N°. 158 IMPROVING PUBLIC HEALTH THROUGH MYCOTOXIN CONTROL

EDITED BY JOHN I. PITT, CHRISTOPHER P. WILD, ROBERT A. BAAN, WENTZEL C.A. GELDERBLOM, J. DAVID MILLER, RONALD T. RILEY, AND FELICIA WU

IARC SCIENTIFIC PUBLICATION N°. 158

IMPROVING PUBLIC HEALTH THROUGH MYCOTOXIN CONTROL

EDITED BY JOHN I. PITT, CHRISTOPHER P. WILD, ROBERT A. BAAN, WENTZEL C.A. GELDERBLOM, J. DAVID MILLER, RONALD T. RILEY, AND FELICIA WU

INTERNATIONAL AGENCY FOR RESEARCH ON CANCER LYON, FRANCE 2012 Published by the International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon Cedex 08, France

©International Agency for Research on Cancer, 2012

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Cover art: “Groundnuts and Mealies” (Jackie Carstairs 1989). This oil and gouache painting is one of a series entitled “Women and Children”, by Zimbabwean artist Jackie Carstairs. This scene depicts the traditional life of the women and children of Zimbabwe. Reproduced with the kind permission of the artist.

IARC Library Cataloguing in Publication Data

Improving public health through mycotoxin control / edited by John I. Pitt … [et al.]

(IARC Scientific Publications ; 158)

1. Mycotoxins 2. Food Contamination – prevention & control 3. Public Health – standards 4. Risk Assessment I. Pitt, John I II. Series

ISBN 978-92-832-2214-9 (NLM Classification: W1) ISSN 0300-5085 Table of contents

Contributors...... v Dedication: Professor Wally Marasas...... vii Executive summary...... ix

Chapter 1...... 1 Fungi producing significant mycotoxins

Chapter 2...... 31 Chemical and physical characteristics of the principal mycotoxins

Chapter 3...... 39 Sampling and sample preparation methods for determining concentrations of mycotoxins in foods and feeds

Chapter 4...... 53 Analysis of mycotoxins

Chapter 5...... 59 Effects in food-producing animals

Chapter 6...... 87 Mycotoxins and human health

Chapter 7...... 105 Risk assessment and risk management of mycotoxins

Chapter 8...... 119 Economics of mycotoxins: evaluating costs to society and cost-effectiveness of interventions

Chapter 9...... 131 Practical approaches to control mycotoxins

Subject index...... 147

Contributors

Editors Dr F. Wu Dr A.W. Schaafsma Department of Environmental and Department of Plant Agriculture, The editors wrote most of the Occupational Health, University of University of Guelph material in the book and worked Pittsburgh Ridgetown, Ontario N1G 2W1, jointly on editing the overall text. Pittsburgh, Pennsylvania 15260, USA Canada

Dr J.I. Pitt Other Contributors Dr K.A. Seifert CSIRO Food and Nutritional Sciences Eastern Cereal and Oilseed North Ryde, New South Wales 2113, The following people provided Research Centre, Agriculture and Australia draft material at different stages Agri-Food Canada of development of the book or Ottawa, Ontario K1A 0C6, Canada Dr C.P. Wild reviewed earlier versions. Their International Agency for Research contributions are gratefully ac- Dr G.S. Shephard on Cancer knowledged. Programme on Mycotoxins and 69372 Lyon Cedex 08, France Experimental Carcinogenesis Dr M. Castegnaro (PROMEC Unit), South African Dr R.A. Baan Département Bioprocédés et Medical Research Council International Agency for Research Systèmes Microbiens, École Tygerberg 7505, South Africa on Cancer Nationale Supérieure Agronomique 69372 Lyon Cedex 08, France de Toulouse The original idea for this book 31326 Auzeville-Tolosane, France came from the IARC Monographs Dr W.C.A. Gelderblom meeting in 2002 on “Some Programme on Mycotoxins and Dr R. Coker Traditional Herbal Medicines, Experimental Carcinogenesis ToxiMet Limited Some Mycotoxins, Naphthalene (PROMEC Unit), South African Kent Science Park, Sittingbourne, and Styrene”. Manuscripts were Medical Research Council Kent ME9 8AZ, United Kingdom first commissioned at a meeting Tygerberg 7505, South Africa at IARC in 2003. The following Dr M. Pineiro people provided comments or Dr J.D. Miller (Food and Agriculture Organization materials that were not used in Department of Chemistry, Carleton of the United Nations, retired) the final version of the book. University M. Barreiro 3119, Montevideo, We acknowledge their support Ottawa, Ontario K1S 5B6, Canada Uruguay and encouragement during the development phase of the project. Dr R.T. Riley Dr J. Rheeder United States Department of Programme on Mycotoxins and Dr A.M. Cheraghali Agriculture, Agricultural Research Experimental Carcinogenesis Department of Pharmacology, Service, Richard B. Russell (PROMEC Unit), South African Baqyyatollah University of Medical Agricultural Research Center Medical Research Council Sciences Athens, Georgia 30605, USA Tygerberg 7505, South Africa Tehran, Iran

Contributors v Dr P. Cotty Dr H. Njapau Dr S. Trujillo United States Department of Center for Food Safety and Applied Center for Food Safety and Applied Agriculture, Agricultural Research Nutrition, United States Food and Nutrition, United States Food and Service Drug Administration Drug Administration Tucson, Arizona 85721, USA College Park, Maryland 20740, USA College Park, Maryland 20740, USA

Dr J.M. Fremy Dr D.L. Park Dr E. Welzig Agence Française de Sécurité Center for Food Safety and Applied Center for Analytical Chemistry, Sanitaire des Aliments Nutrition, United States Food and Institute for Agrobiotechnology 94701 Maisons-Alfort, France Drug Administration 3430 Tulln, Austria College Park, Maryland 20740, USA Dr Y. Grosse Dr G.E. Wood International Agency for Research Dr J.M. Rice Center for Food Safety and Applied on Cancer Department of Oncology, Lombardi Nutrition, United States Food and 69372 Lyon Cedex 08, France Comprehensive Cancer Center, Drug Administration Georgetown University Medical College Park, Maryland 20740, USA Dr S. Henry Center (United States Food and Drug Washington, DC 20007, USA Dr H. Yazdanpanah Administration, retired) Toxicology Department, Shaheed 8004 Craddock Road, Greenbelt, Dr K. Straif Beheshti University of Medical Maryland 20770, USA International Agency for Research Sciences on Cancer Tehran, Iran Dr R. Krska 69372 Lyon Cedex 08, France Center for Analytical Chemistry, Production Team Institute for Agrobiotechnology Dr M.H. Taniwaki 3430 Tulln, Austria Instituto de Tecnologia de Alimentos K. Müller Campinas SP 13073-001, Brazil English Editor Dr R. Lopez-Garcia Logre International Food Science Dr M.W. Trucksess S. Lesage Consulting Center for Food Safety and Applied Editorial Assistant Popotla DF 11400, Mexico Nutrition, United States Food and Drug Administration Dr W.F.O. Marasas College Park, Maryland 20740, USA Programme on Mycotoxins and Experimental Carcinogenesis (PROMEC Unit), South African Medical Research Council Tygerberg 7505, South Africa

vi Dedication: Professor Wally Marasas

Walter Friedrich Otto Marasas Wally moved to the South was born on 25 October 1941 African Medical Research Council in Boksburg, South Africa. After (MRC) in 1975 and remained obtaining his B.Sc. and M.Sc. there until his retirement in 2006, from the University of Pretoria, he when he was the Unit Director of received a Ph.D. in Plant Pathology the Programme on Mycotoxins from the University of Wisconsin, and Experimental Carcinogenesis USA, in 1969. Dr Marasas returned (PROMEC Unit). Throughout this to South Africa with the Plant period, he made major inroads into Protection Research Institute and, understanding the health effects of together with Dr Fanie Kellerman mycotoxins, based on his expertise of the Onderstepoort Veterinary in fungal taxonomy, specifically Research Institute, investigated of Fusarium species and the equine leukoencephalomalacia, a toxic effects of their mycotoxins. disease that causes brain lesions Notably, he investigated the high Photograph courtesy of South African in horses. Mouldy maize was incidences of oesophageal cancer Medical Research Council. a suspected cause, and Wally in the former Transkei region of identified Fusarium verticillioides Eastern Cape Province, at first the Transkei. He published more (then known as F. moniliforme) in relation to the occurrence of F. than 300 scientific articles, and the as the culprit. This launched a verticillioides and later in relation Institute for Scientific Information in decades-long examination of this to fumonisins, which Wally’s team 2002 rated him as one of the most fungus and its impact on animal and characterized from F. verticillioides cited researchers worldwide in two human health. (strain MRC 826) brought back from categories.

Dedication vii Professor Marasas enjoyed South African Academy for Science extraordinary professor at the Univer- wide international recognition and and Arts, and the distinguished sity of Pretoria, South Africa. was admitted to membership of service award from Kansas State Aside from his own academic the prestigious National Academy University. Professor Marasas was achievements, Wally was a great of Sciences of the USA. Among only the second South African to encourager and a scientific enthusiast his many awards were the Gold be elected a Fellow of the American who inspired others. He supervised Medal from the South African Phytopathological Society. He held theses by more than 60 postgraduate Society for Plant Pathology, the several visiting academic positions, students and was a warm and Wellcome Gold Medal from Wellcome at Pennsylvania State University, generous collaborator with many Pharmaceuticals, the African Acad- USA; the University of Sydney, more colleagues across the world. His emy of Sciences/CIBA Prize for Australia; and Kansas State Univer- friendship combined with his scien- Agricultural Biosciences, the MRC sity, USA. He was also appointed as tific excellence and strong motivation Silver Medal for Excellence, the honorary professor at the University for public health distinguished him as MT Steyn Gold Medal from the of the Orange Free State and as a remarkable colleague.

viii Executive summary

Mycotoxins are fungal toxins minimize exposure in the populations and other foods for export must that contaminate staple foods at the greatest risk. comply with quality requirements consumed by many of the poorest The knowledge and methodologies and sanitary regulations, which are and most vulnerable populations exist to control mycotoxins in food. established by the target markets. in the world. These toxins have the However, these are currently only Contaminated commodities may potential to contribute to a diversity applied with any rigour in some also be exported from high- of adverse health effects in humans, high-income countries with well- income countries to those where even at low concentrations. Given established analytical methods to regulations either are not in place the ubiquitous nature of exposure screen for mycotoxins and with or are ineffective or unenforced. in many countries, an urgent need strong regulatory controls. In low- Because of a lack of resources exists for a coordinated international income countries, the situation for an efficient control system, response to the problem of mycotoxin is often in marked contrast: these contaminated imports can contamination of food. The topic has subsistence or small-scale farmers be diverted to the poorest sectors been marginalized for far too long. produce foods for local consumption of society. One man’s meat is truly One priority is development of a better that may be heavily contaminated another man’s poison. As a result, understanding of the consequences with mycotoxins; these foods are for example, some populations of consuming large quantities of untested; and regulatory controls are exposed daily to aflatoxins toxin-contaminated food daily, as is either do not exist or are not enforced. at levels well above those legally often the case in subsistence farming In low-income countries, the permitted in other countries; these areas in developing countries. A main consequence of mycotoxin are toxins that the international second priority is development of awareness may be the commercial health community has recognized effective, accessible, and practical sale of the best quality food and local for two decades as being human intervention measures to prevent or consumption of the worst. Grains carcinogens.

Executive summary. Improving public health through mycotoxin control ix In low-income countries, exposure is widespread. The types of fungi – where it infects small grains. F. to mycotoxins at high levels is often and therefore mycotoxins – found in graminearum is the major producer closely associated with inequality different regions depend on climatic of deoxynivalenol and zearalenone and poverty. Therefore, affordable conditions. The main mycotoxins of in maize, wheat, oats, and barley and feasible solutions should be a significance for human disease are and produces these toxins whenever part of the international development aflatoxins, fumonisins, ochratoxin A, it infects these grains before harvest. agenda. Much can be done; much deoxynivalenol, and zearalenone. Indeed, wheat can be highly affected should be done. Failure to act in a These toxins are produced by just a by deoxynivalenol in southern China timely manner could pose a grave few species of fungi from the common and parts of South America as well risk to consumers in low-income genera Aspergillus, Penicillium, and as in cooler, damper climates in countries where basic commodities Fusarium. These fungi may either Europe and Canada. Maize is at like wheat, maize, and groundnuts grow on the crop or invade the crop considerable risk for contamination by are potentially contaminated with after harvest and produce toxins aflatoxin, fumonisin, deoxynivalenol, harmful levels of mycotoxins and are during drying and storage. and zearalenone, sometimes all four consumed in large quantities. Wheat is the most important at once. Significant contamination This book from the International cereal for human consumption, of maize with aflatoxins and Agency for Research on Cancer, as the staple food for nearly half fumonisins occurs in the tropical the specialized cancer agency of of the world’s population, followed and subtropical growing areas of the World Health Organization, by rice and maize. With some the world. Rice is not infected before aims to sensitize the international exceptions (e.g. parts of West Africa), harvest by any of the major toxigenic community to the mycotoxin problem consumption of the remaining staple fungi and seldom, if ever, contains in a format that is accessible to crops, including sorghum and millet, significant mycotoxins immediately a wide audience and is useful to is minor by comparison. Natural after harvest, drying, and hulling. decision-makers across a broad contamination of the three major However, rice can be contaminated spectrum of disciplines, including crops by mycotoxins can be a serious by aflatoxins if post-harvest storage agriculture, public health, marketing, food safety problem in some regions, systems are inadequate. and economics. The editors hope but the crops differ in susceptibility that this book will be a stimulus to to the various fungi that produce Mycotoxin measurement governments, nongovernmental and toxins. The incidence of mycotoxins international organizations, and the also depends on a wide variety of Investigating the health and eco- private sector to initiate measures agronomic and climatic conditions nomic impacts of mycotoxins requires designed to minimize mycotoxin as well as on whether a particular measuring their occurrence. How- exposure in high-risk populations. cultivar is grown within the area to ever, accurately assessing the level The book not only provides a scientific which it is adapted. of one or more mycotoxins in a description of the occurrence and Of the Aspergillus species occurring crop or food commodity is a major effects of mycotoxins but also goes in warmer climates, A. flavus produces challenge because the mycotoxins further by outlining approaches to aflatoxins in maize, groundnuts, are heterogeneously distributed, reduce mycotoxin exposure aimed tree nuts, and, less frequently, other which makes representative sam- at improving public health in low- commodities, whereas A. parasiticus is pling extremely difficult. Therefore, income countries. This executive common in groundnuts. Fumonisins considerable care and planning are summary briefly outlines the book’s are produced mainly by Fusarium needed in sampling and sample prep- content and provides some context verticillioides, a fungus that is aration to ensure the accuracy of the to its preparation. ubiquitous in maize. Ochratoxin A is estimated levels of contamination. produced mainly by A. carbonarius, In any investigation of mycotoxin Mycotoxin occurrence which is commonly found in grapes, levels in relation to health effects dried vine fruits, wine, and coffee, or for regulatory purposes, careful Mycotoxins are secondary fungal and by A. ochraceus and related attention must be paid to the need for metabolites that contaminate many of species, which produce this toxin accurate estimates of contamination. the most frequently consumed foods in coffee and sometimes in stored Representative sampling can pose and feeds worldwide. Therefore, grains. Penicillium verrucosum also particular problems in the context of human and animal exposure to one produces ochratoxin A but occurs small-scale farmers or subsistence or more of this broad group of toxins only in cool temperate climates, farming communities, where only

x smaller sample sizes can be obtained, Aflatoxins can cause acute defined. For aflatoxins, a strong and thus sampling approaches must toxicity in many species, most notably causal association with liver cancer be adapted accordingly. evident in poisoning outbreaks in has been identified, with a particularly Once an appropriate sample poultry. In addition, chronic exposure elevated risk in people chronically has been prepared, an analytical leads, among other effects, to infected with hepatitis B virus procedure must be selected to reduced weight gain, decreased egg (HBV). Acute aflatoxin poisoning, measure the mycotoxin(s). Many production, impaired immunity, and termed aflatoxicosis, is also a known modern methods require highly so- altered susceptibility to infectious consequence of exposure to high phisticated technology and skilled agents. Fumonisins were first dietary toxin levels. The recently technicians. Although these tech- identified through the occurrence identified association between afla- niques are applicable in settings of equine leukoencephalomalacia toxin exposure early in life and of developed economies handling and, later, the occurrence of impairment of child growth is an bulk commodities, a need still exists porcine pulmonary oedema. The important observation that, together for methods that are applicable to main target organ of ochratoxin A with possible immunomodulatory smaller-scale farms, where resources is the kidney, and nephropathy is effects, demands greater scrutiny are limited and rapid decisions are accompanied by renal dysfunction to evaluate its importance in many needed about the contamination and oedema; this is most common vulnerable populations worldwide. If of foods to be consumed locally. in pigs but has also been reported the link between aflatoxin exposure Validated thin-layer chromatography in horses. Ochratoxicosis in poultry and child growth impairment is and immunoassays are existing is associated with feed refusal and causal, this will transform the technologies capable of meeting high mortality. Deoxynivalenol can public health impact of minimizing this challenge. Indeed, one of the provoke feed refusal, e.g. in cattle, exposure. outstanding requirements in the pigs, and chickens, and pigs are For fumonisins, studies indicate a management of mycotoxins is the particularly sensitive; high intakes possible role in oesophageal cancer production of reliable and easily are also associated with emesis. and in neural tube defects, but applicable tests for use in low-income Zearalenone causes a variety further investigation is needed as a countries. Commercial companies of reproductive problems as a priority. For deoxynivalenol and other and development agencies should consequence of estrogenism. trichothecenes, exposure has been consider production of appropriate In relation to cancer, aflatoxins linked to acute poisoning outbreaks testing kits, sampling equipment are hepatocarcinogenic in several in large numbers of subjects. The (e.g. grinders), and training models different species, and fumonisins effects of chronic exposure need specifically tailored for use in these have been shown to induce to be defined, given the extent of regions. kidney tumours in mice and liver exposure occurring worldwide. For tumours in rats. Ochratoxin A ochratoxin A and zearalenone, the Effects in food-producing is also carcinogenic in rats and human health effects remain largely animals mice, inducing kidney carcinoma undefined and also merit greater in rats and both liver and kidney attention. However, investigating Animals suffer a variety of adverse tumours in mice. In contrast, there the effects of mycotoxins on effects from consuming mycotoxin- is little evidence of carcinogenicity human health is challenging, not contaminated feeds, and these in of deoxynivalenol. Zearalenone least because of the difficulties in turn may pose problems to those treatment increased incidences of measuring exposure, and this is also communities that rely on the animals liver cell and pituitary tumours in reflected in the relative paucity of for food. Several indices can be used mice, consistent with a hormonal epidemiological studies on the toxins to associate a disease in animals mode of carcinogenic action; no mentioned above. Newly established with a particular mycotoxin through carcinogenic effect was seen in rats. biomarkers of exposure at the awareness of the outward adverse individual level are proving valuable signs typical of exposure to a given Effects in humans in improving exposure assessment mycotoxin. As well as the direct effect and should be prioritized in terms in animals, mycotoxicoses in domestic Despite the fact that the potential of development and validation as animals can be a warning sign of a adverse effects of mycotoxins on well as application in studies of risk of high-level contamination of human health have been recognized etiology. foods for human consumption. for centuries, much remains to be

Executive summary. Improving public health through mycotoxin control xi As with other environmental risk assessments have had a food or feed, and (ii) the human toxins, individual differences in variety of impacts in relation to the health losses from the adverse susceptibility to mycotoxins are global burden of diseases related effects mentioned above. likely. Susceptibility may be influ- to mycotoxins as well as regulatory Losses related to markets occur enced by genetic, demographic, measures on food supply and within systems in which mycotoxins nutritional, infectious, or immune international trade. Overall, in line are being monitored in the food factors. For example, individuals with the limited evidence from and feed supply. Such monitoring may differ in the way in which epidemiology referred to already, systems can result in a commodity mycotoxins are metabolized or, many uncertainties remain. The with excessively high mycotoxin alternatively, other co-exposures health end-points used to set levels being rejected for sale or may be critical, as mentioned above regulatory levels need to be having a lower market value, but for aflatoxin exposure and HBV periodically reviewed, given that human exposure to the toxins is chronic carriers. The consequence mycotoxins may cause a diverse thus reduced. Losses related to of these interactions is that the set of effects. For example, with health occur when mycotoxins are same mycotoxin exposure may aflatoxins the risk assessments present in food at levels that can have different effects in different have been dominated by the cause illness, and such losses populations or individuals. The in- association with liver cancer, but can be measured using disability- fluence of malnutrition on sus- the recent studies on child growth adjusted life years (DALYs) or ceptibility to toxicity induced by impairment, if confirmed, may quality-adjusted life years (QALYs). mycotoxins has been little studied. In provide an important additional It is estimated that aflatoxin-related developing countries, most people parameter for consideration. These liver cancer alone may cause more consuming maize as a monocereal adverse effects may occur at than 2 million DALYs annually staple diet also lack sufficient exposure levels below those that worldwide. The total socioeconomic levels of micronutrients and thus induce cancer, in which case the burden of all mycotoxins and all may have enhanced susceptibility current regulatory levels may associated diseases and conditions to adverse effects of contaminating provide inadequate protection, is likely to be much higher. From mycotoxins. Young children may even if adhered to. a market standpoint, in the USA be particularly susceptible, as Many differences are also alone, mycotoxins cost crop may be the fetus. In fact, there is apparent in risk management growers more than 1 billion US a dearth of data on the possible strategies and government regu- dollars annually. Similar losses are effects of mycotoxins in women lations and technologies to reduce sustained by producers in three and children in populations where levels of mycotoxins in the food- Asian countries – Thailand, the mycotoxin exposures are highest. chain. However, it is evident that Philippines, and Indonesia – from Increased awareness of the risks regulatory measures have very aflatoxin alone. posed to vulnerable populations little impact on remote rural and An important component, by mycotoxins should stimulate subsistence communities. which merits more research, is the more research on the interactions economic assessment of costs of with other risk factors and elicit Economics of mycotoxins interventions to reduce mycotoxins caution when risk assessments are compared with benefits, in terms conducted. In the past, the biology and of either improved markets or economics of mycotoxins have improved human health. The cost- Risk assessment and been considered separately, effectiveness of an intervention is management but if the mycotoxin problem evaluated by comparing the cost of is to be addressed, a rounded implementing the intervention in a The well-documented toxicity of consideration is needed of the given population, per DALY saved, mycotoxins in animals and humans broader consequences of these with the per capita gross domestic has provided the impetus for the environmental contaminants. There- product for the country in which the conduct of risk assessments on all fore, the economic impacts of intervention is applied. Aside from the major mycotoxins (aflatoxins, mycotoxins should be thought of in direct economic considerations, fumonisins, ochratoxin A, deoxy- two ways: (i) the direct market costs interventions that are feasible in nivalenol, and zearalenone) by associated with lost trade or reduced low-income countries should be various regulatory agencies. These revenues due to contaminated prioritized.

xii Reducing exposure to gastrointestinal tract, thus reducing of the world mycotoxin levels are mycotoxins absorption. An alternative approach, relatively high but exposure is also for aflatoxins, has involved the modest because of a varied diet. Approaches to minimize mycotoxin use of chemopreventive agents In other areas, a similar level of levels in food are varied, encom- (e.g. chlorophyllin) that increase the contamination may translate into passing good agricultural and detoxification of reactive metabolites a much higher exposure because manufacturing practices as well in the body. Although these the diet is more uniform. This is as approaches at the household approaches can be used to target why communities relying on dietary or individual level. In general, exposed individuals, their application staples such as wheat, groundnuts, interventions at the different stages as a broader, sustainable public or maize, which are frequently include pre-harvest, post-harvest, health intervention is unclear. contaminated by mycotoxins, are and dietary approaches. What is A wide range of agricultural particularly vulnerable. These needed most is the evaluation and strategies, both pre-harvest and communities tend to be in regions adaptation of these approaches post-harvest, exist that may reduce where national regulations on into cost-effective, simple, and the quantity of aflatoxin in food. mycotoxins either do not exist or are sustainable intervention methods, Genetic modification of crops to seldom implemented. predominantly at the population level, reduce insect damage or enhance While facing mycotoxin contami- suitable for low-income countries. fungal resistance offers a promising nation and high levels of exposure, One possibility to reduce opportunity for some toxins. these same regions often face mycotoxin exposure is avoiding Biocontrol by inoculating fields challenges of adequate food produc- contaminated foodstuffs by modi- with non-toxigenic fungi is another tion and preservation combined with fying the diet. However, this is approach, already in use in some a generally poor nutritional status of dependent on having the necessary high-income countries. Post-harvest the population. Food availability may wealth and access to different interventions include the removal have a strong seasonality, which foods to enable choice. It also of infected and/or insect-damaged threatens the adequacy of nutrition presupposes that lack of food is not food components by hand sorting, at the household and national driving the need to eat contaminated preferably combined with correct level. Importation of food may be food. Some examples exist where drying and storage conditions, which constrained by currency problems, economic development has resulted avoid fungal proliferation and toxin among other things. This is the in a change in diet and lower production. Significant reductions in context in which many countries have mycotoxin levels, notably in parts exposure can be achieved by simply to face the challenge of mycotoxins of China where a shift from maize removing and discarding visibly as they balance the adequacy of the to rice consumption has lowered mouldy maize or groundnuts, for food supply with the safety of food. aflatoxin intake. This option is not an example. In addition, under some One key problem is the general obvious one for other populations to circumstances the food commodity lack of awareness about the presence follow but does demonstrate both the can be processed to reduce myco- and health effects of mycotoxin detrimental effects of overreliance toxin levels; an example is chemical contamination of food, compared on a potentially contaminated crop deactivation via nixtamalization. Each with the immediacy of not having and the positive effects of resources of these agricultural and individual enough to eat. As a consequence, flowing to low-income countries, approaches is more or less accessi- in many cases food security simply permitting a greater dietary diversity. ble to different populations, depending translates to having sufficient food Nevertheless, any such changes are upon available resources and cultural for everybody throughout the year, likely to be slow in coming. acceptance of the practice. as viewed from both household The possibility has been explored and government perspectives. of using dietary interventions that Food security versus food This constant concern about food either reduce mycotoxin bioavail- sufficiency sufficiency is itself, therefore, a ability or modulate metabolism in barrier to addressing the issue ways that reduce the harmful effects Human exposure to mycotoxins is of mycotoxins as a component of of ingested toxins. For example, determined by the level of contami- food security. At the household and some enterosorbent clays and nation in a given food commodity community level, misconceptions micronutrients have been added to and the quantity of that commodity about food security result from a the diet to adsorb aflatoxins in the consumed. Thus, in some areas lack of information and nutritional

Executive summary. Improving public health through mycotoxin control xiii education. At the government level, produce mycotoxins and that these we propose the following seven the information may also be lacking, can have adverse health effects priority areas for mycotoxin control but the overriding focus is on food remains uncommon. worldwide. supply, whereas the problem of A limited awareness of the 1. Education leading to improved food security is a matter that awaits danger of mycotoxins in foodstuffs awareness of mycotoxins economic development. is also apparent among the local across different sectors of At an international level, the traders who, in some regions of society, including householders, responsibility to safeguard the the world, may purchase from local farmers, traders, governments, health of a specific population growers and sell to customers and nongovernmental and from mycotoxins is placed on the or larger businesses. In fact, international organizations. importing governments because of regulations applied by importing 2. Better description of the the way mycotoxin regulations are developed countries to crop exports prevalence and level of myco- applied. This leads to heterogeneity from developing countries are often toxin exposure in the regions as a result of economic factors, the point where governments and of the world most affected, as a international trade agreements, and large traders are brought face to face basis for prioritizing control and other factors, such as the protection with the mycotoxin problem. estimating risk. of certain agricultural commodities Overall, therefore, it is important, 3. Development of accurate and by government subsidies. The through education, to improve applicable mycotoxin testing result of these varying pressures awareness of the problems of kits, sampling equipment, and is that mycotoxin regulation varies mycotoxin contamination of foods training models for use in low- among countries, from strict and to promote the potential income countries, to permit implementation based on the latest solutions within different sectors rapid assessment of mycotoxin risk assessments through to a total of society, including householders, contamination of staple foods. lack of implementation and control. farmers, and traders as well as 4. Conduct of epidemiological Therefore, the various influences governments and nongovernmental studies to assess the acute on regulatory decisions in a and international organizations. and chronic health effects of specific country are often played mycotoxin exposure; priorities out against a background of tension Responses to the mycotoxin include the impact of mycotoxins between the health authorities and problem – seven priorities on child growth and immune the political and economic powers status. controlling international trade and This executive summary provides a 5. Identification of individuals and industry. flavour of how this book addresses the groups who are particularly Despite the political, structural, major issues and of the complexity of susceptible to mycotoxins so and economic challenges to myco- the mycotoxin problem in low-income that the risk assessment can be toxin control outlined here, the means countries. However, mycotoxins are focused on those at greatest risk and the motivation for change already one problem embedded within many for adverse health effects. exist. Subsistence and small-scale other challenges to health, resulting 6. Development and evaluation of farmers are familiar with problems from poverty and inequality. cost-effective, simple, and sus- of the quality of seed varieties, pests Therefore, mycotoxins can only really tainable intervention methods (e.g. insects, rodents), and storage be addressed by taking into account suited to low-income countries. management. The understanding that the local situation with respect to food 7. Detailed economic assessments fungi damage crops exists and sufficiency, economic pressures, of interventions, in terms of solutions are welcomed, even competing health problems, and the either improved markets or though an understanding that fungi social environment. In this context, improved human health.

xiv CHAPTER 1 CHAPTER

chapter 1. Fungi producing significant mycotoxins

Summary Aspergillus species, occurring in and zearalenone, is pathogenic warmer climates, are A. flavus on maize, wheat, and barley and Mycotoxins are secondary metabolites and A. parasiticus, which produce produces these toxins whenever it of microfungi that are known to aflatoxins in maize, groundnuts, infects these grains before harvest. cause sickness or death in humans tree nuts, and, less frequently, other Also included is a short section on or animals. Although many such commodities. The main ochratoxin Claviceps purpurea, which pro- toxic metabolites are known, it is A producers, A. ochraceus and A. duces sclerotia among the seeds generally agreed that only a few carbonarius, commonly occur in in grasses, including wheat, barley, are significant in causing disease: grapes, dried vine fruits, wine, and and triticale. The main thrust of the aflatoxins, fumonisins, ochratoxin A, coffee. Penicillium verrucosum also chapter contains information on deoxynivalenol, zearalenone, and produces ochratoxin A but occurs the identification of these fungi and ergot alkaloids. These toxins are only in cool temperate climates, their morphological characteristics, produced by just a few species from where it infects small grains. F. as well as factors influencing their the common genera Aspergillus, verticillioides is ubiquitous in maize, growth and the various susceptible Penicillium, Fusarium, and Claviceps. with an endophytic nature, and commodities that are contaminated. All Aspergillus and Penicillium produces fumonisins, which are Finally, decision trees are included species either are commensals, generally more prevalent when crops to assist the user in making informed growing in crops without obvious are under drought stress or suffer choices about the likely mycotoxins signs of pathogenicity, or invade excessive insect damage. It has present in the various crops. crops after harvest and produce recently been shown that Aspergillus toxins during drying and storage. niger also produces fumonisins, In contrast, the important Fusarium and several commodities may be and Claviceps species infect crops affected. F. graminearum, which is before harvest. The most important the major producer of deoxynivalenol

Chapter 1. Fungi producing significant mycotoxins 1 1. Introduction and aflatoxicosis, which killed 100 000 it), or a storage fungus, and it young turkeys in the United Kingdom produces mycotoxins under all three Mycotoxins have been defined as in 1960 and has caused disease conditions. “fungal metabolites which when in- and death in many other animals, While not of worldwide signif- gested, inhaled or absorbed including humans (Rodricks et al., icance, a fourth genus is of sufficient through the skin cause lowered 1977; Lubulwa and Davis, 1994). importance to be included here. performance, sickness or death in By general consent, the name Claviceps is pathogenic on a wide man or animals, including birds” “mycotoxin” is usually restricted to toxic variety of cereals and other crops, (Pitt, 1996). This definition is widely compounds produced by microfungi producing resting structures called accepted, although interpretation and excludes toxins formed by the ergots, which often contain toxins. of “animals” is still under discussion Basidiomycetes, the mushrooms or in some quarters. The definition as macrofungi consumed as foods in 2.2 Which mycotoxins are it stands may be taken as including many parts of the world. important? lower (invertebrate) animals; as it Many thousands of metabolites seems likely that some mycotoxins have been described from microfungi; As noted above, several hundred are indeed aimed at insects, such even the large number dealt with compounds are known to be toxic as the mites that prey on fungi, this in extensive reviews (Cole and to humans or animals. However, interpretation is reasonable. However, Schweigert, 2003a, 2003b; Cole et many of these cause little concern most mycotoxicologists consider that al., 2003) are only a fraction of those because they are produced by mycotoxins are of relevance only when known. It seems likely that many fungi that are rarely encountered they affect humans and domestic of these metabolites are produced in foods or feeds. Many species of animals, and this much narrower not randomly but in attempts to alter Penicillium, for example, are found definition is used here. the ecology surrounding the fungus, almost exclusively in soils and rarely, It has been known for a long time by inhibiting growth of competitor if ever, in foods or feeds. Therefore, that it is hazardous to eat some species microorganisms, insects, etc. Only a many highly toxic compounds of the larger fungi, i.e. mushrooms and limited number of compounds, a few produced by Penicillium species “toadstools”, but until comparatively hundred, are known to be toxic to have not been found in foods or feeds recently the occurrence of common humans or domestic animals. in appreciable quantities. These moulds on foods has generally been include verruculogen, produced considered an aesthetic problem, 2. Mycotoxigenic fungi by P. simplicissimum and related not a health hazard. The realization species; janthitrems, produced by P. that metabolites of some common 2.1 Which genera are janthinellum; rugulosins, produced foodborne fungi were responsible important? by P. rugulosum and P. variabile; and for animal disease and death came many others. only in the 1960s, despite a few If we look at the worldwide occurrence In a second category are com- excellent studies in the first half of fungi in foods, and at which might pounds that are demonstrably toxic of the 20th century. It is now well be capable of mycotoxin production, under some test conditions, e.g. established that mycotoxins have three genera stand out: Aspergillus, by injection, but are not toxic when been responsible for major epidemics Penicillium, and Fusarium (Pitt and taken by a natural route. Compounds in humans and animals during recent Hocking, 2009). Fusarium species of this type may be inactivated by historical times. The most important are destructive pathogens on cereal stomach acids or are so insoluble epidemics have been ergotism, which crops and other commodities, and pro- as to be excreted without harm. has killed hundreds of thousands duce mycotoxins before, or immediately Sterigmatocystin, produced by the quite of people in Europe during the last after, harvest. Certain species of common storage fungus Aspergillus millennium (Smith and Moss, 1985); Aspergillus and Penicillium are also versicolor, is so insoluble in water, or alimentary toxic aleukia (ATA), which plant pathogens or commensals, but acid, that its true toxicity to mammals was responsible for the deaths of at these genera are more commonly has been difficult to measure, and it least 100 000 people in the USSR associated with commodities and has not been known to cause illness. between 1942 and 1948 (Joffe, 1978); foods during drying and storage. In a third category, of potentially stachybotryotoxicosis, which killed Aspergillus flavus is an exception: greater human health significance, tens of thousands of horses in the it can be a pathogen, a commensal are compounds that are produced by USSR in the 1930s (Moreau, 1979); (growing in a plant without affecting fungi known to occur in foods but that

2 CHAPTER 1 CHAPTER under normal conditions are present Some other mycotoxins are im- 3. Taxonomic overview of in such low concentrations that they portant in limited areas of the world. the fungal genera producing present no real hazard, i.e. their Sporidesmin is a mycotoxin that important mycotoxins effects, if any, are not measurable. causes facial eczema in sheep. It is In most cases the reason is that produced in pasture by the fungus The genera Aspergillus, Fusarium, although the fungi are readily isolated Pithomyces chartarum in some areas and Penicillium all reproduce by from some types of foods, they do not of New Zealand and Australia, and asexually produced spores, known normally grow to the extent required can cause large economic losses in as conidia, which are formed from to produce hazardous levels of toxin. local areas. The fungus Phomopsis specialized cells called phialides, Many examples exist: cyclopiazonic leptostromiformis produces the my- where mitosis takes place and from acid, from Aspergillus flavus; citrinin, cotoxin phomopsin in lupin plants which conidia are generated rapidly from Penicillium citrinin and several and seeds in Western Australia, and and in great numbers. Some species other species; citreoviridin, produced phomopsin is of great importance in each genus also produce a sexual by P. citreonigrum and Eupenicil- to the cattle raising and lupin seed stage defined by the production of lium ochrosalmoneum; roquefortine, export industry in that state. However, asci, specialized cells that result produced by P. roqueforti and its global impact is minimal. from meiosis, usually in well-defined related species; penitrem A, Patulin is sometimes included in macroscopic bodies (up to 1 mm in from P. crustosum; many of the lists of important mycotoxins, and diameter) called cleistothecia (Kirk trichothecenes produced by various concentrations in foods are subject to et al., 2001). A few species produce Fusarium species; and tenuazonic regulatory control in some countries. hard resting cells called sclerotia, acid, from Alternaria species. Under Patulin is produced by the growth of essentially immature cleistothecia. favourable growth conditions, how- Penicillium expansum in apples and In Aspergillus and Penicillium, ever, the fungi in this category are pears. The production of significant phialides are borne in clusters, while capable of extensive growth and levels of patulin is accompanied by conidia are single-celled, more or significant toxin production, so these visible rotting of the fruit, so patulin is less spherical, and very small, usually and some other toxins should be primarily of concern in juices. Nearly not exceeding 5 µm in diameter. The kept in mind when fungal spoilage of all the toxin can be removed if rotting two genera are closely related and foods and feeds occurs. fruit are rejected by visual inspection are distinguished by the way in which Some toxins are produced by or rotten parts are removed by hand phialides are grouped. In Aspergillus, rare species. For example, the trimming or by washing them out with phialides are always borne in tight species that produces rubratoxins is high-pressure water jets. Hence, clusters around the swollen apices known from only a few isolates and patulin in apple juices and other (vesicles) of long stalks (stipes), does not even have a recognized products is controllable by simple with or without an intermediate row name. Rubratoxin A is known to food technological procedures, and of supporting cells called metulae have caused one disease outbreak, its occurrence does not warrant (Raper and Fennell, 1965; Pitt and in two people who consumed mouldy consideration here. Hocking, 2009; Samson et al., 2010); home-made rhubarb wine (Richer The mycotoxins treated in detail see Figs 1.1–1.5. In Penicillium, et al., 1997). However, rubratoxin in this book are based on those phialides are usually borne in finger- can be ignored when overviewing considered by Miller (1995) to be like clusters on more diminutive mycotoxin occurrence worldwide. the most important on a worldwide stipes, again with or without one or A few mycotoxins are considered basis: aflatoxins, ochratoxin A, two intermediate rows of supporting to be significant in feeds but not fumonisins, specific trichothecenes cells (metulae and rami) (Pitt, 1979; foods. These are of known toxicity (deoxynivalenol and nivalenol), and Pitt and Hocking, 2009); see Fig. to birds, in particular, and are mainly zearalenone. These toxins are pro- 1.6. Colonies of Penicillium species water-soluble toxins. The reason duced in foods and feeds by species of grown on identification media in Petri appears simple: whereas mammals Aspergillus, Penicillium, and Fusarium. dishes are usually green, the colour excrete water-soluble toxins, often A limited taxonomic treatment of these of Penicillium conidia, and often have with little ill effect, birds excrete fungi and the species producing other pigments from the mycelium only solid waste, so are unable to important mycotoxins is given in this or excreted from the colonies. In get rid of these toxins so readily. In chapter. Also included is Claviceps Aspergillus, colony colours are this category are cyclopiazonic acid, purpurea, the species that produces those of the conidia, which may be citrinin, and tenuazonic acid. ergot and ergot toxins in small grains. black, yellow, brown, white, or green.

