10

20 minutes at 10,000 g. From this filtered extract, 100 µL was diluted with 100 µL of the injection solvent in a HPLC vial for analysis (Ediage et al., 2015).

3.2.4. Apparatus Following the multi-mycotoxin LC-MS/MS method developed by Ediage et al. (2015) for the detection and quantification of mycotoxins, the same settings and conditions were used. A Waters Acquity UPLC apparatus coupled to a Waters Micromass Quattro Premier XE tandem mass spectrometer was used to detect and quantify the mycotoxins in the samples. Separation of the mycotoxins was performed on a Symmetry C18 column, (5 µm, 150 mm × 2.1 mm i.d.), with a Symmetry C18 guard column (3.5 µm, 10 × 2.1 mm) (both Waters, Zellik, Belgium). Solvent A (methanol/water/acetic acid (94/5/1, v/v/v) and 5 mM ammonium acetate) and solvent B (methanol/water/acetic acid (97/2/1, v/v/v) and 5 mM ammonium acetate) were used as mobile phase. The applied gradient program at a flow rate of 0.3 mL/min is shown in Table 3.3 and Figure 3.3. The injection volume of the sample was set at 10 µL. The analytical run time was 28 minutes per sample (Ediage et al., 2015).

Table 3.3. The applied gradient program for LC-MS/MS Quattro Premier XE.

Time Mobile phase A Mobile phase B Flow (minutes) (%) (%) (mL/min) 0.00 95 5 0.3 7.00 35 65 0.3 11.00 25 75 0.3 13 .00 0 100 0.3 14 .00 0 100 0.3 14.10 95 5 0.3 17.60 35 65 0.3 18.60 0 100 0.3 19.80 0 100 0.3 19.90 95 5 0.3 22.40 35 65 0.3 23.40 25 75 0.3 25.00 0 100 0.3 26 95 5 0.3 28 95 5 0.3

21 100

gradient (%) 40

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Time (minutes)

mobile phase A mobile phase B

Figure 3.3. The applied gradient program for the detection of the different mycotoxins on LC-MS/MS Quattro Premier XE.

Table 3.4. Limit of detection and quantification for the different mycotoxins

Mycotoxin Limit of detection (µg/kg ) Limit of quantification (µg/kg ) NIV 100 175 DON 20 40 FX 62.5 125 NEO 6.25 12.5 3-ADON 31.75 62.5 15 -ADON 62.5 125

AFG 2 5 7.5

AFG 1 2.5 5

AFB 2 2.5 5

AFB 1 5 10 DAS 2.5 5 ROQC 6 12.5 HT -2 12.5 25

FB 1 12.5 25 AOH 40 80 T-2 5 7.5

FB 3 25 50 OTA 2.5 5 ZEN 6.25 12.5 STERIG 2.5 5

FB 2 25 50 AME 6.25 12.5

22 Table 3.5. LC-MS/MS parameters applied for the determination of mycotoxins.

Predicted Ionisation Precursor ion Product ions Collision Cone Voltage Component retention time mode (m/z) (m/z) Energy (eV) (V) (min) 124.96* 10 NIV ESI + 313.0 23 3.18 177.21 15 203.34* 12 DON ESI + 297.0 26 4.29 249.47 13 175.28* 11 FX ESI + 355.1 22 5.26 247.27 9 305.26* 13 NEO ESI + 400.1 20 5.39 365.07 10 109.21* 18 DOM ESI + 281.2 23 5.48 137.19 25 231.23* 10 3-ADON ESI + 339.2 23 6.47 203.18 10 137.10* 10 15-ADON ESI + 339.1 24 6.49 321.20 8 313.15* 24 AFG ESI + 331.0 46 6.99 2 245.30 30 311.17* 25 AFG ESI + 328.9 43 7.21 1 243.36 20 287.30* 25 AFB ESI + 315.0 50 7.60 2 259.40 29 285.27* 21 AFB ESI + 313.0 47 7.87 1 241.42 34 307.28* 11 DAS ESI + 384.2 19 7.97 247.27 15 193.30* 26 ROQC ESI + 390.2 40 8.77 322.24 21 215.27* 10 HT-2 ESI + 441.9 16 9.26 323.00 12 334.36* 37 FB ESI + 722.5 51 9.35 1 352.26 36 185.17* 30 AOH ESI + 259.1 53 10.02 213.15 25 305.21* 12 T-2 ESI + 484.3 12 10.17 245.21 12 336.30* 35 FB ESI + 706.4 51 10.53 3 354.00 29 239.13* 22 OTA ESI + 404.0 24 11.14 101.90 35 303.20* 14 ZAN ESI + 321.2 27 11.19 189.22 21 301.24* 12 ZEN ESI + 319.2 22 11.50 238.34 18 310.16* 35 STERIG ESI + 325.0 44 11.73 281.33 25 336.26* 35 FB ESI + 706.4 51 11.94 2 318.00 38 258.14* 26 AME ESI + 273.0 54 12.66 199.20 25

