Assessment of aflatoxin b1 contamination in maize and awareness of aflatoxins in son la province

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE NGUYEN THI THANH XUAN ASSESSMENT OF AFLATOXIN B1 CONTAMINATION IN MAIZE AND AWARENESS OF AFLATOXINS IN SON LA PROVINCE Supervisor : 1.. THE

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

NGUYEN THI THANH XUAN

ASSESSMENT OF AFLATOXIN B1 CONTAMINATION

IN MAIZE AND AWARENESS OF AFLATOXINS

IN SON LA PROVINCE

Supervisor : 1 Dr Hu Suk Lee

2 Dr Nguyen Thi Thanh Thuy

AGRICULTURAL UNIVERSITY PRESS - 2017

Nguyen Thi Thanh Xuan

ACKNOWLEDGEMENTS

First of all, I am really grateful my supervisors Dr Hu Suk Lee from International Livestock Research Institute (ILRI) for his patience, enthusiasm, motivation and immense knowledge, and to Dr Nguyen Thi Thanh Thuy from Vietnam National University of Agriculture, who gave me the opportunity to carry out this thesis and always had a piece of advice ready regarding the practical aspect of my work Their guidance supported me during this research

I wish to express my gratitude to Prof Nguyen Viet Hung, a Southeast Asia regional representative at ILRI in Vietnam, for giving me a profound insight into the research and sharing his valuable experience with me I always bear in mind the enthusiasm Prof Delia Grace and Dr Johanna Lindahl at ILRI in Nairobi I highly appreciate Ms Nguyen Le Thanh and Ms Le My Hanh for their help in the administrative aspect Eventually, I would like to thank to all the members at ILRI in Vietnam for giving me the opportunity to become temporarily a part of their team

I owe my thanks to all staffs in Plant Protection Research Institute They helped

me so much for conducting sampling, interview, data collection and laboratory analysis Many thanks particularly to the whole team, who went to Son La for their logistical support and suggestion

I also express my admiration to all members in Son La who introduced me to the locals and supervised our team during sampling and interview process I am also very thankful to Son La residents who warmly welcomed our team in their homes and participated in the survey

Last but not least, I want to convey my gratefulness to my family and friends for their unremitting support, patience and understanding

Hanoi, May 10th, 2017 Master candidate

Nguyen Thi Thanh Xuan

LIST OF CONTENTS

Declaration i

Acknowledgements ii

List of contents iii

List of abbreviations v

List of tables vii

List of figures viii

Thesis abstract ix

Part 1 Introduction 1

1.1 Introduction 1

1.2 Research objectives 2

Part 2 Literature review 3

2.1 Situation in the maize production in vietnam – case of Son La 3

2.1.1 Situation in the maize production in Vietnam 3

2.1.2 Study location - Son La province 4

2.2 Aflatoxins and their impacts on human and animal health 8

2.2.1 Aflatoxins 8

2.2.2 The impact of aflatoxins on human and animal health 9

2.3 Factors influencing aspergillus spp infection and aflatoxin development in maize 13

2.4 Situation in the aflatoxin contamination in maize 15

2.4.1 Situation in the aflatoxin contamination in the world 15

2.4.2 Situation in the aflatoxin contamination in Vietnam 16

2.5 Methods to detect aflatoxins 17

2.6 Regulations for aflatoxins in the world and Vietnam 18

2.6.1 Regulations for aflatoxins in the world 18

2.6.2 Regulations for aflatoxins in Vietnam 21

Part 3 Materials and methods 24

3.1 Materials 24

3.1.1 Maize samples 24

3.1.2 The Sigma-Aldrich® Aflatoxin B1 Low Matrix ELISA kit 24

3.2 Methods 24

3.2.1 Sample size calculation and selection 24

3.2.2 Sampling 25

3.2.3 Aflatoxin B1 analysis in Lab 25

3.2.4 Questionnaire survey 27

3.2.5 Survey data analysis 27

Part 4 Results and discussions 30

4.1 Aflatoxin B1 contamination in maize in Son La 30

4.2 Awareness of aflatoxins in Son La 36

Part 5 Conclusions and recommendations 46

5.1 Conclusions 46

5.2 Recommendations 46

References 47

Appendix 58

LIST OF ABBREVIATIONS

Acronym Abbreviations

A flavus Aspergillus flavus

A nomius Aspergillus nomius

A parasiticus Aspergillus paraciticus

EFTA European Free Trade Association

ELISA Enzyme Linked Immunosorbent Assay

F moniliforme Fusarium moniliforme

FAO Food and Agriculture Organization of the United Nations

FB1 Fumonisin B1

FDA Food and Drug Administration

GAPs Good agricultural practices

GPS Global positioning system

HBV Hepatitis B virus

HCC Hepatocellular Carcinoma

HPLC High-performance liquid chromatography

IARC International Agency for Research on Cancer

LOD Limit of detection

µg/kg Microgram per kilogram

LIST OF TABLES

Table 2.1 Area under maize, maize production and yield of districts in Son La

in 2013 7

Table 2.2 The property of aflatoxins 8

Table 2.3 Limits of aflatoxin growth and production by A flavus and A parasiticus 14

Table 2.4 The FDA action levels for aflatoxins in human food and livestock feed 20

Table 2.5 Vietnamese limits for aflatoxins in human food 22

Table 4.1 Awareness of moldy contamination in maize in Son La 30

Table 4.2 Prevalence of AFB1 contamination in maize in Son La 32

Table 4.3 Awareness of molds among 107 respondents in each category corresponding questions with answers 39