Chapter 1. Fungi producing significant mycotoxins 3 Colonies of most Aspergillus species 4. Genus Aspergillus bullet-shaped sclerotia. A. nomius show no other colours. Fusarium is associated with insects, and not colonies generally consist of loose, Aspergillus is a large genus, with 100 usually with foods, but recently has fluffy mycelium, coloured white, pink, or more recognized species, most been shown to be a common cause or purple, and often show similar of which grow and sporulate well on of aflatoxin production in Brazil nuts colours in the colony reverse. In common synthetic or semisynthetic (Olsen et al., 2008). Fusarium, phialides are not clustered, media. The most widely used tax- Enumeration. Satisfactory enu- and conidia may be of two types: onomy is by Raper and Fennell (1965), meration of A. flavus and A. para- those characteristic of the genus are although some of their concepts siticus can be achieved on any large and crescent-shaped (although are out of date. For more modern antibacterial enumeration medium sometimes formed only under natural taxonomic concepts, see Samson and that contains appropriate inhibitors to conditions or on special media), Pitt (1990) or Klich (2002). A minority reduce colony spreading. Dichloran whereas the second type, produced of Aspergillus species make a sexual rose bengal chloramphenicol agar by only some species, are small stage (known as a teleomorph) in (DRBC) or dichloran 18% glycerol and usually cylindrical (Marasas et which spores (ascospores) are borne agar (DG18) are recommended (Pitt al., 1984; Pitt and Hocking, 2009; in asci, in turn borne in cleistothecia. and Hocking, 1997, 2009; Samson et Samson et al. 2010); see Figs 1.7–1.9. Species with teleomorphs are correctly al., 2010). Relatively rapidly growing, The taxonomy of all three genera classified in teleomorph genera, of moderately deep, yellow green is complex. Overall taxonomies for which Eurotium, Neosartorya, and colonies exhibiting “mop-like” fruiting mycotoxigenic fungi occurring in Emericella are the best known. Few of structures under the stereomicro- foods are given in Fungi and Food the important mycotoxigenic species scope can be presumptively counted Spoilage (Pitt and Hocking, 2009) produce teleomorphs. as A. flavus plus A. parasiticus. and Food and Indoor Fungi (Samson The most significant mycotoxigenic Microscopic examination of colonies et al., 2010). The most useful intro- species in Aspergillus are A. flavus and can provide supporting evidence, ductions to the major species in each A. parasiticus, which make aflatoxins, but representative colonies must genus are found in laboratory guides: and the species that make ochratoxin be grown on standard identification A Laboratory Guide to Common A: A. ochraceus and related species, media for confirmation. Penicillium Species (Pitt, 2000), the black species A. carbonarius, and A more effective medium for enu- Identification of Common Aspergillus (uncommonly) A. niger. A. flavus and merating these species is Aspergillus Species (Klich, 2002), and The A. parasiticus are very closely related flavus and parasiticus agar (AFPA; Fusarium Laboratory Manual (Leslie and are treated together. Pitt et al., 1983), a medium formulated and Summerell, 2006). specifically for this purpose. AFPA has Claviceps differs from the three 4.1 Fungi producing aflatoxins: two major advantages: enumeration genera mentioned above because A. flavus and A. parasiticus can be carried out after incubation at most species cannot be cultivated in 30 °C for 2 days, and both species are the laboratory. Species of Claviceps Taxonomy. Aflatoxins are now readily recognized, even by untrained grow on a wide variety of grasses, known to be produced by at least eyes, by intense orange yellow colours where they infect only the ovaries, 10 Aspergillus species. However, in the reverses of the colonies. Under forming hard bodies called sclerotia most are rare or are rarely found in the incubation conditions specified that replace normal seed heads. foods: the principal fungi producing for AFPA (30 °C for 42–48 hours), Conidia are released in droplets aflatoxins remain A. flavus and A. the presence of the bright orange called honeydew, attractive to insects, parasiticus. Of some importance is yellow reverse is diagnostic for these which then disseminate the fungus a new species found in groundnuts species. throughout the crop. in the southern hemisphere, called Descriptions. The descriptions of The taxonomy (classification) of A. minisclerotigenes. This species Aspergillus species given here are these genera is described in more looks like a variant of A. flavus that taken from Fungi and Food Spoilage detail below. produces unusually small sclerotia, (Pitt and Hocking, 2009). The fungi but like A. parasiticus it produces both are grown as colonies on Czapek B and G aflatoxins. The other species yeast extract agar (CYA) and malt of some importance is A. nomius, extract agar (MEA) at 25 °C and which also makes B and G aflatoxins CYA at 37 °C for 7 days. Fungi are and looks like A. flavus but produces inoculated onto these plates at three

4 CHAPTER 1 CHAPTER points, equidistant from each other Fig. 1.1. Aspergillus flavus (a) colonies on CYA (left) and MEA (right), 7 days, 25 °C; and midway between the rim and (b, c) heads, bars = 20 µm; (d) conidia, bar = 5 µm. Source: Pitt and Hocking (2009), Fig. 8.13, p. 305; reproduced with kind permission from Springer Science+Business centre of the Petri dish. Inoculation Media B.V. is facilitated by first dispersing a needle point of conidia in small vials containing 0.2 mL of 0.2% agar and 0.05% Tween 80 or similar detergent (Pitt and Hocking, 2009), as this reduces colonies from stray spores. Formulations for these and other media are given in the Annex (p. 29). Aspergillus flavus Link. See Fig. 1.1. Colonies on CYA 60–70 mm in diameter; conidial heads usually borne uniformly over the whole colony but sparse or absent in areas of floccose (cotton wool) growth or sclerotial production, characteristically coloured greyish green but sometimes pure yellow, becoming greenish in age; sclerotia produced by about 50% of isolates, at first white, becoming deep reddish brown, density varying from inconspicuous to dominating colony appearance and almost entirely suppressing conidial production. Colo- nies on MEA 50–70 mm in diameter, similar to those on CYA, although usually less dense. At 37 °C, colonies usually 55–65 mm in diameter, similar to those on CYA at 25 °C. Sclerotia spherical, usually 400– 800 μm in diameter. Teleomorph developing from sclerotia, but only Aspergillus parasiticus Speare. three quarters of the surface, mostly after selected isolates are mated. See Fig. 1.2. Colonies on CYA 50– bearing phialides only, but in some Structures bearing conidia 400 μm to 70 mm in diameter, conidial heads isolates up to 20% of heads bearing 1 mm or more long; vesicles (terminal in a uniform, dense layer, coloured metulae as well; conidia spherical, swellings) spherical, 20–45 μm in dark yellowish green; sclerotia mostly 4.0–6.0 μm in diameter, with diameter, fertile over three quarters occasionally produced. Colonies distinctly roughened walls. of the surface, typically bearing on MEA 50–65 mm in diameter, The teleomorph is Petromyces both phialides and metulae (cells generally similar to those on CYA. flavus (Horn et al., 2011), but in culture supporting phialides), but in some At 37 °C, colonies covering the is seen only after suitable strains are isolates a proportion of, or even most, available area, similar to those on mated (Horn et al., 2009a). heads bear phialides alone; conidia CYA at 25 °C. Distinctive features. Aspergillus spherical or nearly, usually 3.5–5.0 μm Sclerotia occasionally produced, flavus and A. parasiticus together are in diameter, with relatively thin walls, white at first, becoming black, distinguished by their rapid growth at finely roughened or, rarely, smooth. spherical, 400–800 μm in diameter. both 25 °C and 37 °C and their bright The teleomorph is Petromyces Teleomorph developing from sclerotia, yellow green (or, less commonly, flavus (Horn et al., 2011), but in culture but only after selected isolates are yellow) conidial colour. The definitive is seen only after suitable strains are mated. Structures bearing conidia difference between the two species mated (Horn et al., 2009b). 250–500 μm long; vesicles spherical, is that A. flavus produces conidia that 20–35 μm in diameter, fertile over are rather variable in shape and size,

Chapter 1. Fungi producing significant mycotoxins 5 Fig. 1.2. Aspergillus parasiticus (a) colonies on CYA (left) and MEA (right), 7 days, 25 °C; predictive model for A. flavus growth (b, c) heads, bars = 10 µm; (d) conidia, bar = 5 µm. Source: Pitt and Hocking (2009), in relation to aw and temperature was Fig. 8.18, p. 321; reproduced with kind permission from Springer Science+Business Media B.V. derived from those data (Gibson et al., 1994). Growth of A. flavus occurred over the pH range 2.1–11.2 (the entire range examined) at 25, 30, and 37 °C, with optimal growth over a broad pH range of 3.4–10 (Wheeler et al., 1991). The heat resistance of A. flavus has been studied under various conditions by several authors. The

most reliable figures indicate a D45 value (the time required at 45 °C to kill 90% of the population) of more

than 160 hours, a D50 of 16 hours, a

D52 of 40–45 minutes, and a D60 of 1

minute, at neutral pH and high aw, with z values (the increase in temperature required to reduce the D value by 90%) of 3.3–4.1 C° (ICMSF, 1996). The addition of phosphine, used to control insects, to grain at 0.80

or 0.86 aw reduced growth of A. flavus while having little effect on the survival of conidia (Hocking and Banks, 1991, 1993). Available data indicate that the influence of physical factors on the growth of A. parasiticus is very similar to that on A. flavus (Pitt and Hocking, 2009). However, A. parasiticus grows at somewhat lower temperatures, up

to 42 °C. The effect of aw is similar to that found for A. flavus (Pitt and have relatively thin walls, and range coconut cream agar (Dyer and Miscamble, 1995). from smooth to moderately rough, McCammon, 1994). Recently, the the majority being finely rough. In use of a cyclodextrin, incorporated 4.1.1 Commodities and foods at contrast, conidia of A. parasiticus are into any standard medium, has risk from aflatoxin contamination spherical and have relatively thick, been proposed (Jaimez Ordaz et rough walls. In addition, vesicles of al., 2003). Visualization of aflatoxin Aspergillus flavus and to a lesser A. flavus are larger, up to 50 μm in production is by examination of the extent A. parasiticus have been diameter, and usually bear metulae, reverse of colonies on Petri dishes isolated from a very wide range of whereas vesicles of A. parasiticus under ultraviolet (UV) light. food commodities (Pitt and Hocking, rarely exceed 30 μm in diameter and Factors influencing growth. 2009). Indeed A. flavus may be metulae are uncommon (Klich and Growth temperatures for A. flavus regarded as truly ubiquitous in foods Pitt, 1988). The teleomorphs are not most often reported are a minimum of produced in tropical and subtropical seen in pure culture of single isolates. 10–12 °C, a maximum of 43–48 °C, countries. Although evidence of A. Differentiating toxigenic from and an optimum of about 33 °C (Pitt flavus at low levels in foods cannot be non-toxigenic isolates of A. flavus or and Hocking, 2009). The minimum taken as an indicator of the presence

A. parasiticus can also be of value. A water activity (aw) permitting growth is of aflatoxins, high levels of infection, variety of media have been proposed 0.82 at 25 °C, 0.81 at 30 °C, and 0.80 i.e. plate counts of greater than to achieve this, one of which is at 37 °C (Pitt and Miscamble, 1995). A 105/g, or infection levels of more than

6 CHAPTER 1 CHAPTER 50% of grains when direct plated, Aspergillus flavus is capable of factors are drought stress (Sanders provide reasonable presumptive causing spoilage of some kinds of et al., 1981) and soil temperatures evidence that aflatoxin may be fresh fruit and vegetables, including around 30 °C (Blankenship et al., present. However, quantification of citrus, tomatoes, peppers, litchis, 1984; Sanders et al., 1984; Cole et al., the association between levels of pineapples, and pomegranates 1985; Cole, 1989; Dorner et al., 1989) A. flavus infection and aflatoxin (Snowdon, 1990, 1991), but aflatoxin during the last 30–50 days before production is not possible, so these production is unlikely. harvest (Sanders et al., 1985). figures can only serve as guidelines. Maize. Maize is usually infected Although it is possible to induce 4.1.2 Formation of aflatoxins in only by A. flavus, not by A. parasiticus aflatoxin production in a very wide susceptible crops (Lillehoj et al., 1980; Angle et al., 1982; range of foods or raw materials under Horn et al., 1995). It appears probable experimental conditions, research Groundnuts. Groundnuts are sus- that the most important route for entry and experience have shown that only ceptible to infection by both A. flavus of A. flavus to maize is through insect certain commodity types are likely to and A. parasiticus (Hesseltine et damage (Lillehoj et al., 1982; Bilgrami contain aflatoxin in the absence of al., 1973; Diener et al., 1987; Pitt et al., 1992). Invasion down the silks obvious signs of fungal growth or and Hocking, 2009). The primary is also possible (Marsh and Payne, other deterioration in appearance. source of these fungi is soil, where 1984; Diener et al., 1987). High Based on the results of many high numbers may build up because temperature stress increases infection surveys, commodities most at risk in some groundnuts are not harvested (Jones et al., 1980), the critical time international trade are groundnuts, but remain in the ground and act as for infection being between 16 and 24 maize, and cottonseed. Lesser, but a nutrient source (Griffin and Garren, days after inoculation at silking (Jones still substantial, risk is associated 1976a). Uncultivated soils contain very et al., 1980; Payne, 1983). with tree nuts of all types, especially low numbers of A. flavus, but soils Cottonseed. A. flavus is also a Brazil nuts, pistachio nuts, and semi- in groundnut fields usually contain commensal in the cotton plant (Klich processed coconuts (i.e. copra). 100–1000 propagules/g (Pitt, 1989). et al., 1984). Infection occurs through Walnuts, hazelnuts, and cashews Under drought stress conditions, this the nectaries, natural openings in are only occasionally affected. number may rise to 104–105/g (Horn the cotton stem that are important in Spices from tropical countries are et al., 1995). Large numbers of A. pollination (Klich and Chmielewski, also a frequent source of aflatoxin, flavus spores are also airborne over 1985), or through cotyledonary leaf but these spices are usually present susceptible crops (Holtmeyer and scars (Klich et al., 1984). Upward at only low levels in foods. Oilseeds Wallin, 1981). movement occurs in the stem towards of all kinds are affected from time to Direct entry to developing ground- the boll, but not downwards from boll time (Pohland and Wood, 1987), and nuts through the shell by A. flavus to stem (Klich et al., 1986). Insect figs may carry a substantial risk (Le in the soil appears to be the main damage is also a potential cause of Bars, 1990). method for nut infection (Diener et infection (Lee et al., 1987), but insects Aflatoxins have been reported al., 1987). Infection can also occur are often well controlled in cotton from smoked and dried or cured fish through the pegs and flowers (Wells crops. As in groundnuts and maize, in Sierra Leone (Jonsyn and Lahai, and Kreutzer, 1972; Griffin and temperature appears to be a major 1992) but are not considered to be a Garren, 1976b; Pitt, 1989). A. flavus environmental factor in pre-harvest problem in salted dried fish produced sometimes grows within groundnut infection of cottonseed (Marsh et under South-East Asian conditions plants themselves: growth in plant al., 1973; Simpson and Batra, 1984). (Pitt and Hocking, 1996). Other tissue is not pathogenic but High minimum temperatures, above meat products, including prepared commensal. The seedpod (Lindsey, 24 °C, appear to lead to high aflatoxin hams (Rojas et al., 1991), are not 1970) or the plant (Pitt, 1989; Pitt formation (Diener et al., 1987). considered to be at risk. et al., 1991) show no visible sign of Cereals, legumes, and pulses colonization by the fungus. 4.1.3 Formation of aflatoxins in may also be infected with A. flavus A variety of factors influence other crops (Pitt and Hocking, 2009), but invasion of developing groundnuts unacceptable levels of aflatoxin occur by A. flavus. Infection before harvest With other crops, Aspergillus flavus is only under poor storage conditions occurs only if substantial numbers not associated with the plant, so entry and are rarely of concern. of fungal propagules (perhaps 103/g) to nuts or seeds or other food parts exist in the soil. Other important is opportunistic and usually occurs

Chapter 1. Fungi producing significant mycotoxins 7 only after the crop matures. Entry of low, and oil content high. Sorption reports refer to OTA production by A. flavus into pistachio nuts depends isotherms of these commodities are other, often unspecified, Penicillium on the time of splitting of hulls. Nuts similar (Iglesias and Chirife, 1982). species, but these reports are known in which hull splitting occurs early A. flavus and A. parasiticus cannot to be erroneous (Frisvad 1989; are much more susceptible to A. grow below about 0.80 aw, equivalent Frisvad and Filtenborg, 1989; Pitt flavus invasion on the tree (Doster to about 10% moisture content and Hocking, 2009). and Michailides, 1994). It is known in these commodities. However, Recently, Aspergillus carbon- that some cultivars are more prone storage above 8% moisture content arius has been identified as a third to early splitting than others, and this (about 0.7 aw) can lead to fungal major source of OTA, together with is especially important where nuts spoilage. Fungal growth may result a low percentage of isolates of the are harvested from the ground, after in a moisture increase, creating closely related species A. niger contact with the soil. conditions under which A. flavus (Abarca et al., 1994; Téren et al., Brazil nuts are harvested from can grow, so 8% moisture must be 1996). It is now clear that OTA is the ground, beneath trees growing considered the safe moisture content produced by two closely related naturally in Amazonian forests. Har- for these commodities. Penicillium species, P. verrucosum vesting is intermittent, up to a month Such a low moisture content can and P. nordicum, and by a rather apart, providing time for the ever be difficult to maintain in practice. remarkable range of Aspergillus present A. flavus and other related Shipment of nuts in containers species. The following sections deal species to infect the nuts (Johnsson et across the tropics is a particular with these species in more detail. al., 2008). A. nomius appears to be an hazard as unsuitable stowage, on important source of aflatoxins in Brazil decks or near engines, can lead 4.2.2 Aspergillus ochraceus and nuts (Olsen et al., 2008). to moisture migration sufficient to related species In other tree nuts, formation cause sporadic spoilage or even total of aflatoxins occurs sporadically, loss. Cases of rampant growth of A. Taxonomy. Recent work has shown usually as the result of insect damage flavus accompanied by high aflatoxin that Aspergillus ochraceus is not or poor storage practices. production have been observed a common producer of OTA. Most Figs are sometimes infected by under these conditions. OTA in foods originally attributed to A. flavus. The unique structure of the Storage of nuts in tropical A. ochraceus is now known to be due fruit evolved to enable fertilization countries is sometimes inadequate, to A. westerdijkiae and A. steynii, by insects, and insects may carry A. again leading to spoilage or aflatoxin newly described species very similar flavus spores into the seed cavity. production. morphologically to A. ochraceus Also, figs are harvested from the (Frisvad et al., 2004). Apart from ground in some countries. Immature 4.2 Ochratoxin A these species, this group of OTA figs are not colonized by A. flavus, but producers includes two ascosporic once they are ripe infection occurs 4.2.1 Formation of ochratoxin A fungi, Neopetromyces muricatus readily and fungal growth continues (asexual state A. muricatus) and during drying (Buchanan et al., 1975; Ochratoxin A (OTA) was originally Petromyces alliaceus (asexual Le Bars, 1990). The proportion of described as a metabolite of state A. alliaceus), and two that the crop infected is low, 1% or less Aspergillus ochraceus from labora- do not produce a teleomorph, A. (Steiner et al., 1988). The problem tory experiments (van der Merwe sclerotiorum and A. sulphureus. N. has been serious for some exporting et al., 1965), and subsequently muricatus is the correct name for countries (Sharman et al., 1991) but is from several related Aspergillus isolates that produce OTA, previously now well controlled by examination of species. However, the first report of identified as A. melleus. Both N. individual figs under UV light. natural occurrence, and the potential muricatus and P. alliaceus are importance, of OTA was from a uncommon species. A. sclerotiorum 4.1.4 Formation of aflatoxins in Penicillium species (Scott et al., isolates produce OTA only rarely, and storage 1970; Krogh et al., 1973), reported as although isolates of A. sulphureus are P. viridicatum, but later corrected to usually OTA producers, this is a rare Crops susceptible to aflatoxin P. verrucosum (Pitt, 1987). Larsen et species. Apart from A. westerdijkiae formation are mostly nuts and al. (2001) reported that P. nordicum, and A. steynii, all of these species oilseeds, where soluble solids closely related to P. verrucosum, are very uncommon in foods and are (sugars) in the dried commodity are is also a producer of OTA. Many not known to cause food spoilage.

8 CHAPTER 1 CHAPTER Fig. 1.3. Aspergillus ochraceus (a) colonies on CYA (left) and MEA (right), 7 days, 25 °C; sparsely sporing. At 37 °C, colonies of (b) heads, bar = 20 µm; (c) conidia, bar = 5 µm. Source: Pitt and Hocking (2009), 25–30 mm in diameter produced. Fig. 8.17, p. 318; reproduced with kind permission from Springer Science+Business Structures bearing conidia 1.0– Media B.V. 1.5 mm long, with yellowish to pale brown walls, finely to conspicuously roughened; vesicles spherical, 25– 50 μm in diameter, bearing tightly packed metulae and phialides over the entire surface; conidia spherical or near, 2.5–3.5 μm in diameter, with smooth to finely roughened walls. Distinctive features. Aspergillus ochraceus and closely related spe- cies producing ochratoxin all grow moderately slowly on standard identification media such as CYA and MEA (Pitt and Hocking, 2009). Colonies are coloured pale brown to yellow brown from the conidia. A. ochraceus grows strongly at 37 °C, whereas the closely related species A. westerdijkiae and A. steynii do not grow at that temperature. Apart from that distinction, A. westerdijkiae produces spherical, finely roughened conidia, whereas those of A. steynii are smooth-walled and ellipsoidal, not spherical. Factors influencing growth. Asper- gillus ochraceus and the closely related species described here are mesophilic xerophiles. Growth occurs between 8 °C and 37 °C, with the optimum at 24–31 °C (Pitt and Hocking, 2009). Optimal conditions

Enumeration. Aspergillus ochra- by growth in pure culture is necessary. for growth are 0.95–0.99 aw, while ceus, A. westerdijkiae, and A. steynii Satisfactory enumeration should the lower limit for growth is 0.79 aw on grow slowly on media of high aw as usually be possible also on DRBC, media containing sugars and down they are all xerophilic. Enumeration a selective medium of higher aw to 0.81 aw on media based on NaCl. on a medium of reduced aw, such (King et al., 1979). Dilute media, such A. ochraceus grows slowly at pH 2.2 as DG18, is recommended (Pitt and as potato dextrose agar (PDA) or and well between pH 3 and 10 (Pitt Hocking, 2009). bacteriological enumeration media, and Hocking, 1977). Colonies of A. ochraceus and and incubation temperatures above Commodities and foods at risk. closely related species can be 25 °C, are unsatisfactory. Aspergillus ochraceus has been presumptively recognized by relative- Aspergillus ochraceus Wilhelm. reported from a wide range of food ly deep colonies, uniformly coloured See Fig. 1.3. Colonies on CYA 40– products, more commonly in dried pale brown to yellow brown, that under 55 mm in diameter; conidial heads and stored foods than elsewhere. the low-power stereomicroscope closely packed, coloured light to golden However, it is likely that many of exhibit long fruiting stalks bearing yellow; sclerotia sometimes produced, these reports relate to the recently radiate Aspergillus heads, with spore white when young, later pink to purple. described A. westerdijkiae or A. chains splitting into two or three dense Colonies on MEA 40–55 mm in diameter, steynii. Stored foods from which these columns in age. Confirmation of identity plane, similar to those on CYA but quite species have been isolated include

Chapter 1. Fungi producing significant mycotoxins 9 smoked or salted dried fish and meat, that Aspergillus ochraceus, and no japonicus can be achieved on any beans, chickpeas, and nuts, especially doubt its close relatives A. westerdijkiae antibacterial enumeration medium pecans and pistachios. These species and A. steynii, are major sources of that contains appropriate inhibitors have been reported (usually as A. OTA in coffee (Taniwaki et al., 1999, to reduce colony spreading. DRBC ochraceus) from cereals and cereal 2003; Pitt et al., 2001; Batista et al., or DG18 is recommended (Pitt products but, rather infrequently, also 2003). Other known OTA producers, and Hocking, 1997; Samson et al., from cheese, spices, black olives, A. niger and A. carbonarius, have 2010). Rapidly growing, very dark and cassava. However, these species also been isolated from coffee (Frank, brown to black colonies exhibiting rarely cause spoilage, and are often 2001; Pitt et al., 2001). As detailed “mop-like” fruiting structures under found in foods at only low levels, so mycological studies have not yet the stereomicroscope can be their presence is not a good indicator been conducted in some major coffee presumptively counted as A. niger of significant mycotoxin production growing areas, the relative importance plus A. carbonarius. Microscopic ex- (Pitt and Hocking, 2009). of A. ochraceus and A. carbonarius amination of colonies can provide Several studies have detected as the main source of OTA in coffee is supporting evidence, but repre- A. ochraceus in green coffee beans difficult to assess. sentative colonies must be grown (Levi et al., 1974; Cantafora et al., Available evidence indicates that on standard identification media for 1983; Tsubouchi et al., 1984; Micco the sources of these fungi are environ- confirmation. et al., 1989; Studer-Rohr et al., 1994). mental and that entry to cherries is Aspergillus carbonarius (Bainier) Coffee cherries are usually picked gained during picking and drying Thom. See Fig. 1.4. Colonies on by hand, or sometimes mechanically (Taniwaki et al., 1999). OTA is produced CYA 60 mm or more in diameter, on large farms, and are usually during drying (Taniwaki et al., 1999; usually covering the whole Petri dried in the sun. The beans may be Bucheli et al., 2000; Teixera et al., dish; conidia black or nearly black. dried directly and separated from 2001). Coffee picked and dried under Colonies on MEA 50–60 mm in the hull afterwards, or mechanically good agricultural practice appears to diameter, usually smaller than those dehulled and dried, or dehulled by contain OTA only rarely (Taniwaki et on CYA, otherwise similar. At 37 °C, fermentation before drying. Coffee al., 1999, 2003). colonies 10–20 mm in diameter. beans are stored after drying (as Structures bearing conidia 1.0– “green” coffee), then graded and 4.2.3 Aspergillus carbonarius 3.0 mm long, with heavy, hyaline, shipped to manufacturers. and related species smooth walls; vesicles spherical, Picking cherries and spreading usually 60–85 μm in diameter, bearing them on drying yards frequently Taxonomy. Aspergillus carbonarius closely packed metulae and phialides causes damage, allowing ingress was recognized as a source of OTA over the whole surface; conidia of fungi. If cherries are picked from relatively recently (Horie, 1995; Téren spherical, 7–10 μm in diameter, black, the ground, contamination is likely et al., 1996; Wicklow et al., 1996). It is with walls extremely roughened. to be high. Drying is often a slow now known that most, if not all, isolates Aspergillus niger Tiegh. See Fig. 1.5. process, in particular because of of A. carbonarius produce OTA when Colonies on CYA 60 mm or more in the environment in which coffee grown in pure culture (Heenan et al., diameter, usually covering the whole is grown. Coffee trees will not 1998; Taniwaki et al., 1999), although Petri dish; conidia black. Colonies flower above 19 °C but require high the extent of production is variable. on MEA varying from 30 mm to temperatures to mature, so coffee is The closely related species A. niger 60 mm in diameter, usually smaller than commonly grown on upland areas in has also been reported reliably as a those on CYA and often quite sparse. the tropics. In consequence, drying producer (Ueno et al., 1991; Abarca At 37 °C, colonies 60 mm or more in is often conducted under less than et al., 1994; Heenan et al., 1998; diameter, covering the available space. ideal conditions, with morning mists Taniwaki et al., 1999). However, all Structures bearing conidia 1.0– or rain common in some growing reports agree that OTA production by 3.0 mm long, with heavy, hyaline, areas (Teixera et al., 2001). Fungal A. niger is very uncommon; OTA is smooth walls; vesicles spherical, growth frequently occurs. formed under pure culture conditions usually 50–75 μm in diameter, bearing Although the possibility of signifi- by only 1–2% of isolates. closely packed metulae and phialides cant levels of OTA being present in coffee Enumeration. Satisfactory enumer- over the whole surface; conidia beans has been known for some time, ation of A. niger and A. carbonarius spherical, 4–5 μm in diameter, brown, the fungal cause remained elusive. and the closely related (but with walls conspicuously roughened Only recently has it been established always non-toxigenic) species A. or sometimes striped.

10 CHAPTER 1 CHAPTER Fig. 1.4. Aspergillus carbonarius (a) colonies on CYA (left) and MEA (right), 7 days, 25 °C; optimum of 35–37 °C, and has been (b, c) heads, bars = 40 µm; (d) conidia, bar = 5 µm. Source: Pitt and Hocking (2009), reported to germinate down to 0.77 Fig. 8.10, p. 300; reproduced with kind permission from Springer Science+Business a (Pitt and Hocking, 2009). Media B.V. w Commodities and foods at risk. Some black Aspergillus species, i.e. A. niger, A. carbonarius, and A. japonicus, are common inhabitants of vineyards, as these fungi grow rapidly at relatively high temperatures (above 30 °C) and their pigmentation renders them highly resistant to the deleterious effects of sunlight and UV light. These species appear to have no pathogenicity towards grapes and to be unable to penetrate an intact grape skin. Entry to maturing grapes results from attack by other pathogenic fungi (e.g. Rhizopus stolonifer, Botrytis cinerea, or powdery mildews), from mechanical damage due to cultivating or harvesting equipment, or, in some cultivars, from the splitting of berry skins that results from rain near harvest time. Once entry to a berry is gained, these fungi thrive in the acid, high-sugar environment. Where grapes are dried, the black Aspergilli enjoy a considerable ecological advantage, and will continue to grow and produce OTA until the

grapes dry below 0.8 aw. As grapes are normally dried in the sun, this usually takes several days, allowing ample time for OTA production to occur (Hocking et al., 2003). Distinctive features. Differen- carbonarius and A. japonicus grow None of the three species of tiation of Aspergillus niger, A. much more slowly (less than 20 mm) black Aspergilli appears to enjoy a carbonarius, and A. japonicus from (Mitchell et al., 2003). particular ecological advantage, at nearly all other species is not difficult. It is important to remember that least in Australia, as all three species These species grow rapidly and very few isolates of A. niger produce are commonly recorded from maturing produce very dark brown to black OTA. A. niger is an exceptionally grapes, with proportions of each varying conidia. A. niger and A. carbonarius common species, and recovery of with seasonal factors (Leong et al., produce metulae, whereas A. this species from foods should not 2004). For OTA formation, Aspergillus japonicus does not. A. niger produces be regarded as evidence that OTA is carbonarius is the significant species: conidia that are 4–5 µm in diameter, likely to be present. only a small proportion of A. niger whereas those of A. carbonarius are Factors influencing growth. As- isolates are capable of producing larger, on average 7 µm or more in pergillus carbonarius can grow down OTA, and A. japonicus isolates do not diameter. If these species are grown to 10 °C, with an optimum near 30 °C produce this toxin. on CYA for 7 days at 37 °C, separation and a maximum near 41 °C. The In grapes intended for wine- on colony diameters can be very optimal aw for growth is 0.96–0.98, making, the time interval is usually useful: A. niger grows very quickly with a minimum near 0.85 at 25 °C. short between infection and crushing, (60 mm or more), whereas A. A. niger grows up to 45 °C, with an when fermentation stops fungal growth

Chapter 1. Fungi producing significant mycotoxins 11 Fig. 1.5. Aspergillus niger (a) colonies on CYA (left) and MEA (right), 7 days, 25 °C; The classification of Pitt (1979, (b) head, bar = 15 µm; (c) heads, bar = 10 µm; (d) conidia, bar = 5 µm. Source: Pitt 2000) includes four subgenera: and Hocking (2009), Fig. 8.15, p. 314; reproduced with kind permission from Springer Aspergilloides, in which phialides are Science+Business Media B.V. borne directly on the stalk cells without intervening supporting elements; Furcatum and Biverticillium, in which phialides are supported by metulae; and Penicillium, in which both metulae and rami are usually present. The majority of important toxigenic and food spoilage species are found in subgenus Penicillium. Enumeration. Enumeration pro- cedures suitable for all common Penicillium species are similar. Any effective antibacterial enumeration medium can be expected to give satisfactory results. However, some Penicillium species grow rather weakly on dilute media, such as PDA or dichloran chloramphenicol peptone agar (DCPA), so DRBC is recommended. Penicillia can also be effectively enumerated on DG18 (Pitt and Hocking, 1997; Samson et al., 2010). Identification. For a comprehen- sive taxonomy of Penicillium, see Pitt (1979). Keys and descriptions to common species are provided by Pitt (2000) and to foodborne species by Pitt and Hocking (2009) and Samson et al. (2010). and toxin production ceases. So 5. Genus Penicillium Identification of Penicillium isolates control of OTA in wines relies on good to species level is not easy, preferably vineyard management, i.e. control Taxonomy. Penicillium is a large being carried out under carefully of bunch rots and skin splitting, and genus, with more than 200 recognized standardized conditions of media, short time intervals between harvest species, of which 50 or more are of incubation time, and temperature. and crushing. common occurrence (Pitt, 2000). All As well as microscopic morphology, Occasional contamination of figs common species grow and sporulate gross physiological features, including with OTA has been reported (Özay and well on synthetic or semisynthetic colony diameters, colours of conidia, Alperden, 1991). In a study of Turkish media, and usually can be readily and colony pigments, are used to figs sampled during different stages recognized at genus level. Most distinguish species. of processing, only 3 of 100 samples Penicillium species grow slowly, and contained OTA, and in each case levels have green conidia. 5.1 Ochratoxin A production were between 5 μg/kg and 10 μg/kg. Classification within Penicillium by Penicillium verrucosum Aspergillus niger and A. carbon- is based primarily on microscopic arius also occur in a wide variety of morphology: the genus is divided into Soon after the discovery of OTA from other fruits (Snowdon, 1990, 1991). subgenera based on the number and Aspergillus ochraceus, the formation Generally, other fruits are handled in arrangement of phialides (elements of OTA by a Penicillium species, ways that minimize fungal infection, or producing conidia) and metulae P. viridicatum, was reported (van damaged fruit is discarded, not eaten, and rami (elements supporting Walbeek et al., 1969) and natural so OTA formation is not a hazard. phialides) on the main stalk cells. occurrence confirmed (Krogh et al.,

12 CHAPTER 1 CHAPTER Fig. 1.6. Penicillium verrucosum (a) colonies on CYA (left) and MEA (right), 7 days, mycelium white; conidial production 25 °C; (b, c, d) penicilli, bars = 10 µm; (e) conidia, bar = 5 µm. Source: Pitt and moderate, coloured as on CYA; Hocking (2009), Fig. 7.48, p. 260; reproduced with kind permission from Springer reverse dull brown or olive. No growth Science+Business Media B.V. at 37 °C. Structures bearing conidia 200–500 μm long, with walls finely to conspicuously roughened; fruiting structures variable, with two or three supporting cells beneath phialides; conidia usually spherical, 2.5–3.0 μm in diameter, with smooth walls. Distinctive features. Penicillium verrucosum is characterized by slow growth on CYA and especially on MEA, by conidia coloured relatively bright green, by the absence of other conspicuous pigmentation, and by rough walls on the stalk cells (Pitt, 2000). It is similar in general appearance to P. viridicatum and P. solitum. P. verrucosum and P. viridicatum produce a distinctive violet brown reverse on DRYS (Frisvad, 1983). It should be noted that recognition of this species requires specialist knowledge, or detailed comparison with known cultures (of this and other related species). Only one other species of Penicillium is known to produce ochratoxin A: P. nordicum. This species was segregated from P. verrucosum by small physiological differences (it produces a yellow 1973). The view that P. viridicatum was Enumeration. The media specified reverse on DRYS; Larsen et al., a major source of OTA contamination above for general enumeration of 2001), but it is ecologically distinct, in foods and feeds in some parts of Penicillium species are effective for occurring mainly on meat and the world was accepted for more P. verrucosum. On dichloran rose cheese. Its significance in terms of than a decade. The species involved bengal yeast extract sucrose agar human health is unknown. was later correctly identified as P. (DRYS), a selective medium for the Factors influencing growth. P. ver- verrucosum (Pitt, 1987), and this was enumeration of P. verrucosum and P. rucosum grows from 0 °C to 31 °C, with confirmed (Frisvad, 1989; Frisvad viridicatum, P. verrucosum produces the optimum at 20 °C. The minimum and Filtenborg, 1989). P. viridicatum a violet brown reverse colouration aw for growth is about 0.80 (Pitt and does produce mycotoxins, but these (Frisvad, 1983). Isolation and identi- Hocking, 2009). Growth occurs have only rarely been implicated in fication of P. verrucosum in pure over the pH range 2.1–10.0 at least animal health. culture is essential for confirmation. (Wheeler et al., 1991). The ability of Penicillium verrucosum, and the Penicillium verrucosum Dierckx. P. verrucosum to produce significant closely related P. nordicum, are the only See Fig. 1.6. Colonies on CYA 15– levels of OTA at 4 °C and aw as low Penicillium species that produce OTA. 25 mm in diameter; mycelium white; as 0.86 is noteworthy (Northolt et al., P. verrucosum commonly occurs in conidial formation light to moderate, 1979). The physiology of P. nordicum cereals in temperate climates, whereas grey green to dull green; reverse is likely to be very similar. P. nordicum has been isolated, yellow brown to deep brown. Colonies uncommonly, from processed meats. on MEA 12–15(–20) mm in diameter;

Chapter 1. Fungi producing significant mycotoxins 13 Commodities and foods at risk. 6. Genus Fusarium Taxonomy. The signature micro- The major food habitat for P. morphological characteristics of Fu- verrucosum is cereal crops grown Fusarium is one of the most important sarium species are uncoloured, in cool temperate climates, ranging genera of plant pathogenic fungi, with multiseptate, large (25 µm to 50 across northern and central Europe a record of devastating infections µm or more) curved conidia called and Canada. The occurrence of this in many kinds of economically macroconidia, which are produced species in European cereals has important plants. Fusarium species from phialides. Most species produce two consequences: OTA is present are responsible for wilts, blights, macroconidia in cushion-like structures in many kinds of European cereal root rots, and cankers in legumes, called sporodochia. In some species, products, especially bread and flour- coffee, wheat, maize, carnations, macroconidia are sparsely formed based foods, and in animals that eat pine trees, and grasses. The in Petri dish culture and recognition cereals as a major dietary component. importance of Fusarium species in of these species as belonging to OTA was detected in Danish pig the current context is that infection Fusarium requires experience. meats nearly 40 years ago (Krogh may sometimes occur in developing In addition to macroconidia, some et al., 1973), and its implications seeds, especially in cereals, and also Fusarium species can make one or for human and animal health were in maturing fruits and vegetables. two kinds of smaller, one- or two- recognized at the same time. As An immediate potential for toxin celled conidia called microconidia. bread and other cereal products and production in foods is apparent. Microconidia are usually produced in pig meats are major components The very important role of the aerial mycelium in culture. Most of the European diet, the further Fusarium species as mycotoxin often, microconidia are produced in consequence is that most Europeans producers appears to have remained slimy heads, but some species produce who have been tested have shown largely unsuspected until the them in chains or singly. Microconidia appreciable concentrations of OTA in 1970s. Research has now strongly are also produced from phialides, their blood (WHO, 2007). associated alimentary toxic aleukia which may have a single spore- Recent information indicates that (ATA) with Fusarium species. An bearing opening (monophialides) or P. verrucosum is not a commensal epidemic of this human mycotoxicosis multiple openings (polyphialides). on cereal crops, i.e. it does not grow in the USSR killed at least 100 000 The taxonomy of Fusarium has in grain crops before harvest, but people between 1942 and 1948 been difficult, with several competing its presence is due to post-harvest (Joffe, 1978). ATA outbreaks are taxonomic schemes, recognizing from contamination. The primary sources also known to have occurred in the 9 to 60 species in the genus. The of infection of the grain appear to Russian Federation in 1932 and taxonomy of Nelson et al. (1983), which be from harvesting, processing, 1913, and there is little doubt that accepted 30 species, has met with and storage equipment (Magan and outbreaks occurred in earlier years widespread approval, and is still widely Olsen, 2004; Olsen et al., 2004). as well (Joffe, 1978). Matossian used in conjunction with the laboratory A maximum growth temperature (1981, 1989) has argued persuasively manual of Leslie and Summerell near 30 °C restricts Penicillium that ATA outbreaks occurred in other (2006). Recent molecular studies verrucosum geographically. It ap- countries, including England, in the have suggested that Nelson et al. pears to be uncommon, indeed 16th to 18th centuries at least. (1983) greatly underestimated species almost unknown, in warm temperate Research since 1970 has shown numbers in Fusarium. For example, or tropical climates, or in other kinds that Fusarium species are capable O’Donnell et al. (1998) recognized of foods. P. verrucosum is never a of producing a bewildering array of 36 phylogenetic species (species source of OTA in foods from warmer mycotoxins. Foremost among these recognizably different by molecular climates, such as coffee, wines, or are the trichothecenes, of which at techniques) in a grouping corresponding other grape products. least 50 are known; the majority are to Fusarium section Liseola in which Penicillium nordicum has been produced by Fusarium. The most four species had been recognized by isolated quite commonly from meats, notorious trichothecene is T-2 toxin, Nelson et al. (1983). Nirenberg and especially refrigerated products. which was linked to ATA. Of no less O’Donnell (1998) described 10 new importance in modern times are the species in this section. However, for fumonisins, which are especially practical identification of the species toxic to horses, and are suspected important for mycotoxin production, the to be responsible for chronic human manual of Leslie and Summerell (2006) diseases also. is recommended.

14 CHAPTER 1 CHAPTER Table 1.1. Media of value for isolation and enumeration of Fusarium species advocated the use of PDA made from old potatoes, rather than commercial Medium Advantages Disadvantages Reference formulations, but many laboratories use commercial PDA with satisfactory Pentachloronitrobenzene Often used Carcinogenic; no spore Snyder and results. Oatmeal agar is used in some (PCNB) agar production by Fusarium Hansen species (1940) laboratories. The most commonly Dichloran chlorampheni- Sporulation allows Little pigmentation for Andrews used weak media are carnation col peptone agar recognition of differentiating Fusarium and Pitt leaf agar (CLA) and Synthetischer (DCPA) Fusarium species species (1986) Nährstoffarmer Agar (often now Czapek–Dox iprodione Pigmentation helpful Sterile colonies do Abildgren called synthetic nutrient agar [SNA]). dichloran agar in distinguishing not permit ready et al. (CZID) Fusarium species identification of (1987) SNA has the advantage of being a Fusarium species defined medium, but CLA supports Dichloran 18% glycerol Sporulation allows Low-aw medium, not Hocking superior sporodochial production agar (DG18) recognition of ideal for Fusarium and Pitt Fusarium species growth (1980) in some species. However, use of CLA requires access to a source of gamma-irradiated carnation leaves. A direct consequence of confusion carcinogen. DCPA is to be preferred Banana leaf agar (autoclave-sterilized in taxonomy has been confusion over as it contains pentachloronitroaniline banana leaves on half-strength corn- species–mycotoxin associations. Fu- (dichloran), a molecule with similar meal agar) is also a good medium for sarium isolates producing a particular properties to PCNB but that is not stimulation of sporulation, but is not yet toxin have often been given different carcinogenic. widely used. To maintain uniformity for names. However, Desjardins (2006) Recognition of Fusarium col- descriptions of all foodborne fungi, Pitt has provided a comprehensive onies on these media requires and Hocking (2009) provided Fusarium clarification of the important myco- careful observation and experience. descriptions on PDA, together with toxigenic species and the mycotoxins Presumptive identification to genus media used in Aspergillus and they each produce. The species judged level can usually be made from colony Penicillium identification. Some readily to be most important from the viewpoint appearance: low to floccose colonies, prepared media of value in Fusarium of human health are discussed here. coloured white, pink, or purple, with identification are given in Table 1.1. Enumeration and isolation. Growth pale to red or purple reverses, are Opinions differ regarding the of Fusarium species is favoured by indicative of Fusarium. Confirmation necessity to make a single conidium dilute media of high aw. Enumeration requires microscopic examination, isolate of Fusarium species before of Fusaria can be effectively carried in which the crescent-shaped mac- identification. Some laboratories out on media such as PDA provided roconidia characteristic of the genus make a new single conidium isolate chloramphenicol or other broad- should be observed. However, at every transfer of the culture. This spectrum antibiotics are added to these are not always produced on ensures a highly reproducible growth suppress bacteria. However, acidified enumeration media, especially PDA. rate. Other laboratories prefer not to PDA, a frequently used antibacterial Differentiation of some species on make single spore cultures because medium, is not recommended enumeration media is possible, but this may decrease the vigour of the because it may inhibit sensitive cells. also requires experience. culture. Because Fusarium species DCPA (Andrews and Pitt, 1986) and Identification. All contemporary often grow in mixed colonies when Czapek–Dox iprodione dichloran agar identification schemes based primarily isolated from soil or plant material, (CZID) (Thrane, 1996) are effective on morphology use two media: a weak at least one generation of single enumeration and isolation media for medium for stimulation of sporulation conidium isolates is advisable for most foodborne Fusarium species. and a richer medium for measuring cultures intended for experimental In addition, half-strength PDA is growth rates and for stimulation of use, especially genetic studies. used by many laboratories isolating diagnostic pigment production. The Traditionally, Fusarium cultures directly from plant tissue, where the most commonly used rich medium is have been cultivated for 7–10 days number of unwanted fungi is much PDA. Potato sucrose agar (PSA) was under mixed fluorescent/near UV lower than in soil or plant debris. It used in the manual of Booth (1971) light at room temperature, near 25 °C. should be pointed out that, although and is still used in some laboratories Recently, particularly for the group pentachloronitrobenzene (PCNB) agar instead of, or in addition to, PDA. of species producing fumonisin, is still widely used, PCNB is a known Nelson et al. (1983) among others the importance of also growing the

Chapter 1. Fungi producing significant mycotoxins 15 same cultures in darkness to allow Fig. 1.7. Fusarium verticillioides (a) colonies on PDA (left) and DCPA (right), 7 days, the development of some diagnostic 25 °C; (b) phialides bearing chains of microconidia, bar = 50 µm; (c) phialides, features has been emphasized. bar = 10 µm; (d) macroconidia and microconidia, bar = 10 µm. Source: Pitt and Hocking (2009), Fig. 5.36, p. 120; reproduced with kind permission from Springer The only definitive taxonomy Science+Business Media B.V for Fusarium is that of Nelson et al. (1983). The most useful and up-to- date guide to important species is that provided by Leslie and Summerell (2006). Pitt and Hocking (2009) and Samson et al. (2010) provide keys and descriptions for common foodborne species. The descriptions of species given here have been provided by Dr K.A. Seifert (Agriculture and Agri-Food Canada, Ottawa) and are based on the protocols and media described above.