23 3.2.5. Statistics and data analysis Analysis and quantification of the data was performed using Waters MassLynx V4.1 and Waters QuanLynx V4.1 (Waters Laboratory Informatics, Zellik, Belgium), respectively. Microsoft Office Excel 2007 was used for statistical analysis of the obtained data. The independent t-test was used to determine significance of the difference between datasets at a confidence level of 95% ( https://www.mccallum- layton.co.uk/tools/statistic-calculators/independent-t-test-calculator/ ).

24 4. RESULTS AND DISCUSSION

4.1. INVESTIGATION OF THE CONTAMINATION LEVEL OF MYCOTOXINS IN MAIZE A total of 158 samples were tested for the presence of 22 different mycotoxins. The general contamination of the samples with mycotoxins (regardless of moment of sampling) is shown in Table 4.1. Of the 158 samples tested, 46% were positive for at least one mycotoxin. FX, 15-ADON, DAS, and AFG 1 each only occurred in one sample, at contents of 130.00 µg/kg, 233.00 µg/kg, 9.60 µg/kg, and 14.80 µg/kg respectively. Two out of 158 samples (1%) were contaminated with NIV, with a mean of 2.64 µg/kg (± 23.44 µg/kg, CI95 ± 3.65 µg/kg), and a maximum of 220.60 µg/kg. Zearalenone (ZEN) was detected in five samples (3%), with a mean of 0.94 µg/kg (± 5.75 µg/kg, CI95 ± 0.90 µg/kg) and a maximum level of 44.60 µg/kg. Also STERIG occurred in five out of 158 samples (3%), with a mean level of 0.24 µg/kg (± 1.97 µg/kg, CI95 ± 0.31 µg/kg) and a maximum of 23.70 µg/kg. Furthermore, DON was found in 10 samples (6%), at a mean contamination level of 7.32 µg/kg (± 33.19 µg/kg, CI95 ± 5.18 µg/kg) and a maximum level of 246.50 µg/kg. None of the samples were quantified to be contaminated with the mycotoxins NEO, 3-ADON, HT-2, T-2, AFB 1,

AFB 2, AFG 2, OTA, AOH, AME, or ROQC.

The mycotoxins most abundantly present were the fumonisins. Sixty-nine of the samples (44%) contained at least one fumonisin. Of all three fumonisins, FB 1 was most abundantly present, in 63 out of 158 samples (40%), with a mean of 243.30 µg/kg (± 630.68 µg/kg, CI95 ± 98.34 µg/kg) and a maximum of 3,865.89 µg/kg. Next in ranking was FB 2, which was detected in 38 samples (22%). The mean contamination for this mycotoxin was 52.22 µg/kg (± 144.23 µg/kg, CI95 ± 22.49 µg/kg), and the maximal amount detected was 819.01 µg/kg. Fumonisin B 3 was only detected in 18 of the samples (11%), and at the lowest levels. The mean level of contamination was 13.67 µg/kg (± 51.61 µg/kg, CI95 ± 8.05 µg/kg), and the highest amount detected in the samples was 472.20 µg/kg. Total fumonisin content was calculated, and results showed a mean contamination level of 309.18 µg/kg (± 804.97 µg/kg, CI95 ± 125.52 µg/kg), and a maximum of 4,737.53 µg/kg.