Table 4.5 Practices associated with consumption of maize, meat and milk 42

Table 4.6 Practices related to the medical examination 45

LIST OF FIGURES

Figure 2.1 Agro-ecological regions in Vietnam 3

Figure 2.2 The administrative map in Son La province 4

Figure 2.3 Area under maize, maize production and yield in Son La province 6

Figure 2.4 Chemical structure of aflatoxins 9

Figure 3.1 The map shows sampling sites in Son La province 25

Figure 4.1 Difference in the prevalence of AFB1 contamination in maize 33

Figure 4.2 Socio- demographic characteristics of respondents 37

THESIS ABSTRACT

Master candidate: Nguyen Thi Thanh Xuan

Thesis title: Assessment of aflatoxin B1 contamination in maize and awareness of aflatoxins in Son La province

Major:Food Technology Code: 60540103

Educational organization: Vietnam National University of Agriculture (VNUA) Research Objectives

This research was conducted to evaluate the prevalence of aflatoxin B1

contamination in maize and residents’ awareness of aflatoxins in Son La province Materials and Methods

Materials included maize samples and the Sigma-Aldrich® Aflatoxin B1 Low Matrix ELISA kit

Maize samples were randomly collected at the end of the rainy season from 25 communes in 5 districts in Son La with recording GPS location All maize samples were analysed for AFB1 contamination in Lab, using the Sigma-Aldrich® Aflatoxin B1 Low Matrix ELISA kit Besides, questionnaire survey was simultaneously carried out with maize collection using face-to-face interviews Eventually, all data were imported into Microsoft Excel 2010 and analyzed using STATA (version 14.0, StataCorp, College Station, TX, USA) ArcGIS version 10.4.1 ArcMap (ESRI, Redlands, CA, USA) was used to create the map

Main findings and conclusions

The prevalence of AFB1 contamination in maize in Son La was lower when harvested in the field; however, this prevalence was higher in the rainy season Noticeably, most people had adequate awareness of molds and their impacts on human and animal health, but they lack insight of aflatoxins and the impacts of aflatoxins, particularly ethnic minority groups

In conclusion, the findings can conclude that maize in Son La is exposed to AFB1

to varying degree, and there may be a risk that maize production could contain AFB1 Therefore, we believe that this study produces evidence on potential risk to humans and animals in Son La and will inform science-based messages for reducing risk Addtionally, the findings underline the need to enhance public's awareness about aflatoxins and health risks associated with aflatoxins on human beings and animals

PART 1 INTRODUCTION

1.1 INTRODUCTION

Vietnam has an extremely advantageous geographical location in Southeast Asia and Asia It is located in both tropical and sub-tropical areas Thus, its climate features a tropical monsoon climate with high temperature and humidity which is a favorable condition for fungal growth, especially mycotoxins produced from Aspergillus spp (CAST, 2003) Among mycotoxins, aflatoxins, particularly aflatoxin B1 (AFB1) produced primarily by two species of ubiquitous fungus Aspergillus (A.) including A flavus and A parasiticus have attracted general public attention because it is the most toxic and potent carcinogen even in small quantities (FAO, 1979 and De Campos, 1987) According to The International Agency for Research on Cancer (IARC), AFB1 is classified as group I carcinogens (Humans et al., 2002) Epidemiologically, AFB1 can lead to be genotoxicity and carcinogenic toxicity for both human and animal, especially the development of liver cancer (hepatocellular carcinoma) and higher risk to hepatic B virus infected individuals (WHO, 1998; Hansen et al., 2011 and Shephard, 2008) In fact, AFB1 is very susceptible in nuts and cereal, especially maize and groundnut during the harvest, or graduallysusceptible while storing and processing (Reddy et al., 2009) Therefore, it is extremely important to detect aflatoxins in food and animal feed for assurance of human and animal health as well as food hygiene and safety

In Vietnam, maize is the second only to rice as staple food for the consumption of humans and animals, especially in the rural and mountainous areas It is regarded as the major energy feed source for Vietnam's livestock industry (Ha et al., 2004) Mountainous North-west provinces, such as Son La, are still listed as belong to the poorest in Vietnam with limited development opportunities over the past years Therefore, maize is also considered as one of the most important crops It grows in different ecological regions to alleviate poverty in the provinces with difficult economic conditions Nevertheless, maize was reportedly extremely good substrate for fungal development producing mycotoxins, especially aflatoxins (Wang and Liu, 2006 and Zinedine et al., 2007) Meanwhile, Son La province is the largest maize producing area in Vietnam but few comprehensive AFB1 investigations had been carried out to

establish the prevalence of AFB1 contamination in maize in Son La Previously, some studies on the occurrence of AFB1 in maize in Vietnam have provided some insight; however, these were based on small sample sizes or limited-scale geographical areas or relied on the data from one point in the maize value chain For instance, AFB1 was surveyed in 32 maize samples in a Wang et al.’s study (1995), Trung et al (2008) only conducted in 25 maize samples whereas a bigger sample size (97 samples) were researched by Thieu et al (2008) and Phuong et

al (2015) (Wang et al., 1995; Trung et al., 2008; Thieu et al., 2008 and Phuong

et al., 2015) This would be difficult to generalize the population Moreover, few comprehensive AFB1 contamination research to establish the prevalence of AFB1

contamination in maize and unique study about perception and knowledge of aflatoxins had been conducted in Son La (Lee et al., 2017) Therefore, an investigation extended greater sampling was conducted to assess AFB1

contamination in maize and awareness of aflatoxins in Son La province

1.2 RESEARCH OBJECTIVES

General objectives

This research conducted is to evaluate the prevalence of aflatoxin B1

contamination in maize and residents’ awareness of aflatoxins in Son La province

Specific objectives

- To evaluate the prevalence of aflatoxin B1 in maize in Son La province in

an attempt to give recommendations to mitigate moldy contamination in maize

- To evaluate the awareness of aflatoxins among people so as to improve their understanding of health risks for both human beings and animals relating to aflatoxins contamination by consuming maize in Son La