6.1 Fumonisin production by F. verticillioides and F. proliferatum

Fusarium verticillioides (Sacc.) Niren- berg. See Fig. 1.7. Colonies on rich media (PDA or PSA) at 25 °C grow moderately rapidly, 3.5–5.5 cm in diameter in 4 days. Abundant aerial mycelium is produced, and the reverse usually has rays or large patches of violet or purple. On weak media (CLA or SNA) at 25 °C, sporodochia are The teleomorph of F. verticillioides name F. moniliforme is predated by F. sparsely produced or not present in is Gibberella moniliformis. verticillioides and also cannot reliably most isolates. When present they are Distinctive features. Fusarium be linked to modern species concepts. inconspicuous and almost colourless, verticillioides is recognized by the Fusarium thapsinum (teleomorph: on the agar surface beneath the often combination of purplish colours on Gibberella thapsina) is a closely related dense aerial mycelium. Macroconid- PDA, and the production of long, species, found primarily on sorghum. It ia are usually 3–5 septate, mostly curly chains of microconidia from has similar micromorphology, but PDA 30–45 µm long, straight or variably monophialides in the aerial mycelium. colonies lack purplish pigmentation curved, with more or less parallel walls Although macroconidia are found and tend to be yellow to yellow brown. and with the widest point near the in some cultures, many strains do Both F. verticillioides and F. thapsinum middle. Microconidia are abundantly not produce them. Therefore, an produce longer microconidial chains produced in the aerial mycelium on experienced eye can be necessary to and lack the polyphialides that highly branched conidiophores. The recognize those strains as belonging to characterize F. proliferatum. conidiogenous cells are monophialides Fusarium. The teleomorph, Gibberella Factors influencing growth. The that often collapse before the cultures moniliformis, is produced in culture maximum temperature for growth are about 10 days old. Microconidia only when strains of opposite mating of Fusarium verticillioides has been are produced in long, dry chains type are crossed under appropriate reported as 32–37 °C, the minimum visible with the stereomicroscope; conditions. as 2.5–5 °C, and the optimum near these chains, which are often coiled, Until recently, Fusarium verticil- 25 °C. The minimum aw for growth is give the colonies a distinctive texture lioides was known as F. moniliforme, about 0.87 (Pitt and Hocking, 2009). similar to curly hair. Individual conidia a name that is no longer used on Values of these parameters for F. are ellipsoidal, 0–1 septate, 4–19 × the recommendation of an expert proliferatum are essentially identical. 1.5–4.5 µm. committee (Seifert et al., 2003). The

16 CHAPTER 1 CHAPTER Fig. 1.8. Fusarium proliferatum (a) colonies on PDA (left) and DCPA (right), 7 days, proliferatum. Although macroconidia 25 °C; (b) phialides bearing microconidia in chains and false heads in situ, bar = 50 µm; are found in some cultures, many (c) macroconidia and microconidia, bar = 10 µm; (d) polyphialides, bar = 10 µm; strains do not produce them. (e) monophialides, bar = 10 µm. Source: Pitt and Hocking (2009), Fig. 5.31, p. 111; reproduced with kind permission from Springer Science+Business Media B.V. Therefore, an experienced eye can be necessary to recognize those strains as belonging to Fusarium. The teleomorph, Gibberella intermedia, is produced in culture only when strains of opposite mating type are crossed under appropriate conditions. Fusarium proliferatum is distin- guished from F. verticillioides by the shorter chains of microconidia and the occurrence of polyphialides in the aerial mycelium. Another frequently isolated species that occupies the same ecological niche is F. subglutinans, which does not produce fumonisins. It produces slimy heads rather than chains of microconidia from a mixture of monophialides and polyphialides; strains of this species are more likely to produce macroconidia than either F. verticillioides or F. subglutinans. F. nygamai is a similar species that also produces fumonisin in some strains; it can be distinguished from F. proliferatum by the production of chlamydospores. Other species making fumonisins. Several other Fusarium species are known to produce fumonisins, including F. anthophilum, F. beo- miforme, F. dlamini, F. globosum, Fusarium proliferatum (Matsush.) near the middle. Microconidia are F. napiforme, F. nygamai, F. Nirenberg. See Fig. 1.8. Colonies on abundantly produced in the aerial oxysporum, F. polyphialidicum, and rich media (PDA or PSA) at 25 °C mycelium on divergently branched F. subglutinans (IARC, 2002). None grow moderately rapidly, 3.5–5.5 cm in conidiophores. The cells producing is of major importance in fumonisin diameter in 4 days. There is abundant conidia are predominantly mono- production in foods. white to pink or slightly orange aerial phialides, but up to 20% may be Commodities and foods at risk. mycelium, and the reverse usually has polyphialides. Microconidia are pro- Fusarium verticillioides and F. rays or large patches of violet or purple. duced in short, dry chains visible with proliferatum, the major sources of On weak media (CLA or SNA) at 25 °C, the stereomicroscope, ovoid to ellip- fumonisins, are the most common sporodochia are usually sparsely soidal, 0–1 septate, 7–12.5 × 2–3 µm. fungi associated with maize. F. produced and often are not present. The teleomorph of F. proliferatum verticillioides has been known for When present they are inconspicuous is Gibberella intermedia. many years to occur systemically in and almost colourless, on the agar Distinctive features. The production leaves, stems, roots, and kernels of surface beneath the often dense aerial of purplish pigments on PDA, and the maize (Foley, 1962). These fungi can mycelium. Macroconidia are usually occurrence of short, dry chains of be recovered from virtually all maize 3–5 septate, 30–45 µm long, straight microconidia in the aerial mycelium kernels worldwide, including those or variably curved, with more or less and the sometimes sparse occurrence that are healthy (e.g. Hesseltine parallel walls and with the widest point of polyphialides characterize F. et al., 1981; Pitt et al., 1993; Miller,

Chapter 1. Fungi producing significant mycotoxins 17 1994; Miller et al., 1995; Ramirez European locations and results in the Canada, counties with the highest and et al., 1996; Logrieco et al., 2002). accumulation of moniliformin (Logrieco lowest average concentration after the F. verticillioides has been reported et al., 2002). From limited data, 1993 harvest were 1.4 and 0.4 times to suppress the growth of other ear moniliformin is not commonly found in the state average, respectively. The fungi (Reid et al., 1999), and kernels United States (Gutema et al., 2000) or average temperatures in the counties heat-treated to destroy the fungus Canadian maize (Farber et al., 1988). were similar, at 104% and 107% of germinate but do not thrive (Foley, After genotype susceptibility, the 30-year average, respectively. 1962). Strains of F. verticillioides temperature is the primary determining However, rainfall in the county with isolated from maize have the factor for maize diseases caused the highest fumonisin B1 level was potential to produce fumonisins, by Fusarium species (Miller, 1994; only 49% of normal, whereas in the even for maize from regions where Munkvold, 2003). F. graminearum county with the lowest level, it was fumonisin accumulations in maize are has a very narrow temperature 95% of normal (Miller et al., 1995). historically uncommon. These include window for growth in maize. The After experimental inoculation of Africa, Asia, Europe, North America optimal temperature is between 26 °C 14 maize genotypes in Poland, the (Canada, the USA, and Mexico), and and 28 °C. Its growth rate at 24 °C is average temperature in the year with

South America (WHO, 2000). one quarter that at 26–28 °C, while at the highest fumonisin B1 accumulation Fusarium kernel rot in maize 30 °C it is about one half. In contrast, was 117% of the 30-year average; that due to the growth of F. verticillioides F. verticillioides grows well above 26 °C in the year with the lowest fumonisin and related species causes the (Miller, 2001; Munkvold, 2003). B1 accumulation was 102% of normal. formation of fumonisins, whereas In culture, fumonisin B1 is pro- Rainfall in the year with the highest

Gibberella ear rot or pink ear rot, duced under conditions known to fumonisin B1 accumulation was 6% of mainly caused by F. graminearum, is favour the production of polyketides normal and in the year with the lowest, associated with deoxynivalenol and and sesquiterpenes. The toxin is 65% of normal (Pascale et al., 1997). zearalenone production (see Section optimally produced in media that A study of fumonisin occurrence

6.3). These fungi grow under different have moderate aw and are nitrogen- in hybrids grown in the USA indicated environmental conditions but with limited. Fumonisin is produced under that fumonisins are produced in higher overlap. F. proliferatum, which causes relatively high oxygen tensions concentrations in hybrids grown kernel rot, concurrently produces but apparently has an unusual outside their area of adaptation. fumonisin and moniliformin. Slightly requirement for low pH (about 2) Fumonisin concentrations were in- different environmental conditions for optimal production (Miller et al., versely proportional to June rainfall appear to favour one or the other of 1995); such conditions arise only (Shelby et al., 1994), again suggesting the principal species that produce when the plant is dead or dying. the important role of drought stress fumonisins. For these reasons, it is Fusarium kernel rot is associated (Munkvold, 2003). Data from samples sensible to consider the two diseases with warm, dry years and insect collected in Africa, Italy, and Croatia Fusarium kernel rot and pink ear damage and is caused by F. subglu- also indicate fumonisin accumulation rot together. In North America and tinans (teleomorph: Gibberella subgluti- in lines grown outside their area of Europe, fumonisins can occur in maize nans), F. verticillioides (teleomorph: adaptation, which includes tolerance crops in some seasons, but in others, Gibberella moniliformis), and F. to moisture stress (Doko et al., 1995; deoxynivalenol can occur, sometimes proliferatum (Logrieco et al., 2002; Visconti, 1996). accompanied by zearalenone. In Munkvold, 2003). In warmer parts of Hybrids with an increased other regions, such as Africa or the USA and in the lowland tropics, likelihood of kernel splitting show South-East Asia, all three toxins F. verticillioides is one of the most higher levels of Fusarium kernel can be seen together (Yamashita important ear diseases (De Leon and rot, and kernel splitting is generally et al., 1995; Doko et al., 1996; Ali Pandey, 1989). worse under drought conditions. et al., 1998). These differences in Studies of the occurrence of Drought stress also results in greater mycotoxin occurrence have important fumonisin from natural occurrence insect herbivory on maize; hence toxicological implications. and experimental infections clearly it is not possible to totally separate Fusarium subglutinans is also demonstrate the importance of drought these variables. Further, in studies of common in maize kernels in North stress and insect damage at the same experimental inoculation methods, the America (Miller, 1994; Munkvold, time as temperatures are favourable. severity of Gibberella ear rot is related

2003), but this species apparently The fumonisin B1 concentrations to wound size (Drepper and Renfro, causes a higher level of ear rot in some found in maize from the two Ontario, 1990). It was observed early on that

18 CHAPTER 1 CHAPTER a strong relationship exists between Factors that control insects, confer fumonisins produced by A. niger can be insect damage and Gibberella ear rot resistance to other ear diseases, and expected to be widespread if more than (Lew et al., 1991). Transgenic Bt maize adaptations, including drought and a few isolates are producers. Recent genotypes, which contain a gene from temperature tolerance, are important information indicates that fumonisin the soil bacterium Bacillus thuringiensis in reducing the risk of fumonisin production by A. niger is indeed that results in the accumulation of accumulations in maize. common. In one study of A. niger proteins toxic to key insect pests of Fumonisins are very uncommon in strains from a sample of Californian maize, had lowered levels of recovery commodities or foods other than maize raisins, 50 of 66 strains (77%) produced of F. verticillioides and fumonisin and maize products (IARC, 2002). fumonisins (Mogensen et al., 2010b). (Munkvold et al., 1997, 1999; Bakan In a second study, where isolates were et al., 2002; Munkvold, 2003). Under 6.2 Fumonisin production taken from 13 samples of dried vine conditions of high disease pressure, by Aspergillus niger and fruits from several countries, 20 of 30 the Bt hybrids can make the difference Alternaria arborescens (67%) produced fumonisins (Varga et between a crop being fit or unfit for al., 2010). Of an unstated number of human consumption (Hammond It has been known for 20 years that isolates from Thai coffee, 67% were et al., 2004; De La Campa et al., one particular race of Alternaria able to produce fumonisins (Noonim 2004). In an examination of fumonisin alternata, described as Alternaria et al., 2009). A. niger isolates do not concentration in relation to various alternata f. sp. lycopersici, produces usually produce fumonisin B1; the major climate variables under moderate fumonisins (Chen et al., 1992). This metabolite is fumonisin B2, sometimes insect pressure, most of the variation particular race is a host-specific with lower amounts of fumonisin B4. was explained by temperatures above pathogen that causes a stem canker So far, only a few studies have 30 °C (about 40%), followed by insect disease on tomato plants. For that examined food products that are pressure (about 20%) and hybrid reason, fumonisin production by this frequently infected by A. niger for the (about 10%; De La Campa et al., 2004). species has been largely ignored in presence of fumonisins. Low levels Maize infected by other patho- general discussions of fumonisins (1–9.7 µg/kg) were found in 7 of 12 gens that damage ears (such as F. in foods and feeds. This taxon has coffee samples (Noonim et al., 2009). graminearum) may be predisposed been re-identified as Alt. arborescens Levels of up to 7.8 mg/kg were found to F. verticillioides damage and fumo- (Frisvad et al., 2007). in inoculated dried fruits (Mogensen nisin accumulation. Ears inoculated Recently, however, the picture et al., 2010b). More alarming, seven with F. graminearum, F. verticillioides, has become more complicated. commercial dried fruit samples posi- and F. subglutinans by wounding Studies on the genome sequence tive for A. niger were all positive for produced visible symptoms on a 1–9 of Aspergillus niger showed, totally fumonisins B1 to B4: the average total scale of 7.3, 4.4, and 4.7, respectively. unexpectedly, the presence of the fumonisin level was 7.2 mg/kg, with the Despite the fact that F. graminearum genes for fumonisin (Baker, 2006), range 4.6–35.5 mg/kg (Varga et al., and F. subglutinans do not produce and this was independently verified by 2010). It is not surprising that wines can fumonisin, ears inoculated with Pel et al. (2007). It was soon confirmed also contain fumonisins. Of 51 market these fungi contained 42 µg/g and that this gene cluster (consisting of at samples of Italian wines, 9 (18%)

3 µg/g fumonisin B1, respectively least 15 genes) was active and that contained fumonisin B2, with levels (Schaafsma et al., 1993). at least some strains of A. niger can ranging from 0.4 µg/L to 2.4 µg/L Breeding for resistance to indeed produce fumonisins (Frisvad (Logrieco et al., 2010). Seventy-seven Fusarium kernel rot has not been et al., 2007). wine samples from 13 countries effective. Within areas of adaptation, The implications are vast. As were examined by Mogensen et al. there are apparent differences in described in Section 4.2.3, A. niger is (2010a), and 18 (23%) were found to symptom response (Miller, 2001; a very common fungus, of which a few contain fumonisins, with a range of Munkvold, 2003). In a large trial at strains produce ochratoxin A. Foods 1 µg/L to 25 µg/L. These levels are the International Maize and Wheat in which OTA is found, produced by A. low, but wine has a high consumption Improvement Center, in Mexico, niger and the closely related species rate in some areas. slight improvements in symptom A. carbonarius, include grapes, dried It has been found that fumonisin expression in some tropical maize vine fruits, wines, and coffee. A. niger production in culture by A. niger genotypes were observed after is also common in some fresh fruits, is enhanced at a slightly reduced many cycles of selection (De Leon particularly berries, and on onions aw, about 0.99, by the addition of and Pandey, 1989). (Pitt and Hocking, 2009). Therefore, 5% NaCl to CYA (Mogensen et

Chapter 1. Fungi producing significant mycotoxins 19 al., 2010c). This additive reduced Fig. 1.9. Fusarium graminearum (a) colonies on PDA (left) and DCPA (right), 7 days, fumonisin production by Fusarium 25 °C; (b) Gibberella zeae perithecium and ascospores, bar = 25 µm; (c) macroconidia, bar = 10 µm. Source: Pitt and Hocking (2009), Fig. 5.27, p. 103; reproduced with kind species. Fumonisin was produced permission from Springer Science+Business Media B.V. optimally by A. niger at 25–30 °C, whereas optimal temperatures for production were lower in Fusarium species (20–25 °C). Clearly, some evolution has occurred since the genes were transferred to A. niger (Mogensen et al., 2010c). At this time, it is difficult to assess the relative importance of fumonisin production by A. niger, but the range of commodities in which fumonisins are found has been extended considerably. However, given the very high consumption of maize in some countries, production by A. niger will have lesser importance.

6.3 Deoxynivalenol, nivalenol, and zearalenone production by Fusarium graminearum and related species

The most important Fusarium spe- cies that produce the trichothecenes deoxynivalenol (DON) and (less commonly) nivalenol (NIV) in small grains are F. graminearum, F. Fusarium graminearum Schwabe. Sexual fruiting structures (peri- culmorum, and, less frequently, F. See Fig. 1.9. Colonies on rich thecia) are often produced on CLA crookwellense. media (PDA or PSA) at 25 °C grow or SNA as the medium begins to The name Fusarium roseum rapidly, 7.5–9 cm in diameter in desiccate, almost black, about as used by Snyder and Hansen 4 days. There is abundant white, 200 µm in diameter, with a warty (1940) has caused a great deal of reddish or yellowish brown aerial wall, and exuding a light orange confusion in the literature as their mycelium, and the reverse is usually cloud of ascospores. Ascospores very broad species concept included distinctly red. About 5% of isolates are generally fusiform to allantoid several well-known and important have an orange brown reverse. On (slightly bean-shaped), light brown, Fusarium species, including F. weak media (CLA or SNA) at 25 °C, and 20–30 µm long. Ascospores graminearum. The literature before almost colourless sporodochia are are forcibly discharged and can be the mid-1980s is therefore unreliable, produced under a sparse layer of found on the agar surface away from both taxonomically and with respect white aerial mycelium. Structures the perithecia, or on Petri dish lids. to mycotoxin production. Since the bearing conidia have 1–2 levels The teleomorph of F. graminearum is publications by Nelson et al. (1983) and of branching, terminating with Gibberella zeae (Schw.) Petch. Marasas et al. (1984), F. graminearum 1–4 phialides. Macroconidia are Distinctive features. Fusarium and F. culmorum have become well usually 4–6 septate and abundantly graminearum produces straight mac- established and species–mycotoxin produced in fresh isolates, mostly roconidia with parallel walls, which in relationships have become clear. 40–60 µm long, more or less straight combination with rapid growth and F. crookwellense is a widespread with parallel walls. No microconidia usually red pigments on PDA, are species first described in 1982. are produced, although sometimes relatively distinctive. The frequent immature macroconidia are seen and occurrence of perithecia of the could be confused with microconidia. teleomorph in culture is also a

20 CHAPTER 1 CHAPTER reliable characteristic, occurring in is abundant aerial mycelium, white but F. graminearum appears to be about 90% of fresh isolates under to pink, and the reverse is usually displacing it (Waalwijk et al., 2003). appropriate lighting conditions. F. distinctly red. On weak media (CLA Concerning the other species pseudograminearum (teleomorph: or SNA) at 25 °C, sporodochia are involved in Fusarium head blight, Gibberella coronicola) is an ecologi- usually abundant and distinctly F. avenaceum is also common in cally and phylogenetically distinct reddish brown. Structures bearing wheat from all regions studied. F. species that causes crown rot of wheat. conidia, arising from the sporodochia, crookwellense is relatively common The macroconidia are similar to those have 1–4 levels of branching, in Australia and South Africa, but of F. graminearum, but are reportedly terminating with 1–6 phialides. is rare in wheat from Canada and broadest above the middle; the growth Macroconidia are usually 5 septate, the USA. F. poae, F. langsethii, F. rate on PDA is slower. Diagnostic PCR 35–60 × 4.5–6.5 µm, fairly uniform equiseti, and F. sporotrichioides are primers for F. pseudograminearum in shape, with a more or less straight also isolated from wheat kernels at were designed by Aoki and O’Donnell ventral wall and curved dorsal wall, low to moderate frequencies, more (1999) based on β-tubulin sequences. with the widest point near the middle, commonly under cooler conditions This species also produces DON and of medium size. Microconidia are (Bottalico and Peronne, 2002). zearalenone (ZEA). not produced, although sometimes The distribution of head blight Fusarium culmorum (W. G. Sm.) immature macroconidia give this species is affected by pathogenicity, Sacc. Colonies on rich media (PDA impression. with a relative pathogenicity of F. or PSA) at 25 °C grow rapidly, 7.5– Distinctive features. Distinctive graminearum > F. culmorum >> F. 9 cm in diameter in 4 days. There is macroconidia, with straight inner walls crookwellense > F. avenaceum. The abundant white to slightly orange, and curved outer walls, characterize regional and annual variation of the brown or reddish aerial mycelium, Fusarium crookwellense. pathogenic species is most affected and the reverse is usually distinctly Factors affecting growth. The op- by temperature; species ranked red. About 5% of isolates have timal temperature for growth of from coldest to warmest areas are an orange brown or tan reverse. Fusarium graminearum is 25 °C, F. culmorum > F. crookwellense > F. On weak media (CLA or SNA) at and the maximum below 37 °C. avenaceum > F. graminearum (Miller,

25 °C, reddish brown or orange The minimum aw for growth is near 1994; Bottalico and Peronne, 2002). sporodochia are produced under a 0.90 (Pitt and Hocking, 2009). F. Isolates of F. graminearum and sparse layer of white aerial mycelium. culmorum is a psychrotroph, growing F. culmorum produce a fairly large Structures bearing conidia have up down to 0 °C but up to only 31 °C (Pitt number of other compounds as well to 4 levels of branching, terminating and Hocking, 2009). as DON and ZEA. Isolates from with 1–4 phialides. Macroconidia are Trichothecenes in small grains: North and South America produce usually 3–6 septate and abundantly Fusarium graminearum and related 15-acetyl deoxynivalenol, the precur- produced in fresh isolates, mostly species. Fusarium head blight is an sor to DON. If isolates from Asia 30–45 µm long, with the widest important plant disease in temperate and Europe produce DON or NIV, point above the middle and hence regions that affects small grains, they also produce the respective somewhat wedge-shaped, often mainly wheat, but also barley and 3-acetate, i.e. deoxynivalenol mono- appearing short and fat. No micro- triticale. Five or six Fusarium species acetate or fusarenon-X. Strains of F. conidia are produced. are consistently isolated from small crookwellense produce NIV regardless Distinctive features. Broad, grains affected by this disease, and of geographical origin (Miller et al., wedge-shaped macroconidia with the most pathogenic species, F. 1991; Bottalico and Peronne, 2002, short apical cells and basal cells graminearum and F. culmorum, are under the name F. cerealis). Some are distinctive for F. culmorum. The the most common. These two species of the minor metabolites are found in related species F. sambucinum are closely related and produce DON small grains along with DON. produces narrower macroconidia or NIV and ZEA, depending on the Although increased rainfall than F. culmorum. The wedge geographical origin of the isolate promotes Fusarium head blight, shape of the conidia distinguishes F. (Miller et al., 1991; Waalwijk et al., incidence is most affected by moisture culmorum from F. crookwellense. 2003). F. graminearum is common at anthesis as long as the temperature Fusarium crookwellense Burgess in wheat from North America, remains in the favourable range et al. Colonies on rich media (PDA or South America, and China (Miller, for growth (Miller, 1994). Cultivar PSA) at 25 °C grow rapidly, 7.5– 1994). In cooler parts of Europe, susceptibility and rainfall at anthesis 9 cm in diameter in 4 days. There F. culmorum has been dominant, explain most variability in infection,

Chapter 1. Fungi producing significant mycotoxins 21 but crop rotation also has a large to the membrane-damaging effects of poisonous to humans and animals, effect. Growing wheat following maize DON than susceptible cultivars (Miller which have also found major use in increases disease under favourable and Ewen, 1997). pharmaceuticals. weather conditions (Schaafsma et al., Many Claviceps species are 2002). Reduced tillage is an equivocal 7. Genus Claviceps restricted to a few grass genera source of variation in the amount of as hosts. However, the species disease observed (Miller et al., 1998; The following descriptions of Clav- most important from the mycotoxin Schaafsma et al., 2002). iceps species are taken from viewpoint, C. purpurea, has a host Fusarium graminearum is a Alderman et al. (1999). range of more than 200 grass species. necrotrophic pathogen, i.e. it invades The genus Claviceps, an Asco- It is distributed worldwide in temperate plants by killing host cells in advance. mycete with a conidial state, includes climates and is responsible for the This was reported by the earliest several species that are parasitic disease called ergotism in humans investigators (see Schroeder and on grasses, including cultivated and domestic animals. Christensen, 1963). Trichothecenes cereals throughout the temperate Identification. Species of Claviceps were recognized to be phytotoxic world. Claviceps species infect only cannot be grown in culture, so must be compounds at the time of their the flowers of susceptible hosts. identified by natural characteristics. C. discovery (Brian et al., 1961). It Infection involves replacement of the purpurea is a pathogen on grasses, was realized much later that large ovary by a specialized structure that but not on sorghum or maize, whereas differences exist in the responses develops into a sclerotium, a hard, C. africana, also of importance in of wheat cultivars to Fusarium head compact mass of fungal tissue. The terms of mycotoxins, is a cause of a blight. Coleoptile tissue of cultivars that sclerotia are usually white, black, or serious disease resulting in male- were resistant to Fusarium head blight tan and are 1–4 times as large as sterile sorghum seed. was 10 times as resistant to necrosis the seeds they replace. The sclerotia Claviceps purpurea (Fr.: Fr.) Tul. in the presence of DON than was that and diseases caused by Claviceps The sclerotium of Claviceps purpurea of disease-susceptible cultivars (Wang species go by the general name ergot. comprises a compact mass of fungal and Miller, 1988). This difference was Ergots formed by the most important tissue encased in a dark pigmented shown to be due to the presence species, C. purpurea, are dark purple outer rind. This overwintering stage of a modified peptidyl transferase to black, and are most prevalent on apparently requires 2 months of involved in protein synthesis (Miller rye, but also occur to some extent on cold weather (0–10 °C) to induce and Ewen, 1997) and to unknown barley, oats, and wheat, as well as germination. In warmer regions, functional changes in the membranes wild and cultivated grasses. sclerotia do not survive well. of more resistant types (Snijders and During ergot development, conidia In spring, the sclerotia germinate, Krechting, 1992; Cossette and Miller, are produced by the fungus and are producing stalked ascocarps, in 1995; Miller and Ewen, 1997). Earlier immersed in plant sap to produce a which small, thread-like ascospores studies had shown that cultivars of sugary liquid known as honeydew, are produced. The ascospores are wheat in the field appeared to be able which drips from the infected plant ejected forcibly from the ascocarp to metabolize DON, and this was later as large drops. These are attractive and are carried by air currents to shown to be the case in vitro in cultivars to insects, which act as vectors for grass flowers. resistant to head blight (Miller and dispersing the conidia and spreading The period of susceptibility for Arnison, 1986). Strains that produce infection throughout the crop. most grasses is very brief, from flower high concentrations of DON in the field Sclerotia are the resting stage of opening to fertilization, as fertilized were more virulent (Snijders, 1994; Claviceps species between seasons. ovaries are resistant to infection. Mesterhazy et al., 1999). This implied Under favourable conditions scle- Environmental conditions that delay that one component of resistance rotia germinate, producing the pollination, such as cool temperatures, to Fusarium head blight is related ascomycete stage of the fungus. increase the infective period. Male- to reducing the phytotoxic impact of Ascospores are formed in closed sterile lines of grasses are especially DON. In addition, DON has been bodies on stalks arising from the susceptible to Claviceps infection found to appear in wheat kernels in sclerotia, and in many species because they are not pollinated. advance of fungal mycelia (Snijders these provide the initial inoculum for Within a week after infection, and Perkowski, 1990; Snijders and infection of the next season’s crop. conidia are produced in abundance, Krechting, 1992). The wheat cultivar The sclerotia of many Claviceps present in the sticky honeydew that Frontana is substantially more resistant species contain toxic alkaloids, drips from the flowers. The honeydew

22 CHAPTER 1 CHAPTER acts as the main infective stage, being 8. Decision trees spread by insects, rain splash, or contact with uninfected flower heads. The major commodities susceptible Within about two weeks after to mycotoxin formation are sum- infection, sclerotia begin to appear. marized in Figs 1.10 and 1.11, Maturity of the sclerotia coincides together with the major fungal with maturity of the infected grass species involved. seed heads.

Fig. 1.10. Decision tree for directing risk management decisions or actions based on environmental considerations and probability of fungal contamination in warm climates. Expected toxic effects in susceptible animals are given for each group of mycotoxins.

Warm to hot humid conditions

Sorghum Small grains Maize Tree nuts Cottonseed Groundnuts

F. graminearum F. verticillioides A. flavus Claviceps spp. A. ochraceus? A. flavus F. culmorum F. proliferatum A. parasiticus

Ergot alkaloids Deoxynivalenol Fumonisins Ochratoxin A Aflatoxins and zearalenone

Fig. 1.11. Decision tree for directing risk management decisions or actions based on environmental considerations and probability of fungal contamination in cool climates. Expected toxic effects in susceptible animals are given for each group of mycotoxins.

Cool to warm temperate conditions

Grapes, Coffee, Small grains Maize Small grains Tree nuts and figs wine cocoa

F. culmorum F. verticillioides A. carbonarius A. ochraceus Claviceps spp. A. ochraceus? A. flavus F. graminearum F. proliferatum (A. niger) A. carbonarius

Ergot alkaloids Deoxynivalenol Fumonisins Ochratoxin A Aflatoxins and zearalenone

Chapter 1. Fungi producing significant mycotoxins 23 References

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26 Pascale M, Visconti A, Pronczuk M et al. Pitt JI, Hocking AD, Bhudhasamai K et al. Schaafsma AW, Hooker DC, Baute TS, CHAPTER 1 CHAPTER (1997). Accumulation of fumonisins in maize (1993). The normal mycoflora of commodities Illincic-Tamburic L (2002). Effect of Bt-corn hybrids inoculated under field conditions from Thailand. 1. Nuts and oilseeds. Int J Food hybrids on deoxynivalenol content in grain at with Fusarium moniliforme Sheldon. J Sci Microbiol, 20:211–226. doi:10.1016/0168- harvest. Plant Dis, 86:1123 –1126. doi:10.1094/ Food Agric, 74:1–6. doi:10.1002/(SICI)1097- 1605(93)90166-E PMID:8110599 PDIS.2002.86.10.1123 0010(199705)74:1<1::AID-JSFA752>3.0.CO;2-5 Pitt JI, Taniwaki MH, Teixiera AA, Iamanka BT Schaafsma AW, Miller JD, Savard ME, Ewing Payne GA (1983). Nature of field infection of corn (2001). Distribution of Aspergillus ochraceus, R (1993). Ear rot development and mycotoxin by Aspergillus flavus. In: Diener UL, Asquith A. niger and A. carbonarius in coffee in four production in corn in relation to inoculation RL, Dickens JW, eds. Aflatoxin and Aspergillus regions in Brazil. In: Proceedings of the 19th method and corn hybrid for three species of flavus in Corn. Auburn, AL: Department of International Scientific Colloquium on Coffee, Fusarium. Can J Plant Pathol, 15:185–192. Research Information, Alabama Agricultural Trieste, Italy, 14–18 May, 2001 [compact disc]. doi:10.1080/07060669309500821 Experiment Station, Auburn University Lausanne, Switzerland: International Coffee Scott PM, Lawrence JW, van Walbeek W (Southern Cooperative Series, Bulletin 279), pp. Science Association. (1970). Detection of mycotoxins by thin-layer 16–19. Pohland AE, Wood GE (1987). Occurrence of chromatography: application to screening of Pel HJ, de Winde JH, Archer DB et al. mycotoxins in food. In: Krogh P, ed. Mycotoxins fungal extracts. Appl Microbiol, 20:839–842. (2007). Genome sequencing and analysis in Food. London: Academic Press, pp. 35–64. PMID:5485087 of the versatile cell factory Aspergillus niger Ramirez ML, Pascale M, Chulze S et al. Sharman M, Patey AL, Bloomfield DA, Gilbert CBS 513.88. Nat Biotechnol, 25:221–231. (1996). Natural occurrence of fumonisins and J (1991). Surveillance and control of aflatoxin doi:10.1038/nbt1282 PMID:17259976 their correlation to Fusarium contamination in contamination of dried figs and fig paste im- Pitt JI (1979). The Genus Penicillium and commercial corn hybrids growth in Argentina. ported into the United Kingdom. Food Addit Its Teleomorphic States Eupenicillium and Mycopathologia, 135:29–34. doi:10.1007/ Contam, 8:299–304. doi:10.1080/026520391093 Talaromyces. London: Academic Press. BF00436572 PMID:20882450 73979 PMID:1778266

Pitt JI (1987). Penicillium viridicatum, Penicillium Raper KB, Fennell DI (1965). The Genus Schroeder HW, Christensen JJ (1963). Factors verrucosum, and production of ochratoxin Aspergillus. Baltimore, MD: Williams and affecting resistance of wheat to scab caused by A. Appl Environ Microbiol, 53:266–269. Wilkins. Gibberella zeae. Phytopathology, 53:831. PMID:3566267 Reid LM, Nicol RW, Ouellet T et al. (1999). Seifert KA, Aoki T, Baayen RP et al. (2003). The Pitt JI (1989). Field studies on Aspergillus Interaction of Fusarium graminearum and F. name Fusarium moniliforme should no longer flavus and aflatoxins in Australian groundnuts. moniliforme in maize ears: disease progress, be used. Mycol Res, 107:643–644. doi:10.1017/ In: McDonald D, Mehan VK, eds. Aflatoxin fungal biomass, and mycotoxin accumulation. S095375620323820X Phytopathology, 89:1028–1037. doi:10.1094/ Contamination of Groundnut: Proceedings of Shelby RA, White DG, Burke EM (1994). PHYTO.1999.89.11.1028 PMID:18944658 the International Workshop, 6–9 October l987, Differential fumonisin production in maize ICRISAT Center, India. Patancheru, India: Richer L, Sigalet D, Kneteman N et al. (1997). hybrids. Plant Dis, 78:582–584. doi:10.1094/ International Crops Research Institute for the Fulminant hepatic failure following ingestion PD-78-0582 Semi-Arid Tropics, pp. 223–235. of moldy homemade rhubarb wine (Abstract). Simpson ME, Batra LR (1984). Ecological Gastroenterology, 112:A1366. Pitt JI (1996). What are mycotoxins? Aust relations in respect to a boll rot of cotton caused Mycotoxin Newsletter, 7:1. Rodricks JV, Hesseltine CW, Mehlman ME, by Aspergillus flavus. In: Kurata H, Ueno Y, Pitt JI (2000). A Laboratory Guide to Common eds (1977). Mycotoxins in Human and Animal eds. Toxigenic Fungi: Their Toxins and Health Penicillium Species, 3rd ed. North Ryde, NSW: Health. Park Forest South, IL: Pathotox Hazard. Amsterdam: Elsevier, pp. 24–32. Publishers. Food Science Australia. Smith JE, Moss MO (1985). Mycotoxins: Pitt JI, Hocking AD (1977). Influence of solute Rojas FJ, Jodral M, Gosalvez F, Pozo R Formation, Analysis and Significance. and hydrogen ion concentration on the water (1991). Mycoflora and toxigenic Aspergillus Chichester, UK: John Wiley. flavus in Spanish dry-cured ham. Int J Food relations of some xerophilic fungi. J Gen Snijders CHA (1994). Breeding for resistance Microbiol, 13:249–255. doi:10.1016/0168- Microbiol, 101:35–40. PMID:19558 to Fusarium diseases in wheat and maize. In: 1605(91)90082-Z PMID:1911081 Miller JD, Trenholm HL, eds. Mycotoxins in Pitt JI, Hocking AD (1996). Current knowledge Grain: Compounds Other than Aflatoxin. St of fungi and mycotoxins associated with food Samson RA, Pitt JI, eds (1990). Modern Paul, MN: Eagan Press, pp. 37–58. commodities in Southeast Asia. In: Highley E, Concepts in Penicillium and Aspergillus Classification. New York: Plenum Press. Johnson GI, eds. Mycotoxin Contamination Snijders CHA, Krechting CF (1992). Inhibition in Grains. Canberra: Australian Centre for Samson RA, Hoekstra ES, Frisvad JC, of deoxynivalenol translocation and fungal International Agricultural Research, pp. 5–10. eds (2010). Food and Indoor Fungi. colonization in Fusarium head blight resistant wheat. Can J Bot, 70:1570–1576. doi:10.1139/ Pitt JI, Hocking AD (1997). Fungi and Food Utrecht, Netherlands: Centraalbureau voor b92-198 Spoilage, 2nd ed. London: Blackie Academic Schimmelcultures. and Professional. Sanders TH, Hill RA, Cole RH, Blankenship Snijders CHA, Perkowski J (1990). Effects of head blight caused by Fusarium culmorum Pitt JI, Hocking AD (2009). Fungi and Food PD (1981). Effect of drought on occurrence of on toxin content and weight of wheat kernels. Spoilage, 3rd ed. New York: Springer. Aspergillus flavus in maturing peanuts. J Am Oil Chem Soc, 58:966A–970A. doi:10.1007/ Phytopathology, 80:566–570. doi:10.1094/ Pitt JI, Miscamble BF (1995). Water relations of BF02679302 Phyto-80-566 Aspergillus flavus and closely related species. J Snowdon AL (1990). A Colour Atlas of Post- Food Prot, 58:86–90. Sanders TH, Blankenship PD, Cole RJ, Hill RA (1984). Effect of soil temperature and drought harvest Diseases and Disorders of Fruits and Pitt JI, Hocking AD, Glenn DR (1983). An on peanut pod and stem temperatures relative Vegetables. 1. General Introduction and Fruits. improved medium for the detection of Aspergillus to Aspergillus flavus invasion and aflatoxin London: Wolfe Scientific. flavus and A. parasiticus. J Appl Bacteriol, contamination. Mycopathologia, 86:51–54. Snowdon AL (1991). A Colour Atlas of Post- 54:109–114. doi:10.1111/j.1365 -2672.1983. doi:10.1007/BF00437229 PMID:6429541 harvest Diseases and Disorders of Fruits and tb01307.x PMID:6406419 Sanders TH, Cole RH, Blankenship PD, Hill RA Vegetables. 2. Vegetables. London: Wolfe Pitt JI, Dyer SK, McCammon S (1991). Systemic (1985). Relation of environmental stress duration Scientific. invasion of developing peanut plants by to Aspergillus flavus invasion and aflatoxin Aspergillus flavus. Lett Appl Microbiol, 13:16– production in preharvest peanuts. Peanut Sci., 20. doi:10.1111/j.1472-765X.1991.tb00558.x 12:90–93. doi:10.3146/pnut.12.2.0011

Chapter 1. Fungi producing significant mycotoxins 27 Snyder WC, Hansen HN (1940). The species Tsubouchi H, Yamamoto K, Hisada K, Sakabe Wang Y-Z, Miller JD (1988). Effects of concept in Fusarium. Am J Bot, 27:64 – 67. Y (1984). A survey of occurrence of mycotoxins Fusarium graminearum metabolites on wheat doi:10.2307/2436688 and toxigenic fungi in imported green coffee tissue in relation to Fusarium head blight beans. Proc Jpn Assoc Mycotox, 19:14–21. resistance. J Phytopathol, 122:118–125. Steiner WE, Rieker RH, Battaglia R (1988). doi:10.1111/j.1439- 0434.1988.tb00998.x Aflatoxin contamination in dried figs: distribution Ueno Y, Kawamura O, Sugiura Y et al. (1991). and association with fluorescence. J Agric Food Use of monoclonal antibodies, enzyme-linked Wells TR, Kreutzer WA (1972). Aerial invasion Chem, 36:88–91. doi:10.1021/jf00079a022 immunosorbent assay and immunoaffinity of peanut flower tissue by Aspergillus flavus column chromatography to determine under gnotobiotic conditions (Abstract). Studer-Rohr I, Dietrich DR, Schlatter J, ochratoxin A in porcine sera, coffee products Phytopathology, 62:797. Schlatter C (1994). Ochratoxin A and coffee. and toxin-producing fungi. In: Categnaro Mitteil Gebiete Lebesmittel Hyg, 85:719 –727. M, Plestina R, Dirheimer G et al., eds. Wheeler KA, Hurdman BF, Pitt JI (1991). Influence of pH on the growth of some Taniwaki MH, Pitt JI, Urbano GR et al. (1999). Mycotoxins, Endemic Nephropathy and Urinary Tract Tumours. Lyon: International Agency toxigenic species of Aspergillus, Penicillium Fungi producing ochratoxin A in coffee. In: and Fusarium. Int J Food Microbiol, 12:141– Proceedings of the 18th International Scientific for Research on Cancer (IARC Scientific Publications Series, No. 115), pp. 71–75. 149. doi:10.1016/0168-1605(91)90063-U Colloquium on Coffee, Helsinki, Finland, PMID:2049282 2–6 August, 1999. Lausanne, Switzerland: PMID:1820356 International Coffee Science Association, pp. van der Merwe KJ, Steyn PS, Fourie L et Wicklow DT, Dowd PF, Alfatafta AA, Gloer JB 239 –247. al. (1965). Ochratoxin A, a toxic metabolite (1996). Ochratoxin A: an antiinsectan metabolite from the sclerotia of Aspergillus carbonarius Taniwaki MH, Pitt JI, Teixeira AA, Iamanaka BT produced by Aspergillus ochraceus Wilh. Nature, 205:1112–1113. doi:10.1038/2051112a0 NRRL 369. Can J Microbiol, 42:1100 –1103. (2003). The source of ochratoxin A in Brazilian doi:10.1139/m96-141 PMID:8941986 coffee and its formation in relation to processing PMID:5833211 methods. Int J Food Microbiol, 82:173– van Walbeek W, Scott PM, Harwig J, WHO (2000). Environmental Health Criteria 179. doi:10.1016/S0168-1605(02)00310-0 Lawrence JW (1969). Penicillium viridicatum 219: Fumonisin B1. Marasas WFO, Miller JD, PMID:12568757 Westling: a new source of ochratoxin A. Can J Riley RT, Visconti A, eds. Geneva: United Nations Environment Programme, International Teixera AA, Taniwaki MH, Pitt JI, Martins CP Microbiol, 15:1281–1285. doi:10.1139/m69-232 PMID:5358203 Labour Organization, World Health (2001). The presence of ochratoxin A in coffee Organization. Available at http://libdoc.who.int/ due to local conditions and processing in four Varga J, Kocsubé S, Suri K et al. (2010). ehc/WHO_EHC_219.pdf. regions in Brazil. In: Proceedings of the 19th Fumonisin contamination and fumonisin International Scientific Colloquium on Coffee, producing black Aspergilli in dried vine fruits of WHO (2007). Ochratoxin A. In: Evaluation of Trieste, Italy, 14–18 May, 2001 [compact disc]. different origin. Int J Food Microbiol, 143:143– Certain Food Additives and Contaminants: Lausanne, Switzerland: International Coffee 149. doi:10.1016/j.ijfoodmicro.2010.08.008 Sixty-eighth Report of the Joint FAO/WHO Science Association. PMID:20826035 Expert Committee on Food Additives. Geneva: Food and Agriculture Organization of the Téren J, Varga J, Hamari Z et al. (1996). Visconti A (1996). Fumonisins in maize United Nations, World Health Organization Immunochemical detection of ochratoxin A genotypes grown in various geographic (WHO Technical Report No. 947), pp. 169–180. in black Aspergillus strains. Mycopathologia, areas. Adv Exp Med Biol, 392:193–204. 134:171–176. doi:10.1007/BF00436726 PMID:8850617 Yamashita A, Yoshizawa T, Aiura Y et al. (1995). PMID:8981783 Fusarium mycotoxins (fumonisins, nivalenol, Waalwijk C, Kastelein P, de Vries I et al. (2003). and zearalenone) and aflatoxins in corn from Thrane U (1996). Comparison of three selective Major changes in Fusarium species in wheat in Southeast Asia. Biosci Biotechnol Biochem, media for detecting Fusarium species in foods: the Netherlands. Eur J Plant Pathol, 109:743– 59:1804–1807. doi:10.1271/bbb.59.1804 a collaborative study. Int J Food Microbiol, 754. doi:10.1023/A:1026086510156 PMID:8520126 29:149–156. doi:10.1016/0168-1605(95)00040-2 PMID:8796416

28 CHAPTER 1 CHAPTER annex. media adaptation of the original published Add minor ingredients and agar to formulation (Abildgren et al., 1987), about 800 mL of distilled water. Steam The formulations given below are made from basic ingredients rather to dissolve agar, then make up to 1 L from Pitt and Hocking (2009). than using commercial Czapek–Dox with distilled water. Add glycerol; note broth. Chloramphenicol (100 mg/L) that the final concentration is 18% Aspergillus flavus and replaces the original combination w/w, not w/v. Sterilize by autoclaving parasiticus agar (AFPA) of chlortetracycline (50 mg) and at 121 °C for 15 minutes. Final aw is Peptone, bacteriological: 10 g chloramphenicol (50 mg). 0.955; final pH is 5.5–5.8. Yeast extract: 20 g Ferric ammonium citrate: 0.5 g Czapek yeast extract Dichloran rose bengal Chloramphenicol: 100 mg agar (CYA) chloramphenicol agar (DRBC)

Agar: 15 g K2HPO4: 1 g Glucose: 10 g Dichloran (0.2% in ethanol, 1.0 mL): Czapek concentrate: 10 mL Peptone, bacteriological: 5 g

2 mg Trace metal solution: 1 mL KH2PO4: 1 g

Water, distilled: 1 L Yeast extract, powdered: 5 g MgSO4.7H2O: 0.5 g Sucrose: 30 g Agar: 15 g Sterilize by autoclaving at 121 °C for Agar: 15 g Rose bengal (5% w/v in water, 15 minutes. Final pH is 6.0–6.5. Water, distilled: 1 L 0.5 mL): 25 mg Dichloran (0.2% w/v in ethanol, Czapek concentrate Refined table grade sucrose is 1.0 mL): 2 mg

NaNO3: 30 g satisfactory for use in CYA provided Chloramphenicol: 100 mg KCl: 5 g it is free of sulfur dioxide. Sterilize by Water, distilled: 1 L

MgSO4.7H2O: 5 g autoclaving at 121 °C for 15 minutes.

FeSO4.7H2O: 0.1 g Final pH is 6.7. Sterilize by autoclaving at 121 °C Water, distilled: 100 mL for 15 minutes. Final pH is 5.5–5.8. Dichloran chloramphenicol Store prepared medium away from Czapek concentrate will keep indef- peptone agar (DCPA) light; photoproducts of rose bengal initely without sterilization. The pre- Peptone: 15 g are highly inhibitory to some fungi, cipitate of Fe(OH)3 that forms in time KH2PO4: 1 g especially yeasts. In the dark, the can be resuspended by shaking MgSO4.7H2O: 0.5 g medium is stable for at least 1 month before use. Chloramphenicol: 0.1 g at 1–4 °C. The stock solutions of Dichloran (0.2% in ethanol, 1.0 mL): rose bengal and dichloran need no Czapek–Dox iprodione 2 mg sterilization, and are also stable for dichloran agar (CZID) Agar: 15 g very long periods. Sucrose: 30 g Water, distilled: 1 L Yeast extract: 5 g Dichloran rose bengal yeast Chloramphenicol: 100 mg Sterilize by autoclaving at 121 °C for extract sucrose agar (DRYS) Dichloran (0.2% in ethanol, 1.0 mL): 15 minutes. Final pH is 5.5–6.0. Yeast extract: 20 g 2 mg Sucrose: 150 g Czapek concentrate: 10 mL Dichloran 18% glycerol Dichloran (0.2% in ethanol, 1.0 mL): Trace metal solution: 1 mL agar (DG18) 2 mg Agar: 15 g Glucose: 10 g Rose bengal (5% w/v in water, Water, distilled: 1 L Peptone: 5 g 0.5 mL): 25 mg

Iprodione suspension: 1 mL KH2PO4: 1 g Chloramphenicol: 50 mg

MgSO4.7H2O: 0.5 g Agar: 20 g Sterilize by autoclaving at 121°C for Glycerol, A.R.: 220 g Water, distilled: to 1 L 15 minutes. Add iprodione suspen- Agar: 15 g Chlortetracycline (l% in water, filter- sion (0.3 g Roval 50WP [Rhône- Dichloran (0.2% w/v in ethanol, sterilized, 5.0 mL): 50 mg Poulenc Agrochimie, Lyon, France] 1.0 mL): 2 mg in 50 mL sterile water, shaken Chloramphenicol: 100 mg Sterilize all ingredients except before addition to medium) after Water, distilled: 1 L chlortetracycline by autoclaving autoclaving. This formulation is an at 121 °C for 15 minutes. Add

Chapter 1. Fungi producing significant mycotoxins 29 chlortetracycline after tempering should be made for the additional Potato dextrose agar (PDA) to 50 °C. Chloramphenicol at twice water. Sterilize by autoclaving at Potatoes: 250 g the concentration specified (i.e. 121 °C for 15 minutes. Final pH is 7.0. Glucose: 20 g 100 mg/L) adequately controls Agar: 15 g bacteria in most situations, and Malt extract agar (MEA) Water, distilled: to 1 L this avoids the need for a second Malt extract, powdered: 20 g antibiotic that must be filter- Peptone: 1 g PDA prepared from raw ingredients is sterilized. Glucose: 20 g more satisfactory than commercially Agar: 20 g prepared media. Wash the potatoes, 25% Glycerol nitrate Water, distilled: 1 L which should not be of a red skinned agar (G25N) variety, and dice or slice, unpeeled,

K2HPO4: 0.75 g Commercial malt extract used for into 500 mL of water. Steam or boil Czapek concentrate: 7.5 mL home brewing is satisfactory for for 30–45 minutes. At the same Yeast extract: 3.7 g use in MEA, as is bacteriological time, melt the agar in 500 mL of Glycerol, analytical grade: 250 g peptone. Sterilize by autoclaving water. Strain the potato through Agar: 12 g at 121 °C for 15 minutes. Do not several layers of cheesecloth into Water, distilled: 750 mL sterilize for longer as this medium the flask containing the melted will become soft on prolonged or agar. Squeeze some potato pulp Glycerol for G25N should be of high repeated heating. Final pH is 5.6. through also. Add the glucose, mix quality, with a low (1%) water content. thoroughly, and make up to 1 L If a lower grade is used, allowance with water if necessary. Sterilize by autoclaving at 121 °C for 15 minutes.