Of the 158 samples tested, 18% (n = 29) contained just one mycotoxin. It concerns

FB 1 (n = 22), DON (n = 3), STERIG (n = 2), ZEN (n = 1), and FB 2 (n = 1). 43 samples

25 (27%) were contaminated with two or more mycotoxins. 23 samples (15%) contained three or more mycotoxins, and nine samples (6%) were contaminated with four mycotoxins. Co-occurrence of the mycotoxins found in the tested samples is shown in Figure 4.1. The mycotoxins most often co-occurring in the samples were the fumonisins. FB 1 and FB 2 were found together in 9% of the samples (n = 15), and all three fumonisins together (FB 1, FB 2, and FB 3) occurred in 7% of the tested samples (n = 11). As can be seen in Figure 4.1, also the other combinations of mycotoxins mostly contained at least one fumonisin.

Figure 4.1. General co-occurrence of mycotoxins in subsistence farmed maize from Zimbabwe.

The European Union has set up a legislation to keep contaminants at levels which are toxicologically acceptable in order to protect public health. Since Zimbabwe has no legislation of its own, it generally adopts the European legislation. The maximum levels set for mycotoxins are shown in Table 1.4. Of all the samples tested, only two exceeded a limit. One sample exceeded the limit of 4000 µg/kg for the sum of FB 1 and

FB 2 in unprocessed maize (4,265.33 µg/kg). The other sample exceeded the limit of 10 µg/kg for aflatoxins. In this last sample, this exceedance was solely due to the

26 presence of AFG 1 (14.80 µg/kg). The batches of maize from which these samples were collected, should be rejected.

4.2. DETERMINATION OF MYCOTOXIN LEVELS AT THREE STAGES (HARVEST, THREE MONTHS POSTHARVEST AND SIX MONTHS POSTHARVEST), AND IN TWO DIFFERENT AGRICULTURAL ZONES (MANICALAND AND MASHONALAND WEST) All the samples tested, were collected at different stages, namely at harvest (n = 62), three months postharvest (n = 58), and at six months postharvest (n = 38). Table 4.2, Table 4.3, and Table 4.4 represent the mycotoxin contamination of the samples at harvest, three months postharvest, and six months postharvest, respectively. Figure 4.2 and Figure 4.3 show the changes over time in average mycotoxin contamination for the mycotoxins that were detected in the tested samples. The only mycotoxins that showed a significant change over time were the fumonisins. The mean content of FB 1 at harvest was 136.51 µg/kg (± 536.40 µg/kg) and increased to 374.99 µg/kg (± 768.92 µg/kg) at three months postharvest. This increase was significant (P < 0.05). At six months postharvest, the mean content significantly decreased again to

216.52 µg/kg (± 505.54 µg/kg, P < 0.05). The same trend was seen for FB 2. The sample taken at harvest contained on average 16.50 µg/kg (± 74.79 µg/kg), significantly rising to 92.76 µg/kg (± 203.80 µg/kg, P < 0.05), and thereafter significantly lowering to 48.62 µg/kg (± 101.66 µg/kg, P < 0.05). For FB 3, the average amount was 10.78 µg/kg (± 62.22 µg/kg) at harvest, compared 18.85 µg/kg (± 51.06 µg/kg) three months after harvest, and 10.48 µg/kg (± 28.94 µg/kg) six months after harvest. The differences between all three stages of sample taking were significant (P < 0.05).

Physiological stress during the period just before harvest, due to drastic oscillations in rainfall and relative humidity, is likely to create favorable conditions for fumonisin production (Visconti, 1996). Fusarium verticillioides and Fusarium proliferatum only grow at relatively high water activities (a w > 0.9), so they are normally formed in maize prior to harvest or during the early stage of storage, and their concentration does not increase during storage (Marin et al., 2013). However, this study shows that, overall, fumonisin content still increased until three months after harvest. The occurrence of extreme conditions, such as improper storage facilities and/or weather conditions

27 (periods of rainfall, great humidity) can explain this phenomenon. Unfortunately, no data on storage or weather conditions were available.

Not only changes in weather and inappropriate storage affect the development of Fusarium verticillioides and Fusarium proliferatum , and the synthesis of fumonisins. It has been reported by Bilgrami et al. (1998) that late planting of maize with harvesting in wet conditions also favors disease caused by these fungi, and that repeated planting of maize and other cereal crops in the same or in nearby fields favors fungal infection by increasing the fungal inoculums and insect population that attack maize plants (Bilgrami et al., 1998). Therefore, crop rotation with a nonhost crop of Fusarium is recommended, since these crops are less favorable to Fusarium outbreak than maize (Lipps et al., 1991).