PART 2 LITERATURE REVIEW 2.1 SITUATION IN THE MAIZE PRODUCTION IN VIETNAM – CASE

OF SON LA

2.1.1 Situation in the maize production in Vietnam

As in most of the Asian countries like China, Thailand, Indonesia, Vietnam, maize production have been dramatically increasing during the last decade along with the rapidly increasing demand for animal feed The Vietnamese maize production boosted from 1,177.2 thousand tons in 1995 to 2,005.9 thousand tons

in 2000 and drastically increased 5,281.0 thousand tons in 2015 (GSO, 2017) This rapid increase leads to both

expansion in area and yield Maize

production system was extended in

all the eight Vietnamese

agro-ecological zones included North

West, North East, Red River Delta,

North Central Coast, South Central

Coast, Central Highlands, South East

and Mekong River Delta (Figure

2.1) In 2000, maize was grown on

730.2 thousand hectares and obtained

1,179.30 thousand hectares in 2015

(GSO, 2017) Maize developed in all

agro-ecological zones, especially in

Northern mountainous provinces

The recent development of

production particularly comes from

intensive areas of maize production

such as Son La, Dong Nai, Nghe An,

Thanh Hoa and so on, where maize

is grown on sloping and wet land as

well as invested in new seeds

dissemination (especially hybrid

seeds like LVN10, LV885, DK88

and so on)

Figure 2.1 Agro-ecological regions in

Vietnam Source: Quyet (2013)

2.1.2 Study location - Son La province

Son La province is one of the largest maize producing areas in Vietnam (GSO, 2017 It belongs to Northwestern agro-ecological zone It is the fifth largest province in Vietnam with the area of 14,174.4 km2 (GSO, 2017) It is located in the northwestern region; it is bordered by Laos to the south, with Yen Bai, Lao Cai, Lai Chau provinces to the north, Dien Bien province to the west and Phu Tho and Hoa Binh provinces to the east (Figure 2.2) The provincial capital is Son La city, which is located approximately 300 km from Hanoi There are 11 rural districts: Quynh Nhai, Muong La, Thuan Chau, Phu Yen, Bac Yen, Mai Son, Song Ma, Yen Chau, Moc Chau, Sop Cop and Van Ho Nearly 80% is mountainous and its average elevation is 600 -700m above sea level

Figure 2.2 The administrative map in Son La province

Source: GSO (2017)

The climate in Son La province is influenced by the tropical monsoon climate, it is therefore charactered by both hot summer and cold winter with a small amount of rain Dry winter lasts from October to next March while rainy summer lasts from April to September and shows its humidity and heat Most of the precipitation in years concentrates in June, July, August and September (Appendix 2) The average annual temperature is 23°C (highest 35°C, lowest 11.5°C) The average rainfall in recent years ranges from 120 to 150 mm, and the average humidity is 79% (GSO, 2017)

Son La province had a population of nearly 1,182.4 thousand people in

2005 with the density of 83 people/km2 (GSO, 2017) Similar to other Northwest provinces, Son La’s population comprises many ethnic minorities: 54% are Thai, 13% are H'Mong, whereas Kinh or Viet (the major ethnic group nationwide) accounts for only about 18% (Zohova, 2011) Other ethnics are Ma, Dao, Muong, Khmer, Tay and Tai

The province has a total land area of about 1.4 million hectares whereas 64% is agricultural land Son La has a unique topography as it is partitioned deeply by a high mountain range Mountains and plateaus cover 75% of the province (Karimov et al., 2016) Maize area and production in Son La have increased significantly and now occupy a large part of the total maize production area of the entire Vietnam, owing to its advantageous geography and climate Maize production in Son La increased mostly because of maize field expansion and with a slight boost in maize yield The province production satatistics based

on the area cropped and the corresponding quantity produce from 2004 to 2014 was shown in Figure 2.3 and Appendix 2.1

Figure 2.3 Area under maize, maize production and yield in Son La

province (2004 - 2014)

Source: GSO (2017)

Maize is planted in Son La in two main seasons Maize is grown in swidden fields in the summer season (it is planted in April or May and harvested in July and August or maybe extended until September) while in the winter season maize

is cultivated in alluvial lands near rivers or stream banks Furthermore, it has also been grown in fields during the spring (Tran, 2005) Basically, there are two kinds of maize that are cultivated Local maize has been grown for years and hybrid maize has been newly introduced as a part of the province’s overall agricultural development strategy Hybrid maize yields have higher productivity than local ones but it is more vulnerable to the climate Nevertheless, it is more suitable to produce livestock feed High domestic demand for maize leads to a tremendous increase in maize imports Therefore, it should be noted that there is

a high demand for maize in Son La because, according to the stakeholders at the value chain assessment workshop, it is considered to be of better quality in contrast to imported maize from China It is one of the most important cash crops for ethnic minorities and a big number of poor households in the uplands

The maize grown area is not evenly distributed across Son La province, partly due to inadequate agricultural land The output is highest in Moc Chau, Mai Son and Song Ma district, which also have the highest share of planted area (Table 2.1) A majority of maize producers are small-scale farmers who depend

on maize sales for their livelihoods In 2013, maize yields in the districts of Son

La ranged between 3 and 5 tonnes/ ha on average (Table 2.1) Farmers use fertilizers to keep yields above average

Table 2.1 Area under maize, maize production and yield of districts

Source: Statistic Yearbook of Son La (2013)

Note: Van Ho district was recently separated from Moc Chau district

Maize products particularly hybrid maize produced in Son La are mainly supplied to enterprises in the livestock sector (for example CP Thailand joint venture company) Moreover, Tran (2005) revealed that Son La maize was sold

in many markets such as Ha Tay, Thanh Hoa, Nam Dinh, Nghe An and so on That also means that maize in Son La became a highly market-oriented product