30 chapter 2. CHAPTER 2 CHAPTER Chemical and physical characteristics of the principal mycotoxins

Summary try number : 1162-65-8. Molecular formu- Aflatoxin M1. CA name : (6aR,9aR)-

la : C17H12O6. Molecular weight : 312.3. 2,3,6a,9a-tetrahydro-9a-hydroxy-4-

This chapter provides information Aflatoxin B2. CA name : (6aR,9aS)- methoxycyclopenta[c]furo-(3′,2′:4,5]- about the chemical and physical 2,3,6a,8,9,9a-hexahydro-4-methoxy- furo[2,3-h][l]benzopyran-1,11-dione. properties of the mycotoxins consid- cyclopenta[c]furo(3′,2′:4,5]furo[2,3-h]- CAS registry number : 6795-23-9. ered in this book: aflatoxins; fumonisins; [l]benzopyran-1,11-dione. CAS registry Molecular formula : C17H12O7. Molec- ochratoxin A; trichothecenes, espe- number : 7220-81-7. Molecular formula : ular weight : 328.3. cially deoxynivalenol and nivalenol; C17H14O6. Molecular weight : 314.3. Structures of aflatoxins are shown zearalenone; and ergot alkaloids. This Aflatoxin G1. CA name : (7aR,10aS)- in Fig. 2.1. information about structures reveals 3,4,7a,10a-tetrahydro-5-methoxy-1H, the chemical diversity of mycotoxins, 12H-furo-(3′,2′:4,5]furo[2,3-h]pyrano- 1.2 Physical data which is relevant to the wide range of [3,4-c][l]benzopyran-1,12-dione. CAS toxicological effects in animals and registry number : 1165-39-5. Molecular Descriptions. Colourless to pale- humans discussed later in the book. formula : C17H12O7. Molecular weight : yellow crystals. Fluorescence in ultra-

328.3. violet (UV) light : aflatoxins B1 and

1. Aflatoxins Aflatoxin G2. CA name : (7aR,10aS)- B2, blue; aflatoxins G1 and G2, green;

3,4,7a,9,10,10a-hexahydro-5-methoxy- aflatoxin M1, blue–violet. 1.1 Formulae and structures 1H,12H-furo-(3′,2′:4,5]furo[2,3-h]pyrano- Melting-points. See Table 2.1. [3,4-c][l]benzopyran-1,12-dione.CAS Spectral properties. For UV ab-

Aflatoxin B1. Chemical Abstracts (CA) registry number : 7241-98-7. Molecular sorption, see Table 2.1. Fluorescence name : (6aR,9aS)-2,3,6a,9a-tetrahydro- formula : C17H14O7. Molecular weight : excitation and emission data are 4-methoxycyclopenta[c]furo-(3′,2′:4,5]- 330.3. not listed in Table 2.1 because they furo[2,3-h][l]benzopyran-1,11-dione. depend on the type of instrument, the Chemical Abstracts Service (CAS) regis- solvent, and the supporting media

Chapter 2. Chemical and physical characteristics of the principal mycotoxins 31 Fig. 2.1. Structures of aflatoxins Fumonisin B2. CA name : 1,2,3- propanetricarboxylic acid, 1,1′-[1-(12- amino-9,11-dihydroxy-2-meth- yltridecyl)-2-(1-methylpentyl)-1,2- ethanediyl] ester. CAS registry number: 116355-84-1. Molecular

formula: C34H 59NO14. Molecular weight : 705. Structures of fumonisins are shown in Fig. 2.2.

2.2 Physical data

Unless otherwise noted, data are from WHO (2000). Description. White hygroscopic powder. Melting-point. Not known (com- pounds have not been crystallized). Spectral properties. For mass and NMR spectral data, see Bezuidenhout et al. (1988), Laurent et al. (1989), Plattner et al. (1990), Savard and Blackwell (1994), and Cole et al. (2003a). used. For those data, see Wogan Stability. Unstable to UV light in 2.3 Chemical data (1966), Robertson and Pons (1968), the presence of oxygen. Unstable to Kiermeier and Kroczek (1974), and extremes of pH (< 3 or > 10). Unstable Solubility. Soluble in methanol, in ace- Uwaifo et al. (1977). in the presence of oxidizing agents tonitrile–water, and in water (at least For mass and nuclear magnetic (Castegnaro et al., 1980, 1991). 20 g/L) (NTP, 2001). resonance (NMR) spectral data, see Reactivity. Under alkaline condi- Stability. Stable in acetonitrile– Bycroft et al. (1970), Stubblefield et tions, the lactone ring opens and the water (1:1) at 25 °C. Unstable in meth- al. (1970), and Cole and Schweikert aflatoxins are apparently absent. anol at 25 °C, forming monomethyl (2003). However, the reaction is reversible and dimethyl esters (Gelderblom

Specific rotation. [α]D in chloro- upon acidification. et al., 1992; Visconti et al., 1994). form, –558° (aflatoxin B1), –430° Ammoniation at high temperature Stable in methanol at –18 °C

(aflatoxin B2), –556° (aflatoxin G1), and high pressure opens the lactone (Visconti et al., 1994). Stable in

–473° (aflatoxin G2); [α]D in dimeth- ring and results in decarboxylation. buffer solutions over the pH range ylformamide, –280° (aflatoxin M1) This reaction is not reversible. 4.8–9 at 78 °C (Howard et al., 1998). (Cole and Schweikert, 2003). Octanol – water partition coeffi-

2. Fumonisins cient for fumonisin B1. log P = 1.84 1.3 Chemical data (Norred et al., 1997). 2.1 Formulae and structures Solubility. Insoluble in non-polar 3. Ochratoxin A solvents. Slightly soluble in water Fumonisin B1. CA name : 1,2,3-propane- (10–20 µg/mL). Freely soluble in tricarboxylic acid, 1, 1′-[1-(12-amino- 3.1 Formula and structure moderately polar organic solvents 4,9,11-trihydroxy-2-methyltridecyl)-2- (e.g. chloroform, methanol), espe- (1-methylpentyl)-1,2-ethanediyl] ester. Ochratoxin A. CA name : N-[(5-chlo- cially in dimethyl sulfoxide (Cole and CAS registry number : 116355-83-0. ro-3,4-dihydro-8-hydroxy-3-methyl-1-

Cox, 1981; O’Neil et al., 2001). Molecular formula : C34H59NO15. Molec- oxo-1H-2-benzopyran-7-yl)carbonyl]-L- ular weight : 721. phenylalanine. CAS registry number:

32 Table 2.1. Melting-points and ultraviolet absorption of aflatoxins

Ultraviolet absorption Aflatoxin Melting-point (°C) –1 –1 –3 λmax(nm) ε (L.mol .cm ) × 10

B1 268–269 (decomposition) 223 25.6

(crystals from CHCl3) 2 CHAPTER 265 13.4

362 21.8

B2 286–289 (decomposition) 265 11.7

(crystals from CHCl3–pentane) 363 23.4

G1 244–246 (decomposition) 243 11.5

(crystals from CHCl3–methanol) 257 9.9

264 10.0

362 16.1

G2 237–240 (decomposition) 265 9.7 (crystals from ethyl acetate) 363 21.0

M1 299 (decomposition) 226 23.1 (crystals from methanol) 265 11.6

357 19.0

Data from O’Neil et al. (2001).

Fig. 2.2. Structures of fumonisins

Chapter 2. Chemical and physical characteristics of the principal mycotoxins 33 303-47-9. Molecular formula : C 20H18ClNO6. medium (Subirade, 1996; Van der When DON was gamma- Molecular weight : 403.8. Stegen et al., 2001). irradiated on maize, breakdown of The structure of ochratoxin A Reactivity. The lactone ring DON began only after irradiation to (OTA) is shown in Fig. 2.3. opens under alkaline conditions, but 20 kGy, and 80–90% of the DON the reaction is reversible. Solutions remained after irradiation to 50 kGy 3.2 Physical data of OTA are completely degraded by (O’Neill et al., 1993). treatment with an excess of sodium No significant decomposition of Description. White odourless crys- hypochlorite. DON was observed when stored in talline solid (Pohland et al., 1982). ethyl acetate for 24 months at 25 °C Intensely fluorescent in UV light, 4. Deoxynivalenol or 3 months at 40 °C (Widestrand emitting green and blue fluorescence and Pettersson, 2001). DON was in acid and alkaline solutions, 4.1 Formula and structure relatively stable in buffer solutions respectively, due to two different over the pH range 1–10 (Lauren and forms, i.e. closed or open lactone Deoxynivalenol. CA name: 12,13-ep- Smith, 2001). ring, respectively. oxy-3,7,15-trihydroxy-(3α,7α)-trichothec- Melting-point. 159 °C when re- 9-en-8-one. CAS registry number: 5. Nivalenol crystallized from benzene–hexane 51481-10-8. Molecular formula: C15H20O6. (Natori et al., 1970); 169 °C when Molecular weight: 296.32. 5.1 Formula and structure recrystallized from xylene (Van der The structure of deoxynivalenol Merwe et al., 1965a, 1965b); 168– (DON) is shown in Fig. 2.4. Nivalenol. CA name : 12,13-epoxy- 173 °C after drying for 1 hour at 60 °C 3,4,7,15-tetrahydroxy-(3α,4β,7α)-tricho- (Pohland et al., 1982). 4.2 Physical data thec-9-en-8-one. CAS registry num- 20 Specific rotation. [α] D –118° (c = ber : 23282-20-4. Molecular formula :

1.1 mmol/L in chloroform) (Van Description. White needles. C15H20O7. Molecular weight : 312.32. der Merwe et al., 1965a, 1965b); Melting-point. 151–153 °C. The structure of nivalenol (NIV) is 21 20 [α] D –46.8° (c = 2.65 mmol/L in Specific rotation. [α] D +6.35° (c = shown in Fig. 2.4. chloroform) (Pohland et al., 1982). 0.07 mmol/L in ethanol).

UV spectrum. At λmax of 214, 282, Spectral properties. IR, UV, 5.2 Physical data and 332 nm, extinction coefficients of NMR, and mass spectral data have –3 –3 37.2 × 10 , 0.89 × 10 , and 63.3 × been reported (Cole and Cox, 1981; Description. White crystals. –3 . –1. –1 10 L mol cm , respectively, have Cole et al., 2003c). Melting-point. 222 –223 °C (with been reported (Cole and Cox, 1981). decomposition, after drying in the pres-

Other spectral properties. For 4.3 Chemical data ence of P2O5 at reduced pressure). 20 infrared (IR) spectra, see Van der Specific rotation. [α] D +21.54° (c = Merwe et al. (1965a, 1965b), Steyn Solubility. Soluble in chloroform, 1.3 mmol/L in ethanol). and Holzapfel (1967), and Pohland ethanol, methanol, and ethyl acetate. Spectral properties. IR, UV, NMR, et al. (1982). For NMR spectra, see Stability. Autoclaving creamed and mass spectral data have been Pohland et al. (1982) and Cole et maize reduced DON content by only reported (Cole and Cox, 1981; Brumley al. (2003b). For mass spectra, see 12% (Wolf-Hall et al., 1999). At pH et al., 1982; Cole et al., 2003c). Pohland et al. (1982) and Cole et al. 4.0, DON appeared to be very stable, (2003b). showing no destruction at 100 °C or 5.3 Chemical data 120 °C and only partial destruction 3.3 Chemical data at 170 °C after 60 minutes. At pH Solubility. Soluble in chloroform, 7.0, DON was still stable but showed ethanol, methanol, and ethyl acetate; Solubility. Moderately soluble in polar more destruction at 170 °C after slightly soluble in water; soluble in organic solvents (e.g. chloroform, 15 minutes. At pH 10.0, DON was polar organic solvents (Budavari, ethanol, methanol). partially destroyed at 100 °C after 60 1989). Stability. OTA is partially de- minutes and was totally destroyed Stability. No significant decompo- graded under normal cooking at 120 °C after 30 minutes and at sition of NIV was observed when conditions (Müller, 1983). The sta- 170 °C after 15 minutes (Wolf and stored in ethyl acetate for 24 months bility of OTA to heating conditions Bullerman, 1998). at 25 °C or for 3 months at 40 °C. depends on the water activity of the A significant decrease of NIV stored

34 Fig. 2.3. Structure of ochratoxin A Fig. 2.4. Structures of major trichothecenes CHAPTER 2 CHAPTER

Fig. 2.5. Structure of zearalenone Fig. 2.6. Structure of ergotamine

as a thin film was observed after Spectral properties. IR, UV, Less than 23% of ZEA was lost when 9 months at 25 °C (Widestrand and proton NMR, and mass spectral heated in aqueous buffer solution to Pettersson, 2001). NIV is relatively data have been reported (Cole and 125 °C for 60 minutes, but 34–68% stable in buffer solutions over the pH Cox, 1981). The molar absorptivities was lost after 60 minutes at 150 °C, range 1–10 (Lauren and Smith, 2001). of ZEA in acetonitrile at 236, 274, depending on the pH of the buffer. and 314 nm were established, and More than 92% was lost after 60 6. Zearalenone a common reference wavelength minutes at 175 °C, and complete of 274 nm with molar absorptivity loss was observed in < 30 minutes at 6.1 Formula and structure of 12 623 ± 111 L.mol–1.cm–1 was 225 °C, regardless of pH. ZEA was recommended for ZEA in acetonitrile most stable at pH 7, and the greatest Zearalenone. CA name : 3,4,5,6,9,10- (Josephs et al., 2003). losses occurred above 175 °C (Ryu hexahydro-14,16-dihydroxy-3-methyl- et al., 2003). 1H-2-benzoxacyclotetradecin-1,7(8H)- 6.3 Chemical data Extrusion cooking of maize grits dione. CAS registry number: 17924- resulted in significant reductions

92-4. Molecular formula: C18H22O5. Solubility. Solubilities at 25 °C in of ZEA with either mixing screws Molecular weight: 318.4. percent by weight are: water, 0.002; or non-mixing screws, but use of The structure of zearalenone n-hexane, 0.05; benzene, 1.13; aceto- mixing screws was somewhat more (ZEA) is shown in Fig. 2.5. nitrile, 8.6; dichloromethane, 17.5; effective (66–83% reduction) overall methanol, 18; ethanol, 24; and than non-mixing screws (65–77%). 6.2 Physical data acetone, 58 (Hidy et al., 1977). Greater reduction of ZEA content was Stability. ZEA was stable when observed at either 120 °C or 140 °C Description. White crystals. heated at 120 °C; 29% decomposed than at 160 °C (Ryu et al., 1999). Melting-point. 164–165 °C. when heated at 150 °C for 60 minutes ZEA content was not reduced by 25 Specific rotation. [α] D –170.5° and 69% when heated at 200 °C for heating at 110 °C for 12 days after 21 (c = 1.0 mmol/L in methanol); [α] D 60 minutes (Kuiper-Goodman et al., treatment with a sodium bicarbonate –189° (c = 3.14 mmol/L in chloroform). 1987). Stable to hydrolysis in neutral solution (Lauren and Smith, 2001). or acid buffer solutions (Müller, 1983).

Chapter 2. Chemical and physical characteristics of the principal mycotoxins 35 7. Ergot alkaloids 3′,6′,18-trione. CAS registry number: 113- Řehāček (1975), and the [1H]-NMR

15-5. Molecular formula: C33H35N5O5. spectrum was reported by Pierri et Ergots, the sclerotia produced by Molecular weight: 581.66. al. (1982). 20 Claviceps purpurea and related The structure of ergotamine is Specific rotation. [α] D –160°. species, contain a remarkable variety shown in Fig. 2.6. of compounds, which can be divided 7.3 Chemical data into three groups: derivatives of 7.2 Physical data lysergic acid, derivatives of isolysergic Solubility. Some data on recrys- acid, and clavines. The most impor- Description. White powder. tallization, appearance, and solubility tant of these is ergotamine. Melting-point. 180 °C. were reviewed by Hofmann (1964). Spectral properties. UV, IR, and 7.1 Formula and structure fluorescence spectral data were reviewed by Hofmann (1964). The Ergotamine. CA name: 12′-hydroxy- electron mass spectrum of ergot- 2′-methyl-5′-(phenylmethyl)-ergotaman- amine was described by Vokoun and

36 References

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38 chapter 3. Sampling and sample preparation methods for determining concentrations of 3 CHAPTER mycotoxins in foods and feeds

Summary data to be generated from sur- characteristic curves of such plans veillance studies, representative are also detailed. The constraints Sample variation is often the largest samples should be collected from and issues related to the sampling of error in determining concentrations carefully selected populations harvested crops within subsistence of mycotoxins in food commodities. (batches or lots) of food that, in turn, farming areas are also discussed in The worldwide safety evaluation should be representative of clearly this chapter, as are the essential rules of mycotoxins requires sampling defined locations (e.g. a country, a of sample labelling and storage. The plans that give acceptably accurate region within a country). Although chapter concludes with a short section values for the levels of contamination sampling variability is unavoidable, it on sample preparation methods. in specific batches or lots of a is essential that the precision of the commodity. Mycotoxin concentrations sampling plan be clearly defined 1. Introduction show a skewed or uneven distribution and be considered acceptable by in foods and feeds, especially in those responsible for interpreting Mycotoxin concentrations in foods whole kernels (or nuts), so it is and reporting the surveillance data. and feeds are controlled in various extremely difficult to collect a sample The factors influencing variability ways; the most important are good that accurately represents the mean are detailed here, with reference to agricultural practice and good batch concentration. Sample variance both major mycotoxins and major manufacturing practice accompanied studies and sampling plans have commodities. Sampling of large by end-product testing, product been published for select mycotoxins bag stacks, bulk shipments, and monitoring by regulators, and safety such as aflatoxin, fumonisin, and domestic supplies are all discussed. evaluation of foods on a national deoxynivalenol, emphasizing the Sampling plans currently accepted or worldwide basis. Each of these importance of sample selection, in international trade are outlined. approaches requires efficient sam- sample size, and the number of Acceptance sampling plans and pling and sample preparation incremental samples. For meaningful the variabilities that affect operating methods. End-product testing, for

Chapter 3. Sampling and sample preparation methods for determining 39 concentrations of mycotoxins in foods and feeds example, requires the collection of aflatoxin. They showed that the aration, and analytical variances. For representative samples from defined collection of a sample of 1 kg from a example, increasing the sample size batches, whereas product monitoring cottonseed batch contaminated with beyond a certain point may be less by regulators is more likely to focus on 100 μg/kg aflatoxin was associated cost-effective than increasing the the safety of a particular batch (e.g. a with a standard deviation of 87 μg/ subsample size and/or the number 20 t batch of groundnut kernels) or the kg, i.e. replicate 1 kg samples that of replicate samples. It is important to safety of an individual retail pack (e.g. fell within 2 standard deviations of the realize that variability in sampling is a jar of groundnut butter). The safety mean had aflatoxin concentrations much higher than variability in sample evaluation of mycotoxins requires within the range 0–271 μg/kg preparation or analyses. Adequate sampling plans that give acceptably (Table 3.1). However, the collection sampling is extremely important. accurate values for the levels of of a 32 kg sample reduced the mycotoxin contamination in a variety standard deviation of the sample 2.2 Aflatoxin M1 of foods throughout the world. concentrations to 19 μg/kg and the When sampling is undertaken, it is range for samples that fell within 2 A European Commission (EC) Direc- essential that the following are clearly standard deviations to 64–136 μg/ tive (European Commission, 1998a), defined: the aim of the sampling kg. In general, the sampling variance together with an EC Decision (Euro- exercise, the nature of the population was halved each time the sample size pean Commission, 1991), specifies being sampled, the sampling method, was doubled. Similarly, the sample the sampling method to be used to the efficiency of the sampling method, preparation variance was halved by determine aflatoxin M1 levels in liquid and the sample preparation method. doubling the subsample size and milk. After the batch of milk is mixed, In addition, the sampling plans may the analytical variance was halved by manual or mechanical means, a vary according to the kind of mycotoxin by doubling the number of replicate minimum sample of 0.5 kg or 0.5 L being analysed. analyses (Whitaker et al., 1976). is collected, composed of at least 5 For the total variability to be increments. The batch is accepted

2. Sampling variability reduced to an acceptable level, a if the concentration of aflatoxin M1 associated with testing cost-effective balance is needed in the sample does not exceed the for mycotoxins between the sampling, sample prep- permitted limit.

2.1 Aflatoxins Table 3.1. Effect of sample size on the range of results bracketing the 95% confidence limits in a cottonseed lot containing 100 μg/kg aflatoxins In sampling of commodities for aflatoxins, sampling variance is Concentration of aflatoxins in sample (μg/kg) Standard known to be a major contributor to Sample size deviation (kg) the total variance associated with an (μg/kg) Lowa Highb Range analysis. Aflatoxin concentrations show a skewed distribution in foods and feeds, especially in whole kernels, 1 87 0 271 271 so it is extremely difficult to collect a sample that accurately represents the 2 62 0 222 222 mean batch concentration (Whitaker and Wiser, 1969). This problem 4 45 13 187 174 has been extensively investigated: Whitaker et al. (1995) reported on the 8 32 37 163 126 sampling, sample preparation, and analytical variances associated with 16 24 53 147 94c the testing of groundnuts (during farm storage and after shelling), shelled 32 19 64 136 72 maize, and cottonseed.

Whitaker et al. (1976) studied a The low concentration value was calculated as 100 – (1.96 * standard deviation); a value of 0 was recorded if a the impact of sample size on the negative result was obtained. b The high concentration value was calculated as 100 + (1.96 * standard deviation). precision of sampling plans used c CAST (2003) number was 95. to test batches of cottonseed for Adapted, by permission of the publisher, from CAST (2003).

40 The United States Food and Drug these values were independent of the in wheat. However, it was recognized

Administration/Office of Regulatory fumonisin type (B1, B2, B3, or total). that further studies were required to Affairs’ Compliance Policy Guide The CV associated with the total confirm the results of this preliminary (FDA/ORA, 1996) stipulates that test procedure (sampling, sample investigation. samples of milk for the determination preparation, and analysis) was 21%, of aflatoxin M1 should consist of at which was similar to that associated 2.5 Deoxynivalenol least 10 lb (4.5 kg), composed of with the testing of shelled maize for not less than 10 randomly selected aflatoxin with a similar test procedure. The variability associated with the portions. If it is considered desirable to testing of barley for deoxynivalenol Although the distribution of understand sampling variability at (DON) was studied by Freese et al. aflatoxin M1 in liquid milk may be locations with quite different climate (2000). Bulk samples of 225 kg were expected to be reasonably homo- than the USA, the type of testing collected from 6 batches of barley, geneous, no studies have been detailed above should be carried out and each was riffle divided into 16 reported that evaluated the sampling using the methods and calculations test samples each of 0.1 kg, 0.8 kg, CHAPTER 3 CHAPTER variance associated with this provided by Whitaker et al. (1998). and 7 kg. Each test sample was mycotoxin–commodity combination. comminuted in a Romer mill, and 2.4 Ochratoxin A 50 g portions were taken from each 2.3 Fumonisins sample for the determination of DON No sampling plans have been concentration. An evaluation of the The sampling variability associated published for the determination analytical results indicated that the with the testing of shelled maize for of ochratoxin A (OTA) in foods. A variability associated with the sample fumonisin was analysed by Whitaker preliminary study commissioned preparation and analytical steps was et al. (1998). A bulk sample of 45 kg by the Food Standards Agency of more significant than the sampling was taken from each of 24 batches the United Kingdom evaluated the variance for all sample sizes, and of shelled maize, which had been application of the United States, that variability was not significantly harvested from 24 different fields United Kingdom, and Dutch sam- reduced by increasing the sample in North Carolina, USA. Each pling plans (see Table 3.5) and size. This finding is in contrast to the bulk sample was riffle divided into the EC sampling plan (European situation with groundnuts and maize 32 test samples of 1.1 kg, which Commission, 1998a), which are because of the smaller grain size of were comminuted in a Romer mill all plans for the determination of barley. (Romer Labs, Union, MO, USA). aflatoxins in foods, to the deter- In a further experiment by Freese A nested design was used to mination of OTA in green coffee and et al. (2000), 10 samples of about determine the variability associated wheat (Food Standards Agency, 2.5 kg were taken from each of 10 with each step (sampling, sample 2000). The green coffee study truckloads of barley, using sampling preparation, and analysis) of the was performed on two batches methods prescribed by the Grain fumonisin test procedure. Briefly, containing low levels of OTA (0.9 μg/ Inspection, Packers and Stockyards 10 of the 24 batches were selected, kg and 0.4 μg/kg), whereas the Administration (GIPSA, 1995). Each covering a wide range of fumonisin wheat study was performed on a 2.5 kg sample was comminuted in concentrations. For each selected single batch containing 5.6 μg/kg a Romer mill, and two 50 g portions batch, 10 comminuted test samples OTA. The distribution of OTA in the were taken from each sample. A single were randomly selected, and two two coffee batches was relatively determination of DON concentration 25 g portions were taken from each uniform, whereas the distribution was performed on the extract from sample by riffle division. Finally, the in the single wheat batch was far the first portion, whereas duplicate concentrations of fumonisins B1, B2, more heterogeneous. The sampling determinations were performed and B3 were determined with AOAC simulation exercise indicated that on the second portion, using an methods (Sydenham et al., 1996). At the United Kingdom sampling plan enzyme-linked immunosorbent assay a batch contamination level of 2 mg/ (United Kingdom, 1992) would (ELISA) procedure. In this instance, kg, the coefficient of variation (CV) accurately predict the OTA con- an evaluation of the variances associated with sampling, sample centration in batches of coffee associated with the sampling, preparation (Romer mill and 25 g but that a plan based on the EC sample preparation, and analytical analytical portion), and analysis was sampling plan was required for the steps indicated approximately equal 16.6%, 9.1%, and 9.7%, respectively; determination of OTA concentration contributions from each step. It was

Chapter 3. Sampling and sample preparation methods for determining 41 concentrations of mycotoxins in foods and feeds concluded that sample sizes of 100– CV associated with the sampling, proportion of this population is 200 g were adequate, assuming that sample preparation, and analytical accessible for sampling without these samples were obtained by the steps was 6.3%, 10.0% and 6.3%, dismantling the complete stack; the riffle division of a large bulk sample. respectively. The sampling variance sample collected is representative However, it was also concluded that was specific to a 0.45 kg sample, only of outer bags that are readily batches may be stratified to different the sample preparation variance to accessible. An evaluation of the degrees, depending upon the amount a Romer mill and a 25 g analytical mycotoxin content of the inner bags of mixing that occurred during sample, and the analytical variance can be performed only during the handling, and that stratification could to the Romer FluoroQuant method. construction or dismantling of the have a significant impact on sampling The CV associated with the total stack. Although the maize crib (Fig. variance. If stratification is suspected, test procedure was 13.4%. The 3.1b) contains far less material than the larger sample sizes are desirable. low variance associated with the bag stack, a sample that represents A similar approach was used sampling step (relative to that for other the whole population can be obtained in a study of the sampling, sample mycotoxins and other commodities) is only by dismantling the complete crib. preparation, and analytical variances partly due to the high kernel count of The very large bulk consignment of associated with the testing of wheat wheat (about 30 kernels/g), which is oilseed meal (Fig. 3.1c) is composed for DON (Whitaker et al., 2000). A about 10 times that for shelled maize of tens of thousands of tonnes 20 kg bulk sample was taken from and about 30 times that for shelled of material, which is discharged, each of 24 commercial batches, and groundnuts. ultimately, into a barge with a capacity each sample was riffle divided into of about 500 t. The discharge is a 32 test samples of 0.45 kg each. 2.6 T-2 and HT-2 toxins convenient sampling point where the Each 0.45 kg sample was finely meal may be sampled as it flows into comminuted in a Romer mill, which No sampling plans have been the barge from the weighing tower. was set to automatically produce a published for the determination of In this case, the population being representative, comminuted 25 g T-2 and HT-2 toxins in foods, and tested is equivalent to the material portion. DON concentration was details of the sampling variability of contained in the barge, and sampling determined in each of 768 (24 × these toxins have not been reported. from a flowing stream ensures that 32) 25 g portions using the Romer However, the sampling plans outlined the sample is representative of the FluoroQuant fluorometric procedure, above for DON should be applicable. whole population. and the analytical data were used Once a population has been to determine the total variability 3. Sampling and surveillance selected, it is equally important that (sampling plus sample preparation samples are collected using a clearly plus analytical). Next, 20 commi- Surveillance studies involve the defined sampling plan designed to nuted samples, with a wide range of collection of information on the produce a reasonably representative DON levels, were selected from the mycotoxin contamination of selected sample. Although sampling variability residual 768 comminuted samples, batches of food or feed within is unavoidable, it is essential that the and eight 25 g portions were taken specified regions. precision of the sampling plan be from each of these samples by riffle For meaningful data to be clearly defined and be considered division. The Romer FluoroQuant generated from surveys or sur- acceptable by those responsible method was then used to determine veillance studies, representative for interpreting the surveillance the DON concentration in four samples should be collected from data. If the samples are too small, replicate extracts prepared from each carefully selected populations of food a wide range of estimated levels of 25 g portion. The combined sample (e.g. batches or lots, marketplaces, contamination with mycotoxins will be preparation and analytical variance, farmers’ stores) that, in turn, should obtained for a given population, and and the analytical variance alone, be representative of clearly defined there will be a strong probability that were estimated using SAS procedures locations (e.g. a country, a region the average value will be significantly (SAS, 1997). The CV associated with within a country). Three examples lower than the true value. It is equally the total test procedure varied from of different types of materials to be important that enough samples be 261.8% (for a batch concentration sampled are shown in Fig. 3.1. collected from each population to of 0.02 mg/kg DON) to 7.9% (for The large bag stack of maize ensure that infrequently occurring, 14.38 mg/kg DON). For a batch (Fig. 3.1a) consists of several thou- highly contaminated samples are concentration of 5.0 mg/kg DON, the sand bags. However, only a small included in the surveillance data.

42 Fig. 3.1. Examples of materials to be sampled: (a) large bag stack of maize; (b) crib 3.2 Sampling processed of maize cobs; (c) very large bulk consignment of oilseed meal. (a, b) Photographs commodities courtesy of Raymond Coker, Founder & Director at Raymond Coker Consulting Limited. (c) Source: Coker (1997);reproduced with permission from the Natural Resources Institute of the University of Greenwich. The very heterogeneous distribution of aflatoxins in whole groundnuts is illustrated in Table 3.2. After the batch of raw, ungraded nuts was crushed, aflatoxin was more uniformly dis- tributed: every sample contained aflatoxin, and the highest level was 2.5 times the mean concentration of the samples (Coker, 1998). In general, the distribution of aflatoxins in ground or comminuted CHAPTER 3 CHAPTER commodities has been shown to be relatively homogeneous compared with that in whole kernels. Coker et al. (2000) found that the aflatoxin concentration of large (500 t) batches of crushed oilseeds, including copra cake, copra meal pellets, and palm kernel cake, could be determined using quite small samples composed of a small number of primary 3.1 Sampling whole kernels bags. However, if the stack is samples. For example, a sampling reasonably new (and, consequently, plan that had a precision (CV) of The sampling variance associated spoilage is not likely to have occurred), 9% involved the collection of just 20 with different numbers of grains or nuts the accessible population may be primary samples of 100 g to produce in a sample was studied by Whitaker assumed to be representative of the a 2 kg composite sample. and Wiser (1969). They showed that, inner bags if the stack was assembled for a batch of groundnuts contamina- in an unbiased manner. 3.3 Sampling point ted with 30 µg/kg aflatoxins, the The distribution of aflatoxins in sampling variance increased very groundnuts and groundnut products An ideal point for sampling occurs significantly if the sample was smaller is illustrated in Table 3.2. Bags from while the batch is being transferred, than 40 000 kernels but changed stacks were sampled systematically for example from one holding vessel very gradually for larger samples. by collecting 100 g samples and to another. Examples of this approach The recommended weight of bulk then determining the aflatoxin level to sampling are shown in Fig. 3.3. samples thus depends on the grain of each sample (Coker, 1998). Of or kernel size of the commodity. The 204 samples of raw, ungraded nuts, 3.4 Proposed surveillance sample weight should be 30 kg for 15% contained aflatoxins, and the sampling plans groundnuts, 10 kg for maize, and highest aflatoxin level was about 5 kg for rice (European Commission, 20 times the mean concentration of Some examples of sampling plans 1998a). Ideally, a bulk sample is the samples. However, in a different proposed for surveillance purposes composed of 100 primary samples. set of samples, from graded and are given in Table 3.3. In Table 3.3, The sampling of groundnut ker- roasted nuts, only 1% of the samples “sample size” refers to samples nels, typically from a 20 t batch, is were contaminated, but the highest produced by riffle division of large illustrated in Fig. 3.2. Here, a 20 kg level was about 200 times the mean (e.g. 40 kg), unground, bulk samples, sample is being produced by concentration of the samples. as described by Freese et al. (2000) collecting 200 g primary samples from and Whitaker et al. (2000) when 100 bags. As mentioned previously, evaluating the sampling variance the population being evaluated is associated with the testing of barley composed of only the accessible and wheat, respectively, for DON.

Chapter 3. Sampling and sample preparation methods for determining 43 concentrations of mycotoxins in foods and feeds Table 3.2. Distribution of aflatoxins in groundnuts and groundnut products

Product No. of samples of No. of samples Range of aflatoxin Batch mean aflatoxin (total weight) 100 g containing aflatoxin (%) concentrations (µg/kg) concentration (µg/kg)

Raw kernels, ungraded 84 13 (15%) < 1–4000 200 (10 t)

Roasted kernels, graded 200 2 (1%) 7–600 3 (15 t)

Ground kernels 204 204 (100%) 90–250 100 (5.6 t)a a Produced by crushing the 10 t batch of raw, ungraded kernels shown in the first row. Adapted, by permission of the publisher, from Coker (1998).

Table 3.3. Summary of minimum sample sizes suggested for interpretation of surveillance data

Commodity Increments (n × y grams) Minimum sample size (kg)

AFLATOXIN M1

Milk (liquid and dried) (e.g. raw, pasteurized, homogenized, 5 × 100 0.5 UHT, skimmed, semi-skimmed, evaporated, infant formula)a

Milk products 5 × 100 0.5

FUMONISINS

Maize Whole maizeb 50 × 100 5.0 Maize on the cobc 50 cobs 7.5 Maize flour 10 × 100 1.0 Maize meal 10 × 100 1.0 Maize grits 10 × 100 1.0 Brand 10 × 100 1.0 Processed maize foods (e.g. cornflakes, tortilla chips, 10 × 100 1.0 popcorn, muffin mix, starch)e

OCHRATOXIN A

Maize Whole maizef 50 × 100 5.0

Maize on the cobc 50 cobs 7.5 Maize grits 10 × 100 1.0 Processed maize foods (e.g. cornflakes, tortilla chips, 10 × 100 1.0 popcorn, muffin mix, starch)d

Wheat 30 × 100 3.0

Barley 30 × 100 3.0 Rice (including dehusked and polished rice) 30 × 100 3.0 Peas and beans (including coffee beans)f 30 × 100 3.0

44 Table 3.3. Summary of minimum sample sizes suggested for interpretation of surveillance data (continued)

Commodity Increments (n × y grams) Minimum sample size (kg)

OCHRATOXIN A

Dried fruit (e.g. raisins, currants, sultanas, figs, dates, 30 × 100 3.0 apricots) Flour, meal, and bran of all origins 10 × 100 1.0 Breadd 10 × 100 1.0 Ground and instant coffee 10 × 100 1.0

Cocoa powder 10 × 100 1.0

Beverages (e.g. coffee, wine, grape juice)a 5 × 100 0.5 CHAPTER 3 CHAPTER DEOXYNIVALENOL AND T-2 AND HT-2 TOXINSg

Maize

Whole maizeh 20 × 100 2.0 Maize on the cobc 20 cobs 3.0

Maize grits 10 × 50 0.5

Processed maize foods (e.g. cornflakes, tortilla chips, 10 × 50 0.5 popcorn, muffin mix, starch)i

Wheatj 20 × 50 1.0

Barleyk 20 × 50 1.0

Oats 20 × 50 1.0

Rye 20 × 50 1.0

Flour, meal, and bran of all originsi 10 × 50 0.5

Bread 10 × 50 0.5

UHT, ultra-high-temperature pasteurized. a European Commission (1998a). The sampling variance for mycotoxins in beverages is assumed to be similar to that for aflatoxin M1 in milk. b Whitaker et al. (1998). Sampling variance (CV, %) for fumonisins in maize is similar to that reported for aflatoxins in maize. c Assuming that the core of a cob contributes about 30% of the total weight of a cob and that a cob yields about 100 g of kernels. d Coker et al. (2000). Sampling variance in these commodities is assumed to be similar to that for aflatoxin in comminuted feeds. The suggested sampling plan is associated with a sampling precision (CV) of 12.5% for aflatoxin in comminuted feeds. e The minimum sample size for comminuted/processed foods is set at half the sample weight required for estimating fumonisins in whole kernels. f The sampling variability for ochratoxin A is assumed to be similar to that for fumonisins (and aflatoxins; see footnote b). g No data on sampling variance for T-2 or HT-2 toxins; assumed to be similar to sampling variance for deoxynivalenol. h The minimum sample size for maize is set at double the sample weight required for estimating deoxynivalenol in wheat and barley. i The minimum sample size for deoxynivalenol in cereal products is arbitrarily set at half the sample weight required for estimating fumonisins and ochratoxin A in cereal products. j Whitaker et al. (2000). k Freese et al. (2000).

Chapter 3. Sampling and sample preparation methods for determining 45 concentrations of mycotoxins in foods and feeds Fig. 3.2. Sampling a 20 t batch of groundnuts: (a) collecting primary samples; (b) a sampling probe. Source: Coker (1997); reproduced with permission from the Natural Resources Institute of the University of Greenwich.

Fig. 3.3. Sampling of mobile batches: (a) copra cake emerging from a hopper; (b) sampling a 500 t batch of oilseed meal as it flows into a barge. Photographs courtesy of Raymond Coker, ToxiMet Limited.

46 The values shown are minimum Table 3.4. Number of populations required to achieve a 95% probability of capturing sample sizes and minimum numbers at least one population with a mycotoxin concentration that is significantly greater of incremental samples for effective than the acceptable level for that toxin sampling. Because these values True proportion of populations with are based on currently available mycotoxin concentrations above the No. of populations (e.g. batches) required information, these sampling plans acceptable level may change as more comprehensive 0.3 9 data become available. As batches of foods move along the food-chain, the heterogeneity 0.2 14 of their mycotoxin distribution will 0.1 29 decrease with increased han- dling. Therefore, it may be more 0.05 59 appropriate to collect smaller samples CHAPTER 3 CHAPTER directly from batches of foods of approximately the size of the samples 0.03 99 shown in Table 3.3. The number of incremental samples proposed 0.01 298 reflects the distribution (or anticipated distribution) of the mycotoxin in that particular commodity. subsistence farmers is problematic. 1.5–2.0 kg) that is representative It is important that enough No elaborate technical sampling plan of the overall amount of grain. This batches are sampled to ensure will work in such situations because technique is also applicable to other that infrequently occurring, highly there are too many variables to small grain crops, including sorghum contaminated batches are included take into consideration. These and millet. in the surveillance data. The number include limited sample size, varying Maize on the cob is more difficult of populations required in a given grades of post-harvest sorting, and to sample because the larger region depends on the variability different varieties and/or hybrids. the individual unit (here, the cob) in mycotoxin concentration among It is, nevertheless, imperative that within the lot, the more skewed the the populations within the region. It sampling and surveillance studies distribution of mycotoxins within that is suggested that there should be a be conducted among subsistence lot (Whitaker et al., 1969; Coker et al., 95% probability of capturing at least farmers to determine to what extent 2000). Sampling whole cobs should one population with a mycotoxin they are being exposed to mycotoxin be avoided as far as possible. If no concentration that is significantly contamination. Such data can then alternative is available, given time and greater than the acceptable level for be used to plan detailed intervention other constraining factors in a rural that toxin. The number of populations studies, which are better suited to area, the aim should be to collect at required in a survey will increase as the needs of rural communities than least 10 cobs, randomly selected, the proportion of highly contaminated are mycotoxin regulations. from the top and as far down as populations decreases. Table 3.4 Subsistence maize farmers will possible in the pile of maize cobs. shows the relationship between have the following available: un- Once shelled, 10 cobs will result in a the proportion of populations with shelled maize cobs hung on walls sample size of ≤ 1.5 kg, which will need mycotoxin concentrations above the or stored in wooden huts or cribs, to be milled and then effectively mixed acceptable level and the number of in metal or plastic containers, or in before being ground for analysis. To populations required in a survey. heaps on homestead floors (Fig. obtain a composite sample that is 3.4a–c) and/or shelled maize kept in representative of a specific region 3.5 Sampling in subsistence hessian or nylon bags or in metal or (e.g. a village), it is suggested that farming areas plastic containers (Fig. 3.4d, e). subsamples of equal size (100–250 g) Sampling shelled maize is be taken from each of the collected Unlike the statistically based sam- relatively easy because a small samples in a specific region, pooled, pling plans used for bulk foods and probe can be used to take samples thoroughly mixed, and then analysed feeds, the sampling of harvested from bags and open containers, to give a location value (R.D. Coker, crops, particularly maize, from with a sample size (a minimum of personal communication).

Chapter 3. Sampling and sample preparation methods for determining 47 concentrations of mycotoxins in foods and feeds Sampling groundnuts is also Fig. 3.4. Storage of maize in subsistence farming areas of Guatemala: (a) maize difficult. As noted previously, vari- cobs stored outdoors in a highland community; (b) maize cobs stored in a wooden crib in the lowlands; (c) maize cobs for shelling and (background) shelled kernels for ability in aflatoxin levels among storage; (d) metal silo for long-term storage of shelled maize; (e) shelled maize kept individual groundnut kernels can be in bags for immediate sale. Photographs courtesy of Mario Roberto Fuentes Lopez, very high. It is unlikely that samples Guatemala. from a single farm will be meaningful. Composite samples from an area such as a village will be essential to provide useful data. Other important issues need to be taken into consideration when sampling in subsistence farming areas; five such issues are discussed here. First, it is unethical to take large samples from households without some sort of appropriate compensation in the form of com- mercial maize meal or rice. Second, very little grain may be available because of poor harvests. To maintain good relationships with rural communities, it is vital that farmers are not left with the impression that their food is being stolen when samples are taken. Before sampling is done, the farmers’ consent should be obtained, accompanied by adequate mycotoxins, the hazards associated 4. Sampling and regulation explanations that it is in their best with them, and the usefulness of interest to cooperate with the study sorting if supplies permit that. Most studies have focused on the and that they will receive feedback on Finally, in most subsistence development of sampling plans for the its outcome. households in southern Africa, har- regulation of aflatoxins, as described Third, rural areas with extreme vested maize cobs are sorted before here, and little or no work has focused levels of poverty and/or low crop yields storage and processing. Maize cobs specifically on acceptance sampling may have locally produced food crops are sorted by hand into two lots: visibly plans for other mycotoxins. available for only a limited period each non-mouldy maize (referred to as year. For example, in southern Africa good maize) and mouldy maize. The 4.1 Acceptance sampling plans subsistence maize farmers may only good maize consists of cobs that the have enough of their own maize people regard as acceptable for direct Acceptance sampling plans, an to last them for 4–6 months after human consumption. Once these essential part of surveillance and harvest, before becoming dependent cobs are shelled, any visibly mouldy regulation of mycotoxins, are de- on what little commercial maize meal maize kernels are sometimes removed signed to assess whether a lot is they can afford until the next cropping before food preparation. The sorted “acceptable”, i.e. has a high probability season. Sampling should, therefore, mouldy portion of maize cobs is kept of containing a level of a particular be undertaken as soon after harvest separate from the good maize and is toxin below some specified limit, after as possible to obtain a reasonable used predominantly as animal feed. analysis of a specified number of number of samples. Sampling protocols need to take this samples of a specified size from that lot. Fourth, care should be taken to scenario into consideration because The efficacy of any plan depends, ensure that subsistence farmers are only sorted grain intended for human among other, usually less important, never told that their crops should not consumption should be sampled for factors, on the variance associated be eaten or that their food is bad for inclusion in surveillance studies. with sampling. The sampling variance them. A culturally sensitive approach associated with the determination is to explain the occurrence of of aflatoxins has been studied in a

48 Table 3.5. United States, United Kingdom, and Dutch sampling plans for the determination of aflatoxins in raw shelled groundnuts

No. of Country Mc (μg/kg)a χc (μg/kg)b Sample size (kg) Type of mill Subsample size (g) Analytical method samples

USA 20 15 3 21.8 Hammer 1100 TLC

United Kingdom 10 10 1 10.0 VCM 50 HPLC

The Netherlands 9 5 4 7.5 VCM 50 HPLC

HPLC, high-performance liquid chromatography; TLC, thin-layer chromatography; VCM, vertical cutter mill. a Mc is the maximum permitted level of total aflatoxins; for the Dutch plan, an Mc of 5 μg/kg aflatoxin B1 is assumed to be equivalent to 9 μg/kg total aflatoxins. b χc is the critical concentration of aflatoxin in the sample (i.e. the maximum level of aflatoxin permitted in the sample if the batch is to be accepted); for the Dutch plan, a χc of

3 μg/kg of aflatoxin B1 is assumed to be equivalent to 5 μg/kg total aflatoxins.