Mechanical damage during and after harvest may offer entry to the fungal spores either in the maize cobs or grains (Fandohan et al., 2013). Damage can also be caused by insects, which play an important role in the infection of maize by fungi (Dowd, 1998; Fandohan et al., 2013). They can act as wounding agents, or as vectors spreading the fungus from origin of inoculums to plants (Dowd, 1998). Any action to avoid insect infestation therefore is useful for reducing infection of maize with mycotoxigenic Fusarium species (Riley et al., 1999).

All of the above factors may explain the abundant presence of fumonisins in the tested samples. However, proving the influence of these factors is not a part of this thesis.

28 0.00 2.00 4.00 6.00 8.00 10.00 12.00

NIV

DON

FX mean harvest 15ADON mean 3MPH

DAS mean 6MPH

ZEN

AFG1

STERIG

Figure 4.2. The trend in mean mycotoxin content (excluding fumonisins) over time in maize from Zimbabwe (3MPH – 3 months postharvest, 6MPH – 6 months postharvest).

0.00 100.00 200.00 300.00 400.00 500.00

FB1

FB2 mean harvest mean 3MPH mean 6MPH FB3

FB ALL

Figure 4.3. The trend in mean fumonisin content over time in maize from Zimbabwe (3MPH – 3 months postharvest, 6MPH – 6 months postharvest).

The co-occurrence of mycotoxins at the different stages of sampling are shown in Figure 4.4. It is clear that all three fumonisins are present together throughout the time of this study.

29 FB2

70 ZEN

3 FB1 + ZEN 3 60 STERIG 3

5 FB1 + FB2 + FB3 + STERIG

50 FB1 + FB2 + FB3 + DAS 2 5 2 2 3 FB1 + DON 3 5 FB1 + FB2 + DON + AFG1 40 3 3 % FB1 + FB2 + DON + 15ADON 3 14 3 30 FB1 + FB2 + FB3 + DON

FB1 + NIV 2 2 2 16 FB1 + FB2 + FB3 + ZEN 20 2 14 DON 13 10 FX + NIV + ZEN

16 DON + FB1 + FB2 5 12 0 FB1 + FB2 + STERIG

Harvest 3 FB1 + FB2 3 MPH FB1 6 MPH FB1 + FB2 + FB3

Figure 4.4. Co-occurrence of mycotoxins over time in subsistence farmed maize from Zimbabwe (3 MPH – 3 months postharvest, 6 MPH – 6 months postharvest)

Samples were not only collected at different time periods, but also in two different agricultural zones in Zimbabwe: Mashonaland West and Manicaland provinces. This allows to compare mycotoxin contamination between geographical zones. In Mashonaland West, 93 samples were collected: 35 at harvest, 33 three months postharvest, and 25 six months postharvest. In Manicaland, 65 samples were collected: 27 at harvest, 25 at three months postharvest, and 13 at six months postharvest.

Since fumonisins are the biggest problem in Zimbabwe, comparison between Mashonaland West and Manicaland will be restricted to these mycotoxins. Figure 4.5 and Figure 4.6 display the total fumonisin content in the positive maize samples per time period and per household in Mashonaland West and Manicaland, respectively. Contamination of maize with fumonisins occurs more in Mashonaland West than in Manicaland. From the 38 households in Mashonaland West where samples were collected, 71% (n = 27) tested positive for one or more fumonisins. In comparison, out of the 29 households in Manicaland, only in 59% (n = 17) of them fumonisins were found. From the samples collected at harvest, 29% (n = 10) of the ones from Mashonaland showed the presence of fumonisins, compared to 15% (n = 4) in Manicaland.

30 Not only was the prevalence of fumonisins higher in Mashonaland West, also the content of fumonisins was significantly higher in Mashonaland West compared to Manicaland. The mean content of total fumonisins in Mashonaland West (for the three time periods of sampling, n = 93), was 392.95 µg/kg (± 190.65 µg/kg, 95% CI) versus 189.33 µg/kg (± 133.12 µg/kg, 95% CI) in Manicaland (n = 65). This difference was significant (P < 0.05).