2.2 AFLATOXINS AND THEIR IMPACTS ON HUMAN AND ANIMAL HEALTH

2.2.1 Aflatoxins

Aflatoxins are toxic secondary metabolites produced by filamentous, which can occur in host crops contaminated by some species of Aspergillus (Calvo et al., 2002) Among Aspegillus spp., two strains such as A flavus and A parasiticus can produce aflatoxins B1, B2, G1, G2, and M1 (Busby and Wogan, 1984) Worldwide, around 25% of agricultural products were contaminated with aflatoxins (Wild and Gong, 2010 and Yard et al., 2013), while a number of agricultural products are contaminated such as groundnut, maize, and cereal grains with cereals, and groundnuts being the most susceptible (Wilson and Payne, 1994)

 Properties of aflatoxins

Hell (1997) stated that four major groups of aflatoxins are identified: B1,

B2, G1 and G2 These abbreviations are indicative of the colours these fluorescence under the ultraviolet light (385 nm); thus B is for blue and G is for yellow-green The M is a hydroxylated metabolic product of B (Bankole and Adebanjo, 2003)

Aflatoxins are crystalline substances They not only dissolve freely in some moderately polar solvents such as methanol, chloroform, dimethyl sulfoxide but they also dissolve in water to the extent of 10-20 mg L-1 Several important physical and chemical properties of aflatoxins are shown in Table 2.2 (Cole and Cox, 1981)

Table 2.2 The property of aflatoxins

Property Aflatoxins

Chemical Formular C17H12O8 C17H14O8 C17H12O7 C17H14O7 C17H12O7

Molecular weight 312 314 328 330 328 Melting point ( oC ) 268-269 (D) 287-289 (D) 244-249 (D) 230 299 (D) Sorbent, + Pentane Chloroform Chloroform Chloroform

Ethyl acetate Methanol Flourescence 425 nm 425 nm 450 nm 425 nm 425 nm

 Chemical structures of aflatoxins

The chemical structures of some aflatoxins are shown in figure 2.4 (Cole and Cox, 1981)

Figure 2.4 Chemical structure of aflatoxins Aflatoxins are highly carcinogenic and can be acutely toxic or fatal if ingested in sufficient quantities for both livestock and humans (Shephard, 2008) 2.2.2 The impact of aflatoxins on human and animal health

 On human health

Aflatoxins are genetoxic, carcinogenic and immunosuppressive substances, and cause both acute and chronic toxicity Associated health problems are difficult to diagnose, mainly due to cryptic, long-term and chronic exposures Nevertheless, in 2004 and 2005, the Kenyan government showed and recognized that hundreds of human death cases were ascribed to the consumption of aflatoxin contaminated maize products (Lewis et al., 2005)

Among worldwide countries, populations of developing countries are the most susceptible to aflatoxicosis sickness This is because security blankets of developed countries in crops at pre-harvest and post-harvest level are quite strict The same occurs with milk derivates, because developing countries have

not accepted and assumed amenities as quick as developed countries Shephard (2003) and Williams et al (2004) reported in developing countries worldwide that there were more than 5 billion people at risk of chronic exposure to aflatoxins through contaminated food Because of being an alarming number, aflatoxins have been recently considered as an important public health issue Adult humans usually have a high tolerance of aflatoxins, and, in the reported acute poisonings, there are usually children who die (Cullen and Newberne, 1994)

The adverse impacts of aflatoxins in human beings and animals have been categorized in two common forms:

 Acute aflatoxicosis

It is produced when moderate to high levels of aflatoxins are consumed Specific, acute episodes of disease include hemorrhage, acute liver damage which manifests as severe hepatotoxicity with a case fatality rate of approximately 25%, edema, absorption and/or metabolism of nutrients and alteration in digestion The early symptoms of hepatotoxicity from aflatoxicosis can include anorexia, malaise, and low-grade fever Acute high-level exposure can progress to potentially lethal hepatitis with vomiting, abdominal pain, jaundice, fulminant hepatic failure and death (Walderhaug, 1992; Cullen and Newberne, 1994 and Strosnider et al., 2006)

 Chronic aflatoxicosis

It results from ingestion of low to moderate levels of aflatoxins The effects are usually subclinical and difficult to recognize Some of the common symptoms are impaired food conversion and slower rates of growth with or without the production of an overt aflatoxin syndrome (Walderhaug, 1992) Aflatoxicosis is not only caused by inhalation, but also, as mentioned before, is caused by aflatoxin ingestion In places like Brazil and Abu Dhabi, there have been found lots of cases in which infants were exposed to aflatoxin M1

from mother’s breast milk Aflatoxins have also been found in infant formula (Aksit et al., 1997; Saad et al., 1995 and Navas et al., 2005) There are lots of earlier studies reporting the presence of aflatoxins and derivatives in human urine, blood, and human cord blood that apparently can enter the developing fetus in humans and animals (Denning et al., 1990) The 80’s and 90’s were

globally fatal decades In India, at least 400 people were affected by eating infected corn, and 104 of them died In Kenya, 12 people were also killed by high consumption of aflatoxins (Mehan and Mc Donald, 1991) In Southeast Asia, 19 patients after eating rice and pasta became jaundiced and sick within hours; 17 of them presented symptoms of hepatitis, and in total, 14 died because

of liver failure and 7 because of renal failure In biopsies, there were found high concentrations of aflatoxin in liver, lungs and other organs (Hendrickse, 1999)

It has been well documented that chronic aflatoxin exposure causes Hepatocellular Carcinoma (HCC), generally in association with hepatitis B virus (HBV) or other risk factors That’s why the International Agency for Research on Cancer (IARC) recognized aflatoxins as carcinogenic in 1976 (Chen et al., 2001; Henry et al., 2002; Omer et al., 2004; Qian et al., 1994 and Wang et al., 1996) HCC is the sixth most prevalent cancer worldwide Developing countries have a higher incidence rate, with approximately 82% of the 600,000 new cases each year occurring in developing countries (Parkin et al., 2005)