Data summarized from Whitaker et al. (1995). 3 CHAPTER variety of commodities (Coker, 1998; with a sample size of 21.8 kg, gave 4.2 Consumer and producer Coker et al., 2000; Johansson et al., the greatest percentage of correct risks 2000a, 2000b, 2000c; Park et al., decisions (95.6%), followed by the 2000). For example, Johansson et United Kingdom plan, with a sample The efficacy of acceptance sampling al. (2000b) evaluated the efficacy size of 10.0 kg (91.1%) and the Dutch plans may be evaluated by calculating of three sampling plans for shelled plan, with a sample size of 7.5 kg their consumer and producer risks. maize, involving sample sizes of 5, 10, (82.4%). The consumer risk associated with a and 20 kg and a specified maximum In addition, a sampling plan specified plan is the probability that permitted level of contamination (the for the determination of aflatoxins the plan will accept an unacceptable sample acceptance level, also called in edible nuts and dried fruit was batch (i.e. a batch that exceeds the the critical concentration) of 20 μg/kg. specified by an EC Directive (Euro- permitted level of toxin), whereas the The efficacy of each sampling plan pean Commission, 1998a). The producer risk is the probability that was determined by simulating the required sample size and the number the plan will reject an acceptable performance of the plan as applied to of incremental samples depend on batch (i.e. a batch that does not a collection of batches of maize with both the batch size and the type of exceed the permitted level). A well- aflatoxin concentrations within the commodity. For example, for batches designed sampling plan will attain an range 0–150 μg/kg. The percentage of groundnuts, pistachios, Brazil acceptable balance between the two of correct decisions, i.e. acceptance nuts, or dried figs of > 15 t, a 30 kg types of risk. of the lot when the true mean was sample composed of 100 incremental For any given sampling plan, < 20 μg/kg, increased slightly with samples is required. If the commodity the magnitude of the consumer risk increasing sample size, from 97.2% is not intended for further processing, and producer risk can be estimated for 5 kg samples to 97.8% for 20 kg the unground 30 kg sample must be by constructing an operating char- samples. mixed and divided into three 10 kg acteristic (OC) curve, which is a plot Published sampling plans for subsamples. The 30 kg sample (or the of the probability of the sampling plan the determination of aflatoxins in three 10 kg subsamples) should then accepting a batch as a function of the groundnuts, developed for use in be comminuted and mixed before mean concentration of mycotoxin in the USA, the United Kingdom, and the analytical sample is withdrawn. the batch. The shape of the OC curve the Netherlands, were examined For unprocessed commodities, the will be determined by the sample size, by Whitaker et al. (1995). The plans 30 kg sample should contain no the size and degree of comminution of are summarized in Table 3.5. The more than 15 μg/kg total aflatoxins. the subsample, the type and number percentage of correct decisions of For those commodities intended for of analyses performed, and the each sampling plan was determined direct human consumption, the batch critical concentration of the sample. A by evaluating a collection of groundnut is accepted if each of the three 10 typical OC curve is shown in Fig. 3.5, lots with a specific distribution of kg samples contains no more than where the maximum permitted level aflatoxin, produced in the USA during 4 μg/kg total aflatoxins (European of contamination is 20 µg/kg. An ideal 1976–1985. The United States plan, Commission, 1998b). sampling plan would be represented

Chapter 3. Sampling and sample preparation methods for determining 49 concentrations of mycotoxins in foods and feeds by the vertical line. Any practical OC Fig. 3.5. An operating characteristic curve defining the probability that the curve, such as the one shown here, concentration of a sample (Cs) will compare to the acceptable concentration (Ca) of 20 µg/kg. Source: Whitaker et al. (2010), Fig. 10.2, p. 42; reproduced with kind rejects some acceptable batches permission from Springer Science+Business Media B.V. (designated as false positives, or producer risk) and accepts some unacceptable batches (designated as false negatives, or consumer risk). The consumer risk can be reduced by increasing the sample size (thereby also reducing the producer risk) and/or by decreasing the critical concentration (thereby increasing the producer risk).

5. Sample labelling and storage

An equally important facet of the whole sampling procedure is the accurate labelling and storage of samples, especially if they have been collected in the field and need to be transported to a laboratory for analysis. The underlying principle is infestation or moisture damage (e.g. whether the grain was damaged to handle samples in such a way as in transit and in the laboratory. If in any way, how long it had been in to prevent spoilage before analysis possible, the samples should be kept storage, whether it had been exposed and thus ensure that analyses reflect in cold storage at < 5 °C until ready for to heat treatment or dusted with any the mycotoxin levels in the commodity analysis. In the case of visibly moist fungicides or insecticides). at the time of sampling. There are samples, it is preferable to place these no universal guidelines for handling at –20 °C as soon as possible after 6. Sample preparation methods samples because the best practice collection and to maintain the samples will depend on the condition of the at this temperature until ready for Because representative samples of samples when collected and on the analysis. In handling the specimens foods and feeds are typically between climate in which the work is being subsequently, containers should be 3 kg and 30 kg in size, it is essential performed. allowed to reach room temperature that sampling procedures allow the If samples are already dry, it is before opening. Lengthy storage of preparation of laboratory samples that advisable to store them in polyethylene samples (> 3 months in cold storage, are representative of the original lot. or glass bottles so that no moisture and > 6 months at –20 °C) before Representative laboratory samples enters the sample. Storage of moist analysis should be avoided. require successive comminution and samples should be avoided. Samples Care should be taken that dividing of the total sample, using should be dried as quickly as possible, samples are labelled with the correct either static or rotary dividers. Ideally, after a subsample has been taken sample number and other relevant the comminution and division steps for moisture measurement (if this is information, such as the place and should be performed simultaneously to be determined). If drying cannot date of collection. A separate record using a subsampling mill. Such mills will be carried out immediately, samples of all collected samples should be convert large samples of edible nuts, should be refrigerated, frozen, or kept. A copy of this record should oilseeds, or grains into comminuted, sampled for mycotoxin analysis im- be stored with the samples at all representative subsamples in a single mediately. The samples should be times and must include additional operation. transported to the laboratory as soon information about the quality of the as possible and kept free of insect commodity at the time of collection

50 References

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Chapter 3. Sampling and sample preparation methods for determining 51 concentrations of mycotoxins in foods and feeds

chapter 4. Analysis of mycotoxins CHAPTER 4 CHAPTER

Summary less infrastructure are described disadvantages of these methods here. The first approach is thin-layer include the need to refrigerate the kits The analytical methods for mycotoxin chromatography (TLC), which has before use and the limited shelf-life. determination used in fully developed been used for more than 50 years to It has been proposed that companies countries require sophisticated infra- analyse mycotoxins. The advantages and development agencies could be structure, stable electricity, ready of TLC include simplicity and solicited to develop packages of kits, availability of supplies, and qualified proven reliability. Accuracy may be sampling equipment (e.g. grinders), and experienced technicians for improved by using precision spotters and training models for deployment instrument maintenance. Simple to apply precise amounts of sample in the many areas where mycotoxins and appropriately validated tools to TLC plates and optical readers. are a chronic problem. analogous to those used for the The costs of these refinements to management of contaminated bulk TLC are far lower than those of gas 1. Introduction commodities at the grain elevator or liquid chromatography systems. level are needed at the rural level The disadvantages of TLC include Determination of mycotoxin con- in developing countries. These the need for stable supplies of centrations in staple crops is a tools are needed to promote public solvents and standards as well as challenging exercise because health and to manage emergency safe conditions for their storage. The of the problems associated with situations in subsistence farming second approach described here is sampling heterogeneously distributed communities with an immediate based on immunological methods compounds (see Chapter 3) and the and severe problem of mycotoxin using anti-mycotoxin antibodies. fact that the analytical methods contamination of food grains, with These tests are available as kits, need to have low limits of detection, the goal of working towards feasible have the necessary standards built generally in the mg/kg (ppm) or µg/ reductions in exposure. Two general in, use little or no organic solvent, kg (ppb) range, depending on the analytical approaches that require and are generally easy to use. The individual mycotoxin being analysed.

Chapter 4. Analysis of mycotoxins 53 Analysis at these levels needs to official methods are based on high- 3. Analytical methods useful be very specific to avoid analytical performance liquid chromatography in developing countries interferences and can produce (HPLC) with various detectors, and large uncertainties. Given the many the most recent of these methods The techniques used in developed advances in analytical science since use immunoaffinity columns (IACs) countries require sophisticated infra- the discovery of the aflatoxins in the for sample extract clean-up before structure, stable electricity, ready 1960s, it is not surprising that a wide the HPLC analysis. More recently, availability of supplies, and qualified array of analytical methods have the advances made in coupling mass and experienced maintenance been used for mycotoxin testing; spectrometry to HPLC have enabled technicians. The facilities are most fall into the general categories analytical chemists to combine expensive to build, require highly of either chromatographic methods analytical steps with a confirmatory trained personnel, and generally or immunological methods based test by measuring the mass spectrum have a low throughput unless on antibody technology (for a tutorial of the HPLC peak. The highly staff numbers are large and spare review, see Shephard, 2008). All specific nature of mass spectrometry instruments are available. The these analytical methods require has also been used to avoid extract difficulties in meeting the challenges solvent extraction of the mycotoxin purification and to develop multitoxin associated with mycotoxin testing of interest from the matrix, followed methods, which can be applied in Africa, namely a lack of political by key analytical steps, which, to mixtures of mycotoxins in one commitment, infrastructure and for chromatographic determination, analytical experiment. However, in trained personnel, sustainable typically involve extract clean-up and addition to these official methods, supplies, instrument maintenance concentration before final deter- rapid screening methods have been and repairs, and laboratory quality mination. Another component of the developed for situations where quick control, have been discussed by complexity of mycotoxin analysis decisions are required, such as at Waliyar et al. (2008). For these is the fact that the varied chemical granaries, silos, and factories, and reasons, the extent to which such structures of mycotoxins mean that such methods can be adapted for methods can be transferred to specific methods are required for transfer to developing countries. Most developing countries depends individual toxins. This constraint is of these rapid methods are based on on the country’s exact stage of now being overcome by the use of immunological principles and use development and the importance expensive and sophisticated mass antibodies raised against specific attached to analytical determinations spectrometric methods. Because of mycotoxins. They include quantitative of mycotoxins. Usually, developing this plethora of methods and their enzyme-linked immunosorbent as- countries rely on less sophisticated individual characteristics, when select- says (ELISAs), fluorometric methods, methods, such as thin-layer ing a method for mycotoxin analysis, lateral flow devices, and a range chromatography (TLC) and antibody- one should consider the purpose for of tests that give a yes/no result for based methods. TLC remains a which the results are needed, the contamination above or below a set useful tool in developing countries, matrix to be analysed, the detection control level. Whereas a full review of can be semi-automated with limit required, and the expertise and these methods lies outside the scope sample spotters and ultraviolet (UV) infrastructure available. of this chapter, many recent reviews scanners for detection, and has the exist of methods for the analysis advantage of testing several samples 2. Analytical methods used in of mycotoxins in fully developed simultaneously. Its potential use in rural developed countries countries or in export certification settings is discussed in Section 4.1. laboratories set up in countries to The wide range of commercial For the survey and control of myco- certify bulk commodities for export immunological methods has found toxin levels and the implementation of (Krska et al., 2008; Solfrizzo et al., broad application in laboratories that mycotoxin regulations in developed 2009; Maragos and Busman, 2010; lack sophisticated instrumentation countries, several official analytical Shephard et al., 2012). or in which access to such in- methods have been validated by strumentation is limited by high interlaboratory collaborative studies demand. Of these, quantitative or conducted under the auspices of semiquantitative ELISAs, which do not international bodies such as AOAC require sample extract purification, are International and the European Com- widely used and have the advantage mittee for Standardization (CEN). Most of handling many samples in a

54 single experiment. The purification analysis methods is pursued with tank, and solvents. Chromatography of sample extracts via IACs has considerable urgency.” Unfortunately, was carried out using strips of silica- also been commercialized for direct the agenda set by these authors has coated plastic sheets for dipsticks fluorescence measurements using not yet been addressed. and visualization with a handheld UV proprietary calibrated fluorometers. Appropriate and useful tools lamp. The author reported a detection

In recent years, the technique of analogous to those used for the limit of 10 ppb for aflatoxin B1. fluorescence polarization (FP) im- management of contaminated bulk TLC methods are useful for the munoassay has been successfully commodities at the grain elevator key toxins discussed in this book: applied to mycotoxin determination. level are needed at the rural level in aflatoxins and the Fusarium myco- Rather than measuring the total developing countries. These tools are toxins fumonisins, deoxynivalenol, fluorescence, FP measures the needed to promote public health and to and zearalenone (Lin et al., 1998; orientation of the fluorescence manage emergency situations in rural Schaafsma et al., 1998; Shephard, emission, which is related to the rate of areas with an immediate and severe 1998; Shephard and Sewram, 2004). molecular rotation. The advantage of problem of mycotoxin contamination AOAC International has approved FP is that it is performed entirely in the of food grains. This is not a question several TLC methods for aflatoxins in extract solution (Lippolis et al., 2006). of meeting Codex standards, but groundnuts and maize as well as for Lateral flow devices can provide a rather of working towards feasible ochratoxin A in barley, deoxynivalenol yes/no result for contamination above reductions in exposure. in wheat, and zearalenone in maize or below a set control level, and they Analytical methods applied in (Table 4.1). These procedures are have also been commercialized with rural settings need to relate to a more accurate and reliable if carried optical readers to provide quantitative comprehensive risk management out with precision spotters and CHAPTER 4 CHAPTER results. It needs to be recognized strategy designed to address optical readers (Nawaz et al., 1992); that all antibody-based methods are and reduce exposure to relevant these units are relatively expensive liable to cross-reactivities and matrix mycotoxins. Therefore, the methods but are far less costly and are effects, but they are extremely useful must be rapid and easy to perform easier to maintain than HPLC or gas as a first line of analysis. Where it is and should require a minimum of chromatography (GC) instruments, possible, problematic samples can local or transportable infrastructure. particularly because of the absence be confirmed by HPLC methods. Methods should have a wide analytical of precision pumps and, in the case of range because determinations at the GC, a constant supply of carrier gas. 4. Analytical methods useful rural level mostly require a focus on Like all chemical analyses, TLC in rural areas removing or managing contaminated requires trained staff; individuals lots as opposed to determining small with college-level education need Methods of analysis suitable for use differences in contamination that several weeks of intensive training at the rural level are still a challenge to might be relevant for compliance with a in chemical analysis to perform analytical scientists. Citing Sashidhar regulatory limit. For this purpose, TLC reliable TLC analyses. Experience (1993), Fernández-Surumay et al. and some immunological methods has been gained in providing such (2000) commented (in a rural Latin would be suitable. training in Asia and Africa (FAO, American context) that methods used 1990; Boutrif, 1995; Cardwell, 1996). in fully developed countries “require 4.1 Thin-layer chromatography Potential problems with TLC analysis highly qualified personnel, as well as include the acquisition of the essential sophisticated equipment in ad- Cognizant of the constraints in de- mycotoxin standards, the preparation vanced laboratories.... [therefore,] veloping countries, Sashidhar (1993) of fresh standards in solution, and simpler methods must be developed described a portable mycotoxin the stability of the resulting solutions. that do not require such infrastructure, analysis kit housed in a large fibreglass Pure standards as solids are ex- are easier to manipulate, and at the (or metal) suitcase-sized package pensive and are perishable unless same time do not compromise the as a suitable method for use at the stored under very carefully controlled quality of the analysis.” A decade village market level. The approach conditions. The preparation of earlier, in commenting on mycotoxin used was a simple, reliable, and standards in solution requires analysis in developing countries, inexpensive TLC method. The main access to an accurate balance as Coker (1991) wrote: “It is therefore components of the kit were a portable well as weighing conditions with imperative that the development of sample grinder, a robust domestic appropriately conditioned air (ca. efficient, cost-effective sampling and blender for toxin extraction, a TLC 25 °C, 30–40% relative humidity).

Chapter 4. Analysis of mycotoxins 55 Table 4.1. AOAC International official TLC methods for some mycotoxins in cereals

Mycotoxin AOAC method Commodity Remarks

Aflatoxins 968.22 Groundnuts and groundnut products CB method

970.45 Groundnuts and groundnut products BF method

998.03 Groundnuts Alternative BF method

993.17 Maize and groundnuts

975.37 — Aflatoxin B1/aflatoxin G1 confirmation method

985.17 — Aflatoxin B1 confirmation method

Ochratoxin A 973.37 Barley

Deoxynivalenol 986.17 Wheat

Zearalenone 976.22 Maize

BF, United States Food and Drug Administration Best Foods; CB, United States Food and Drug Administration Contaminants Branch; TLC, thin-layer chromatography.

More typically, standards are products from several companies Institute for the Semi-Arid Tropics purchased as certified solutions or are quite effective (Schaafsma et (ICRISAT) has developed a simple, access to a UV spectrophotometer is al., 2009). A comparison of TLC and robust, versatile, and cost-effective required for accurate determination antibody-based methods showed ELISA for the determination of afla- of toxin concentration. The stability that the training needs are similar toxins in groundnuts (ICRISAT, 2009). of standards in solution is limited for the two kinds of systems but After appropriate validation, the unless they are kept refrigerated or that for antibody-based methods United States Department of frozen. Therefore, the challenges of the equipment and supply costs are Agriculture Grain Inspection, Packers TLC are training, solvent supplies, lower, and the problems associated and Stockyards Administration acquisition of standards, standard with standards are eliminated. Cross- (GIPSA) has approved several preparation, and standard stability. reactivity of related mycotoxins antibody-based tests for use as occurs with most ELISA methods first-action tools at silos. Several 4.2 Antibody-based methods and precludes their use as regulatory companies produce very good tools (Tangni et al., 2010). Although dipstick methods based on antibodies As noted previously, many pri- antibody-based tests can also suffer with simple-to-use and relatively mary testing laboratories in both from the occurrence of various matrix inexpensive readers. It would seem developed and developing countries effects, especially if they are used plausible to build on the portable use antibody-based methods to inappropriately or in matrices for which mycotoxin analysis kit of Sashidhar assay mycotoxins. The sampling, they were not validated, they could (1993), replacing the TLC method grinding, blending, and extraction supply the rapid analyses needed in with antibody-based dipsticks. The steps are similar to those for TLC, rural settings. The local development sensitivity of these methods can be except that solvent use is much of antibodies and immunoassay improved by using nanoparticles reduced and solvents are usually kits has been proposed to obviate to support the capture antibody less expensive and less hazardous. the commercial costs, but care (Posthuma-Trumpie et al., 2009; Several studies have examined needs to be exercised in adequately Maragos and Busman, 2010). antibody-based tests and have validating locally developed kits. shown that commercially available The International Crops Research

56 5. Conclusions and robust analytical equipment and improved agronomy, crop varieties, methods are needed to identify the greater crop diversity, and improved The capacity to perform mycotoxin most severely affected rural areas storage. Many commercial ELISAs analysis is needed to manage emer- and to provide feedback on the are available for mycotoxins, each gency situations and to promote effectiveness of the management with its strengths and weaknesses, public health. When a rural region has practices instituted. In addition to including sensitivity, cross-reactivity, an immediate and severe problem mycotoxin analysis for managing and shelf-life. Evaluating such tests of mycotoxin contamination of food emergency situations, general public for applicability for field use in rural grains, appropriate risk management health can be promoted by regular areas of developing countries would practices (see Chapter 7) need to monitoring of mycotoxin in rural areas, be a useful and important project. be implemented. Suitable portable which may also identify priorities for CHAPTER 4 CHAPTER

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58 chapter 5. Effects in food-producing animals CHAPTER 5 CHAPTER Summary ergot alkaloids. In all cases the effect possible involvement of a mycotoxin of mycotoxins on animal performance in an animal production problem. Unexplained disease outbreaks in is potentially a major problem for In conjunction with the information farm and domestic animals have farmers regardless of their scale provided in the other chapters, this suggested the likely presence of of operation. Reduced growth, information will assist farmers in mycotoxins in feeds for many years. decreased egg and milk production, making decisions that will minimize The manifestations of mycotoxicoses lower reproductive efficiency, and losses due to diseases induced by in the field are frequently nondescript increased susceptibility to stress are all mycotoxins. and potentially have many contrib- potentially devastating consequences uting factors, which are often of mycotoxin exposure. Thus, being 1. Introduction difficult to define. Nevertheless, aware of the outward signs that might toxigenic moulds were implicated signal the involvement of a mycotoxin In this chapter, we discuss the in, and sometimes proven to be the in an animal performance problem is effects in farm and domestic animals cause of, animal disease in field the first step to minimizing potential of the most economically important outbreaks long before the toxins adverse impacts. The target organ feedborne mycotoxins. The chapter were discovered. The development affected can provide important clues begins with a summary of field of methods for the chemical analysis to involvement of a specific mycotoxin, outbreaks, followed by sections of mycotoxins in feeds and animal in which case understanding the that describe the toxicokinetics tissues has contributed to an toxicokinetics and toxicology will assist and metabolism of each mycotoxin improved understanding of the dose– in minimizing the cost and maximizing or group of mycotoxins. Of the response relationships of farm animal the effectiveness of interventions. trichothecenes, only deoxynivalenol diseases associated with exposure The primary objective of this chapter is covered in depth; however, it should to aflatoxins, fumonisins, ochratoxin is to provide information that will be recognized that the predominant A, deoxynivalenol, zearalenone, and aid in the field identification of the trichothecene encountered in con-

Chapter 5. Effects in food-producing animals 59 taminated feeds may differ in different to cause animal disease. A major the case for aflatoxins, fumonisins, geographical locations (Starkey et al., problem for the veterinarian in the field deoxynivalenol, zearalenone, and 2007; Miller, 2008; Sugita-Konishi and is that disease expression is seldom ergot alkaloids, where mould- Nakajima, 2010). pathognomonic, i.e. with a sign or contaminated feed was associated Additional information relevant symptom that is so characteristic of a with disease outbreaks before the to the toxicology in animals can be disease as to be diagnostic. Instead, toxins were identified. For example, found in Chapter 6, which focuses multiple interacting factors often in the 1960s the outbreak of a on effects and toxicology in humans. occur that can modify the expression disease known as turkey “X” disease Potential interventions to prevent field of toxicity. Thus, the manifestations led to the discovery of aflatoxins. outbreaks or minimize their adverse of mycotoxicoses in the field are However, before this disease was effects are described in Chapter 9. frequently nondescript and have reported, outbreaks of liver cancer Numerous extensive review articles many potential contributing factors, in farm-raised rainbow trout fed diets and monographs provide additional which are often difficult to define. containing cottonseed (Butler, 1974) information on effects in farm An attempt to apply Koch’s and of liver toxicity in pigs and cattle fed animals (WHO, 2001, 2011; CAST, postulates to demonstrate causality maize contaminated with Aspergillus 2003; Cousin et al., 2005; Roberts requires a bioassay that reproduces flavus were documented in 1935 and et al., 2005; Fink-Gremmels and the disease when a pure compound 1953, respectively (Raisbeck et al., Malekinejad, 2007; Morgavi and is used. Unfortunately, reproducing 1991). Equine leukoencephalomalacia Riley, 2007a, 2007b; Voss et al., the exact conditions that existed (ELEM) was first linked to mouldy 2007; Fribourg et al., 2009; Pestka, in the field is confounded by the maize in 1891 (Haliburton and Buck, 2010a, 2010b; Eaton et al., 2010). potential presence of multiple 1986). ELEM is now known to be To aid in the field identification of the contributing factors. These include, caused by fumonisins. Effects from main observations characterizing but are not limited to, environmental the consumption of maize on which exposure to a particular mycotoxin, stress, multiple toxigenic fungi Fusarium verticillioides had been Table 5.1 summarizes the expected and mycotoxins, nutrient/vitamin cultured were the first indication that toxic effects, species sensitivity, and deficiencies, infectious agents, and mouldy maize might cause porcine potentially useful biomarkers in farm pre-existing conditions. These co- pulmonary oedema (PPE) syndrome animals for each of the major groups occurring factors can influence (Kriek et al., 1981). This was of mycotoxins. the clinical signs, severity, and confirmed only after the discovery progression of the disease (CAST, of fumonisins in 1988 and after the 2. Field outbreaks 2003) in ways that can confound cause of outbreaks of PPE in the both diagnosis and replication of USA in 1989–1990 was confirmed The interest of the scientific and the observed adverse effects. Also, by inducing PPE with pure fumonisin regulatory communities in mycotoxins mycotoxins in feeds are not evenly B1 (Marasas, 2001). began in earnest when aflatoxins were distributed (see Chapter 3); therefore, Feed refusal syndrome in the USA, found to be potent carcinogens that reproducing disease at the dosages most likely caused by deoxynivalenol occurred in several important food found in feed samples analysed from and other trichothecenes, led to an and feed commodities. However, even field outbreaks can be difficult. It has embargo of United States barley by in the absence of any known causal been suggested that 100–200 kg Germany in the 1930s (Hamilton, agent, unexplained disease outbreaks or more of suspect feed should be 1982). The association between in farm and domestic animals, which saved for confirmatory studies in consumption of mouldy feed and often involved mortality or evidence of experimental animals (Osweiler, estrogenism in pigs has been known acute toxicity, served as an indicator 2000). In addition, experimental since 1928 (McNutt et al., 1928), and of the likely presence of mycotoxins in confirmation can require a large estrogenism has been attributed to foodstuffs (Forgacs and Carll, 1962). number of animals if the incidence consumption of feeds contaminated Definitively linking a disease of the disease in the suspected field with zearalenone. The biological outbreak in the field to a specific outbreak is low. activity of ergot (the sclerotia of mycotoxin is very difficult (Hamilton, Despite these difficulties, toxi- Claviceps spp.) was known in China 1982), even though a great deal genic moulds were implicated as more than 5000 years ago even of experimental evidence exists the cause of animal disease in though its involvement in animal to characterize the potential of field outbreaks long before the disease was probably not reported mycotoxins and mouldy foodstuffs toxins were discovered. This was until the Middle Ages (Christensen,

60 Table 5.1. Expected toxic effects, species sensitivity, and potentially useful biomarkers in farm animals for each of the major groups of mycotoxins

Relative sensitivity of Mycotoxin Target organs and major effects Biomarkers species

Aflatoxins Reduced performance, jaundice, pale liver, hepatotoxicity with Ducklings > turkeys Aflatoxin–albumin adducts fatty changes, coagulopathy, and increased susceptibility to > chicks > quail. in serum and DNA adducts internal bruising during handling. Rabbits > swine > cattle > in urine.

Liver tumours in trout. sheep. Aflatoxin M1 in milk and Dogs and mink can also be urine. affected. Young animals > mature animals.

Fumonisins Liver in all species and kidney in many. Brain in horses (ELEM) Horses and rabbits > pigs Fumonisin B1 in urine and and lung in pigs (PPE). and catfish > ruminants and faeces. poultry. Elevated sphinganine and Breeding animals sphinganine-1-phosphate > animals being raised for in tissues, urine, and serum slaughter. and elevated sphinganine- 1-phosphate in red blood cells.

Ochratoxin A Pale and grossly enlarged kidney. Fatty liver in poultry. Pigs and dogs > poultry Ochratoxin A or its Altered performance, including decreased feed consumption, > calves. metabolites in tissues, reduced weight gain, and decreased egg production. Increased Chicks > turkeys > quail. blood, and urine. susceptibility to bruising, decreased tensile strength of the large Mature cattle are intestines, reduced pigmentation, decreased immune response, considered resistant. increased susceptibility to infection, and glycogen accumulation in the liver.

Deoxynivalenol Feed refusal in swine and reduced weight gain and vomiting. Pigs > dogs and cats Deoxynivalenol and its Gastrointestinal problems, soft stools, diarrhoea, increased > cattle, sheep, and glucuronide conjugate in susceptibility to other diseases, and decreased performance. poultry. urine.

Zearalenone Estrogenic effects. Swollen red vulva, vaginal prolapse, rectal Pre-pubertal pigs Zearalenone or metabolites 5 CHAPTER prolapse, and fertility problems in swine. >> cattle and sheep (including glucuronic acid > poultry. conjugates) in urine or faeces.

Ergot alkaloids In cattle, dry gangrene aggravated by cold weather and heat Cattle > horses and sheep. Lysergic acid in urine. intolerance in warm weather. Poultry and swine can also In cattle and horses, neurotoxicity, including tremors, be affected. convulsions, and agalactia. In swine, reproductive problems and agalactia.

ELEM, equine leukoencephalomalacia; PPE, porcine pulmonary oedema.

1980). Ergot was implicated as suggested that multiple mycotoxins In the remainder of this section, the cause of animal production could have been involved, including we provide some examples of problems in domestic animals (mainly aflatoxins and ochratoxin A (Hamilton, the spectrum of documented or cattle) in the USA in the early 19th 1982). Ochratoxin A, unlike aflatoxins, strongly suspected field outbreaks century (Hesseltine, 1979), but the fumonisins, deoxynivalenol, zearale- for aflatoxins, fumonisins, ochratoxin alkaloids responsible for ergotism none, and ergot alkaloids, was A, deoxynivalenol, zearalenone, and were not identified until the 1930s. isolated and characterized (van der ergot alkaloids. The causative agent or agents for Merwe et al., 1965) before it was haemorrhagic syndrome in the USA proven to cause outbreaks of kidney in the 1950s caused by mouldy feed disease in poultry (Hamilton et al., (Forgacs and Carll, 1962) have never 1977) and pigs (Krogh, 1978a, 1978b). been identified. However, it has been

Chapter 5. Effects in food-producing animals 61 2.1 Aflatoxins cases of ELEM in Egypt, Wilson and Outbreaks of mycotoxic nephropathy Maronpot (1971) reproduced ELEM have also been reported in horses Turkey “X” disease was responsible in horses by feeding them maize on (Krogh, 1978b), and there are a for the deaths of > 100 000 turkeys in which this fungus had been grown. few suspected cases in ruminants the United Kingdom in 1960. Mortality Kriek et al. (1981) induced PPE, (Raisbeck et al., 1991). The first was also documented in ducks, a disease that has been known in well-documented and confirmed chickens, pheasant, calves, and Hungary since 1950 (Fazekas et case of ochratoxicosis occurred in pigs (Butler, 1974). In the USA and al., 1998), by feeding pigs maize young turkeys in the south-eastern elsewhere, field outbreaks causing on which F. verticillioides had been USA (Hamilton et al., 1977). In 1976, mortality have been well documented grown. After isolating and chemically about 80% of the marketed turkeys in turkeys, laying hens, pigs, cattle, characterizing the fumonisins, Mara- in North Carolina displayed signs rainbow trout, and dogs (Butler, 1974; sas et al. (1988) induced ELEM in a of ochratoxicosis, and all cases

Hamilton, 1982). In the case of poultry, horse by using purified fumonisin B1. involved maize contaminated with pigs, and farm-raised trout, large Serendipitously, in 1989–1990 there OTA (Schaeffer and Hamilton, 1986). numbers of animals were involved were numerous outbreaks of ELEM In a turkey house with 16 000 turkeys, (Butler, 1974; Hamilton, 1982). Acute and PPE syndrome in the USA. mortality was 59%. The first toxicity is easily recognized, but the In 1990, PPE was induced using indication of intoxication was feed more subtle effects are probably pure fumonisin B1 (Harrison et al., refusal (Schaeffer and Hamilton, of greater concern to farmers. 1990). Field outbreaks of PPE have 1986). Death was most commonly For example, decreased rates of not occurred in the USA since the seen in younger birds, whereas weight gain, decreased milk or egg early 1990s, but reports of ELEM older birds developed infections production, increased susceptibility to have persisted. In poultry, reduced with Escherichia coli (air-sacculitis) bruising during processing of poultry performance associated with feeds that did not respond to antibiotics. and pigs, underpigmentation of contaminated with fumonisins Interestingly, in outbreaks in chickens, meat in poultry, and altered immune has been reported, but the effects feed refusal did not occur, whereas function have all been associated seldom involve increased mortality poor pigmentation was common with exposure in the field (Hamilton, (WHO, 2000). (Schaeffer and Hamilton, 1986). 1982; Raisbeck et al., 1991). Pet food Other field cases in chickens have recalls due to aflatoxin contamination 2.3 Ochratoxin A reported decreased egg production resulting in liver toxicity and death in and eggshell quality, increased dogs are not uncommon in the USA. In pigs, mycotoxic nephropathy was susceptibility to intestinal rupture first described in 1928 in Denmark during processing, decreased 2.2 Fumonisins and was reproduced experimentally bone strength, and haemorrhagic using mouldy barley or oats (Krogh, episodes (Schaeffer and Hamilton, In the USA, field outbreaks of ELEM 1992). This disease has also been 1986). In all species, effects on the caused by mouldy maize have been reported in Norway, Sweden, Ireland, kidney were apparent. reported for more than 100 years Finland, Germany, Hungary, Poland, (Haliburton and Buck, 1986). It was the former Serbia and Montenegro, 2.4 Deoxynivalenol reported that about 5000 horses died and Bulgaria (Krogh, 1978a, 1978b; of “mouldy corn poisoning” in Illinois Marquardt and Frohlich, 1992; Stoev Trichothecenes have been implicated in 1934–1935 (Haliburton and Buck, et al., 1998). The main clinical signs in feed refusal by cattle, pigs, and 1986). The disease has also been are renal dysfunction and oedema. In chickens; however, pigs appear to be reported in South America, Hungary, pigs, excessive thirst (polydipsia) and the most sensitive to deoxynivalenol China, Greece, France, Mexico, passage of large volumes of urine (DON) (Rotter et al., 1996; Haschek New Caledonia, Egypt, South Africa, (polyuria) are characteristic signs of et al., 2002). Outbreaks in animals and Germany (Magnol et al., 1983; this disease under field conditions. of “red mould poisoning” from Haliburton and Buck, 1986; Laurent The association of ochratoxin A (OTA) cereals in Japan as far back as 1890 et al., 1998; Rosiles et al., 1998). After with field outbreaks of nephropathy (Miller, 2008) led to the discovery of identifying Fusarium verticillioides in poultry was first reported in DON. It was first isolated in Japan (then known as F. moniliforme) as 1975 (Krogh, 1992) and has been and was originally called Rd-toxin the predominant fungal contaminant reported in many countries since (Moorooka et al., 1972). It was given of mouldy maize that had caused then (Marquardt and Frohlich, 1992). the name vomitoxin (Vesonder et al.,

62 1973) after its isolation from maize Reproductive problems have been problems and neurological effects contaminated with F. graminearum reported in sheep grazing on grasses in horses (staggers, “drunkenness”, because it is associated with emesis contaminated with ZEA (CAST, 2003), and sleep) and reduced reproductive in pigs. Numerous confirmed cases and ZEA was found to interfere with performance, decreased milk produc- have been reported of feed refusal the induction of parturition by oxytocin tion, and reduced growth in cattle or reduced feed intake in pigs in gilts and sows (Alexopoulos, 2001). (Cross, 2003; White et al., 2003). consuming feeds contaminated with In maize contaminated with ZEA, co- In cattle and sheep, increased DON (Osweiler, 2000). The most contamination with DON is likely. body temperature due to peripheral frequently observed effect of DON vasoconstriction is often seen in in farm animals is feed refusal, which 2.6 Ergot alkaloids animals that have ingested grasses may explain why toxic effects are to infected by endophytes (Cross, 2003). a great extent self-limiting (Osweiler, Poisoning resulting from the con- Field cases of fescue foot (necrosis of 2000). There have been unconfirmed sumption of foods contaminated by the extremities) in cattle and laminitis reports of feed refusal in dogs, cats, ergots, which are fungal sclerotia that in horses have been reported to be due and rabbits consuming pet foods replace grass seeds, has been known to peripheral vasoconstriction caused naturally contaminated with DON since the Middle Ages, but it was by ergot alkaloids (Cross, 2003). (Bohm and Razzazi-Fazeli, 2005). not until the 20th century that ergot Environmental conditions appear In ruminants, field disease outbreaks alkaloids from Claviceps purpurea to influence the vasoconstrictive attributed to DON are rare (Raisbeck were determined to be the causative effects of ergot alkaloids in grasses; et al., 1991). agent (Barger, 1931). Ergot alkaloids episodes of fescue foot are most associated with or suspected to common in autumn and early winter, 2.5 Zearalenone be involved in diseases of farm and the condition known as summer animals include ergoline alkaloids, slump is most common in warm Estrogenism in pigs consuming which contain the lysergic acid ring weather (Raisbeck et al., 1991). mouldy feed was first reported in structure, and ergopeptine alkaloids, 1928 (McNutt et al., 1928); since which contain a tripeptide moiety. 3. Toxicokinetics and then, field outbreaks of reproductive In farm animals, ergot poisoning is metabolism problems, including vulvovaginitis usually associated with grazing on CHAPTER 5 CHAPTER and anestrus, and enlargement of seed heads of grasses contaminated Mycotoxins are chemically diverse, the mammae in males have been with ergots of Claviceps spp. or and therefore their uptake, distribution, attributed to consumption of feeds grasses colonized by groups of fungi metabolism, and excretion are equally contaminated with zearalenone that occur as endophytic symbionts diverse. Galtier (1998) summarized (ZEA) (Aucock et al., 1980; Raisbeck (Neotyphodium spp. and Epichloë the kinetics and biological fate of et al., 1991), which was previously spp.; Roberts et al., 2005) or with the most of the economically important called F-2 toxin (Mirocha et al., consumption of feeds contaminated mycotoxins. Uptake from the 1968). Field outbreaks of estrogenic with ergot (CAST, 2003). Outbreaks gastrointestinal tract depends to a syndrome in pigs have been reported of ergot poisoning have also been large extent on the water solubility in North America, Europe, Africa, reported in pigs and cattle consuming or lipophilicity of the particular Asia, and Australia (Christensen, sorghum-based feeds. Consumption mycotoxin. Metabolism by microbes 1979). ZEA-induced abortions in of feeds based on small grains, in the gut can also have a profound pigs have also been reported and such as rye and barley, or foraging effect on uptake and toxicity. are probably a result of embryo on infected grasses contaminated Some mycotoxins, like OTA, are implantation failure (Osweiler et al., with ergots has caused lameness well absorbed, whereas others,

1986). Suggestions that reproductive or necrosis of ears, tails, and feet like fumonisin B1, are very poorly problems in ruminants can be attri- in cattle (CAST, 2003). Endophytes absorbed. Binding of mycotoxins buted to ZEA are considered to be (Neotyphodium spp. and Epichloë to plasma proteins can influence controversial (Raisbeck et al., 1991). spp.) that occur in pasture grasses uptake, distribution, and the half-life The semisynthetic ZEA analogue such as tall fescue and ryegrass are in the blood. Again, some mycotoxins, zeranol is used in cattle as a growth known to produce ergot alkaloids, including OTA, are tightly bound, promoter, and its estrogenic effects which have caused a diverse array whereas others, including fumonisin have caused reduced performance of toxic syndromes in grazing B1, are not bound. Metabolism and in bulls (Raisbeck et al., 1991). animals, including reproductive excretion are important considerations

Chapter 5. Effects in food-producing animals 63 for determining whether residues 3.1.3 Bioavailability 1993a). Low levels of aflatoxin B1 and will remain in tissues and also for aflatoxin M1 (< 1 µg/kg) were detected developing biomarkers for exposure. Aflatoxin B1 is rapidly absorbed from in the liver and kidneys of lactating

For example, aflatoxin B1 is extensively the small intestines, and the rate of cows fed diets containing 1250 µg/ metabolized in the liver and elsewhere, absorption is much higher in suckling kg aflatoxin B1 for 2 weeks (Busby and significant amounts can be and young animals than in adults and Wogan, 1981a). Pigs fed diets excreted as metabolites in milk. For (Kumagai, 1985). The process of containing 100–400 µg/kg aflatoxin

DON, most of the absorbed toxin is absorption from the small intestines is B1 for 4 weeks had detectable levels excreted in urine, whereas only very passive and complete. For example, of aflatoxins B1 and M1 in the liver, small amounts of fumonisin B1 are the time needed to reduce aflatoxin B1 muscle, kidney, and blood. At 400 µg/ excreted in urine. Fumonisin B1 is in the intestinal lumen by 50% is about kg aflatoxin B1 in feed, the detected not metabolized in the liver or other 7 minutes (Ramos and Hernández, levels of aflatoxins B1 and M1 were tissues and therefore is recovered 1996). Aflatoxins B1 and B2 are more as high as 4 µg/kg and 1.5 µg/kg, intact or partially hydrolysed (probably rapidly absorbed than are aflatoxins respectively (Busby and Wogan, by microbes in the gastrointestinal G1 and G2, ensuring that their 1981a). In chickens, maximal levels tract) in faeces. Although mycotoxin bioavailability is high (Ramos and were attained in plasma and the liver residues can be carried over into Hernández, 1996). Aflatoxin B1 is also within 6 hours after oral dosing, and milk and eggs (Galtier, 1998), only absorbed slowly through the skin, and levels declined rapidly thereafter aflatoxin M1 and OTA have been intratracheal absorption is more rapid (Hirano et al., 1994). After either a detected naturally in cow’s milk. than via the oral route (IARC, 1993a). single oral dose or daily oral dosing

Once absorbed, aflatoxin B1 is non- for 2 weeks, aflatoxin residues were 3.1 Aflatoxins covalently bound to albumin and is detected in various organs and in transported to other tissues. muscle tissue in laying hens (Busby 3.1.1 Absorption and Wogan, 1981a). Aflatoxins can 3.1.4 Distribution cross the placenta and accumulate

Aflatoxin B1 absorption, distribution, in developing fetuses (IARC, 1993a). and elimination is rapid. Aflatoxin B1 Early distribution studies focused on Unmetabolized aflatoxin B1 can is well absorbed; it accumulates in the extraction of the parent compounds accumulate in tissues rich in melanin liver and is extensively metabolized in or non-polar aflatoxin metabolites and pigment and the upper respiratory the liver and other tissues. The binding ignored the water-soluble metabolites tract (Larsson and Tjälve, 1993). of metabolites to macromolecules, (Busby and Wogan, 1981a). In sheep including proteins and nucleic acids, and other animals, aflatoxin B1 is 3.1.5 Excretion also occurs soon after absorption. immediately transported to the liver Unbound water-soluble metabolites after absorption from the gut (Wilson After intraperitoneal or intravenous are excreted in urine and other fluids, et al., 1985). The half-life of aflatoxin dosing of aflatoxin B1, excretion and the parent compound is excreted B1 in plasma after intravenous is rapid and most of the dose is in faeces; however, some metabolites dosing is < 1 hour (Wong and Hsieh, eliminated in faeces and urine (Wong bound to nucleic acids can persist for 1980), whereas after intratracheal and Hsieh, 1980; Busby and Wogan, relatively long periods in tissues. or oral dosing it is about 90 hours 1981a). The relative retention of

(Coulombe and Sharma, 1985). aflatoxin B1 is greater than that of

3.1.2 Gastrointestinal As with absorption from the small aflatoxin B2 due to greater urinary metabolism intestines, aflatoxin B1 is rapidly taken excretion of aflatoxin B2 (Busby and up by the liver with a half-life of < 5 Wogan, 1981a). A large percentage Little published information is avail- minutes (Busby and Wogan, 1981a). of the intraperitoneal dose is able about the ability of micro- Aflatoxin B1 is widely distributed in recovered in bile (Busby and Wogan, organisms in the gastrointestinal the body, with most accumulating in 1981a). In sheep, aflatoxin residues, tract to metabolize or bind aflatoxins. the liver, kidney, and lung. Maximal mainly aflatoxin M1, are excreted It is known, however, that certain levels in tissues, and especially primarily in urine within 48 hours bacteria can bind aflatoxin B1 in vitro, the liver, are reached quickly, and after oral dosing (Busby and Wogan, and it has been suggested that they the relative retention of aflatoxin B1 1981a). Aflatoxin M1 is the main may aid intestinal excretion (Oatley is greater than that of aflatoxin B2 unconjugated metabolite of aflatoxin et al., 2000). (Busby and Wogan, 1981a; IARC, B1 excreted in the milk of sheep,

64 goats, and cows. In sheep and cattle, microsomal systems to aflatoxins P1, al., 2010). For example, domestic aflatoxin B1 is the main component in M1, and Q1 and, most importantly, turkeys, one of the most susceptible faeces. In laying hens, aflatoxin B1 the highly reactive aflatoxin B1-8,9- species to aflatoxicosis, are known to excretion is primarily in faeces and epoxide. In the liver, cytochrome be deficient in the GST that mediates is maximal 24 hours after a single P450 enzymes are responsible for detoxification of the aflatoxin B1-8,9- oral dose, a finding similar to that activation of aflatoxins B1, M1, and epoxide (Klein et al., 2000), and that seen in laboratory animals dosed P1, all of which can form nucleic deficiency results in high levels of intravenously (Wong and Hsieh, acid adducts or undergo conjugation hepatic aflatoxin B1 epoxidation (Yip 1980). In laying hens, the body to glutathione, conversion to and Coulombe, 2006; Rawal et al., half-life of aflatoxin B1 was 67 hours dihydrodiols, or binding to serum 2009). after a single oral dose (Busby and proteins or other macromolecules.