The same trend was seen for the three different periods of sampling. At harvest, mean total fumonisin quantity in the samples obtained from households in Mashonaland West was 191.16 µg/kg (± 267.18 µg/kg, 95% CI, n = 35), compared to 128.31 µg/kg (95% CI ± 156.31 µg/kg, n = 27). This difference was also significant (P < 0.05). After three months of storage, the mean total fumonisin content showed a significant increase, both in Mashonaland West (609.77 µg/kg (± 392.90 µg/kg, 95% CI)) and in Manicaland (324.01 µg/kg (± 297.11 µg/kg, 95% CI)). After six months of storage, the mean quantity of total fumonisin decreased significantly to 389.25 µg/kg (± 293.23 µg/kg, 95% CI) in Mashonaland West, and to 57.08 µg/kg (± 61.80 µg/kg, 95% CI) in Manicaland.

The higher prevalence and levels of fumonisins in Mashonaland West are in accordance with other worldwide surveys proving that levels of fumonisins are higher in warmer and drier climates (Shephard et al., 1995). Mashonaland West has a somewhat warmer climate (average annual temperature 19.8°C versus 18°C, respectively) and less rainfall (803 mm/year versus 963 mm/year) than Manicaland (http://en.climate-data.org/ ). Hove et al. (2016) found similar results in maize sampled one year previously in the same regions.

31 Table 4.1. General mycotoxin contamination in maize sampled from subsistence farming households in Manicaland and Mashonaland West provinces of Zimbabwe

N = 158 Mycotoxin contamination Alternaria Penicillium Fusarium spp. toxins Aspergillus spp. toxins spp. toxins spp. toxins NIV DON NEO FX 3ADON 15ADON DAS HT2 T2 ZEN FB1 FB2 FB3 AFB1 AFB2 AFG1 AFG2 OTA STERIG AOH AME ROQC Min (µg/kg) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Max 220.60 246.50 n.d. 130.00 n.d. 233.00 9.60 n.d. n.d. 44.6 3,865.89 819.01 472.20 n.d. n.d. 14.8 n.d. n.d. 23.7 n.d. n.d. n.d. (µg/kg) Median n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. (µg/kg) % positive 1.27 6.33 0.0 0.63 0.0 0.63 0.63 0.0 0.0 3.16 39.87 22.15 11.39 0.0 0.0 0.63 0.0 0.0 3.16 0.0 0.0 0.0 Mean 2.64 7.32 0.0 0.82 0.0 1.47 0.06 0.0 0.0 0.94 243.30 52.22 13.67 0.0 0.0 0.09 0.0 0.0 0.24 0.0 0.0 0.0 (µg/kg) S.D. 23.44 33.19 0.0 10.34 0.0 18.54 0.76 0.0 0.0 5.75 630.68 144.23 51.61 0.0 0.0 1.18 0.0 0.0 1.97 0.0 0.0 0.0 (µg/kg) 95% CI ± 3.65 ± 5.18 ± 0.0 ± 1.61 ± 0.0 ± 2.89 ± 0.12 ± 0.0 ± 0.0 ± 0.90 ± 98.34 ± 22.49 ± 8.05 ± 0.0 ± 0.0 ± 0.18 ± 0.0 ± 0.0 ± 0.31 ± 0.0 ± 0.0 ± 0.0 n.d. – not detected S.D. – Standard Deviation 95% CI – 95% Confidence Interval

Table 4.2. Mycotoxin contamination in maize sampled at harvest from subsistence farming households in Manicaland and Mashonaland West provinces of Zimbabwe.

N = 62 Mycotoxin contamination Alternaria Penicillium Fusarium spp. toxins Aspergillus spp. toxins spp. toxins spp. toxins NIV DON NEO FX 3ADON 15ADON DAS HT2 T2 ZEN FB1 FB2 FB3 AFB1 AFB2 AFG1 AFG2 OTA STERIG AOH AME ROQC Min n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. (µg/kg) Max 221 184 n.d. 130 n.d. n.d. n.d. n.d. n.d. 16.3 3,866 431 472 n.d. n.d. n.d. n.d. n.d. 2.50 n.d. n.d. n.d. (µg/kg) Median n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. (µg/kg) % positive 1.61 1.61 0.0 1.61 0.0 0.0 0.0 0.0 0.0 1.61 22.6 9.68 4.84 n.d. n.d. n.d. n.d. n.d. 1.61 n.d. n.d. n.d. Mean 3.56 2.96 0.0 2.10 0.0 0.0 0.0 0.0 0.0 0.26 137 16.5 10.8 0.0 0.0 0.0 0.0 0.0 0.04 0.0 0.0 0.0 (µg/kg) S.D. (µg/kg) 28.0 23.3 0.0 16.5 0.0 0.0 0.0 0.0 0.0 2.07 536 74.8 62.2 0.0 0.0 0.0 0.0 0.0 0.32 0.0 0.0 0.0 95% CI ± 6.97 ± 5.81 ± 0.0 ± 4.11 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.52 ± 133 ± 18.6 ± 15.5 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.08 ± 0.0 ± 0.0 ± 0.0 n.d. – not detected S.D. – Standard Deviation 95% CI – 95% Confidence Interval