 On animal health

There are no differences in the effects of aflatoxin consumption among all animals; however, the species’ s susceptibility varies by species, age, and individual variation Symptoms of acute aflatoxicosis comprise of depression, anorexia, weight loss, disease, gastrointestinal bleeding, pulmonary edema and liver damage Signs of acute hepatic injury are seen as coagulopathy, increased capillary fragility, hemorrhage, and prolonged clotting times Blood pigments may occur in the urine and mucous membranes are icteric The liver indicates gross changes brought about by centralobular congestion and hemorrhage and fatty changes of surviving hepatocytes The animals may die within hours or several days Symptoms of prolonged exposure to moderate to aflatoxins may be reflected in a decline in feed consumption and production (growth and production of eggs and milk) The quality of milk and milk products are likely to

be influenced, and pose a threat to the presence of AFM1 as derived from AFB1

consumed by lactating females In chronic aflatoxin poisoning, the majority of effects are still referable to hepatic injury, but on a lighter scale The most sensitive clinical sign of chronic aflatoxicosis is reduced rate of growth of young animals There are also other indicators such as prolonged clotting time,

increases in serum glutamic oxalacetic trans-aminase, ornithine carbamyl transferase, and cholic acid levels Hepatic pathology includes a yellow to brassy color, enlarged gall bladder, dilute bile, histologic signs of fatty changes in the hepatocytes, and bile duct proliferation In fact, the signs of chronic aflatoxins are so protean that the situation goes undiagnosed for such a long period of time Chronic aflatoxin poisoning, however, is the manner in which animals are most frequently affected and the economic drawbacks are often significant (Pier, 1992 and Denli and Pérez, 2006)

AFB1 is absorbed via the gastrointestinal tract into the portal blood system and is transported to the liver to be metabolized A portion of aflatoxin is activated and set in hepatic tissues Some water-soluble conjugated metabolites

of AFB1 are excreted into the bile and go to the stool Other water-soluble conjugated metabolites, AFB1 degradation products and non conjugated metabolites are excreted into the blood circulatory system and distributed in a systemically Finally, these residues are referred to milk, eggs, muscle and edible tissues (Dennis and Hsieh, 1981) AFM1 is one of those metabolic derivatives which make milk tainted Other metabolites are formed from AFB1, including aflatoxicol (18 times less toxic than AFB1) and aflatoxin B2a (not toxic) The animal organism usually produces those metabolic products as an autodetoxification system (Gimeno, 2004)

AFB1 mainly affects birds, pigs and other monogastric animals Ruminants are less vulnerable to aflatoxin ingestion In monogastric animals, clinical symptoms may occur after consumption of feed contaminated with concentrations above 50 µg/kg while the symptoms in cattle occur at concentrations above 1.5 to 2.23 mg/kg Depending on the presence of other concurrent factors, small amounts of AFB1 (greater than 20 µg/kg) can cause toxic effects In these conditions, an aflatoxin level above 100 µg/kg may be also toxic in ruminants (Denli & Pérez, 2006)

Evidence obtained from experimental animal unveils that chronic exposure

to aflatoxins may cause impaired immunity and decreased uptake of nutrients from the diet too (Hall and Wild, 1994 and Miller and Wilson, 1994) Furthermore, diseases occurred by aflatoxins can lead to subclinical losses in production, and increase the risk and incidence of other diseases (Denli and Pérez, 2006)

2.3 FACTORS INFLUENCING ASPERGILLUS SPP INFECTION AND AFLATOXIN DEVELOPMENT IN MAIZE

Aspergillus spp is a fungus that basically belongs to grains storage flora It develops optimally at 25°C with a minimum water activity of 0.75 It begins to produce secondary metabolites at 10 - 12ºC, but the most toxic ones are produced

at 25°C with a water activity of 0.95 (Hesseltine, 1976) Those toxic secondary metabolites named aflatoxins are chemical metabolites which are naturally produced by a large number of Aspergillus species, especially A flavus and A parasiticus (or less commonly by A nomius) Summarizing some characteristics

of aflatoxins growth and production by two species of fungi is given in Table 2.2 There are three primary categories of factors influencing growth of Aspergillus spp and formation of aflatoxins They are climate factors, agronomic factors and biotic factors (Diener et al., 1987)

For climate factors, spores of the fungus will develop and produce aflatoxins in case of favorable conditions This is due to mycoflora of stored cereal is affected by environmental elements, particularly temperature, water activity, pH and gas atmosphere (Magan and Lacey, 1988) According to Ross et

al (1979), the results unveiled that if both temperature and moisture are favorable for A flavus, aflatoxin can be produced within 48 hours Another study carried out by Thompson et al (1980) indicated that during the growing season, especially during the grain filling period, the weather was closely related to pre-harvest infection and subsequent fungal development Chang and Markakis (1981) showed that considerably higher moisture contents may be optimal for growth and toxin production There will be no infection with A flavus if moisture content is less than 17% However, researchers in Thailand observed that moisture content of maize samples ranged from 16.8 to 30.7% depending on harvest time and maize contaminated the highest levels of aflatoxins due to an early havesting (113 days) (Kawasugi et al., 1988) Futhermore, aflatoxins can

be formed at a relative humidity of 88, 90 and 99% (Lillehoj, 1983) Growth of different fungi on media with a specific pH showed that A parasiticus was more tolerant of acidic pH than A flavus with optimum growth at a pH from 5 to 8 (Table 2.3)

Table 2.3 Limits of aflatoxin growth and production by A flavus and

oC 10-12 12 33 32 43 42 Water activity 0.8 0.80-0.83 0.98 0.99 > 0.99 > 0.99

oC 13 12 16-31 25 31-37 40 Water activity 0.82 0.86-0.87 0.95-0.99 0.95 > 0.99 > 0.99

Source: ICMSF (1996)