Wogan, 1981a). Aflatoxin M1 is the main unconjugated 3.2 Fumonisins metabolite in the urine of cows, 3.1.6 Transmission pigs, and sheep. In rodents, and 3.2.1 Absorption presumably in farm animals,

Approximately 1–15% (IARC, 2002a) aflatoxin B1–nucleic acid adducts are In most animals, fumonisin B1 of the aflatoxin B1 dose is recovered also found in urine, and 80% of the absorption, distribution, and elim- as aflatoxin M1 in the milk of sheep, depurinating adducts are excreted ination is rapid. Fumonisins are goats, and cows (IARC, 1993a). within 48 hours after dosing. A close poorly absorbed, and although some The conversion rate is > 1% when correlation has been established evidence exists that fumonisins can the aflatoxin B1 dose is low (IARC, between levels of adducts in urine be partially metabolized in the gut, 2002a). Extensive evidence has and levels in the liver (IARC, 1993a), metabolism by the liver or other been found for the lactational transfer and correlations have also been tissues has not been convincingly of aflatoxin M1 and its accumulation observed between dietary intake and demonstrated (WHO, 2000, 2001, in the livers and lungs of offspring levels of adducts in urine and serum 2012; IARC, 2002b; Shephard et al., (IARC, 1993a). Low levels of aflatoxin (IARC, 2002a). 2007; Voss et al., 2007). metabolites can be detected in The relative sensitivity of animals the liver and other tissues several to the toxic effects of aflatoxin B1 3.2.2 Gastrointestinal CHAPTER 5 CHAPTER weeks after animals are exposed is closely linked to differences metabolism to high levels of aflatoxin B1 (Busby in metabolism among species and Wogan, 1981a; Coulombe (IARC, 2002a). The susceptibility of Microbial metabolism most likely and Sharma, 1985). However, the animals to aflatoxin B1 toxicity and occurs in the gut of monogastric levels retained in edible tissues are carcinogenicity depends to a large animals, because partially hydrolysed generally low; feed-to-tissue ratios extent on the type of metabolites fumonisin B1 (lacking one tricarballylic range from 800:1 to 14 000:1 (IARC, produced and the rate of formation acid side chain) and, to a lesser

2002a). Aflatoxin B1 can be detected and detoxification of the aflatoxin B1- extent, fully hydrolysed fumonisin in eggs of laying hens fed diets 8,9-epoxide. Risk factors contributing B1 (lacking both side chains) were containing aflatoxin B1, with a feed- to an individual’s sensitivity to liver recovered in faeces but not in bile of to-tissue ratio ranging from 2200:1 tumours and hepatotoxicity include vervet monkeys (WHO, 2000). Most to 5000:1 (Oliveira et al., 2000; level of exposure to aflatoxin B1; (60–90%) of the total fumonisin B1 IARC, 2002a). With the exception of expression of enzymes in the found in ruminant faeces was present milk, transmission to edible animal aflatoxin activation and detoxification as the partially hydrolysed form. In products should pose little health risk pathways; nutritional status; co- non-ruminants, the parent compound to consumers. exposure to other mycotoxins, es- was the dominant species present pecially fumonisin; and exposure to (WHO, 2000). However, studies 3.1.7 Metabolism infectious agents (see Chapter 6). in pigs have reported significant Evidence suggests that the critical amounts of fully hydrolysed and

The metabolism of aflatoxin B1 has factor determining species sensitivity partially hydrolysed fumonisin B1 been extensively reviewed (IARC, is the rate at which the aflatoxin B1- in faeces and tissues (Fodor et al., 1993a, 2002a; see also Chapter 6). 8,9-epoxide can be conjugated 2008). Whether the hydrolysed

Briefly, in the liver and other tissues, to glutathione by glutathione fumonisin B1 was produced in the aflatoxin B1 is metabolized in S-transferase (GST) (Eaton et tissues was not determined.

Chapter 5. Effects in food-producing animals 65 3.2.3 Bioavailability been detected in the brains of pigs Several studies have confirmed this (Prelusky et al., 1996a), but little or finding using the persistence of free In all animals studied, including pigs, no fumonisin has been detected in sphinganine as a biomarker in the laying hens, turkey poults, ducks, and the brain tissue of horses, although kidney and liver to show that although dairy cows, the fumonisin absorption the brain is a known target organ fumonisin B1 is rapidly eliminated, that does occur is rapid and the quantity (Haschek et al., 2002). Until recently the biomarker remains elevated for of fumonisin B1 detected in plasma it was believed that fumonisins a much longer period. Although the and tissues after oral administration is could not cross the placenta and half-life after oral dosing is not known, very low (negligible to < 4% of dose) enter the developing embryo (WHO, the oral half-life is probably between (WHO, 2000, 2001, 2012; IARC, 2001). However, recent studies have 8 hours and 48 hours based on what 14 2002b; Fodor et al., 2008; Tardieu et detected [ C]fumonisin B1 in embryos is known from the parenteral routes, al., 2008, 2009). The bioavailability of and placentas, an observation the time required to reach peak levels fumonisin B2 may be less than that of confirmed by the presence of in plasma (1–7 hours) after gavage, fumonisin B1. Feeding studies have elevated levels of free sphinganine, and the estimated time for complete shown that, in the liver and kidney, a biomarker for fumonisin inhibition clearance from the liver and kid- fumonisin B1 is accumulated to a of ceramide synthase (Gelineau-van ney (2 weeks) (WHO, 2000). much greater extent than expected Waes et al., 2005). based on the relative amounts in feed 3.2.6 Transmission of fumonisin B1, fumonisin B2 (Riley 3.2.5 Excretion and Voss, 2006; Fodor et al., 2008; Little evidence exists to suggest Gazzotti et al., 2010), and fumonisin After intraperitoneal or intravenous significant transfer of fumonisins

B3 (Riley and Voss, 2006). Although dosing of fumonisin B1, initial elim- through milk (WHO, 2000). No diets containing predominantly ination from tissues is rapid, with no fumonisin B1 was detected in the milk fumonisin B2 from culture material evidence of metabolism (Shephard of lactating sows fed diets containing induced liver toxicity in both rats and et al., 2007; Voss et al., 2007), but nonlethal levels of fumonisin B1, and horses (Riley et al., 1997; Voss et extensive enterohepatic circulation no evidence was found of toxicosis al., 1998), pure fumonisin B2 did not occurs (Prelusky et al., 1996a). After in their suckling pigs. In a study with induce liver toxicity in mice in one oral dosing, peak plasma levels lactating cows administered fumonisin feeding study (Howard et al., 2002). occur within 1 hour to several hours. B1 intravenously, the carry-over of

In a cultured intestinal epithelial cell Several studies using different routes fumonisin B1 into the milk was either model, hydrolysed fumonisin B1, of exposure and different animal very small or not detected. The fact but not fumonisin B1, was found to species have shown that fumonisins that very little fumonisin B1 is retained cross the monolayer (primarily from are excreted primarily in faeces, in any tissue, milk, or eggs has led basolateral to apical), suggesting a either unchanged or with loss of one to the conclusion that fumonisin carrier-mediated efflux process (De or both of the tricarballylic acid side residues in food products derived

Angelis et al., 2005). chains. Low levels of fumonisin B1 can from animals are insufficient to render be detected in the urine of animals them injurious to consumers (WHO, 3.2.4 Distribution exposed experimentally to fumonisin, 2000). However, in a study in weaned including rabbits (Orsi et al., 2009), piglets fed diets containing fumonisin

Although fumonisins are distributed rats (Cai et al., 2007), pigs (Fodor et B1 at 10 mg/kg or 30 mg/kg diet, the to most tissues, the liver and kidney al., 2008; Dilkin et al., 2010), horses livers contained 306 μg/kg or 830 μg/ retain the highest concentrations of (Tumbleson et al., 2003), and vervet kg of fumonisin B1, respectively, after the absorbed material in all animals monkeys (Shephard et al., 2007). In 28 days (Dilkin et al., 2003). At the studied (reviewed in Voss et al., pigs, < 1% of the oral dose is recovered higher level of contamination, a 70 kg

2007). Fumonisin B1 persists in the in urine (Prelusky et al., 1996a; Dilkin person would need to consume kidney much longer than in plasma or et al., 2010). It has been estimated that 170 g of liver to exceed the Joint the liver, and in male Sprague Dawley pigs exposed to dietary fumonisin B1 WHO/FAO Expert Committee on (Riley and Voss, 2006) and Wistar at 2–3 mg/kg body weight (bw) in feed Food Additives (JECFA) provisional rats (Martinez-Larranaga et al., would require a withdrawal period of maximum tolerable daily intake

1999), the levels of fumonisin B1 in the at least 2 weeks for the fumonisin B1 (PMTDI) of 2 μg/kg bw/day. Pigs fed kidney can be 10 times the amount in to be eliminated from the liver and diets containing low levels (1.66 mg/ the liver. Radiolabeled fumonisin has kidney (Prelusky et al., 1996a, 1996b). animal/day) of fumonisins for 7 weeks

66 had detectable, but much lower, acylation of the free primary amino the intestinal microbial population can levels of fumonisins (15–43 μg/kg) in group prevents toxicity and reduces also reduce the hydrolysis of OTA to the liver (Gazzotti et al., 2010). Levels the ability of the fumonisin to inhibit its non-toxic metabolite, ochratoxin of fumonisin detected in the muscle ceramide synthase (Norred et al., α, leading to increased blood of pigs or poultry orally dosed with 1997, 2001). The two tricarballylic concentrations of OTA (WHO, 2001). fumonisin were either very low or acid side chains are also important for In pigs, ochratoxin B is metabolized below the detection limits (Prelusky toxicity, as demonstrated by reduced much more efficiently to ochratoxin et al., 1996a; Tardieu et al., 2008). or no toxicity, or evidence for disruption β than OTA is to ochratoxin α. The of sphingolipid metabolism, when rate of disappearance of OTA from 3.2.7 Metabolism fumonisin is hydrolysed (Howard et rumen fluids and its gastrointestinal al., 2002; Collins et al., 2006; Voss et metabolism depend on diet (Müller Fumonisins do not appear to be al., 2009; Grenier et al., 2012). et al., 2001) and are affected by the metabolized in vitro or in vivo by animal amount of feed concentrates (feed tissues (WHO, 2001), even though 3.3 Ochratoxin A additives with a high nutrient density) they are clearly excreted in bile, and added to the diet (Höhler et al., 1999). hydrolysed and partially hydrolysed 3.3.1 Absorption fumonisin B1 has been reported in 3.3.3 Bioavailability tissues (Fodor et al., 2008). The source Comprehensive reviews of the of hydrolysed fumonisins in tissues pharmacokinetics and metabolism OTA is well absorbed from the is unknown. However, formation of OTA are available (Marquardt and gastrointestinal tract, presumably of hydrolysed fumonisin during Frohlich, 1992; WHO, 2001; Dietrich from the small intestines, and it is also alkaline processing (nixtamalization) et al., 2005; Pfohl-Leszkowicz and well absorbed from the lungs, with and by microbial metabolism in Manderville, 2007). OTA is rapidly a calculated bioavailability of 98% the gastrointestinal tract has been absorbed. The half-life in plasma (WHO, 2001). Bioavailability from the demonstrated (see Section 3.2.2). One depends on the extent of binding oral route is reported as ranging from study suggested that a cytochrome to plasma proteins. OTA is widely 40% to 70% in chickens, rabbits, and P450 isoform is capable of producing distributed; in pigs, it is accumulated pigs. Absorbed OTA is rapidly and a fumonisin B1 metabolite (Marvasi et in the kidney and other tissues and tightly bound to serum proteins and CHAPTER 5 CHAPTER al., 2006), but no convincing evidence can occur in edible tissues. OTA in that form is only slowly transferred has been reported of in vivo or in vitro and its metabolites are reabsorbed from the bloodstream to the liver and metabolism by cytochrome P450, the by the kidney and excreted in urine kidney. The high affinity of OTA for microsomal esterase, or any other and also undergo enterohepatic serum albumin results in a higher microsomal enzyme (WHO, 2001, circulation and excretion in faeces. concentration in plasma than in the 2012). However, studies have shown The potential exists for extensive gut (Kumagai, 1988). The maximal that cytochrome P450 activity can metabolism by microbes to less toxic concentration in serum after a single be altered as a result of the inhibition metabolites in the gastrointestinal oral dose is attained in < 1 hour in of the enzyme ceramide synthase tract. Ochratoxins A and B can also chickens and in 10–48 hours in pigs by fumonisin (WHO, 2001). Both be metabolized by cytochrome P450 (WHO. 2001). The plasma half-life of B fumonisins and their hydrolysed enzymes in various tissues. OTA is quite variable, ranging from a counterparts can react with ceramide few hours in chickens to 5 days in pigs. synthase; hydrolysed fumonisin B1 3.3.2 Gastrointestinal is a substrate for this enzyme, produc- metabolism 3.3.4 Distribution ing the compound N-palmitoyl-AP1 (Seiferlein et al., 2007). Studies using In cows and sheep, OTA is degraded In pigs, chickens, and goats, the hydrolysed fumonisin B1 have shown by the rumen flora and protozoa (WHO, relative tissue distribution of OTA is that it is much less toxic than the parent 2001). However, some portion is not usually kidney > liver or muscle > fat compound (Howard et al., 2002; metabolized and can accumulate in (WHO, 2001; Biró et al., 2002; Collins et al., 2006; Voss et al., 2009; serum, tissues, and milk (IARC, 1993b; Dietrich et al., 2005). In rabbits, the Grenier et al., 2012). Höhler et al., 1999). The enzymes relative distribution is kidney > liver Even though little evidence responsible for the metabolism > mammary gland > muscle (Ferrufino- has been found for metabolism of are carboxypeptidase A and Guardia et al., 2000). OTA has been B fumonisins in tissues, chemical chymotrypsin. Antibiotics that inhibit shown to cross the blood–brain

Chapter 5. Effects in food-producing animals 67 barrier in rats and the placenta in 3.3.7 Metabolism 3.4.2 Gastrointestinal rats and pigs (WHO, 2001). The rate metabolism of disappearance of OTA from the In addition to gastrointestinal degra- blood is much slower than that from dation by microbes, microsomal A great deal of information has the kidney or liver and other tissues, preparations from rabbits and pigs been published about the ability of indicating the importance of binding containing various cytochrome P450 microorganisms in the gastrointestinal to serum proteins and enterohepatic enzymes can oxidize OTA to less tract to metabolize DON. In some recirculation in the overall fate of OTA toxic hydroxyochratoxin A (WHO, studies, incubation with cultures or once it has been absorbed. In cells, 2001). At least 20 OTA derivatives extracts from the gastrointestinal tract OTA is accumulated via a multispecific have been identified after incubation has resulted in extensive conversion organic anion transporter (O’Brien and with liver microsomes or cultured cells, of DON to the de-epoxy metabolite, Dietrich, 2005). The accumulation and and hydroxylated metabolites have DOM-1 (WHO, 2001). Near-complete persistence of OTA in the kidney is been detected in pig kidney (Pfohl- de-epoxidation has been shown due to its reabsorption by the organic Leszkowicz and Manderville, 2007). using intestinal contents from pigs and anion transporter (Zingerle et al., Metabolism by cyclo-oxygenases and chickens and bovine rumen fluid. Pigs 1997; Welborn et al., 1998). The ability other oxidative enzymes can produce lacking the ability to carry out intestinal of the kidney to accumulate OTA plays reactive oxygen species, leading to de-epoxidation can acquire the ability an important role in its nephrotoxicity. oxidative damage to tissues. OTA through contact with faeces from In pigs fed OTA for 1 month, the half- has been suggested to induce DNA pigs capable of making the de-epoxy life of residues in the kidney, liver, and adducts (see Pfohl-Leszkowicz and metabolite (Eriksen et al., 2003). It has muscle was 3.5–4.5 days (Busby and Manderville, 2007, and Chapter 6). also been shown that de-acetylation Wogan, 1981b). of 3-acetylDON to DON occurs in the 3.4 Deoxynivalenol pig gastrointestinal tract before DON is 3.3.5 Excretion absorbed. Whereas the de-epoxides of 3.4.1 Absorption DON and NIV are less cytotoxic than Elimination of OTA is primarily via the parent compounds, de-epoxidation, the urine, but significant amounts Depending on geographical location, unlike de-acetylation, appears to occur can also be excreted in faeces. the predominant trichothecenes may primarily in the distal portion of the OTA is eliminated slowly because be DON, nivalenol (NIV), or one of digestive tract, where DON absorption it undergoes enterohepatic recir- their acetylated precursors. However, is low (Eriksen et al., 2003). culation, is reabsorbed by the kidney, DON is the focus of this section. For and binds tightly to serum protein more information specific to NIV and 3.4.3 Bioavailability (WHO, 2001). The extent of albumin acetylated derivatives of DON and binding also markedly decreases the NIV, see Pestka (2010a) and Sugita- DON is rapidly absorbed from the uptake of OTA by transporters (Bow Konishi and Nakajima (2010). gastrointestinal tract in sheep, cows, et al., 2006). The elimination half-life Absorption, distribution, and elim- and pigs (WHO, 2001). In sheep, is much shorter for ochratoxins B and ination of DON are rapid after either the bioavailability is low (< 10%). C than for OTA and its metabolites oral or parenteral dosing. No evidence Bioavailability in cows also appears to (Li et al., 1997). has been found for DON accumulation be low, whereas one study estimated in tissues or transmission to eggs that the systemic bioavailability in pigs 3.3.6 Transmission or milk at the DON levels normally was 55%. After a single intragastric encountered in animal feed (Prelusky dose in pigs, the peak plasma level OTA has been shown to be transferred et al., 1994). The potential for extensive occurred at 15–30 minutes and the efficiently to milk in rodents and metabolism in the gastrointestinal plasma half-life was 7.1 hours. The rabbits (WHO, 2001). At high levels tract via de-epoxidation reactions plasma half-life after intravenous of exposure, OTA can accumulate in results in the formation of de-epoxy dosing was 3.9 hours in pigs and eggs of chickens and quail (WHO, DON (DOM-1) (reviewed in WHO, 100–125 minutes in sheep. When 2001). Detectable levels have been 2001; Pestka, 2010a, 2010b). pigs were fed diets containing reported in pig kidney and liver, blood 3-acetylDON for 3 days, DON was products, and other meat products detected in plasma 20 minutes after for human consumption (WHO, 2001; the first feeding (Eriksen et al., 2003). CAST, 2003). The maximal plasma level occurred

68 2.8 hours after feeding, suggesting 3.4.6 Transmission 3.5.2 Gastrointestinal that absorption started in the stomach metabolism or the upper part of the duodenum. DON was not detected in milk from cows fed a diet containing maize naturally ZEA can be degraded in the rumen. 3.4.4 Distribution contaminated with DON at up to 12 mg/ In both sheep and cattle, rumen kg dry diet for 10 weeks (WHO, 2001). fluid reduces ZEA to its more The little information available on the Low levels of DON (< 1.7 mg/egg) were easily excreted metabolites, α- and distribution of DON in tissues suggests detected in eggs from chickens fed β-zearalenol (Raisbeck et al., 1991). that this compound is rapidly and widely diets containing DON at 5.5 mg/kg distributed but is not accumulated. In diet for 65 days. Other studies 3.5.3 Bioavailability chickens, 3 hours after a single oral also indicate that transmission to dose of radiolabelled DON, the relative milk is small in sheep and cows. ZEA is poorly absorbed from the gut accumulation was bile >> kidney > blood/ Transmission of DON to edible animal of chickens (Christensen, 1979). For plasma > liver >> other tissues (WHO, products should pose little health risk example, in chickens, 12 hours after 2001). At later time points (72 and to consumers. oral dosing, 0.08% of the dose was 96 hours), very little or no DON was recovered in tissues and organs, detected. In pigs, 3 hours after a 3.4.7 Metabolism whereas 99% was recovered in single intravenous dose, DON was excreta, bile, and the digestive distributed to all tissues examined and No evidence has been found that tract plus its contents (Christensen, the relative accumulation was kidney DON is metabolized by microsomal 1979). Peak plasma concentrations > plasma > liver > fat >> other tissues enzymes. However, evidence for occurred 2–6 hours after a single (WHO, 2001). At 24 hours after dosing, glucuronide conjugation in sheep oral dose in broilers (Bernhoft et the levels in all tissues were reduced and pigs is well documented (WHO, al., 2001). Oral bioavailability may by > 90% relative to those at 3 hours 2001; Eriksen et al., 2003). In some be much greater in other species; after dosing. studies with sheep, the glucuronide for example, in rats the oral conjugate of DON can account for a bioavailability was reported as 2.7% 3.4.5 Excretion large percentage of the total plasma of the dose (Shin et al., 2009). In pigs DON. In pigs fed diets containing orally dosed with ZEA, the half-life CHAPTER 5 CHAPTER DON is rapidly eliminated in urine and 3-acetylDON, 42% and 33% of in plasma was 87 hours (Biehl et al., faeces in chickens, sheep, and pigs the DON in plasma and urine, 1993) due to extensive enterohepatic (WHO, 2001). In chickens, 79%, 92%, respectively, was conjugated to the recirculation. and 98% of an oral dose could be glucuronide (Eriksen et al., 2003). In accounted for in excreta at 24, 48, and that study, a significant portion of the 3.5.4 Distribution 72 hours after dosing, respectively. In DON in faeces was the de-epoxide. sheep, 36 hours after a single oral dose, In rats orally dosed with ZEA, only 6.9%, 0.11%, and 64% of the dose was 3.5 Zearalenone very low levels were detected in recovered in urine, bile, and faeces, tissues (Christensen, 1979). The respectively. In ewes, 91% and 6% of a 3.5.1 Absorption liver contained the highest levels single intravenous dose was recovered in of ZEA of the tissues examined urine and bile, respectively, after 24 hours, ZEA is rapidly absorbed and elim- (Christensen, 1979; Bernhoft et and a similar result was seen in pigs. inated. It is metabolized in the liver al., 2001). ZEA and its metabolite Pigs consuming wheat naturally (and possibly the intestinal mucosa) α-zearalenol can cross the placenta contaminated with DON excreted 50– and excreted in urine and faeces as and enter the fetus (Bernhoft et al., 62% of the DON in their urine (Goyarts the glucuronide after considerable 2001). and Dänicke, 2006). In a feeding study enterohepatic recirculation. It is with 3-acetylDON, 45% and 2% of metabolized by rumen microbes. 3.5.5 Excretion the dose was recovered in urine and Accumulation in tissues is minimal. faeces, respectively, 48 hours after the Comprehensive reviews of the The half-life of ZEA in pigs dosed pigs were taken off the contaminated toxicokinetics of ZEA in animals either intravenously or orally was diets, and the remainder of the dose are available (Fink-Gremmels and 87 hours (Biehl et al., 1993). When was unaccounted for (Eriksen et al., Malekinejad, 2007; Zinedine et al., bile was removed, the half-life 2003). 2007). was reduced to 3 hours, indicating

Chapter 5. Effects in food-producing animals 69 that enterohepatic cycling of ZEA the substrates for these enzymes fluids are quickly degraded by rumen (principally as the glucuronide) is include natural steroid hormones microorganisms to metabolites such extensive in pigs. Excretion in bile is (reviewed in Fink-Gremmels and as lysergic acid (Moyer et al., 1993; rapid in broilers; peak concentrations Malekinejad, 2007). The intestinal Hill, 2005; Ayer et al., 2009). In cattle, occur 2–6 hours after a single mucosa may also actively reduce the main ergot alkaloid found in urine oral dose (Dänicke et al., 2001). In ZEA to α-zearalenol and mediate is lysergic acid (Stuedemann et al., chickens orally dosed with ZEA, conjugation to glucuronic acid (Biehl 1998). Gastrointestinal metabolism 75% of the administered dose was et al., 1993). The rapid conversion of in monogastrics is probably limited recovered in excreta after 24 hours ZEA to the more easily excreted α- (Hill, 2005). (Christensen, 1979). Zearalenol and β-zearalenol derivatives in cattle, can be detected as the glucuronide along with microbial metabolism in 3.6.3 Bioavailability in urine and faeces of pigs and in the rumen, could explain the relative excreta of chickens (Christensen, resistance of cattle to the reproductive Gastrointestinal absorption of ergot 1979; CAST, 2003). α-Zearalenol effects of ZEA, compared with pigs alkaloids is low (< 5%), and clearance is glucuronide appears to be the major (Raisbeck et al., 1991). rapid (Haschek et al., 2002). However, metabolite detected in pig urine and physiological effects can be persistent, serum after prolonged exposure to 3.6 Ergot alkaloids suggesting either that metabolites are ZEA (Dänicke et al., 2005). tightly bound or that an undiscovered 3.6.1 Absorption reservoir exists in the body. The 3.5.6 Transmission bioavailability of ergot alkaloids appears The absorption and metabolism of to be a function of solubility. The ZEA is poorly transferred to milk as ergot alkaloids may be quite different ergopeptine alkaloids are less soluble zearalenol derivatives (< 1% of dose) in monogastrics and ruminants. than the ergoline alkaloids and when and may induce signs of estrogenism Water-soluble alkaloids appear to administered orally must be dissolved in female piglets (Osweiler, 2000). be more strongly absorbed than in lipophilic carriers or chemically In the liver, α-zearalenol is detected lipophilic ones, and rumen microbes modified to improve solubility (Hill, more frequently than β-zearalenol. appear to play an important role in 2005). In sheep rumen and omasal Trace amounts of ZEA and its the release and intestinal availability tissue, transport of alkaloids appears metabolites can be detected in muscle of soluble alkaloids in the rumen to be an active process, and lysergic tissues of pigs fed oats contaminated (Hill, 2005; Ayer et al., 2009). The acid and lysergol are transported much with ZEA; however, residues do not more soluble alkaloids, such as more effectively than are ergopeptine persist (Zöllner et al., 2002). lysergic acid, are excreted in urine, alkaloids (Hill et al., 2001). and the less soluble alkaloids can 3.5.7 Metabolism undergo enterohepatic recirculation 3.6.4 Distribution and are excreted primarily in bile. After considerable enterohepatic re- Transmission is unlikely, and accu- Ergot alkaloids are widely distributed, circulation, ZEA is metabolized in mulation in tissues, if any, is low. as evidenced by the fact that most of the liver and excreted in urine and Once absorbed, ergot alkaloids such the physiological effects involve direct faeces as the parent compound, as ergotamine can be metabolized interaction with dopamine receptors the metabolites α-zearalenol by cytochrome P450 enzymes. in the peripheral vasculature and, in and/or β-zearalanol, or their some cases, in the brain and other respective glucuronide conjugates 3.6.2 Gastrointestinal neuronal tissue (see Section 4.6). (reviewed in Fink-Gremmels and metabolism Malekinejad, 2007; Zinedine et al., 3.6.5 Excretion 2007). The enzymes responsible Studies in sheep and cattle suggest for the conversion of ZEA to α- that rumen microorganisms can Once ergot alkaloids are absorbed, and β-zearalenol are 3-α- and degrade plant material and release clearance is very rapid. For ex- 3-β-hydroxysteroid dehydrogenase, the more soluble ergoline alkaloids ample, intravenously administered respectively. These two microsomal (Hill, 2005; Ayer et al., 2009). The ergovaline was shown to have a enzymes are important in steroid small amounts of less soluble plasma half-life of 24 minutes in metabolism, so ZEA has the potential ergopeptine alkaloids (ergonovine sheep (Jaussaud et al., 1998) and a to disrupt steroid metabolism because and ergovaline) present in rumen half-life of 56 minutes in horses (Bony

70 et al., 2001). Ergopeptine alkaloids cannot model the uncontrolled reduced weight gain and decreased are excreted in bile and ergoline exposure of farm animals to naturally feed efficiency. The effects on alkaloids in urine (Hill et al., 2001). In contaminated feeds and foods in growth are dose-dependent and at cattle grazing on tall fescue infected the field, where multiple factors low levels may be barely discernible. by endophytes, the urine contained contribute to the expression of Other performance effects include approximately 96% of the total ergot disease. Thus, the potential for tox- decreased reproductive performance, alkaloid excreted, and after 2 days of icity revealed in controlled experi- abortion, and reduced egg or milk grazing on grass free of endophytes, ments is often not predictive of the production. In turkeys, a sensitive the urinary alkaloids were reduced levels of exposure in feeds associated species, reduced weight gain is seen to control levels (Stuedemann et al., with suspected field outbreaks of at a dose of 125 µg/kg diet, impaired 1998). The maximal levels of ergot disease. One explanation for the immune response and increased alkaloids in urine were attained within difficulty in equating dose–response mortality at 250 µg/kg, and acute 48 hours, and the main alkaloid was in laboratory studies with dose– mortality at 500 µg/kg (Norred, lysergic acid. response in the field is the inability 1986). A similar relative dose– to identify all the environmental, response occurs in pigs but at higher 3.6.6 Transmission nutritional, and genetic factors that levels of exposure because they are contribute to disease expression. less affected by aflatoxins. In cattle There are no reports that ergot Nevertheless, information gained and chickens, much higher levels alkaloids are transferred to milk or from in vitro studies and studies are required to induce a decrease accumulate in tissues. with laboratory animals is predictive in performance, and in chickens of the possible contribution of impaired immune response can 3.6.7 Metabolism mycotoxins in altering immune occur at levels that have no effect function (Bondy and Pestka, 2000), on the growth rate. In all species, Ergopeptine alkaloids are metab- thereby contributing to unexplained aflatoxins are hepatotoxic, with fatty olized in the liver via cytochrome animal diseases and performance changes, hepatocyte degeneration, P450 enzymes such as CYP3A, and problems in farm animals (Osweiler, necrosis, and altered liver function. hydroxylated metabolites are excreted 2000). Several excellent reviews A common clinical sign is jaundice. in bile (Moubarak and Rosenkrans, have documented the toxicology Grossly, the liver appears pale and CHAPTER 5 CHAPTER 2000; Haschek et al., 2002). Ergoline of mycotoxins in farm animals and swollen or fatty with variable texture. alkaloids (i.e. lysergic acid), which provided extensive descriptions of In chickens, fatty liver syndrome is are more water soluble, are excreted the clinical manifestations (Oltjen, believed to be caused by aflatoxin in urine (Hill, 2005; Ayer et al., 2009) 1979; Richard and Thurston, 1986; (Norred, 1986), although OTA can and faeces in amounts greater than Raisbeck et al., 1991; WHO, 2001; also cause fatty liver in poultry that consumed, suggesting that Haschek et al., 2002; CAST, 2003; (Trenholm et al., 1988). Liver damage ergovaline and possibly other ergot Cousin et al., 2005; O’Brien and ultimately can lead to coagulopathy, alkaloids are metabolized in tissues Dietrich, 2005; Fink-Gremmels and as evidenced by haemorrhaging and to lysergic acid (Schultz et al., 2006). Malekinejad, 2007; Pestka, 2007, anaemia (Fig. 5.1). In poultry and 2010a, 2010b; Pfohl-Leszkowicz and pigs, coagulopathy contributes to 4. Toxicological effects Manderville, 2007; Voss et al., 2007; the appearance of internal bruising Zinedine et al., 2007; Steyn et al., during handling. In pigs, this may Only a few known mycotoxins pose 2009). In this section, we describe occur at levels as low as 150 µg/kg a measurable health risk to farm the main clinical signs in farm animals diet (Edds, 1979). animals, for several reasons. First, exposed to the levels of mycotoxins The mechanism of action of a fundamental tenet of toxicology encountered in field outbreaks. We aflatoxins involves metabolism to re- is “the dose makes the poison”. also present postulated responses to active intermediates and their binding Thus, even though farm animals are low levels of mycotoxins. to macromolecules (nucleic acids and exposed to mycotoxins every day proteins) and consequent disruption through their feed, the dose is usually 4.1 Aflatoxins of transcriptional and translational insufficient to make the contaminated processes and regulatory pathways feed acutely poisonous. Second, the The overt symptoms of aflatoxin critical for repair of damaged DNA, for doses and routes of exposure used poisoning are not definitive. Animals cell growth, death, and differentiation, in controlled laboratory experiments do not eat well and therefore have and ultimately for toxicity and

Chapter 5. Effects in food-producing animals 71 carcinogenicity (Wild and Gong, head pressing, and decreased feed In equids, the minimum toxic level

2010; Eaton et al., 2010; Kensler et intake, followed by convulsions of fumonisin B1 in feed for inducing al., 2011). Some evidence has been and death after several days. Early ELEM appears to be between 15 mg/ found that aflatoxin B1 produces clinical signs include hindlimb ataxia, kg and 22 mg/kg diet (WHO, 2000, reactive oxygen species, resulting in delayed forelimb placing reactions, 2001). Analysis of feeds from oxidation of DNA bases (Guindon et and tongue paresis (Fig. 5.2), which confirmed cases of ELEM indicated al., 2007). are all mild signs of proprioceptive that a diet containing a fumonisin

The response of an animal to dysfunction (Foreman et al., 2004). B1 concentration of > 10 mg/kg diet aflatoxin depends to a large extent Elevation in levels of serum enzymes presented an increased risk of devel- on the rate of metabolism and the indicative of liver damage occurs oping ELEM (WHO, 2000, 2001). type of metabolites that are produced soon after elevation in levels of free Like ELEM, PPE syndrome is a (see Section 3.1.7 and Chapter 6). sphingoid bases and sphinganine-1- rapid-onset disease that is often fatal For example, quail and turkeys are phosphate in serum. The elevation in to affected animals. Clinical signs sensitive to aflatoxin toxicity and have sphinganine levels is also seen in the typically occur 2–7 days after pigs a high rate of epoxide formation and liver and kidney (Riley et al., 1997) start consuming diets containing large a low rate of glutathione conjugation. and other tissues (Tumbleson et amounts of fumonisins. Clinical signs In resistant species, even if the rate al., 2003). Sphinganine-1-phosphate include decreased feed consumption, of epoxidation is high, a high rate of is also greatly elevated in serum of dyspnoea, weakness, cyanosis, and glutathione conjugation is protective. horses treated with pure fumonisin B1 death. When animals are examined However, the resulting clinical (Constable et al., 2005). The elevated at necropsy, varying amounts of signs are similar although the dose serum and tissue levels of free clear yellow fluid are seen in the dependence may be quite different. sphingoid bases and sphinganine- pleural cavity together with varying Aflatoxin adducts in urine and blood 1-phosphate are biomarkers for degrees of interstitial and interlobular of farm animals may be very useful exposure to potentially toxic levels oedema, with pulmonary oedema as a biomarker for exposure during of fumonisins (Riley et al., 2011) and hydrothorax (Fig. 5.3). Toxic suspected field outbreaks (Riley et and have been used in studies in hepatosis usually occurs concurrently al., 2011). horses, pigs, rabbits, poultry, and with pulmonary oedema, and in some other farm animals (WHO, 2000). In animals that consume high levels of 4.2 Fumonisins horses, serum enzyme levels often fumonisins, toxic hepatosis appears return to near-normal concentrations without signs of pulmonary oedema. Consumption of feeds contaminated but usually increase markedly im- Nodular hyperplasia has been with fumonisins is a proven cause mediately before, or at the first signs observed in some pig livers (WHO, of two farm animal diseases and a of, behavioural changes indicative 2000, 2001). suspected cause of others. ELEM of the onset of ELEM (WHO, 2000, As with ELEM, fumonisin con- is a fatal neurotoxic disease that 2001). centration in maize screenings occurs only in equids (horses and In addition to the brain lesions, obtained from different farms was related species). The disease is histopathological abnormalities in the closely correlated with outbreaks of characterized by the presence of liver and kidney have been reported PPE in the USA in 1989–1990. The liquefactive necrotic lesions in the in horses, and these are also minimum dose necessary to induce white matter of the brain; the grey correlated with elevation in levels this disease has not been clearly matter may also be involved (WHO, of free sphinganine (Tumbleson et established, but in diets containing

2000). All aspects of the disease can al., 2003). ELEM concurrent with fumonisin B1 from culture material, be reproduced experimentally. The significant liver disease and fatal concentrations as low as 17 mg/ brain lesions are caused by vasogenic liver disease in the absence of any kg diet induced pulmonary oedema cerebral oedema (Haschek et al., brain lesions have been observed in in 5 days, whereas concentrations 2002; Foreman et al., 2004) and horses and ponies. The appearance of 150–170 mg/kg diet for up to are accompanied by increased of the clinical disease is likely to 210 days caused liver effects early protein in the cerebrospinal fluid depend on multiple factors, including on but no evidence of pulmonary and other changes consistent with the length of exposure, level of oedema (WHO, 2000). Pigs fed a vasogenic cerebral oedema (Smith contamination, individual animal dif- diet containing fumonisin B1 at 45 et al., 2002; Foreman et al., 2004). ferences, previous exposure, and mg/kg diet for 10 days developed Early symptoms include lethargy, pre-existing liver impairment. mild signs of pulmonary oedema,

72 including the accumulation of fluid in Fig. 5.1. (a) Liver from a chick fed a diet containing 7.5 mg/kg of aflatoxin B1; (b) a the pleural cavity, which persisted in normal liver. Similar results are seen in turkey poults fed the same diets. Photograph courtesy of Timothy Phillips, Texas A&M University. several animals 10 days after removal from the contaminated diets (Fodor et al., 2008). Pigs given a single oral dose of 5 mg/kg bw of fumonisin B1 (equivalent to 83 mg/kg diet) did not develop pulmonary oedema but did show behavioural and clinical signs of toxicity suggestive of its onset (Dilkin et al., 2010). In pigs, tissues other than the liver and lung that have been reported to be targets for fumonisins include the pancreas, heart, kidney, spleen, pulmonary intravascular macro- phages, and oesophagus. Altered Fig. 5.2. (a) A horse in the initial stage of equine leukoencephalomalacia. Photograph growth and changes in selected courtesy of Walter F.O. Marasas, Programme on Mycotoxins and Experimental haematological parameters in pigs Carcinogenesis (PROMEC Unit), South African Medical Research Council. (b) A have been reported at dietary levels weanling Appaloosa filly administered fumonisin B1 intravenously at 0.20 mg/kg bw/ as low as 1 mg/kg diet (WHO, day for 7 days. Note the severe displacement of the tongue after manual exteriorization by the examiner. From day 4 through day 7, this filly developed progressive tongue 2000). In weaned pigs, growth, paresis, proprioceptive deficits, and ataxia in all four limbs, hind limb spasticity and attainment of sexual maturity, and paresis, and forelimb hypermetria. On the day of euthanasia, it kept its tongue out sperm production were impaired in of its mouth for periods of up to 1 minute at a time. Source: Foreman et al. (2004); animals consuming diets containing reproduced with the permission of the publisher. fumonisin B1 at > 5 mg/kg diet (Gbore, 2009). The physiological basis for performance problems induced in CHAPTER 5 CHAPTER pigs by fumonisin is unclear; however, several in vitro and in vivo studies have shown that fumonisin exposure can have deleterious effects on intestinal integrity and function, which can lead to altered intestinal immune responses and possibly other effects on intestinal physiology (Bouhet et al., 2004; Bouhet et al., 2006; del Rio Garcia et al., 2007; Loiseau et al., 2007; Devriendt et al., 2009; Lessard et al., 2009). Oral exposure to feed contaminated with fumonisins also has effects on other immune responses, Clinical features include diarrhoea, (75–644 mg/kg diet) because poultry including sex-specific decreased weight loss, increased liver weight, appeared to be unusually resistant antibody titres after vaccination and poor performance, and increased to dietary fumonisins (WHO, 2000, increased susceptibility to secondary susceptibility to infectious diseases. 2001). However, toxicity and altered pathogens (Halloy et al., 2005; Marin Several studies have confirmed that haematological parameters have et al., 2006). F. verticillioides, F. proliferatum, been observed in broiler chicks fed

Several published reports sug- fumonisin B1, and moniliformin are diets containing only 10 mg/kg of gest the involvement of Fusarium toxic to poultry (broiler chicks, turkeys, pure fumonisin B1 and 30 mg/kg of verticillioides in diseases of poultry, turkey poults, quail, and ducklings). fumonisin B1 from F. verticillioides and by implication the presence of The levels of fumonisin used in many culture material (WHO, 2000, fumonisin contamination in feed. of the early studies were quite high 2001). Like other poultry, ducks are

Chapter 5. Effects in food-producing animals 73 Fig. 5.3. (Left) A lung with severe oede- pathway (Wang et al., 1991). (Haschek et al., 2002). The elevation ma from a pig with porcine pulmonary Fumonisins are structurally similar of free sphingoid bases and sphingoid oedema after being fed fumonisin- to sphingoid bases and especially base 1-phosphates and the depletion contaminated feed and (right) a normal lung. Photograph courtesy of Wanda 1-deoxysphinganine, which lacks a of more complex sphingolipids in Haschek, University of Illinois. hydroxyl group on carbon 1 (Zitomer tissues, serum, and urine have proven et al., 2009). Sphingoid bases are to be useful biomarkers for exposure essential components of the chemical and the effects of fumonisin in farm backbone of all sphingolipids in animals (Riley et al., 2011). animals. When toxicity associated with fumonisins is observed in 4.3 Ochratoxin A laboratory or farm animals, the onset and severity of the pathology The primary effect of OTA in all farm is closely correlated with evidence animals is nephrotoxicity. In pigs of disrupted sphingolipid metabolism and poultry, the proximal tubules are (Riley et al., 1996, 2001; WHO, mainly affected and the kidney is 2000, 2001, 2012; Riley and Voss, pale and grossly enlarged (Fig. 5.4). 2006; Voss et al., 2011). Disrupted Fatty liver can occur in poultry. sphingolipid metabolism can also be The most sensitive indicator of evident at dosages that do not cause acute ochratoxicosis in chickens overt toxicity (NTP, 2001). This is is the reduction in total serum especially true in resistant species proteins and albumin. A decrease in such as ducks (Tardieu et al., phosphoenolpyruvate carboxykinase relatively resistant to the toxic effects 2006). The major biochemical and in the kidney is a sensitive and of fumonisins (Tran et al., 2005). cellular consequences resulting from specific indicator in pigs (Krogh, However, increased mortality and blockage of ceramide biosynthesis 1992; Marquardt and Frohlich, 1992). toxicity have been observed in ducks are the accumulation of free In pigs, large increases in levels force-fed diets containing 20 mg/kg sphingoid bases and sphingoid base of proteins excreted in urine are of fumonisin B1 (Tardieu et al., 2009). 1-phosphates (Riley and Voss, 2006; indicative of glomerular proteinuria As in pigs, fumonisin can also alter Zitomer et al., 2009), the depletion of and correlate with histological obser- immune response and susceptibility more complex sphingolipids (Voss et vations of renal damage. to infectious agents in poultry (Tessari al., 2009), and the global disruption of In poultry and pigs, exposure to et al., 2006; Deshmukh et al., 2007). lipid metabolism (WHO, 2001). The OTA at lower levels can result in altered Other farm animals that have changes in concentrations of impor- performance, including decreased been studied using pure fumonisins, tant lipid mediators lead ultimately feed consumption and reduced weight contaminated maize screenings, to perturbation of the signalling gain, and at higher levels can result in or maize culture material from F. pathways and altered regulatory and delayed response to immunization and verticillioides include carp, catfish, physiological processes (Lemmer increased susceptibility to infection lambs, goats, trout, cattle, mink, et al., 1999; Bondy et al., 2000; (Stoev et al., 2000a, 2000b). Other and rabbits. Rabbits are especially Merrill et al., 2001), which are the effects in poultry include decreased sensitive to nephrotoxicity (Voss basis for the observed clinical signs egg production, coagulopathy (in- et al., 2001; Ewuola, 2009). In all associated with the diseases induced creased susceptibility to bruising during cases where toxicity was evident, it by fumonisins. ELEM is associated processing), decreased bone strength, involved the liver and/or kidney or the with alterations in cardiovascular decreased tensile strength of the large equivalent organs in fish. function induced by sphingolipids, intestines, underpigmentation, and Disruption of lipid metabolism i.e. deregulation of cerebral arteries glycogen accumulation in the liver. appears to be the underlying responsible for autoregulation of blood The mechanism of action in mechanism by which fumonisins flow to the horse’s brain (Haschek et farm animals is unclear. However, cause toxicity to animals (WHO, al., 2002; Foreman et al., 2004). PPE is the structural similarity of OTA to 2000, 2001). The initial mechanism hypothesized to be a result of acute left- phenylalanine and the fact that it of action of fumonisin is inhibition of sided heart failure as a consequence inhibits many enzymes and processes ceramide synthase, a key enzyme in of inhibition of L-type calcium that are dependent on phenylalanine the de novo sphingolipid biosynthesis channels induced by sphingoid bases strongly suggest that OTA acts at least