32 Table 4.3. Mycotoxin contamination in maize sampled 3 months postharvest from subsistence farming households in Manicaland and Mashonaland West provinces from Zimbabwe.

N = 58 Mycotoxin contamination Alternaria Penicillium Fusarium spp. toxins Aspergillus spp. toxins spp. toxins spp. toxins NIV DON NEO FX 3ADON 15ADON DAS HT2 T2 ZEN FB1 FB2 FB3 AFB1 AFB2 AFG1 AFG2 OTA STERIG AOH AME ROQC Min (µg/kg) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Max (µg/kg) 197 247 n.d. n.d. n.d. 233 n.d. n.d. n.d. 30.8 2,842 819 275 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Median n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. (µg/kg) % positive 1.72 6.90 0.0 0.0 0.0 1.72 0.0 0.0 0.0 3.45 48.3 32.8 17.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Mean 3.39 10.2 0.0 0.0 0.0 4.02 0.0 0.0 0.0 0.73 375 92.8 18.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 (µg/kg) S.D. (µg/kg) 25.8 43.5 0.0 0.0 0.0 30.6 0.0 0.0 0.0 4.30 769 204 51.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 95% CI ± 6.65 ± 11.2 ± 0.0 ± 0.0 ± 0.0 ± 7.87 ± 0.0 ± 0.0 ± 0.0 ± 1.11 ± 198 ± 52.5 ± 13.1 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 n.d. – not detected S.D. – Standard Deviation 95% CI – 95% Confidence Interval

Table 4.4. Mycotoxin contamination in maize sampled 6 months postharvest from subsistence farming households in Manicaland and Mashonaland West provinces from Zimbabwe.

N = 38 Mycotoxin contamination Alternaria Penicillium Fusarium spp. toxins Aspergillus spp. toxins spp. toxins spp. toxins NIV DON NEO FX 3ADON 15ADON DAS HT2 T2 ZEN FB1 FB2 FB3 AFB1 AFB2 AFG1 AFG2 OTA STERIG AOH AME ROQC Min (µg/kg) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Max (µg/kg) n.d. 115 n.d. n.d. n.d. n.d. 9.60 n.d. n.d. 44.6 2,709 424 121 n.d. n.d. 14.8 n.d. n.d. 23.7 n.d. n.d. n.d. Median n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 44.5 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. (µg/kg) % positive 0.0 13.2 0.0 0.0 0.0 0.0 2.63 0.0 0.0 5.26 55.3 34.2 13.2 0.0 0.0 2.63 0.0 0.0 10.5 0.0 0.0 0.0 Mean 0.0 10.0 0.0 0.0 0.0 0.0 0.25 0.0 0.0 2.35 217 48.6 10.5 0.0 0.0 0.39 0.0 0.0 0.94 0.0 0.0 0.0 (µg/kg) S.D. 0.0 28.5 0.0 0.0 0.0 0.0 1.56 0.0 0.0 10.1 506 102 28.9 0.0 0.0 2.40 0.0 0.0 3.96 0.0 0.0 0.0 (µg/kg) 95% CI ± 0.0 ± 9.06 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.50 ± 0.0 ± 0.0 ± 3.21 ± 161 ± 32.3 ± 9.20 ± 0.0 ± 0.0 ± 0.76 ± 0.0 ± 0.0 ± 1.26 ± 0.0 ± 0.0 ± 0.0 n.d. – not detected S.D. – Standard Deviation 95% CI – 95% Confidence Interval