For agronomic factors, they are plant stress, irrigation, cropping pattern, variety, time of planting, time of harvest and storage conditions (Cotty, 1994).The timing of growing plays a significant role in the development of the toxin Reductions in AFB1 were associated with early planting (Jones and Duncan, 1981) In respect date of havest, some evidences by Lee and Chuang (1993), Scott and Zummo (1994) and Lynch and Wilson (1991) pointed out that the date

of harvest influenced on the development of aflatoxins Peculiarly, grains harvested late were more significantly than that harvested early (Jones and Duncan, 1981) Also, soil sort, condition and the availability of viable spores are important factors that influence the infection process and the conditions that influence toxin formation (Horn, 2003) Lastly, in storage conditions, Ahmad (1993) revealed that aflatoxins contamination was affected by storage system

In terms of biotic factors, insects are considered as an agent vectors fungi It causes damage that allows fungus access to grains and other crop tissues thereby the chances of aflatoxin contamination increase (Setamou et al., 1997) Specifically, McMillian et al (1990) reported that damage of insect contributed

significantly to boost A flavus sporulation and aflatoxins contamination by transmitting the fungal spores into the kernels Additionally, another agent which affects the evolution of A flavus on its substrate was competition with other organism For example, there are two fungal species, faster species will inhibit slower species (Ramakrishna et al., 1993) An experiment conducted by Wicklow et al (1980) also reported that level of aflatoxins contamination in kernels simultaneously inoculated with Fusarium moniliforme (F moniliforme) and A flavus was 12 times lower than kernels that were wound inoculated with

According to Vargas et al (2001), the result showed that 38.3% of maize samples in Brazil contaminated aflatoxin B1 with an average level of 9.4 µg/kg,

up to 12.9 µg/kg and only 3.7% above 20 µg/kg

In April 2004, one of the large outbreaks associated with aflatoxicosis occurred in rural Kenya, there were 317 cases and 125 deaths Then, market surveys were carried out and highlighted, whereas exposure to aflatoxins was above 100 to 1000 µg/kg compared to regulatory limit of only 20 µg/kg in Kenya (CDC, 2004) Another study by Williams et al (2004) on aflatoxins contamination foods from more than 20 countries showed that the maximum aflatoxin levels found in maize or maize products was 770 µg/kg in Nigeria, and

465 µg/kg in maize in Mexico

Maize is widely used and makes up 24% of the ingredients in commercial feed in Asia (Binder et al., 2007) However, the percentage of raw maize

samples contaminated with mycotoxins and their levels are very high, particularly some important toxins such as AFs, DON, ZEA and FUM (Biomin Newsletter, 2008 and Solovey et al., 1999) Binder et al (2007) reported that 68% of maize samples from Asia contained fumonisin B1 (FB1), while DON had 67% positive samples, 40% of ZON and 19% of AFs According to Rodrigues and Nahrer (2008), FUM are mostly found in this matrix, with 71% positive samples, followed by DON, AFs, ZEA and OTA with 59, 40, 37and 15%, respectively Furthermore, the highest levels of those toxins were AFs (2,483 µg/kg), ZEA (3,112 µg/kg), FUM (9,481 µg/kg) and OTA (197 µg/kg) (Rodrigues and Nahrer, 2008)

In 2008, high levels of aflatoxins contamination in maize in a number of African countries, Benin and Togo in particular had been informed and a third of households with grain containing aflatoxin about five times higher than the limit safe (Wagacha et al., 2008) Similarly, there were high levels of aflatoxins contamination found in maize meal from India, mixed snacks from India and rice from Thailand AFB1 was detected in all samples of cereals in Nigeria from 17.01 to 20.53 µg/kg of wheat, 34 to 40.30 µg/kg of millet, 27.22 to 36.13 µg/kg

of maize Guinea and 40.06 to 48.59 µg/kg of fruit bread (Odoemelam et al., 2009) Last but not least, a report by Ghiasian et al., (2011) announced that AFB1

was detected accounting for 58.3% and 80% in maize samples collected from Kermanshah and Mazandaran province in Iran

2.4.2 Situation in the aflatoxin contamination in Vietnam

Many studies on aflatoxins contamination were conducted in Vietnam on the basis of a hot and humid tropical climate

Initially, Wang et al (1995) had an early study of mycotoxins in maize intended for animal feed in Northern Vietnam in 1995 This report found high incidences of AFB1 in 14 out of 15 maize kernels samples (93.33%) and 14 out

of 17 maize meal samples (82.35%), with an average concentration of 28 and 30 µg/kg, respectively

After that, another by Nguyen et al (2007) reported that there was a coexistence of three mycotoxins in rice, consisting AFB1, citrinin (CIT) and OTA

in which AFB1 contaminated over one half of the total number of samples (51%) Furthermore, Trung et al (2008) revealed in another carried out in Vietnam

on maize intended for both human and animal consumption that the Aspergillus

genus developed in almost all maize samples (90%), and 68% of tested samples was contaminated with AFB1

In 2008, Thieu et al reported that AFB1 was found in 92 % of maize samples with mean concentration of 77.5μg/kg Until 2010, a survey on the contamination of maize with aflatoxins, fumonisins and zearalenone was carried out by Phuong et al., in the Southeastern provinces and Central Highland provinces in Vietnam, with 97 maize kernel samples The result of aflatoxins positive samples in the Southeastern provinces (61.7%) was higher than in the Central Highlands (50%)

Then, Huong et al (2016) investigated about aflatoxins and fumonisins in rice and maize staple cereals in Northern Vietnam and dietary exposure in different ethnic groups This researcher observed that AFs were detected in 27 rice (24.3%) and 27 maize samples (26.4%) at minimum and maximum levels in rice of 2.06 and 77.8 µg/kg and 20.5 and 110 µg/kg in maize, respectively

Recently, Lee et al (2017) revealed that 799 samples (33.71%; 95% confidence interval (95% CI): 31.81-35.66%) and 687 samples (28.98%; 95% CI: 27.17-30.86%) were above 2 μg/kg and 5 μg/kg, respectively in a survey of AFB1 in maize and awareness of aflatoxins in Vietnam