74 Fig. 5.4. Examples of pig kidneys taken at slaughter, showing increasing degrees the mechanism appears to be indirect of mycotoxic nephropathy, with enlarged and mottled or enlarged and pale kidneys. (Miller, 2008; Pestka, 2010a). In pigs, Mycotoxic nephropathy in these animals was attributed to exposure to ochratoxin A feed refusal occurs even when DON and other mycotoxins (primarily, fumonisin and penicillic acid); however, mycotoxic nephropathy has also been reported in pigs exposed primarily to ochratoxin A (Elling is administered intraperitoneally, and Moller, 1973; Krogh, 1974). Source: Stoev et al. (2010); reproduced with the so feed refusal cannot be due permission of the publisher. to taste or learned responses (Prelusky, 1997). Clinical signs include gastrointestinal problems, soft stools, diarrhoea, increased susceptibility to other diseases, and decreased performance. In pigs, mild renal nephrosis, reduced thyroid size, gastric mucosal hyperplasia, increased albumin-to-α-globulin ratio, and sometimes mild changes in other haematological parameters have been reported (WHO, 2001). Numerous studies in laboratory animals have demonstrated alter- ations in immune function induced by DON (Bondy and Pestka, 2000; Pestka, 2010a), but conclusive evidence that DON induces altered resistance to infectious diseases in the field is still lacking (Osweiler, partially by disrupting phenylalanine 4.4 Deoxynivalenol 2000). Nevertheless, the mechanism metabolism (CAST, 2003; Riley of action in laboratory animals et al., 2011). Several studies have Many reviews on the toxicity of DON suggests, and controlled studies in CHAPTER 5 CHAPTER shown that supplementation of feed have been published (Beasley, 1989; the laboratory setting support, the with L-phenylalanine or proteins Prelusky et al., 1994; Rotter et al., 1996, potential for involvement by DON in protects against the toxic effects of WHO, 2001; Pestka, 2010a, 2010b). altered immune response. OTA, including mortality (Marquardt Although DON is not considered to The mechanism of action of DON and Frohlich, 1992; WHO, 2001). be acutely toxic to farm animals, it is complex. Disruption of ribosomal In addition to inhibition of protein is considered to be a major cause and endoplasmic reticulum function synthesis via binding to phenylalanine- of economic losses due to reduced as a consequence of DON binding tRNA synthetase, recent studies performance (Miller, 2008). In the field, to ribosomes is clearly a key event have demonstrated the ability of OTA concentrations as low as 1 mg/kg in disease causation in animals to induce oxidative stress, reduce have been associated with feed (Pestka, 2010a, 2010b). Binding of cellular defence, and alter signalling refusal in pigs; however, more typically DON to ribosomes is known to inhibit pathways involved in various aspects concentrations of > 2–5 mg/kg are translation by preventing polypeptide of cellular and mitotic regulation (Mally required for decreased feed intake and chain initiation or elongation in and Dekant, 2009). The ultimate reduced weight gain and concentra- animals (Ueno, 1984). However, consequence of generalized disruption tions of > 20 mg/kg for vomiting and several other ways exist in which DON of these metabolic and regulatory feed refusal (Trenholm et al., 1988; can interfere with protein synthesis, pathways is increased cell death, and Haschek et al., 2002; Fig. 5.5). all involving disrupted ribosomal the kidney is the most sensitive target Dogs and cats are also sensitive function (Pestka, 2010a). The because of its ability to accumulate to the emetic effects of DON, and downstream cellular consequences OTA to high levels. Because OTA binds acetylated DON also induces of disrupted ribosomal function tightly to albumin and serum proteins, emesis. Feed refusal and emesis appear to be dose-dependent, with serum OTA is a useful biomarker for appear to be due to neurochemical low doses being cytostimulatory exposure in pigs. imbalances in the brain, and although and high doses cytotoxic (Pestka, the emetic centre is clearly involved, 2010a). For example, DON is

Chapter 5. Effects in food-producing animals 75 Fig. 5.5. Reduced weight gain in pigs, an example of the effects of deoxynivalenol. of ZEA to estrogen receptors, These pigs are littermates, but the pig in the foreground was fed a diet containing the complex interacts with genes deoxynivalenol at 5 mg/kg diet for 7 weeks after weaning. Note also the white, rat-like controlling both estrogen-like and hair of the pig fed the deoxynivalenol-containing diet. Source: Trenholm et al. (1988), Fig. 3, p. 15; reproduced with the permission of the Minister of Public Works and antiestrogen-like responses (Boehme Government Services Canada, 2012. et al., 2009; Parveen et al., 2009). ZEA is also a ligand for the pregnane X receptor, which controls the expression of genes in some pathways that regulate biotransformation of endobiotics and xenobiotics (Ding et al., 2006). ZEA, and its metabolites and their conjugates, are detectable in urine and faeces and have been used as exposure biomarkers in animal studies (reviewed in Zinedine et al., 2007). Clinical signs of oestrus can be induced in ovariectomized sows. Doses of ZEA as low as 1–5 mg/ kg bw can induce vulvovaginitis, tenesmus, vaginal prolapse, and rectal prolapse in young female pigs (Osweiler, 1986; Fig. 5.6). Effects on pre-pubertal boars known to increase expression of as mechanism-based biomarkers have also been reported, and these pro-inflammatory cytokines, affect (Amuzie and Pestka, 2010) could include reduced libido, decreased transcription factors for many genes prove useful as markers of DON plasma testosterone, and other related to immunity and inflammation, involvement in disease outbreaks in effects (Osweiler, 1986). In sows, increase mRNA expression, in- farm animals (Riley et al., 2011). dietary levels of 3–10 mg/kg of ZEA crease mRNA stabilization, affect can induce anoestrus, reduced MAP kinase signalling, and induce 4.5 Zearalenone litter size, fetal reabsorption, and the ribotoxic stress response, all implantation failure. Cattle are more of which contribute to its biological The species that is the most sensitive resistant to the estrogenic effects; effects in animals (Pestka, 2010b). to the effects of ZEA is the pig. The however, conception rates can be It has also recently been shown that observed effects of ZEA on pigs reduced. DON disrupts the growth hormone involve primarily the reproductive axis; this finding provides a credible system (Fitzpatrick et al., 1989; Fink- 4.6 Ergot alkaloids explanation for growth retardation in Gremmels and Malekinejad, 2007; animals. Specifically, DON induces Zinedine et al., 2007). The sensitivity Based on major signs, ergotism in expression of pro-inflammatory of the pig appears to involve the way in farm animals can be divided into the cytokines, which leads to upregulation which this species metabolizes ZEA. gangrenous form and the convulsive of suppressors of cytokine signalling, Metabolism depends primarily on form (Robbins et al., 1985; Raisbeck which leads ultimately to reduced two hydroxysteroid dehydrogenases, et al., 1991). Livestock consuming feed levels of both insulin-like growth factor which produce the two main me- grains containing small seed grasses 1 and insulin-like growth factor acid- tabolites α- and β-zearalenol. The or foraging on pasture grasses are at labile subunit in the blood (Amuzie α-zearalenol isomer has a much particular risk. Cattle are at greatest and Pestka, 2010). The combined greater uterotropic activity than ZEA risk, and sheep and horses are less use of urinary DON glucuronide level does, and in pigs the predominant frequently affected. The symptoms of as an exposure biomarker (Turner, form of hydroxysteroid dehydrogenase ergotism and toxic alkaloids (Oliver, 2010) and changes in plasma levels of is the one that yields primarily 2005) include lameness, gangrene, insulin-like growth factor 1 and insulin- α-zearalenol (Fink-Gremmels and agalactia, reduced weight gain, abortion, like growth factor acid-labile subunit Malekinejad, 2007). After binding hypersensitivity, ataxia, convulsions,

76 Fig. 5.6. Vulvas of pre-pubertal gilts fed control diet (left) or zearalenone-contaminated 5. Sensitive or target species feed (right). Note swelling and oedema due to zearalenone consumption. Photograph and confounding factors courtesy of Walter F.O. Marasas, Programme on Mycotoxins and Experimental Carcinogenesis (PROMEC Unit), South African Medical Research Council. Although acute disease outbreaks from exposure to mycotoxins are not uncommon, definitively linking exposure to effects is not easy at the low levels of exposure most commonly encountered in the field. The observed physiological effects usually involve subtle changes in animal performance or behaviour and increased susceptibility to infectious disease, all of which are relatively nonspecific effects. Nevertheless, when changes in animal performance are observed and can be linked to the feed, exposure to low levels of mycotoxins in feed should be suspected as a contributing factor, at the very least. Young animals are more sensitive than adults to the adverse effects of mycotoxins, in part because of a higher metabolic rate. Factors and, in sheep, intestinal inflammation. In much of their time seeking shade or that can influence susceptibility cattle grazing on tall fescue, syndromes standing in ponds (Raisbeck et al., to performance problems and known as fescue foot, summer slump, 1991). Fat necrosis is characterized disease due to mycotoxins are sex, CHAPTER 5 CHAPTER and fat necrosis have been described by hard fat masses in the adipose genetic strain, reproductive status, (Robbins et al., 1985). Fescue foot tissue of the abdominal cavity. Fat pre-existing conditions, nutritional is characterized by gangrene of the necrosis is believed to require long- factors such as vitamin- or protein- extremities (feet, ears, and tail) and is term consumption (for years) of tall deficient diets, environmental caused by vasoconstriction resulting in fescue, whereas summer slump and stress, concurrent exposure to reduced blood flow and ischemia in the fescue foot may occur within weeks infectious agents, and exposure to peripheral tissues (Oliver 2005; Fig. 5.7). of exposure (Raisbeck et al., 1991). other toxins, including mycotoxins. This is usually accompanied by In all cases, it is the vasoconstrictive Target-organ specificity and species increased core body temperature in action of the ergot alkaloids and their sensitivity are known largely from cattle. Necrosis and loss of the tip metabolites (Hill et al., 2001; Ayer et laboratory studies in which a single of the tail is a common sign. Cold al., 2009) that is the underlying cause species has been studied using a weather aggravates the problem of the three common fescue toxicities. single toxin. Comparative studies of fescue foot, whereas summer In horses, reproductive problems are quite rare, so dose–response slump is commonly associated with resulting from consumption of ergot relationships comparing species, warm conditions. Summer slump alkaloids in tall fescue include longer sex, and exposure to multiple toxins is characterized by reduced milk gestation, stillborn foals, agalactia, at low levels – a scenario that is likely production, heat intolerance, rough placental retention, and reduced in field situations – are also rare. In hair coat, increased respiration rate, breeding efficiency (Cross, 2003). this section, we briefly describe the decreased feed intake, reduced Laminitis can also be caused by susceptibility of farm and domestic serum prolactin levels, and reduced consumption of toxic tall fescue and, animals to mycotoxins and the target conception rates (Robbins et al., 1985; like fescue foot in cattle, it is believed organs for aflatoxins, fumonisins, Raisbeck et al., 1991; Spiers et al., to be caused by vasoconstriction OTA, DON, ZEA, and ergot alkaloids 2005). A common sign in herds with in the extremities induced by ergot (summarized from WHO, 2001; summer slump is that the cattle spend alkaloids (Cross, 2003). Haschek et al., 2002; CAST, 2003).

Chapter 5. Effects in food-producing animals 77 Fig. 5.7. A field case of fescue toxicity. acute toxicity is the duckling (death), al., 2001). Studies have shown that The cow’s tail switch has fallen off and and the most sensitive species for pigs treated with fumonisins have a prominent ring has formed, produced chronic toxicity is the rainbow trout increased susceptibility to intestinal by ergot alkaloids in tall fescue. In this animal, the foot is also affected (not (liver cancer). Domestic turkeys infection with Escherichia coli shown) with reddening and swelling; this and quail are also very sensitive. (Oswald et al., 2003) and decreased is common with gangrenous ergotism. Reproductive effects have been specific antibody response during Photograph courtesy of Charles Bacon, reported in mink at low dietary levels vaccination (Taranu et al., 2005). United States Department of Agriculture, Agricultural Research Service. (10 µg/kg), and dogs are also quite Fumonisins frequently co-occur with sensitive to acute toxicity. In poultry, aflatoxins in maize and have been the relative sensitivity is ducklings shown to promote aflatoxin-initiated > turkey poults > goslings > chicks liver tumours in rainbow trout (IARC, > quail. In other farm animals, the 2002b). Fumonisins also cause liver relative sensitivity is rabbits > young toxicity in catfish, poultry, mink, pigs > calves > mature cattle > sheep. goats, and cattle (WHO, 2001). It However, these comparisons depend has been suggested that mycotoxic very much on the experimental nephropathy in pigs and chickens conditions under which the studies is a result of concurrent exposure were carried out. For example, young to multiple mycotoxins, including pigs are much more sensitive than fumonisin, OTA, and penicillic acid older pigs. The susceptibility of a (Stoev et al., 2010). The relative species to aflatoxin toxicity depends sensitivity, based on the United on its ability to metabolize aflatoxin States Food and Drug Administration efficiently and quickly to less toxic or (FDA) Guidance to Industry, is nontoxic metabolites (see Section equids and rabbits > pigs and catfish 3.1.7). Because maize is an important > ruminants, poultry, and mink, and source, exposure to both aflatoxins breeding animals are considered to and fumonisins is likely in animals be more sensitive than animals being consuming maize-based feeds. raised for slaughter (FDA, 2001). 5.1 Aflatoxins 5.2 Fumonisins 5.3 Ochratoxin A The liver is the primary target organ for aflatoxins, for both acute and chronic Fumonisins cause liver damage in The kidney and, to a much lesser toxicity. The kidney can also be all farm animals tested and also extent, the liver are the main targets affected in pigs and goats. However, kidney damage in rabbits, cattle, of OTA. Cattle (but not calves) anecdotally the most commonly and sheep. In addition, fumonisins are considered resistant due to suspected indicator of exposure to induce species-specific toxicities, metabolism of OTA to nontoxic aflatoxins is decreased performance in the brain in horses and the lung ochratoxin α by rumen microbes. and/or increased susceptibility to en- in pigs, that are a consequence of Other ruminants may also show vironmental and microbial stressors. cardiovascular dysregulation (WHO, resistance; however, dietary factors Conversely, undernourished or 2001; Haschek et al., 2002). Because can affect the extent of rumen stressed animals will be more fumonisins inhibit sphingolipid bio- metabolism (Höhler et al., 1999). sensitive to aflatoxin toxicity, and synthesis, it is likely that receptors Pigs and dogs are the most sensitive studies in rodents show that exposure and processes that are dependent domestic animals, and poultry are to bacterial endotoxins augments liver on sphingolipids are affected. For less sensitive than pigs. The relative injury induced by aflatoxin (Barton example, glycosphingolipids are sensitivity in poultry is chicks > turkeys et al., 2000). Cyclopiazonic acid and necessary for the proper functioning > quail (Newberne and Rogers, 1981). aflatoxins frequently occur together of many membrane receptors, in groundnuts and maize, and the including those for some vitamins original outbreaks of turkey “X” (i.e. folate) and for the recognition disease in the United Kingdom in 1960 of numerous microbial pathogens may have involved both toxins (Cole, and microbial toxins (i.e. Shiga-like 1986). The most sensitive species for toxins and cholera toxin) (Merrill et

78 5.4 Deoxynivalenol 5.5 Zearalenone 5.6 Ergot alkaloids

In farm animals, the primary target Pre-pubertal female pigs are the The vasoconstrictive properties organ for DON toxicity is uncertain. most susceptible farm animal to of ergot alkaloids are the primary The reason is that the most sensitive the estrogenic effects of ZEA. The cause of problems in farm animals. effects are reduced weight gain sensitivity of pigs may be due to Sensitive species are those that in the mouse, presumably due to a higher affinity of their estrogen graze on pasture grasses (especially reduced feed intake, and feed refusal receptors for α-zearalenol (Fitz- tall fescue) or consume feeds and emesis in pigs (WHO, 2001). patrick et al., 1989; Fink-Gremmels comprised of small seed grasses There is evidence that feed refusal and Malekinejad, 2007). Cattle and (such as ryegrass). Cattle are and emesis are due to a central sheep are much less sensitive than probably the most sensitive, followed serotoninergic effect. Cattle, sheep, pigs, and poultry are considered to by sheep and horses. Environmental and poultry are resistant to the be resistant (Haschek et al., 2002). factors (especially temperature) are emetic effects of DON, but reduced Cycling mares also appear to be very important in determining the feed intake is seen at 10–20 mg/kg relatively insensitive to the estrogenic nature of the observed effects in diet in ruminants (Osweiler, 2000). effects of ZEA at low doses cattle (Raisbeck et al., 1991; Oliver, Horses are resistant, but shrimp (equivalent to natural contamination) 2005). are very sensitive to the effects on (Juhász et al., 2001). An interaction body weight gain, whereas dogs and between ZEA and oxytocin (an ergot cats are also sensitive to the emetic alkaloid derivative) has been reported effects of DON (WHO, 2001). DON in pigs (Alexopoulos, 2001). Grains and ZEA frequently occur together, contaminated with DON are frequently making exposure to both likely. also contaminated with ZEA. CHAPTER 5 CHAPTER

Chapter 5. Effects in food-producing animals 79 References

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Chapter 5. Effects in food-producing animals 85

chapter 6. Mycotoxins and human health

Summary especially in vulnerable populations. 1. Introduction For fumonisins, studies indicate a Mycotoxins have been investigated possible role in oesophageal cancer This chapter covers the effects on in relation to a wide range of adverse and in neural tube defects, although human health of the major mycotoxins human health effects, but the evidence no definitive conclusions can be occurring in foods. This chapter also for all but a small number of associations drawn at present. For deoxynivalenol includes information on mechanisms is limited. Thus, the full impact on human and other trichothecenes, exposure of action of mycotoxins in humans CHAPTER 6 CHAPTER health of the widespread exposure has been linked to acute poisoning where relevant to the adverse health to mycotoxins remains to be defined. outbreaks in large numbers of effects under consideration. No The main exception is for aflatoxins; subjects. For ochratoxin A and attempt is made at a comprehensive epidemiological, experimental, and zearalenone, the human health review, but at appropriate points we mechanistic studies have contributed effects remain undefined. The limited refer to more extensive accounts. to establishing aflatoxins as a tools available to accurately assess The major source of human cause of human liver cancer, with a human exposure to mycotoxins and exposure to mycotoxins is consump- particularly elevated risk in people the relative paucity of epidemiological tion of contaminated foods. Exposure chronically infected with hepatitis B studies need to be addressed if the full is highest when those foods are dietary virus. In addition, acute aflatoxicosis extent of the adverse effects of these staples, such as maize, groundnuts, or after exposure to high dietary toxin common dietary contaminants is to be various other cereals. Exposures to levels has been demonstrated. The understood and adequate public health metabolites or parent toxins may also impairment of child growth by aflatoxin measures taken. In this respect, newly occur by consumption of contaminated exposure early in life remains an established biomarkers of exposure at milk and milk products. We covered important subject of study. More the individual level are proving valuable the dietary sources of mycotoxin information is also required on the in improving exposure assessment in exposure in some detail in Chapter 1. potential immune effects of aflatoxins, epidemiological studies. In this chapter, we also consider

Chapter 6. Mycotoxins and human health 87 occupational exposures in granaries 2. Aflatoxins but is less mutagenic than AFB1, and other food and feed processing reflecting the stearic chemistry of the

due to mycotoxins contained in dusts Aflatoxins are produced in a wide respective epoxides; the AFB1-8,9- from contaminated grains. The specific range of commodities by Aspergillus epoxide intercalates more readily into effects due to particular mycotoxins are flavus and A. parasiticus, and the DNA double helix than does the

discussed in Sections 2–6. Additional, occasionally other Aspergillus species. equivalent AFG1 molecule, resulting in more general information on health The commodities most at risk are higher levels of DNA adduct formation problems associated with mycotoxins maize and groundnuts in tropical areas for a given dose. Minimal information in grain dusts is covered in Section 7. (see Chapter 1). exists about the importance of The human health effects the reactive epoxide for the non- considered here encompass acute 2.1 Mechanisms mutagenic actions of aflatoxins or poisoning, cancer, other chronic indeed about the biological effects of diseases, and biological effects, Until recently, attention on af- aflatoxins independent of metabolic including growth impairment and latoxins has been focused on activation to the 8,9-epoxide. Unlike

immunomodulation. their carcinogenic effects. For AFM2, AFM1 contains an 8,9 double One of the major limitations more detailed information, see the bond and hence can be bioactivated in assessing the effects of extensive reviews in IARC (2002), to the reactive 8,9-epoxide. mycotoxins on health has been WHO (2002), and Wild and Gong A significant observation in the inability to accurately assess (2010). Consideration is usually given terms of aflatoxin carcinogenicity is exposure at the individual level. to the naturally occurring aflatoxins the association between exposure

The development of validated in the diet – aflatoxins B1 (AFB1), B2 and a specific mutation in the TP53

biomarkers for aflatoxins has greatly (AFB2), G1 (AFG1), and G2 (AFG2) – tumour suppressor gene in liver

assisted epidemiological studies and or to AFB1 alone, or more rarely to cancer (hepatocellular carcinoma

allowed an evaluation of aflatoxins aflatoxins M1 (AFM1) and M2 (AFM2), [HCC]). In HCC tumours from patients

in relation to cancer, aflatoxicosis, the hydroxylation products of AFB1 who are from regions endemic for

child growth impairment, and immune and AFB2, respectively, that occur aflatoxin and who are chronically effects (see Wild and Gong, 2010). in milk. This distinction between the infected with hepatitis B virus (HBV), Development of validated biomarkers type of aflatoxin exposures under a high prevalence exists of a specific for fumonisins (Wild and Gong, 2010; consideration is important but is missense mutation in the gene, Van der Westhuizen et al., 2011) rarely considered, particularly when namely an AGG → AGT (Arg → Ser) and deoxynivalenol (Meky et al., studying adverse health effects other point mutation at codon 249 (codon 2003; Turner et al., 2008a, 2008b, than cancer. 249ser) (IARC, 2002; Hussain et al., 2008c) also offers promise for future Given the focus on mutagenicity 2007). This mutation is extremely rare studies of the human health effects and carcinogenicity, most studies in HCC associated with HBV in areas

of these mycotoxins. However, have been of AFB1, which, due to the where aflatoxins are uncommon, the biomarker field for mycotoxins presence of a double bond at the 8,9 but it is as yet unclear whether HBV also offers a cautionary tale: an position, can be metabolized to the infection influences occurrence of

unvalidated biomarker for ochratoxin reactive AFB1-8,9-epoxide, which the mutation in HCC from aflatoxin- A in plasma or serum has been binds to cellular macromolecules endemic areas. used to assess dietary exposure including DNA (for more detail, see The major human cytochrome to this toxin, but subsequent Wild and Turner, 2002). The major P450 (CYP) enzymes involved in

careful duplicate diet studies have DNA adduct is AFB1-N7-guanine, and aflatoxin metabolism are CYP3A4, shown that this biomarker does not this pro-mutagenic lesion commonly 3A5, and 1A2, and the predominant reflect intake at the individual level results in a G → T transversion site of bioactivation is the liver,

(Gilbert et al., 2001). Nevertheless, mutation. AFB1-N7-guanine can although CYP3A4 expression in the availability of biomarkers to also be detected in the urine and the human intestine means that measure exposure to mycotoxins used as an exposure biomarker in metabolism may also occur in that

provides new opportunities for more epidemiological studies. AFB2 and organ (Wild and Turner, 2002;

systematic monitoring of exposure AFG2 are generally considered to be Kamdem et al., 2006; Thelen and in populations as well as improved far less biologically active due to the Dressman, 2009). The contribution of

etiological studies. absence of the 8,9 double bond. AFG1 these enzymes to AFB1 metabolism in can be bioactivated to the 8,9-epoxide exposed people will depend on both

88 the affinity and level of expression An understanding of the Baumgartner et al., 2005; Lewis et of the different enzymes; CYP3A4 metabolism, DNA damage, and al., 2005). These outbreaks resulted appears to be the most important in induction of mutations in people in several hundred deaths associated generating the exo-8,9-epoxide, and exposed to aflatoxins in the diet with consumption of maize heavily the relative contribution of CYP3A5, has contributed to the overall contaminated with aflatoxin. A case– which also produces the exo-8,9- assessment of their adverse health control study of aflatoxicosis, defined epoxide, varies by individual (Kamdem effects (Groopman et al., 2008; Wild as acute jaundice of unknown origin, et al., 2006). In fact, CYP3A5 and Gong, 2010). The major health found that aflatoxin levels in foods expression is polymorphic and effects linked to aflatoxin exposure from affected households were varies by ethnic group; for example, are described briefly here. much higher than those in foods 40% of African Americans show no from unaffected households. Similar expression due to identified genetic 2.2 Aflatoxicosis differences between cases and polymorphisms. Such polymorphisms controls were found when aflatoxin may affect sensitivity to the toxic Sporadic historical accounts of biomarker levels in blood were effects of aflatoxins (Wojnowski et al., human poisoning with aflatoxins examined (Azziz-Baumgartner et al., 2004). CYP1A2 leads predominantly were reported, but these early 2005; McCoy et al., 2008). to formation of the hydroxylated AFM1 studies were not definitive in The association of aflatoxin metabolite and the AFB1-endo-8,9- assigning causation (Hall and Wild, contamination of maize with acute epoxide, which does not form DNA 1994). In 1974, hepatitis cases due hepatitis and aflatoxicosis is well adducts. to aflatoxicosis in Western India supported by the evidence, most Given the fact that aflatoxin is (Krishnamachari et al., 1975) were notably by the observations in Kenya. known to cross the placenta, it is associated with consumption of It is of interest that aflatoxicosis has also of interest that CYP3A7, a major maize contaminated with A. flavus. been reported only in communities CYP in human fetal liver, can activate Patients exhibited jaundice preceded where maize is the dietary staple.

AFB1 to the 8,9-epoxide (Kamataki by fever, vomiting, and anorexia, with This reflects both high levels of et al., 1995; Wild and Turner, 2002). subsequent progression to ascites aflatoxins in maize and high daily Indeed, aflatoxin adducts have been and oedema in lower extremities. In intakes (300–500 g) of this staple identified in cord blood (Wild et al., maize from households where cases commodity. In addition, however, the 1991; Turner et al., 2007), indicating occurred, the aflatoxin levels were role of co-contaminating mycotoxins, that environmental levels of aflatoxin extremely high, 6.25–15.6 mg/kg, notably fumonisins, has not been are bioactivated to the reactive and the estimated daily ingestion of assessed, and these may contribute metabolites in utero. aflatoxins was 2–6 mg in adults. to the acute toxicity observed. Detoxification of the aflatoxin exo- In Kenya in 1981, another The level of aflatoxin intake and endo-epoxides occurs mainly outbreak of acute hepatitis was associated with aflatoxicosis and through glutathione S-transferase- associated with aflatoxin poisoning death has been estimated (Wild CHAPTER 6 CHAPTER mediated conjugation to reduced (Ngindu et al., 1982). Patients were and Gong, 2010). The intake of glutathione (Guengerich et al., 1998). diagnosed with jaundice preceded total aflatoxins estimated to result The epoxides can also undergo rapid by abdominal discomfort, anorexia, in a risk of fatality was > 1 mg/day, non-enzymatic hydrolysis to AFB1- general malaise, and low-grade i.e. > 20 µg/kg body weight (bw)/ 8,9-dihydrodiol, which in turn forms fever; tachycardia and oedema day in adults. It was considered that a dialdehyde phenolate ion with an were also observed. Maize from two aflatoxicosis without fatality may opened ring. The dihydrodiol can affected households contained up occur with 5–10-fold lower doses. react with the ε-amino group of lysine to 3.2 mg/kg and 12 mg/kg AFB1. Further estimates suggested that in serum albumin to form aflatoxin– An additional report came from an the total intake of AFB1 associated albumin adducts, which are often used incident in Malaysia in 1988, where with half the exposed people dying as exposure biomarkers (Wild and 13 children died from acute hepatic (i.e. the median lethal dose [LD50]) Gong, 2010). In a further metabolic encephalopathy after consuming would be 0.54–1.62 mg/kg bw, a step, aflatoxin aldehyde reductase noodles (Lye et al., 1995). similar magnitude to the LD50 value catalyses the NADPH-dependent In 2004, well-documented reported for rabbits, cats, dogs, pigs, reduction of the dialdehyde phenolate cases of aflatoxicosis occurred in and baboons (Wild and Gong, 2010). ion to a dialcohol (Johnson et al., Kenya, close to the locality of the Therefore, daily exposure to staple 2008). cases reported in 1981 (Azziz- foods consumed at several hundred

Chapter 6. Mycotoxins and human health 89 grams per day and contaminated with between these two factors in relation 2.4 Cirrhosis ≥ 5000 µg/kg of aflatoxins may lead to to increased HCC risk (Qian et al., death in humans. Daily consumption 1994; Wang et al., 1996; IARC, To date, little information exists on of foods with > 1000 µg/kg may lead 2002). In a more recent follow-up of the risk of liver cirrhosis in relation to to aflatoxicosis. the cohort in Taiwan, China, Wu et al. aflatoxin exposure. A case–control It is of great concern that these (2009) conducted the largest nested study in The Gambia (Kuniholm et al., contamination levels in maize that case–control study to date and 2008) reported that increasing lifetime are associated with aflatoxicosis reported that the combined effect of groundnut intake (a surrogate for

and death are only 10–100 times AFB1 exposure and HBV infection aflatoxin consumption) was associated the levels that occur regularly in was more consistent with an additive with a significantly increased risk of many parts of sub-Saharan Africa. model than with the multiplicative cirrhosis, approaching 3-fold with the Despite the demonstration that heavy one observed in the original report highest level of consumption. The contamination of maize with aflatoxins of Wang et al. (1996). However, presence of the codon 249ser mutation does lead to aflatoxicosis and death, after examining the plasma of HCC associated with aflatoxin was also these outbreaks continue to occur. patients, cirrhosis patients, and associated with a similar magnitude of Thus, in affected parts of the world, controls for the TP53 gene mutation increased risk of cirrhosis. However, there is an urgent need for a rapid (codon 249ser; AGG → AGT), Kirk et further studies are needed before field test that, as part of a preventive al. (2005) showed that the increased any conclusions can be drawn about strategy, can detect dangerously high risk associated with the presence aflatoxin and cirrhosis. This is an area levels (e.g. > 1000 µg/kg) of aflatoxins of both the 249ser mutation and that merits more attention, given the in cereals and nuts, as well as an HBV infection was consistent with large burden of cirrhosis worldwide. emergency response analogous a multiplicative effect of exposure to to those in place for outbreaks of aflatoxin and chronic HBV infection. 2.5 Immune effects infectious diseases. The HCC risk from exposure to aflatoxins in the absence of chronic The immunomodulatory effects of 2.3 Liver cancer HBV infection is difficult to assess aflatoxins have been considered in in populations where HBV infection experimental studies in cell models The International Agency for is widespread. A review by Omer and animals as well as in observations Research on Cancer (IARC) has et al. (2004) reported 1.7–3.4- of farm animals (IARC, 1993, 2002; classified naturally occurring mixtures fold increased risks in individuals WHO, 2002; Williams et al., 2004). of aflatoxins as Group 1, carcinogenic exposed to aflatoxins without However, only a few studies have to humans (IARC, 2002) (Table 6.1). chronic HBV infection. Wu et al. considered the association between Before the 1990s, most studies of (2009) reported a similar magnitude aflatoxin exposure and immune aflatoxins and HCC were either of increased HCC risk in subjects parameters in human populations. ecological correlation studies or positive only for aflatoxin exposure Two such studies have been reported case–control studies. The ecological biomarkers, but occult HBV infections from The Gambia (Allen et al., 1992; studies did not always consider in some of the individuals in these Turner et al., 2003). The first provided infection with HBV and hepatitis C studies cannot be ruled out. some evidence that children with virus, had relatively crude estimates The overall evidence from higher aflatoxin exposure were more of aflatoxin intakes, and had limitations epidemiological studies shows a likely to have malaria parasitaemia, in diagnosis and registration of HCC particularly elevated risk of HCC but no significant associations were cases. Despite this, most showed a from aflatoxin exposure in individuals observed with experience of malaria positive correlation between estimated chronically infected with HBV infection, antibody titre to asexual aflatoxin intakes and HCC rates in a and reasonable evidence that an stages of Plasmodium falciparum, given region (IARC, 1993, 2002). increased risk also exists in individuals or lymphoproliferative responses. Prospective cohort studies of exposed to aflatoxins without chronic The second study investigated improved design, begun in the late HBV infection. Given that > 350 million the effect of aflatoxin exposure on 1980s and 1990s in South-East chronic HBV carriers exist worldwide, cell-mediated immunity (skin test), Asia, used biomarkers of exposure many living in aflatoxin-endemic antibody titres (in response to rabies to both HBV and aflatoxins. These areas, the need to reduce aflatoxin and pneumococcal vaccines), and studies provided strong evidence of exposure remains highly relevant for salivary immunoglobulin A (IgA). No a more than multiplicative interaction cancer prevention. associations were found between

90 aflatoxin exposure and either the although the breastfeeding period In summary, growth faltering in skin test or antibody titres, but higher provides some respite from high West African children may occur at the aflatoxin exposure was associated daily intakes. Studies in several time of introduction of solid foods, when with lower salivary IgA, suggesting animal species indicate that aflatoxin high exposure to aflatoxin occurs. The that aflatoxin exposure could exposure can severely affect growth dose–response relationships between modulate mucosal immunity. and development. However, until aflatoxin biomarker levels and growth From Ghana, two studies have recently such effects had not been effects are also consistent with a been reported that compared considered in human populations. causal effect. However, at this time aflatoxin biomarker levels and Early studies explored the link other confounding factors cannot be subsets of peripheral blood cells in between aflatoxin exposure and excluded as explanations for these adults (Jiang et al., 2005, 2008). In kwashiorkor (Hendrickse et al., 1982), associations. The mechanisms of the first, a higher aflatoxin biomarker but no firm conclusions could be action by which aflatoxin may exert level was associated with a lower drawn due to various weaknesses in an effect on growth are currently percentage of CD3+ and CD19+ cells study design (Hall and Wild, 1994). unknown, although the possibility of (B lymphocyte antigens) expressing A study in rural Kenya in the 1980s a compromised intestinal integrity, the CD69+ activation marker and linked aflatoxin detection in mothers’ through altered barrier function as a with lower percentages of CD8+ blood with significantly lower birth consequence of endothelial cell toxic- T cells expressing perforin and weights of female babies (De Vries ity or immune suppression, is a valid granzyme A. In the second study, a et al., 1989). A more recent study in hypothesis that should be explored higher aflatoxin biomarker level was Kisumu District, Kenya, showed a further (Wild and Gong, 2010). associated with lower percentages of significantly greater prevalence of In areas where aflatoxin is CD8+ cells expressing perforin and wasting (low weight for height) in common, namely sub-Saharan of CD19+ cells expressing CD69+. children fed cereals with high aflatoxin Africa and South Asia, 7.1 million In addition, HIV-positive individuals contamination, compared with those children died under the age of 5 with higher aflatoxin biomarker levels whose cereals had lower aflatoxin years in 2008. It is estimated that had significantly lower percentages levels (Okoth and Ohingo, 2004). about 50% (3.55 million) of these of CD4+ T regulatory cells and A series of studies has been deaths are related to undernutrition naive CD4+ T cells compared with conducted in West African children and poor growth (Black et al., 2003). HIV-positive individuals with lower exposed to aflatoxins early in life. In the If aflatoxin exposure were to be aflatoxin biomarker levels. first of these, a cross-sectional study of responsible for even a few per cent Overall, the studies of im- children aged 1–5 years in Benin and of these deaths, the total number munomodulation do not permit Togo, a striking inverse association would be tens of thousands per year. conclusions to be drawn about was found between aflatoxin–albumin the impact of environmental levels adduct level and growth (Gong et 2.7 Occupational exposures of aflatoxin exposure on human al., 2002). In a subsequent 8-month CHAPTER 6 CHAPTER immunity and susceptibility to longitudinal study, a strong negative AFB1 concentrations of up to infectious disease. Nevertheless, correlation was observed between 612 μg/kg have been reported in the data suggest that immune aflatoxin–albumin adduct level and airborne dusts during the handling of parameters could be affected in height increase (Gong et al., 2004). contaminated maize and groundnuts populations exposed chronically to The highest quartile of aflatoxin– (Miller, 1994a; Sorenson, 1999). aflatoxins. If this were to be proven, albumin adducts was associated with Most aflatoxin was contained in the the impact would add greatly to the a mean reduction of 1.7 cm in height < 7 μm and 7–11 μm particle size burden of disease related to cancer increase compared with the lowest ranges. In grain dusts, a substantial and aflatoxicosis. quartile. Recently, an association fraction of the aflatoxin is contained was also found between exposure to in the spores of A. flavus and A. 2.6 Child growth impairment aflatoxin in utero and impaired growth parasiticus (Miller, 1994a, 1994b). during the first year of life in children in Retrospective studies of feed Children are chronically exposed The Gambia (Turner et al., 2007). This processing workers in Denmark to high levels of aflatoxins in areas finding suggests that the consumption reported elevated risks of HCC, gall where food contamination is en- of aflatoxin-contaminated food during bladder cancer, and extrahepatic demic. Exposure begins in utero pregnancy may have effects on the bile duct cancer in this population and continues throughout early life, child after birth. exposed occupationally, with a

Chapter 6. Mycotoxins and human health 91 2-3- fold increased risk after a 10-year damage and lipid peroxidation The role of fumonisins in im- latency period. Inhalation exposure (Stockmann-Juvala and Savolainen, munomodulation has also been to aflatoxin (170 ng/day) was reported 2008). This finding is consistent with highlighted through changes in to be the most likely explanation. increased oxidative DNA damage cytokine levels in vitro and in vivo in Some evidence of elevated aflatoxin and malondialdehyde adducts in animal models (Sharma et al., 2000) biomarker levels related to the rat liver and kidney (Domijan et al., and effects on antibody vaccine

handling of contaminated feeds was 2006) and lipid peroxidation (Abel responses in pigs exposed to FB1 also reported (Olsen et al., 1988; and Gelderblom, 1998) in vivo after (Taranu et al., 2005; Stockmann-

Autrup et al., 1991, 1993). A risk FB1 treatment. Fumonisin-induced Juvala and Savolainen, 2008). assessment model suggested that carcinogenesis in the liver proceeds Animal experiments have shown

exposure to AFB1 in airborne dust through initiation and promotion that fumonisins are rapidly excreted may pose little significant risk during stages in a manner similar to that for unmetabolized from the gut (Shephard maize harvest and elevator loading/ genotoxins and is dependent on the et al., 1994a, 1994b; Martinez- unloading but a relatively high risk dose and time of exposure (Gelderblom Larranaga et al., 1999). It is not known during swine feeding and storage bin et al., 2008a). Nevertheless, no whether the gut microflora metabolize

cleaning (Liao and Chen, 2005). evidence has been found for direct fumonisins, although hydrolysed FB1 interaction of fumonisin with DNA has been detected in faeces of vervet 3. Fumonisins nor for its metabolism to a reactive monkeys (Shephard et al., 1994a, metabolite (WHO, 2001; IARC, 2002). 1994b), pigs (Fodor et al., 2008), and

Fumonisins occur in maize and, FB1 disrupts de novo sphingolipid ruminants (WHO, 2000b). Studies much less commonly, other cereals, biosynthesis by inhibition of the en- using radiolabelled fumonisins failed as a result of infection with Fusarium zyme ceramide synthase (Merrill to show any metabolism by primary verticillioides and related species (see et al., 2001), resulting in many hepatocytes or hepatic esterases and Chapter 1). An Environmental Health effects on signalling pathways and lipases (Cawood et al., 1994).