33 5,000.00

4,500.00

4,000.00

3,500.00

3,000.00

2,500.00 total fumonisins (ppb) fumonisinstotal

2,000.00

1,500.00

1,000.00

500.00

0.00 MSW064 MSW066 MSW067 MSW068 MSW070 MSW072 MSW073 MSW075 MSW076 MSW077 MSW078 MSW079 MSW081 MSW082 MSW083 MSW085 MSW087 MSW088 MSW090 MSW110 MSW093 MSW097 MSW098 MSW101 MSW102 MSW103 MSW104 Harvest 0.00 0.00 0.00 0.00 0.00 4,737.53 0.00 0.00 49.48 38.27 0.00 267.47 37.38 0.00 799.65 0.00 120.96 171.79 431.20 0.00 0.00 0.00 0.00 36.77 0.00 3 MPH 0 35.4 0.00 122.97 1,244.15 3,660.95 2,534.05 3,552.18 130.00 0.00 2,793.86 365.74 0.00 75.53 606.00 145.88 1,585.90 3,134.14 35.29 0.00 0.00 100.31 6 MPH 169.96 526.27 100.88 293.81 0.00 1,593.70 678.74 94.04 0.00 160.82 64.84 711.76 56.35 32.57 0.00 3,254.09 72.42 77.31 227.03 1,616.55

Figure 4.5. Total fumonisin content (in µg/kg) found in maize per household over time in Mashonaland West (3 MPH – 3 months postharvest, 6 MPH – 6 months postharvest). Only households where fumonisins were found are displayed.

34 3,500.00

3,000.00

2,500.00

2,000.00

1,500.00 total fumonisins fumonisins (ppb) total

1,000.00

500.00

0.00 MCL003 MCL004 MCL005 MCL006 MCL011 MCL012 MCL026 MCL027 MCL030 MCL036 MCL040 MCL041 MCL051 MCL061 MCL062 MCL063 MCL064 Harvest 0.00 0.00 0.00 0.00 0.00 1,941.77 0.00 0.00 0.00 0.00 329.99 0.00 995.47 0.00 0.00 0.00 197.25 3 MPH 835.68 110.46 2,394.98 59.62 159.20 523.67 46.60 0.00 270.84 90.79 0.00 0.00 0.00 160.04 381.13 3,067.29 0.00 6 MPH 0.00 0.00 139.62 87.76 73.83 36.48 0.00 404.30

Figure 4.6. Total fumonisin content (in µg/kg) found in maize per household over time in Manicaland (3 MPH – 3 months postharvest, 6 MPH – 6 months postharvest). Only households where fumonisins were found are displayed..

35 5. CONCLUSION

This study showed the presence of fusarenon X, 15-acetyldeoxynivalenol, diacetyoxyscirpenol, nivalenol, sterigmatocystin, aflatoxin G 1, zealarenone, deoxynivalenol, and fumonisins B 1, B 2, and B 3 in subsistence farmed maize from Zimbabwe. Especially the toxicologically relevant fumonisins were the most abundantly present.

Over a quarter of the samples contained two or more mycotoxins. The mycotoxins most often co-occurring were the fumonisins. Also the other combinations (apart from solely fumonisins) contained at least one fumonisin.

Only one sample exceeded the legal limit of 4,000 µg/kg for fumonisins. However, since the tolerable daily intake (TDI) of fumonisins B 1, B 2, and B 3 (alone or in combination) is set at 2 µg/kg bodyweight, and considering that maize is a staple food in Zimbabwe, some of the other samples may also be unacceptable for consumption.

The samples collected in Mashonaland West showed significantly higher contents of fumonisins. This was in accordance with a previous study conducted Hove et al. (2016), where similar results were found. This result was also in accordance with other worldwide surveys proving that levels of fumonisins are higher in warmer and drier climates (Shephard et al., 1995). Compared to Manicaland, Mashonaland West has a somewhat warmer and drier climate.

This study showed that, in general, fumonisin content in maize increases during the first months of storage. From this, it can be concluded that not only prevention of fungal growth on the field is important, but also appropriate postharvest practices are necessary. The application of grain protection chemicals, frequently turning of the maize during storage, and sorting the maize prior to processing were found to significantly reduce fumonisin contamination in maize (Hove et al., 2016). Especially the last two practices are realizable in subsistence farms in Zimbabwe.