2.5 METHODS TO DETECT AFLATOXINS

Tons of foods are contaminated with aflatoxins, it not only has adverse effects on human health but also cause serious economic losses Therefore, determination of aflatoxins concentration in food and feeds is very important However, due to their low concentration in foodstuff and feedstuff, analytical methods for detection and quantification of aflatoxins have to be specific, sensitive, and simple to be carried out Aflatoxins contamination in fresh food processing can be checked by chromatography or in combination with antibodies (Seo et al., 2011) Several methods include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), mass spectroscopy, enzyme-linked immune-sorbent assay (ELISA), and electrochemical immunosensor These have been described for detecting and quantifying aflatoxins in foods, of which TLC, HPLC, ELISA and other immunoassays have been identified as the preferred methods for aflatoxins detection

Chromatography is one of the most common methods for quantifying aflatoxin At the beginning of aflatoxin analysis and research, Gas Chromatography (GC) was frequently used for detection and quantification of compounds Later on, new chromatography-based techniques were developed for aflatoxins Examples of these improvements are Liquid Chromatography (LC), Thin Layer Chromatography (TLC) (Stroka et al., 2000), and High-Performance Liquid Chromatography (HPLC) (Bacaloni et al., 2008) which nowadays is the most commonly used chromatographic technique for detection of a wide diversity of mycotoxins, especially for aflatoxin derivatives (Jinap et al., 2012) described an HPLC method to identify 182 mycotoxins and other fungal metabolites based on their alkylphenone retention indices and diode array spectra Coupling of HPLC with mass spectroscopy or tandem mass spectroscopy allows for highly accurate determination of toxin concentrations and compound identification in one analysis (Sobolev, 2007) Alternatively, fluorescence detection of the unmodified aflatoxins is widely used in HPLC applications as well as in TLC Furthermore, there are combinations of the methods above with pre-process techniques, which can detect the concentration of aflatoxin in a solution in a better way For instance, immunoaffinity column sample clean-up followed by a normal or reverse phase of HPLC separation with fluorometric detection is mostly used for quantitative determination of AFM1 due to the characteristics of specificity, high sensitivity and simplicity of operation (Muscarell et al., 2007)

Immunochemical detection for aflatoxins is based on antibody-antigen reactions (Ab-Ag) (Lee et al., 2004) Since different kinds of aflatoxin molecules can be considered as antigens, it is possible to detect them by developing antibodies against the compounds Most of the immunological methods are based

on enzyme-linked immunosorbent assays (ELISA), which have good sensitivity, speed and simplicity Even though several reports have been published on the immunechemical determination of aflatoxin in food, only a few validation studies are available to show that the results comply with certain regulations because of the requirement for expensive instrumentation

2.6 REGULATIONS FOR AFLATOXINS IN THE WORLD AND VIETNAM

2.6.1 Regulations for aflatoxins in the world

Aflatoxins particularly aflatoxin B1 are considered as unavoidable

contaminants of food and feed, even where good manufacturing practices have been followed To ensure food safety, maximum tolerable levels for aflatoxins in food and feed have been regulated by national and international organizations and these regulations differ from nation to nation for the determination of aflatoxin concentrations in food and feed commodities By the end of 2003, around 100 countries around the world have regulations governing aflatoxins in food and most include maximum permited, or recommended levels for specific commodities, and the number continues to increase Binder et al (2007) reported that AFB1 was presented in most agricultural commodities and it was quite high

in animal feed, not only in Asian and Pacific countries but also in European and Mediterranean countries The AFB1 concentration regulation levels in agricultural commodities in EU countries are lower than those in Asian and Pacific countries This might be a result of the stringent aflatoxins for export and imported food and feed of European Commission (EC) countries

 European Union(EU)

The EU regulates limits for aflatoxin B1 and for total aflatoxins (B1, B2,

G1 and G2) in nuts, dried fruits, cereals and spices Limits vary according to the commodity, but range from 2 - 12 µg/kg for B1 and from 4 - 15 µg/kg for total aflatoxins (Richard Lawley, 2013)

According to Tedesco et al (2008), few countries regulate AFB1 in feedstuffs for dairy cattle This is entirely reasonable and logical because that it is aflatoxin M1, the metabolite of AFB1, which cause health concern As a result, limiting AFB1 in animal feeds is the most effective means of controlling aflatoxin M1 in milk A limit of 5 μg/kg in feed for dairy cow and a level of 20 μg/kg in feed for cattle, sheep, goats, swine and poultry are applied in the European Union (EU) countries This limit is applied by countries in the European Free Trade Association (EFTA), in many of the candidate EU countries and sporadically outside Europe

 United States of America (USA)

The U.S General Accounting Office has concluded that industry, federal, and state programs are effective in detecting and controlling aflatoxins and that

it is doubtful that additional programs or limits would reduce the risk of aflatoxins in the food supply The Food and Drug Administration (FDA) has established specific guidelines on acceptable levels of aflatoxins in human food and animal feed by establishing action levels that allow for the removal of

violative lots from commerce The FDA action levels for aflatoxins are shown

1 Human consumption Milk

0.5 ( AFM1)

20

4 Dairy animals, animals not listed above, or unknown use

Corn, peanut products, cottonseed, and other animal feeds and

ingredients

20

5

Breeding cattle, breeding

swine and mature poultry Corn and peanut products 100

6

Finishing swine 100 pounds or

greater in weight Corn and peanut products 200

7

Finishing (i.e., feedlot) beef

cattle Corn and peanut products 300

8

Beef, cattle, swine or poultry,

regardless of age or breeding

status

Cotton seed meal 300

Source: Henry (2006)

Therefore, the FDA food safety regulatory limits for total aflatoxins (B1, B2,

G1 and G2) in all food except milk are no more than 20 μg/kg and a limit of 0.5 μg/kg for M1 in milk Higher limits are applied in animal feeds

 Others

Both Australia and Canada set a limit of 15 μg/kg for total aflatoxins (B1,

B2, G1 and G2) in nuts This is similar to the international limit recommended for raw peanuts by the Codex Alimentarius Commission (Lawley, 2013)

With AFB1, a limit of 20 μg/kg for dairy animals and a limit of 100 μg/kg intended for breeding beef cattle, breeding swine, or mature poultry are applied

in the United States, Africa and Latin America (Tedesco et al., 2008)

Consequently, the accepted limits of AFB1 and total aflatoxins in foods are

5 and 10 μg/kg, respectively, in more than 75 countries around the world whilst they are 2 and 4 μg/kg in the European Union (López et al., 2003 and Van Egmond and Jonker, 2004) Regulations vary depending on whether country setting limits is an importer or exporter

Moreover, regulations for AFM1 existed in 60 countries at the end of 2003,

a more than threefold increase as compared to 1995 (FAO, 2005) This is because exposure to any level of genotoxic carcinogens as AFM1, may pose a health risk

to consumers, especially for children, so the exposure level should be zero for a zero risk to liver cancer that may be caused by aflatoxins in general Countries which defended an AFM1 maximum level of 0.5 ppb argue that those concentrations they could cause adverse economic consequences due to the difficulty of milk exports to countries that accept only a maximum level of 0.05 ppb Delegates from some other countries argue that the level of 0.05 ppb is difficult to achieve in most regions of the world, so, a level of 0.5 ppb is enough

to promote public health protection Specifically, EU, EFTA, candidate EU countries and some other countries in Africa, Asia and Latin America, apply a maximum level of 0.05 μg/kg in milk and a maximum level of 0.025 μg/kg for AFM1 in infant formula A limit of 0.5 μg AFM1/kg in milk is applied in the United States, several Asian, European countries and in Latin America, where it

is also established as a harmonized Mercosur (a trading block consisting of Argentina, Brazil, Paraguay and Uruguay) limit

2.6.2 Regulations for aflatoxins in Vietnam

Aflatoxins have the highest acute and chronic toxicity of all mycotoxins; hence, the maximal concentration in agricultural food and feed products and their commodities is regulated worldwide included Vietnam These regulations differ

from nation to nation With an aim at controlling the presence of aflatoxins in foods as well as protecting public health, Vietnam also has established specific guidelines on acceptable levels of aflatoxins in human food However, there were

no specific regulations for total aflatoxin as well as aflatoxin B1 in feedstuff (FSI, 2011) The Table 2.4 summarized some Vietnamese action levels for aflatoxins

in human food Therefrom, a limit of 5 µg/kg for aflatoxin B1 in maize (No.7 in Table 2.4) is regulated for human consumption

Table 2.5 Vietnamese limits for aflatoxins in human food

No Food Category

Max limits (µg/kg) AFB1 AFs AFM1

1

Peanuts and other kinds of oil seeds used as raw

materials or need to be processed before using as food or

as food ingredients

8 15 NS

2 Almonds, pistachio, dried apricots used as raw materials

or need to be processed before using as food or as food

ingredients

12 15 NS

3 Chestnuts, brazil nuts used as raw materials or need to

be processed before using as food or as food ingredients

8 15 NS

4

Tree nuts used as raw materials, excluding the products

specified in 2, 3; or need to be processed before using as

food or as food ingredients

5 10 NS

5

Material dried fruits that need to be processed before

using as food or as food ingredients

5 10 NS

Dried fruits and derived products used as food or as food

ingredients

2 4 NS

6 Cereal and derived products including processed

products (except for the products specified in 7, 10, 12)

2 4 NS

7 Maize and rice that need to be processed before using as

food or as food ingredients

5 10 NS

No Food Category

Max limits (µg/kg) AFB1 AFs AFM1

8 Material milk, thermoprocessed milk, milk for producing

dairy

5 NS 0.5

9

Spices:

- Chili: all kinds of chilies, chili sauce, chili powder,

paprika, hot chilies

- Pepper, including both black and white pepper

11 Milk powder for children and milk for newborn babies

(Infant formulae and follow-on formulae)

NS NS 0.025

12

Diet food especially for newborn babies (Dietary foods

for special medical purposes intended specifically for

infants)

0.1 NS 0.025

PART 3 MATERIALS AND METHODS 3.1 MATERIALS

3.1.1 Maize samples

Maize samples were randomly collected in Son La province in September

2016 The majority of maize in Son La is hybrid maize colouring yellow The name of hybrid maize samples are CP501, CP888, DL7328, DK9955, DK8868, DK6919, DK6818, LVN10, LVN885, NK66, NK67, NK6326, DK9901, VPA88, and so on

3.1.2 The Sigma-Aldrich® Aflatoxin B1 Low Matrix ELISA kit

The Sigma-Aldrich® Aflatoxin B1 Low Matrix ELISA kit (Enzyme Linked Immunosorbent Assay) (Helica Biosystems INC; Cat No: 941BAFL01B1) that was provided by Helica producer from the USA The solid-phase direct competitive AFB1 ELISA kit consisted of a 96-well microplate coated with an antibody that had been optimized to cross-react with AFB1 The sensitivity can

go down to as low as 0.1ppb and the specificity of the kit show 100% reactivity to AFB1

cross-Reagents and chemicals included antibody coated microwell plate, dilution well, aflatoxin standards, aflatoxin HRP-conjugate, substrate reagent, stop solution, washing buffer and methanol (70%)

3.2 METHODS

3.2.1 Sample size calculation and selection

A sample size was determined basing on 50% prevalence, a precision level

of 5% and 95% confidence interval (CI) with formular below:

N = Z2 (1-α/2)

P: Prevalence, P = 0.5 (50%)

d: Confident limit around the point estimate, d = 0.05 (5%)

Z: Z score corresponding to the desired statistical significance, 95% CI, side test Z = 1.96

2-Therefore, a total of 385 maize samples were gathered from local retail traders, small farmers, households and maize fields The maize survey in the