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47 APPENDIX A: Raw data

1. RAW DATA OBTAINED FROM THE SAMPLES FROM MASHONALAND WEST IN ZIMBABWE

Household NIV DON NEO Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MSW064 Karoi

I Household FX 3ADON 15ADON Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MSW064 Karoi

II Household DAS HT -2 T2 Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MSW064 Karoi

III Household ZEN OTA STERIG Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MSW064 Karoi

IV Household FB 1 FB 2 FB 3 Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MSW064 Karoi

V Household FB 1 + FB 2 Total FB AME Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MSW064 Karoi

VI Household AFB 1 AFB 2 AFG 1 Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MSW064 Karoi

VII Household AFG 2 ROQC AOH Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MSW064 Karoi

VIII 2. RAW DATA OBTAINED FROM THE SAMPLES FROM MANICALAND IN ZIMBABWE

Household NIV DON NEO FX 3ADON Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MCL001 Chipinge

IX Household 15ADON DAS HT -2 T2 ZEN Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MCL001 Chipinge

X Household FB 1 FB 2 FB 3 OTA STERIG Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MCL001 Chipinge

XI Household FB 1 + FB 2 Total FB AOH AOH ROQC Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MCL001 Chipinge

XII Household AFB 1 AFB 2 AFG 1 AFG 2 Zone code Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH Harvest 3MPH 6MPH MCL001 Chipinge

XIII APPENDIX B: INTERNATIONALIZATION @ HOME

1. ANNEMIE SERLIPPENS – PERSONEN MET EEN DRUGS- PROBLEMATIEK: “KLANT” IN DE APOTHEEK EN BIJ JUSTITIE?

Annemie Serlippens is sinds 1999 Substituut-Procureur-des-Konings te Gent met een specialisatie in georganiseerde misdaad en drugs. In haar lezing schetste ze ons een beeld over de drugs- en geneesmiddelenverslaafde, en gaf ons enkele inzichten betreffende de problematieken waarmee dergelijke personen te kampen hebben, want als apotheker zullen wij in het werkveld hoogstwaarschijnlijk vaak in contact komen met mensen met een drugsprobleem.

Annemie Serlippens stond mee aan de wieg van de Drugsbehandelingskamer in Gent, een initiatief waarbij de drugsverslaafde een intensieve begeleiding krijg, in plaats van strafrechtelijke vervolging. Deze aanpak lijkt veel meer zijn vruchten af te werpen dan de vorige aanpak (waarbij druggebruikers gestraft werden met boetes en soms zelfs gevangenistijd).

Ook was Annemie Serlippens verantwoordelijk voor de implementatie van “Amnesty Bins” bij grote festivals. Dit zijn vuilbakken waar festivalgangers hun drugs kunnen in smijten zonder hiervoor een boete te krijgen.

2. BERNARD VRIJENS – THE PHARMACIST IS A KEY STAKEHOLDER IN MEASURING AND MANAGING PATIENTS’ ADHERENCE TO MEDICATION.

Bernard Vrijens is Chief Science Officer at MWV Healthcare. He leads research programmes investigating the most common dosing errors with patients, and how to optimize these.

Medication inadherence is a huge problem with people on chronic medication therapy. They do not always see the health profit brought by their daily medication, and so they sometimes “forget” to take it. It is our role as a pharmacist to make chronic patients understand the importance of their therapy, and how important it is for them to continue, because even though they don’t always feel the effect, that does not mean there is no effect. To help the patient optimize his medication adherence, we can offer

XIV him some tools, such as a pill organizer, so that the patient can see whether or not he/she already took his dose for the day.

3. WIM BRAECKMANS – GLOBALIZATION @ JANSSEN: WHY WOULD I BE INTERESTED?

Wim Braeckmans works for Janssen Pharmaceutica, a Belgian pharmaceutical company that, in 1961, was incorporated in the multinational company of Johnson & Johnson. Johnson & Johnson is a major producer of healthcare products, medicines and medical devices, and employs over 120,000 people.

Johnson & Johnson is a global company. Although the term “global company” sometimes has a bad connotation, globalization is sometimes necessary to address larger problems. One rule in business is that a company needs to match the complexicity of the world it is interacting with. By being a global company, very diverse people from all over the world are employed, offering the company a large diversity in cultures, and that helps to connect with people’s needs across very different communities.

Global companies have good and bad aspects. The challenge is to make sure the good ones outweigh the bad ones.