Criteria document for fumonisin B1 cell functions that are dependent Analyses of human faeces has

(FB1) has been published (WHO, on ceramide, sphingoid bases, revealed unmetabolized FB1 and

2000a), and the Joint FAO/WHO sphingoid base 1-phosphates, and FB2 (Chelule et al., 2000, 2001). The

Expert Committee on Food Additives complex sphingolipids (Dragan et presence of FB1, FB2, and FB3 in (JECFA) has established a provisional al., 2001; Merrill et al., 2001). These human hair has been demonstrated, maximum tolerable daily intake include effects on apoptosis and suggesting that fumonisins are

(PMTDI) of 2 μg/kg bw/day for FB1, mitosis, thus potentially contributing absorbed from the gut after ingestion

FB2, and FB3 alone or in combination to carcinogenesis through an altered of contaminated maize (Sewram et

(WHO, 2001, 2002). This PMTDI is balance of cell death and replication al., 2003). More recently, FB1 in urine based not on tumorigenicity data (Stockmann-Juvala and Savolainen, has been reported in individuals but on the no-observed-effect level 2008). Disruption of sphingolipid consuming large amounts of (NOEL) for nephrotoxicity in rodents metabolism leads to changes in the maize (Gong et al., 2008). Direct of 0.2 mg/kg bw/day, divided by a sphinganine-to-sphingosine ratio, evidence therefore exists that safety factor of 100 (WHO, 2001). with increased sphinganine tissue human populations are exposed to concentrations, which correlate fumonisins after absorption, although 3.1 Mechanisms closely with the in vivo toxicity and the actual levels and the possible risk carcinogenicity of fumonisins (Riley this poses still need to be quantified. Fumonisins may exert their biological et al., 2001). Such changes were Probable daily intake values effects through several different demonstrated in rat liver and mouse of fumonisins, determined using

mechanisms. FB1 genotoxicity is kidney at carcinogenic doses of FB1 a validated dietary assessment somewhat unclear; negative results (Voss et al., 2002). Disruption of tool, showed that intakes of up to were obtained from several ge- cholesterol, phospholipid, and fatty 10 times the PMTDI can occur in notoxicity assays, but other in vitro acid synthesis and interaction with individuals in a rural, subsistence

studies reported that FB1 induced ceramide have been proposed to play farming community where maize is micronuclei and chromosomal key roles in the differential growth the main dietary staple (Burger et aberrations (Ehrlich et al., 2002; patterns of altered hepatocytes al., 2010). Drinking home-brewed IARC, 2002). The DNA damage may during cancer promotion in the liver maize beer further increased the be a result of stimulation of oxidative (Gelderblom et al., 2008b). level of exposure. Estimates of daily

92 fumonisin intake in rural communities Similar correlations have been development of oesophageal cancer. in Guatemala (Torres et al., 2007) reported in China (Sun et al., 2007). However, confounding by other risk have also shown that total fumonisin Other reports have associated factors and the possible interactions intake can potentially be > 10 times maize consumption per se with of fumonisins with other mycotoxins the recommended PMTDI. high oesophageal cancer incidence should be considered in future studies. rates but did not consider fumonisin Some experimental studies have 3.2 Acute poisoning exposure specifically (Franceschi reported a synergistic interaction

et al., 1990). In these studies, other between AFB1 and FB1 in the No confirmed cases have been fungi and their mycotoxins were development of liver cancer (Carlson reported of acute human poisoning generally also present, and to date et al., 2001; Gelderblom et al., 2002). due to fumonisin exposure. Part of the no analytical studies have been Perhaps due to the focus on difficulty in discerning a specific effect conducted that specifically link FB1 oesophageal cancer, the role of fumonisins in acute poisoning is their to human cancer at any organ site of fumonisins in cancer in other co-occurrence with other mycotoxins, (IARC, 1993, 2002; WHO, 2001). organs has been largely unexplored. notably aflatoxins and trichothecenes. Based on the disruption of However, given the interactions found For example, one poisoning outbreak sphingolipid biosynthesis mentioned experimentally, the co-contamination in India occurred where fumonisin above, the serum sphinganine-to- of crops by aflatoxins and fumonisins, contamination of foods was reported, sphingosine ratio was used as an and the fact that both toxins occur in notably in unleavened bread prepared exposure biomarker in a nested populations with a high prevalence of from mouldy sorghum or maize, and case–control study of oesophageal HBV infection, a role for fumonisins where symptoms were characterized cancer in China (Abnet et al., 2001), in HCC is plausible. Some ecological by abdominal pain, borborygmi, and but no association was found between correlation studies provide support diarrhoea (Bhat et al., 1997). However, biomarker levels and cancer risk. One for this hypothesis (Ueno et al., 1997; assays of other mycotoxins potentially study conducted in China did report Li et al., 2001; Sun et al., 2007). The present were not reported. that the sphinganine-to-sphingosine possible interaction between various An outbreak of human intoxication ratio in urine was significantly mycotoxins (fumonisins, aflatoxins, related to the consumption of increased in males estimated to have trichothecenes) and the microcystins maize gruel prepared from mouldy consumed > 110 µg/kg bw/day of FB1 B (algal toxins) in the development maize powder occurred in Guangxi (Qiu and Liu, 2001). However, of HCC merits more investigation. Province, China, in 1989 (Li et al., in all subsequent reports, no Interactions of fumonisins with dif- 1999). In this case, co-occurrence association was observed between ferent dietary constituents could also with trichothecenes was found, and sphingoid bases or sphinganine- have an impact on the toxicological based on an average maize meal to-sphingosine ratios in the plasma effects (Gelderblom et al., 2004). intake of 200 g/person (60 kg bw) per and urine and individual fumonisin IARC has concluded that there is day, the total daily dietary intake of exposure, suggesting that these inadequate evidence in humans for the CHAPTER 6 CHAPTER deoxynivalenol and FB1 was 80 µg/kg biomarkers are not sufficiently carcinogenicity of toxins derived from bw and 2.3 µg/kg bw, respectively. sensitive for monitoring exposure F. verticillioides (as F. moniliforme), These incidents highlight the in human populations (Nikièma et leading to a classification of Group need for comprehensive analyses of al., 2004; van der Westhuizen et al., 2B, possibly carcinogenic to humans mycotoxins in contaminated foods 2010; Xu et al., 2010). (IARC, 1993). FB1 was also classified and in biological samples when acute Many risk factors exist for the as Group 2B (IARC, 2002) (Table 6.1). poisoning outbreaks occur. development of oesophageal cancer. These differ among geographical 3.4 Neural tube defects 3.3 Cancer regions with respect to demography, ethnicity, genetic susceptibility, Animal studies have demonstrated Ecological studies in the former cultural practices, and socioeco- that fumonisin exposure can cause Transkei region of South Africa nomic and nutritional status. The use neural tube defects, possibly showed that both F. verticillioides of home-grown maize as one of the through the disruption of sphingo- and fumonisin contamination of main dietary staples, coupled with lipid biosynthesis and consequent maize were positively correlated an underlying poor socioeconomic depletion of sphingolipids, which with oesophageal cancer incidence status, could implicate fumonisins are critical for lipid raft functions, rates (Marasas, 2001; IARC, 2002). as a contributing factor in the specifically folate processing via

Chapter 6. Mycotoxins and human health 93 the high-affinity folate transporter (Ncayiyana, 1986; Venter et al., 1995); 4.1 Mechanisms (Stevens and Tang, 1997; Sadler et in Hebei Province, China (Moore et al., 2002; Gelineau-van Waes et al., al., 1997; Marasas et al., 2004); and Recent reviews have extensively 2005). Neural tube defects are known in Guatemala (Marasas et al., 2004) summarized evidence on the to be associated with reduced folate and Mexico, all areas where large absorption, distribution, metabolism, levels, and cell membrane disruption quantities of maize are consumed. and mechanisms of action of OTA induced by fumonisins could lead to (Pfohl-Leszkowicz and Manderville, reduced folate absorption through 3.5 Occupational exposure 2007; Marin-Kuan et al., 2008; Mally damage to the folate receptors on and Dekant, 2009). Wide species the membrane (Marasas et al., 2004). There are no reports of occupational differences have been reported in More recently, elevation in sphingoid exposure to fumonisin and adverse the serum half-life of OTA in vivo. base 1-phosphates induced by health effects. In humans, the elimination of OTA fumonisins has been implicated in the follows a two-phase pattern, a fast induction of neural tube defects in mice 4. Ochratoxin A excretion followed by a slow clearing, (Gelineau-van Waes et al., 2009). with a calculated plasma half-life of A possible link between human Human exposure to ochratoxin A 35 days. Even infrequent exposure neural tube defects and fumonisin (OTA) occurs principally in Europe and (consumption of contaminated food consumption was suggested when Canada, where it comes from eating once a week or even once a month) a high rate of neural tube de- foods made from barley and wheat can result in persistent blood levels fects was recorded in babies of in which Penicillium verrucosum of OTA (Studer-Rohr et al., 2000). Mexican American women living has grown. Minor sources include Blood samples from healthy people in in Texas who conceived during meat, especially pork, from animals European countries show OTA levels 1990–1991 (Hendricks, 1999), fed contaminated grain. In tropical of 0.1–40 ng/mL (WHO, 2008). The soon after the outbreaks of equine and subtropical countries, OTA parent molecule is the major compound leukoencephalomalacia and porcine consumption is much lower, resulting found in blood, whereas ochratoxin α is pulmonary oedema that occurred from contamination due to growth the major component detected in urine in 1989–1990 in the USA (Ross of Aspergillus carbonarius and, less (Studer-Rohr et al., 2000). et al., 1991). In the border region commonly, A. niger in coffee, cocoa OTA is absorbed from the of Texas, exposure to fumonisins and cocoa products, and dried fruit, gastrointestinal tract in mammals and may be elevated due to frequent and sometimes in cereals, including becomes strongly bound to plasma consumption of contaminated maize. sorghum, maize, and millet (see proteins (predominantly albumin) in In a case–control study in this Chapter 1). blood, whereby it is distributed to the region of Texas (Missmer et al., OTA has been the subject of kidneys, with lower concentrations 2006), moderate tortilla consumption an Environmental Health Criteria in liver, muscle, and fat. OTA is in the first trimester of pregnancy was document (WHO, 1990) and JECFA metabolized by several different associated with an increased risk of evaluations (WHO, 1991, 2001, 2002, CYP enzymes, depending on the neural tube defects compared with 2007, 2008). JECFA noted that neither species and tissue involved. In cells low consumption. However, high a conclusive association between expressing human CYP enzymes, the consumption was not associated OTA intake and human cancer nor main metabolite was 4(R)-hydroxy- with increased risk. A similar the mechanism by which OTA is OTA formed by CYP1A2, 2B6, 2C9, result was found using estimates carcinogenic in animals has been 2D6, and 2A6, whereas the 4(S)- for fumonisin intake from tortillas, established. JECFA has confirmed hydroxy-OTA derivative was formed whereas an increased sphinganine- a provisional tolerable weekly intake by only CYP2D6 and 2B6 (Pfohl- to-sphingosine ratio was associated (PTWI) for OTA of 100 ng/kg bw/week Leszkowicz and Manderville, 2007). with increased risk, apart from the (WHO, 2001, 2008). It is noteworthy Identified OTA metabolites include highest category. that risk assessment indicated that not only these two hydroxylated High incidence rates of neural acute toxicity of OTA occurred in species but also 10-hydroxy-OTA tube defects have been recorded in animals at lower levels than did long- and ochratoxin α, which is formed rural areas of Mpumalanga Province, term effects such as carcinogenicity, by hydrolysis of the peptide bond in South Africa, and in the Umzimkulu so this PTWI is based on acute OTA and thus lacks the phenylalanine district of the former Transkei region in toxicity. moiety and consequently is non- Eastern Cape Province, South Africa toxic. OTA may also be metabolized

94 Table 6.1. IARC Monographs evaluations of carcinogenic hazards of mycotoxins to humans

Degree of evidence of Monographs volume carcinogenicity Overall evaluation of Mycotoxin (year) carcinogenicity to humansa In humans In animals

Aflatoxins, naturally 56 (1993), 82 (2002) Sufficient Sufficient Group 1 occurring mixtures of

Aflatoxin B1 56 (1993) Sufficient Sufficient

Aflatoxin B2 56 (1993) Limited

Aflatoxin G1 56 (1993) Sufficient

Aflatoxin G2 56 (1993) Inadequate

Aflatoxin M1 56 (1993) Inadequate Sufficient Group 2B

Toxins derived 56 (1993) Inadequate Sufficient Group 2B from Fusarium verticillioidesb

Fumonisin B1 82 (2002) Inadequate Sufficient Group 2B

Fumonisin B2 56 (1993) Inadequate

Fusarin C 56 (1993) Limitedc

Ochratoxin A 56 (1993) Inadequate Sufficient Group 2B

Toxins derived from 56 (1993) Inadequate Group 3 Fusarium graminearum, F. culmorum, and F. crookwellense Deoxynivalenol 56 (1993) Inadequated

Nivalenol 56 (1993) Inadequate

Zearalenone 56 (1993) Limitede

Citrinin 40 (1986) Inadequate Limitedf Group 3

Patulin 40 (1986) Inadequate Inadequate Group 3 6 CHAPTER

Toxins derived 56 (1993) Inadequate Group 3 from Fusarium (no data) sporotrichioides T-2 toxin 56 (1993) Limitedg a Group 1, carcinogenic to humans; Group 2A, probably carcinogenic to humans; Group 2B, possibly carcinogenic to humans; Group 3, not classifiable as to its carci- nogenicity to humans; Group 4, probably not carcinogenic to humans. b Formerly known as Fusarium moniliforme. Fumonisin B1 is also produced by additional Fusarium species. c Fusarin C caused marginal increases in incidences of papillomas and carcinomas of the oesophagus and forestomach when given by gavage to mice and rats. d More recent carcinogenicity studies of deoxynivalenol in mice have been negative (Iverson et al., 1995; Lambert et al., 1995) and reinforce the conclusion of the 1993 IARC Working Group that there is inadequate evidence in experimental animals for the carcinogenicity of deoxynivalenol. e Zearalenone caused increased incidences of hepatocellular and pituitary tumours in mice of both sexes when given in the diet, but no carcinogenic effect was seen in rats. f Citrinin caused benign epithelial tumours (clear cell adenomas) of the kidney in male rats in one experiment. Whereas this study (Arai and Hibino, 1983) was unequivo- cally positive, other studies in other strains of rat at lower doses were negative (IARC, 1986). A single positive study with only benign tumours as findings is considered limited evidence of carcinogenicity by the IARC Monographs criteria. g T-2 toxin caused increased incidences of liver cell tumours and lung tumours in male mice when given in the diet.

Chapter 6. Mycotoxins and human health 95 by cyclo-oxygenase, lipoxygenase, (reviewed in Marin-Kuan et al., 4.3 Cancer and epoxygenase, particularly in 2008; Adler et al., 2009; Mally and extrahepatic organs such as the Dekant, 2009) and in oxidative stress Evidence for the carcinogenicity kidney, to yield reactive oxygen (Arbillaga et al., 2008; Cavin et al., of OTA is principally from studies species, which in turn may result in 2009). Changes in urinary metabolite in experimental animals. OTA is oxidative damage. profiles, obtained using GC-MS, carcinogenic to laboratory rats and OTA competitively inhibits phenyl- LC-MS, and [1H]-NMR, have been mice, causing HCC in mice and alanine-tRNA ligase, resulting in used as a metabonomic approach to kidney carcinomas in mice and rats. inhibition of protein synthesis as assess the potential of these changes The mechanism of carcinogenic well as RNA and DNA synthesis. In for development of a predictive model action has not been firmly animals, acute toxic effects of OTA for OTA toxicity (Sieber et al., 2009). established. can be inhibited by co-administration Although the results were not specific Reports of increased cancer risk of phenylalanine (FAO/WHO/UNEP, for OTA, they were indicative of in humans who consumed OTA have 1999). kidney damage and general toxicity, been limited to descriptive studies. The formation of DNA adducts by and this approach could prove to be No analytical epidemiological OTA and their potential role in cancer of value for discovery of more specific studies were available to IARC at induction has been investigated mechanisms unique to OTA. the time of the evaluation of OTA (Mally and Dekant, 2009; Mantle (IARC, 1993). The descriptive et al., 2010), and hypotheses for 4.2 Acute toxicity studies generally focused on the the formation include direct adduct co-occurrence of Balkan endemic formation induced by OTA after The kidney is the major target organ nephropathy, a fatal chronic renal metabolic activation via an OTA for adverse effects from OTA (Pfohl- disease, and higher than expected phenoxy radical and indirect DNA Leszkowicz and Manderville, 2007; rates of urinary tract tumours, damage resulting from oxygen radical WHO, 2002). Short-term toxicity including tumours of the kidney and formation as mentioned above (Pfohl- studies in mice, rats, dogs, and urinary bladder, in Bulgaria and Leszkowicz and Manderville, 2007). pigs have shown both time- and other Balkan countries. Available The concentration of OTA- dose-dependent development of studies have not established that this specific transporters in tissues has progressive nephropathy. Significant increased cancer incidence was due been proposed to explain the relative sex and species differences exist, to exposure to OTA. species, sex, and target organ as well as differences due to route IARC has concluded that there sensitivities to OTA toxicity (reviewed of administration. Other toxic effects is sufficient evidence that OTA is in Dietrich et al., 2005). Another include cardiac and hepatic lesions in carcinogenic in experimental animals contributor to selective sensitivity is rats, lesions of the gastrointestinal tract but inadequate evidence that OTA the extent of albumin binding, which and lymphoid tissues in hamsters, increases cancer risk in humans. markedly decreases the uptake of myelotoxicity in mice, and kidney OTA has therefore been classified OTA by transporters (Bow et al., lesions in chickens. Pigs appear as Group 2B, possibly carcinogenic 2006). Mechanisms that account for to be the most sensitive species to to humans (IARC, 1993) (Table 6.1). the toxicity and carcinogenicity of the nephrotoxic effects; the lowest- OTA without invoking the production observed-effect level (8 µg/kg bw) 4.4 Occupational exposure of OTA DNA adducts have also was used as the basis for establishing been proposed (reviewed in Mally the PTWI. OTA is found in the spores of P. and Dekant, 2009) and typically A great deal of work has been verrucosum on grains and also A. involve alterations in expression undertaken recently to elucidate carbonarius as well as some strains of genes regulating rates of cell the likely mechanisms of toxicity. of A. niger on grapes and coffee proliferation and cell death. Potential Degenerative changes in the prox- beans. Some occupational studies biomarkers of effect in target tissues imal tubules of the kidney have been in Europe reported elevated OTA include the development of unique the most common effects seen in levels in plasma in workers exposed gene expression profiles specific animal species studied. However, it to grain dust (Pfohl-Leszkowicz and to alterations of gene expression has not been possible to determine Manderville, 2007). From what was induced by OTA. Genes include just what acute effects, if any, OTA inferred to be a massive exposure those involved in cellular defence, has on humans (WHO, 2008). to OTA from working in a confined cell proliferation, and cell death space with grain contaminated by

96 A. ochraceus, a farmer developed with MAPK activation by DON is the 5.2 Acute toxicity acute renal disease after temporary activation of processes leading to respiratory distress (Di Paolo et the ribotoxic stress response, which In animals, DON has a wide range of al., 1993). Only limited estimates is induced by other translational proven toxicities, including feed refusal, of inhalation exposure to OTA from inhibitors that, like DON, bind to or decreased weight gain, gastroenteritis, occupational exposure are available damage a specific region at the 3′ cardiotoxicity, teratogenicity, and (Mayer et al., 2007). end of the 28S rRNA. The ribosome immunotoxicity (Rotter et al., 1996; plays a key role in the ribotoxic stress Meky et al., 2001; Pestka et al., 5. Deoxynivalenol response by serving as scaffolding for 2004; Pestka and Smolinski, 2005; interactions between various MAPKs Gray and Pestka, 2007; Amuzie and Deoxynivalenol (DON) is produced in (Pestka, 2008). Pestka, 2010). cereals, especially wheat, as the result DON toxicity studies have recently DON can cause acute poisoning in of growth of Fusarium graminearum revealed several possible approaches humans, where severe gastrointestinal and related species (see Chapter 1). for developing useful biomarkers of its toxicity is the primary symptom. JECFA has established a PMTDI for effects. For example, DON exposure in Consumption of cereals contaminated DON of 1 μg/kg bw/day on the basis mice results in upregulation of several with DON has been associated with of the NOEL for body weight reduction suppressors of cytokine signalling. numerous poisoning incidents in China in mice in a 2-year bioassay and a These suppressors are known to between 1961 and 1991 (see Pestka safety factor of 100 (WHO, 2001, impair growth hormone signalling and Smolinski, 2005) and a major 2011). The NOEL in mice was 100 μg/ (Pass et al., 2009). Impairment of outbreak in India (Bhat et al., 1989); kg bw/day (Iverson et al., 1995). the growth hormone axis precedes in some of these episodes, tens of the growth retardation in the mouse thousands of individuals were affected. 5.1 Mechanisms induced by DON (Amuzie and Pestka, In these outbreaks, symptoms were 2010). Oral DON perturbs the growth analogous to those observed in DON is directly toxic via an hormone axis by suppressing two animals, notably a rapid onset, nausea, epoxide moiety and thus does growth-related proteins, IGFALS and vomiting, abdominal pain, diarrhoea, not require metabolic activation to IGF1. Thus, reduced expression of headache, dizziness, and fever. In an exert its biological effects. Low- these two proteins in conjunction with episode in the Kashmir valley, DON level trichothecene exposure in elevated urinary DON levels could levels in wheat ranged from 0.4 mg/ animal models has been shown to potentially serve as biomarkers of kg to 8.4 mg/kg (Bhat et al., 1989), and modulate the expression of several effect. in China DON poisoning was linked cytokines and chemokines that are Detoxification varies by species; to wheat contaminated at DON levels key regulators of immune function metabolism by gut microflora between 0.3 mg/kg and 100 mg/kg (Pestka, 2008). Exposure to DON generates a de-epoxy metabolite (Pestka and Smolinski, 2005). These causes the upregulation of the (DOM-1), and conjugation to gluc- data suggest that acute toxicity may CHAPTER 6 CHAPTER mRNAs responsible for production uronic acid is catalysed by UDP- occur at exposures estimated in the of cytokines, chemokines, and other glucuronyltransferase (reviewed by low µg/kg bw/day range. immune-related proteins and can Pestka and Smolinski, 2005; Wu et al., also induce gene transcription. In 2007). In humans, DON-glucuronide 5.3 Cancer addition, DON modulates numerous has been reported in urine samples in physiological processes controlled several studies (Turner et al., 2008a, Minimal data are available on the by mitogen-activated protein kinases 2008b, 2008c). In contrast, information carcinogenicity of DON in either (MAPKs). These include processes on DOM-1 is limited. An absence of humans or experimental animals. controlling cell growth, differentiation, de-epoxidase activity in a small series A 2-year bioassay in B6C3F1 mice and apoptosis, which are all crucial of human faecal samples was reported of both sexes fed DON in the diet at for signal transduction in the immune (Sundstøl-Eriksen and Pettersson, concentrations of 0, 1, 5, or 10 mg/kg response (Pestka, 2008). Thus, in ad- 2003), whereas, in apparent contrast, showed no increase in the incidence dition to altered cytokine expression, a study in France of farmers exposed of pre-neoplastic or neoplastic lesions alterations in MAPK expression are to grain handling reported detection in the liver or other tissues (Iverson likely to also contribute to the immune of DOM-1 in a proportion of subjects et al., 1995). DON was also tested dysregulation and toxicity of DON and (Turner et al., 2010). for its ability to initiate or promote other trichothecenes. Also associated skin tumours when applied topically

Chapter 6. Mycotoxins and human health 97 to the skin of female SENCAR mice, handing of grain on farms in Finland, a PMTDI for ZEA of 0.5 µg/kg bw/day with negative results (Lambert et including grain drying, milling, and based on the NOEL for hormonal al., 1995). No studies have reported cattle feeding, were similar to those effects in pigs (WHO, 2001). on the carcinogenicity of DON in reported from Canada (Lappalainen humans. Oesophageal cancer in et al., 1996). 6.1 Mechanisms humans has been anecdotally linked Studies of DON concentrations to consumption of grains infected during grain handling in Germany ZEA is metabolized during ab- with Fusarium species that produce reported a median concentration of sorption by the intestinal tissue in DON and other mycotoxins, but no 2 ng/m3 and a maximum of 703 ng/ pigs. Metabolism involves reduction analytical epidemiological studies link m3 (Mayer et al., 2007). In France, of the 6-keto group of ZEA and results DON to the occurrence of any human urinary biomarkers for DON (DON, in formation of α- and β-zearalenol cancer (IARC, 1993). DOM-1) were higher in active farmers, as well as, upon further reduction, IARC has concluded that there particularly those from larger farms, α- and β-zearalanol, all of which can is inadequate evidence in both than in retired farmers, whose be conjugated in turn to glucuronic humans and experimental animals exposure was from diet (Turner et al., acid (WHO, 2001). Few data are for the carcinogenicity of DON. 2010). available in relation to metabolism in DON and other toxins derived from Epidemiological studies have humans. Studies of liver microsomes F. graminearum, F. culmorum, and been conducted in Norway relating in vitro have suggested a high rate of F. crookwellense have therefore to occupational exposures of male α-zearalenol production compared been categorized as Group 3, not and female farmers to mycotoxins. with that of β-zearalenol in pigs and classifiable as to their carcinogenicity Norwegian grains (wheat, oats, humans, a point of importance because to humans (IARC, 1993) (Table 6.1). and barley) are affected mainly by of the greater relative estrogenicity Fusarium head blight, and various of α-zearalenol compared with ZEA 5.4 Occupational exposure trichothecenes and culmorins have (Fink-Gremmels and Malekinejad, been reported as common (Langseth 2007). In other words, the formation Inhalation exposure to DON has been and Elen, 1996; Ghebremeskel of α-zearalenol may be considered a the subject of several health hazard and Langseth, 2001). A longitudinal bioactivation step contributing to the evaluations. Fusarium head blight in survey of farmers over more than two estrogenic effects of ZEA. wheat resulting from infection by F. decades suggested a relationship ZEA and its metabolites can bind to graminearum or F. culmorum begins between grain farming and mid- estrogen receptors, resulting in various at the outside of the grain head and pregnancy deliveries in the families of changes consequent to binding to moves inward. As a result, most DON farmers, possibly linked to mycotoxins elements in the nucleus responsive is found in the outer layers of the kernel (Kristensen et al., 1997). Small to estrogens. In addition, however, and the chaff (Miller, 1994b; Snijders, increased relative risks were observed ZEA is a competitive substrate for 1994). Grain dusts can contain quite in several cancers in female but not enzymes involved in steroid synthesis high concentrations of DON and male farmers (Kristensen et al., 2000). and metabolism and therefore has sometimes of other mycotoxins, which the potential to act as an endocrine are not always present in the kernels. 6. Zearalenone disruptor. ZEA can activate the For example, other fungi, including F. pregnane X receptor by displacement sporotrichioides, can grow on what Zearalenone (ZEA) is a non-steroidal of a co-repressor and recruitment becomes the chaff so that small estrogenic mycotoxin. It is produced of co-activators (Ding et al., 2006). amounts of T-2 toxin can be present. principally by F. graminearum and Thus, ZEA could have widespread Air samples collected in grain related species, and consequently effects on gene expression as a result elevators in Canada contained a occurs wherever DON occurs, most of the modified activity of this nuclear mean airborne concentration of 37 notably as a contaminant of maize, transcription factor. ng/m3 DON and a maximum of 2.59 wheat, barley, oats, rye, sorghum, μg/m3 DON. Airborne dust from the millet, and rice. Distribution of ZEA 6.2 Acute toxicity same source contained 0.5–5.8 mg/ is worldwide (Zinedine et al., 2007). kg DON, 1 mg/kg T-2 toxin, and low Estimates of human exposure from ZEA is considered to be of relatively levels of HT-2 toxin (De Mers, 1994). dietary sources are generally in the low acute toxicity. No reports have Concentrations of dusts, fungal range of 1–30 ng/kg bw/day (Zinedine appeared of acute poisoning due to spores, and DON associated with et al., 2007). JECFA has established ZEA in humans.

98 6.3 Cancer by mycotoxins. Inhalation of silica, Shotwell, 1983). Several interesting allergens, mycotoxins, and triple- toxins have been found in sclerotia ZEA resulted in an increased helical glucans contained in airborne of various Aspergillus species, again incidence of liver cell and pituitary dusts is a potential health risk, rarely at higher concentrations than occur tumours in mice, consistent with a from systemic exposure but from either in culture or in affected crops hormonal mode of carcinogenic action effects on lung biology. Outdoor work (Gloer et al., 1988; Wicklow et al., (IARC, 1993). No carcinogenic effect is normally not a problem, except 1988), some of which are thought to be was seen in rats, however, and overall when handling the most damaged present in conidia along with kojic acid animal carcinogenicity data for ZEA maize, small grains, and groundnuts. and other A. flavus toxins. Spores of F. were considered limited (IARC, 1993). In contrast, handling contaminated graminearum contained 30 µg/g of T-2 No studies of human carcinogenicity grains, especially damaged grains, in toxin and those of F. sporotrichioides have been reported for ZEA (Table 6.1). any confined space (e.g. grain storage, 50 µg/g (both approximately ZEA was measured in endometrial storage bin cleaning, animal feeding 10 –5 moles). The spores of many tissue from a small group of women in barns, indoors) carries a more species of toxigenic fungi have been with endometrial adenocarcinomas, significant risk. demonstrated to contain mixtures of endometrial hyperplasia, or normal The allergic disease hyper- the toxins associated with the species. proliferative endometria (Tomaszewski sensitivity pneumonitis, also called The high-molecular-weight toxic et al., 1998). ZEA in blood samples extrinsic allergic alveolitis or farmer’s compound present in spores and in has also been investigated in some lung, develops through repeated spore and mycelial fragments from small studies of early onset of puberty exposure to allergens. Dust from any the anamorphic Trichocomaceae (i.e. in Hungary (Szuets et al., 1997) and in mouldy crop, such as straw, maize, Penicillium, Aspergillus, and related Italy (Massart et al., 2008). In the study small grains, or groundnuts, can cause hyphomycetes) is (1→3)-β-D-glucan in Italy, there was a suggestion that the condition. Symptoms may include in the triple-helical form (Rand et al., elevated serum ZEA and α-zearalenol shortness of breath, a dry cough, a 2010). In the species tested so far, the levels were associated with early sudden general feeling of sickness, concentration is 1–11 pg/spore (Foto et puberty in 6 of the 17 girls examined fevers and chills, a rapid heart rate, al., 2004; Iossifova et al., 2008). from a rural area, but no positive and rapid breathing. The symptoms Inhalation of intact spores samples were seen in the 15 patients are serious, and once an allergic leads to little net exposure because from an urban area. reaction begins, the person will always their relatively large size enables have the potential for symptoms with entrapment and removal by lung 6.4 Occupational exposure exposure to the offending fungi. Long- defence mechanisms. However, in term exposure can cause permanent outdoor air, exposure is primarily to No studies of occupational exposure of lung damage, physical disability, or spore and mycelial fragments (Green humans have been reported for ZEA. even death (Sorenson and Lewis, et al., 2012) and to dusts (particulate 1996; Schenker et al., 1998; Girard et matter < 2.5 µm in diameter), which CHAPTER 6 CHAPTER 7. Occupational exposures to al., 2009). efficiently penetrate deep into the lung grain and groundnut dusts The reproductive structures of (Buczaj, 2008; see Miller et al., 2010). many fungi are known to contain Inhalation of toxins affects macrophage Grain dusts present an occupational mycotoxins or low-molecular-weight function and other aspects of lung hazard when protection of workers toxic compounds, often in high biology. Pure compounds tested is inadequate, and several health concentrations (Sorenson, 1999). Most for effects in macrophages include consequences are possible, including is known about the fungi that produce fumonisin, aflatoxin (Liu et al., the allergic disease hypersensitivity toxins important in agriculture. The 2002), T-2 toxin (Sorenson et al., pneumonitis, endotoxicosis, and conidia of A. flavus, A. parasiticus, F. 1986), and the aflatoxin precursor organic dust toxic syndrome (Rylander graminearum, and F. sporotrichioides sterigmatocystin (Miller et al., 2010). and Jacobs, 1994; Sorenson and contain very high concentrations DON has been tested for effects in Lewis, 1996). Endotoxicosis is a result of toxins, particularly in the case peritoneal macrophages (Ayral et al., of exposure to bacterial endotoxin, of the species that produce aflatoxins. 1992). and organic dust toxic syndrome (also The spores of the two Aspergillus called pulmonary mycotoxicosis, toxic species have been reported to contain organic dust syndrome, or grain fever), 100–1100 µg aflatoxin/g, or approx- as far as is known, is not caused imately 10–4 moles (Wicklow and

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104 chapter 7. Risk assessment and risk management of mycotoxins

Summary international trade. Apart from the well- 1. Introduction established risk posed by aflatoxins, Risk assessment is the process many uncertainties still exist about This chapter covers two key topics of quantifying the magnitude and risk assessments for the other major related to mycotoxins in human food: exposure, or probability, of a harmful mycotoxins, often reflecting a lack risk assessment and risk management. effect to individuals or populations of epidemiological data. Differences Managing risks of food contaminants from certain agents or activities. Here, exist in the risk management strategies such as mycotoxins is of global impor- we summarize the four steps of risk and in the ways different governments tance. Indeed, two international policy- assessment: hazard identification, impose regulations and technologies making bodies – the United Nations dose–response assessment, expo- to reduce levels of mycotoxins in the Environment Programme (UNEP) sure assessment, and risk char- food-chain. Regulatory measures and the World Health Organization acterization. Risk assessments have very little impact on remote rural (WHO) International Programme on using these principles have been and subsistence farming communities Chemical Safety (IPCS) – have declared conducted on the major mycotoxins in developing countries, in contrast that humans have a right to food free CHAPTER 7 CHAPTER (aflatoxins, fumonisins, ochratoxin to developed countries, where from mycotoxins that could cause A, deoxynivalenol, and zearalenone) regulations are strictly enforced to significant health risk. In highly populated by various regulatory agencies for reduce and/or remove mycotoxin parts of the world, mycotoxins in staple the purpose of setting food safety contamination. However, in the crops remain the most significant guidelines. We critically evaluate absence of the relevant technologies foodborne risk for human health, animal the impact of these risk assessment or the necessary infrastructure, we health, and market access (FAO/WHO/ parameters on the estimated global highlight simple intervention practices UNEP, 1999). burden of the associated diseases to reduce mycotoxin contamination Risk assessment and risk man- as well as the impact of regulatory in the field and/or prevent mycotoxin agement of mycotoxins deserve much measures on food supply and formation during storage. more global attention and action than

Chapter 7. Risk assessment and risk management of mycotoxins 105 they have been given. Wild and Gong identification, dose–response as- individuals exposed to higher levels (2010) identified several reasons sessment, exposure assessment, and of a particular mycotoxin than the rate for current inaction in addressing risk characterization (NRC, 2008). in a group of individuals exposed to mycotoxin risks in developing countries. lower levels of that mycotoxin, or not First, knowledge about mycotoxins and 2.1 Hazard identification exposed at all. A cohort study can be the full range of their risks to health is used to estimate the relative risk (RR) incomplete. Second, risks are poorly Hazard identification is the process of of a particular disease in exposed communicated to policy-makers in determining whether exposure to an versus unexposed populations: regions where mycotoxins are most agent can increase the incidence of prevalent. Third, the perceived value a particular adverse health effect. A RR = (Incidence of disease in the of interventions to reduce mycotoxin variety of different studies can help to exposed population)/(Incidence of risk is low compared with those of determine whether an agent causes disease in the unexposed other medical interventions, such as a specific health effect; these include population). vaccination programmes, malaria con- studies of adverse effects in humans, trol, and improved sanitation. Fourth, animals, and in vitro systems, and In the exposed group, if the intervention to control mycotoxins is mechanistic studies. number of people who develop the required at multiple time points both Epidemiological studies on the disease is a and the number of people before and after harvest. Fifth, regulation relationship between exposure to who do not develop the disease is of mycotoxins has minimal effects on a mycotoxin and particular harmful b, then the incidence of disease in food quality for subsistence farmers. effects obviously provide the best the exposed population is a/(a + b). Finally, mycotoxin contamination is a evidence for identifying a human Likewise, in the unexposed group, if problem that encompasses agriculture, health hazard. Studies that provide the number of people who develop the health, and economics, fields that a quantitative estimate of association disease is c and the number of people traditionally have not interacted at a between a hazard and a disease who do not develop the disease is d, research or policy-making level. include cohort and case–control then the incidence of disease in the This chapter covers both the studies. Other types of human unexposed population is c/(c + d). theoretical and quantitative aspects of studies, which may or may not In a case–control study, an risk assessment, to provide a background provide a quantitative estimate of investigator identifies a group of in how human health risks are assessed association, include studies that are individuals with a particular disease both experimentally and for decision- descriptive (case reports), ecological (cases) and a comparable group making purposes. Section 2 outlines the (geographical comparison), and of individuals without that disease four steps in the risk assessment process cross-sectional (observing disease (controls), and determines what and describes how risk assessments prevalence across different pop- proportion of the cases were exposed have been conducted for five myco- ulations at a single time point). and what proportion were not (Gordis, toxins: aflatoxins, fumonisins, ochratoxin In a cohort study, an investigator 2009). The assumption is that if the A, deoxynivalenol, and zearalenone. selects a group of initially healthy proportion of individuals exposed to Section 3, on risk management, is more individuals (a cohort) exposed to an agent is different between cases practical and focuses specifically on how any number of potentially hazardous and controls, then exposure to the to manage mycotoxin risks in foods. agents, and follows individuals in agent may be associated with an Finally, Section 4 briefly describes the this group over time to determine the increased or decreased occurrence importance of risk communication and incidence of a particular disease that of the disease. A case–control study public education about mycotoxins in may be associated with particular can be used to estimate an odds ratio countries at risk. exposures. Then, the incidence of (OR) to compare the odds that cases the disease in exposed individuals were exposed to the agent with the 2. Risk assessment is compared with the incidence odds that controls were exposed: in unexposed individuals, or the Risk assessment is the process incidence in a highly exposed group OR = (Odds that a case is of estimating the magnitude and may be compared with the incidence exposed)/(Odds that a control is probability of a harmful effect to in a group exposed to lower levels. exposed). individuals or populations from certain For example, it may emerge that over agents or activities. Four steps are time, the incidence rate of a particular Using the same variables de- involved in estimation of risk: hazard cancer is higher in a group of scribed above to estimate RR, the

106 odds that a case is exposed is are chosen for or respond to a equations are most useful in setting a/(a + c) and the odds that a control is study, or information biases such as priorities for further research, thereby exposed is b/(b + d). those associated with interviewing, reducing the number of animal More recently, attention has surveillance, recall, or reporting. experiments required. Requirements focused on whether it is possible to for SAR modelling thus include a take advantage of the benefits of both 2.1.2 Animal studies database of chemicals of known of these study types by combining toxicity or carcinogenicity, information elements of both cohort and case– Animal studies (in vivo) are often used about their chemical and spatial control approaches into a single study. in place of human studies for hazard structure, and information about their For example, a case–control study can identification, for reasons described physicochemical properties. be initiated within a cohort study, i.e. above. The major concern with animal the individuals within the cohort who studies is that extrapolation from 2.2 Dose–response develop a particular disease during the results in another species to results in assessment study period can be considered cases humans is imperfect. However, animal and those who do not develop the experiments have several advantages: The second step in risk assessment, disease can be considered controls. dose levels of the hazardous agent dose–response assessment, deter- In nested case–control studies, the may be varied, other conditions may mines the relationship between controls are a sample of individuals be kept constant across all groups the dose of a toxic agent and the who are at risk for the disease at to reduce confounders, and an occurrence of health effects. This the time each case of the disease animal group can specifically be kept relationship is often graphically repre- develops (Gordis, 2009). unexposed as controls. Then, the sented in a dose–response curve, incidence of a health effect can be which shows the proportion of a 2.1.1 Human studies compared across these groups. given population that experiences an adverse health effect (on the vertical Human studies for the purpose 2.1.3 Cell and tissue culture axis) at different doses of a toxic agent of risk assessment can be studies (on the horizontal axis). The shape of problematic, for several reasons. the dose–response curve is a critical First, epidemiological studies exist Cell and tissue culture assays, unlike component in policy-making to control for only a few hazardous agents. human and animal studies, are in vitro human health risks. Second, circumstances of human assays, and these are increasingly Usually, dose–response data are exposure levels and duration are important experiments for identifying gathered from animal and/or human difficult to measure precisely, and hazards. Isolated cells or tissues (or studies. Animal studies are especially there is often a time interval between microorganisms) are prepared and useful for dose–response assessment when exposure occurs and disease maintained in culture by methods that because the exact doses of a particular symptoms appear. Third, even well- preserve some in vivo properties, and, toxin can be carefully controlled. planned studies cannot always after exposure to a hazardous agent, Different groups of animals are exposed show a clear causative association they can be tested for point mutations, at each dose level through food, water, between an agent and a disease. chromosomal aberrations, DNA air, or dermal contact, and one group Many potential confounders exist repair or damage, gene expression, of animals is not exposed to the toxin that can contribute to the disease cell transformation, and metabolic at all (control group). The prevalence etiology, such as exposure to multiple and other physiological effects. These of a health effect, or the lack thereof, environmental disease agents, varied tests may give information on potential is measured in each animal group at CHAPTER 7 CHAPTER nutritional status, and genetic factors mechanisms of the biological end- each dose level, and these results are that modulate disease susceptibility. point (e.g. an adverse health effect). plotted in a curve. Finally, human studies may be Structure–activity relationship Dose–response assessment ad- susceptible to bias or systematic (SAR) models allow the toxicological dresses toxic (non-carcinogenic) and errors in the design, conduct, or activity of an unknown chemical or carcinogenic effects separately and analysis of the studies that result in agent to be predicted on the basis of its differently, even if an agent can cause mistaken estimates of the effect of chemical and/or structural properties. both types of effects (e.g. aflatoxin). an exposure on disease risk (Gordis, The relationships are computationally These effects are briefly described 2009). For example, there may be derived from information about agents here. selection bias in how individuals of known toxicity. These predictive

Chapter 7. Risk assessment and risk management of mycotoxins 107 2.2.1 Toxic (non-carcinogenic) a model that best fits the overall for policy-making purposes, this type effects shape of the dose–response curve of extrapolation from an animal study in an animal study and then, from to a human PMTDI has often caused For toxic but non-carcinogenic ef- that model, finding the dose that controversy because of the arbitrary fects caused by an agent, it is as- corresponds to a proportion (usually nature of choosing uncertainty factors. sumed that protective mechanisms 10% or 5%) of response in the test Practically speaking, the safety factor must be overcome before the animals. This particular dose is assumes that humans are 10 times as adverse health effect can occur. A called the benchmark dose. Then, sensitive as the most sensitive animal threshold dose of the toxin may exist, the lower bound of the confidence species tested for a particular toxin i.e. humans and animals can be interval around that dose (from and that the most sensitive human exposed to the toxin at doses below uncertainties in the animal study is 10 times as sensitive as the least this particular threshold without itself) is calculated. This benchmark sensitive human. experiencing the health effect in dose lower confidence limit (BMDL10 question. The threshold dose is or BMDL05) is used as the point of 2.2.2 Carcinogenic effects referred to as the no-observed-effect departure for extrapolating to a TDI level (NOEL), sometimes called the for humans. In contrast to most toxic effects, no-observed-adverse-effect level To extrapolate animal toxicity data carcinogenesis may be regarded as (NOAEL). In animal studies, the to humans, a provisional maximum a process in which the presumption NOEL is the highest dose of a toxin tolerable daily intake (PMTDI), also of no threshold may be appropriate at which no significant increases in called a reference dose (RfD) in some if the chemical is (directly acting) or the frequency or severity of adverse cases, is calculated. The PMTDI is the its metabolites are (indirectly acting) effects are observed when an dose below which humans exposed to reactive with genomic DNA (Klaunig exposed group is compared with the agent in question are not expected and Kamendulis, 2008). For such an unexposed group. Sometimes a to experience adverse health effects. carcinogens, theoretically even a single lowest-observed-effect level (LOEL), The PMTDI is derived from the NOEL molecular event could evoke changes the lowest dose in the experiment or BMDL by applying uncertainty in genomic DNA leading to mutations, at which an adverse effect can be factors, which can account for selective cellular proliferation, and observed, is used in place of or in interspecies variability (extrapolating cancer. Hence, it is assumed that addition to the NOEL. from animals to humans), in- there is no safe dose above zero. For The NOEL is one metric used traspecies variability (to protect example, it is assumed that there is to evaluate the toxicity of an agent. sensitive individuals), use of a LOEL no threshold of exposure to aflatoxin

Another is the median lethal dose instead of a NOEL, the chronicity of B1 below which cancer would never

(LD50), the dose of a toxin that, when the study, and other terms reflecting occur, because aflatoxin B1 has a administered to a group of animals the professional assessment of reactive metabolite that interacts over a specified period of time, is lethal additional uncertainties in the data. directly with DNA. Experiments in to 50% of the animals. LD50 values are The PMTDI is calculated by dividing rats exposed to aflatoxin B1 in their used to rank toxicity across multiple the NOEL or BMDL by the product of drinking-water showed that the level agents. Other metrics include the the uncertainty factors: of DNA adducts in the liver was

10% lethal dose (LD10), the dose of a linear over 6 orders of magnitude toxin lethal to 10% of test animals, and PMTDI = NOEL/(product of after both single and chronic dosing; the median effective dose (ED50), the uncertainty factors), or dose levels reached those seen in dose that causes a particular health exposed human populations (Buss et effect – not necessarily death – in PMTDI = BMDL/(product of al., 1990). Additional mechanisms of 50% of test animals. uncertainty factors). carcinogenicity may exist that do not More recently, benchmark dose involve genotoxicity, which could be modelling – which uses the entire Often, the uncertainty factors relevant for other mycotoxins. dose–response curve from an animal for interspecies and intraspecies For policy-making purposes, car- study – has been used to provide variability are each given the value cinogens are evaluated in two parts. a different point of departure from of 10. Therefore, the human PMTDI First, a weight of evidence (WOE) is the NOEL in calculating a tolerable for a non-carcinogen is usually 0.01 designated, and second, a slope factor, daily intake (TDI). Benchmark times the NOEL or BMDL found in or cancer potency factor, is calculated dose modelling involves finding animal studies. It is worth noting that on the basis of the dose–response

108 curve. WOE is a term that refers to the Table 7.1. IARC classification of agents as to carcinogenicity to humans based on strength of the evidence that a particular the weight of evidence agent causes cancer in humans. Category Significance Two organizations that provide WOE evaluations for carcinogens are the Group 1 Carcinogenic to humans International Agency for Research Group 2A Probably carcinogenic to humans on Cancer (IARC) and the United States National Toxicology Program Group 2B Possibly carcinogenic to humans

(NTP). IARC classifies agents as Group 3 Not classifiable as to its carcinogenicity to humans to carcinogenicity to humans by considering the WOE (Table 7.1). Group 4 Probably not carcinogenic to humans For example, IARC has classified naturally occurring mixtures of is also a critical component of all daily dose (ADD) or intake. This term aflatoxins as Group 1, carcinogenic epidemiological studies and is often is usually expressed as the mass of to humans, and fumonisin B1 and used to identify control options or substance in contact with the body ochratoxin A as Group 2B, possibly technologies to reduce risk. per unit body weight (bw) per unit carcinogenic to humans. time, such as in mg/kg bw/day, for For carcinogenic agents that are 2.3.1 Calculating exposure ingestion exposures, or as the mass of reactive with DNA, a slope factor, or substance per cubic meter of ambient cancer potency factor, is calculated by Three steps are involved in air, such as in mg/m3, for inhalation estimating the slope of the linearized calculating exposure: (i) charac- exposures. If the agent being studied is dose–response curve. Sometimes, terizing the exposure setting, (ii) a carcinogen, a lifetime average daily for policy-making purposes, the identifying exposure pathways, and dose (LADD) is calculated, with an slope factor is actually the upper 95% (iii) quantifying exposure. averaging time equal to the expected confidence limit of the dose–response To characterize an exposure lifetime of the individual (e.g. the United slope. In practical terms, the slope factor setting, both the physical setting States Environmental Protection or cancer potency factor estimates the and the exposed populations must Agency assumes an averaging time increase in probability of developing the be understood. The physical setting for cancer risk assessment of 70 years, particular cancer per unit dose of the includes factors such as climate, although the average lifespan in the agent over a human lifetime. Thus, the geographical setting, vegetation, soil USA is now longer than this). steeper the dose–response curve for a type, and location of water. Potentially To calculate ADD or LADD, the carcinogenic agent, the more potent it is exposed populations include both exposure quantity E must first be in causing cancer. For carcinogens that humans and animals: those living estimated. It is the concentration of do not directly damage DNA, the NOEL nearest the risky agent; those with an agent as a function of time t, can be used for setting a PMTDI. For diets, water, or air supply containing over an exposure duration. The total example, the long-term nephrotoxicity the agent; those who come to an exposure quantity during that given of fumonisin B1 is a prerequisite for the area near the risky agent for work time is expressed as the integral renal carcinogenicity of fumonisin B1 or play; and any other demographic (sum) of concentrations C over the (Dragan et al., 2001). In this case, the groups (e.g. based on age or sex) exposure duration: renal carcinogenicity is subsumed by that would be disproportionately the dose–response relationship for exposed to the agent. E = ∫C(t) dt. the nephrotoxicity, which is clearly a Characterizing an exposure CHAPTER 7 CHAPTER threshold event (Bolger et al., 2001). pathway involves identifying sources However, the integrated concen- and points of contact, media that tration can be difficult to obtain, so 2.3 Exposure assessment transport the agent to the population, instead one can estimate the arithmetic

ways in which the agent may react or average of the concentration Cave over The third step in risk assessment, change in transport media, and other the exposure duration ED to estimate exposure assessment, is the pro- physical and chemical properties of the the total exposure: cess of estimating the intensity, fre- agent that explain its fate as it moves quency, and duration of human or along its pathway to a target population. E = Cave * ED. animal exposures to an agent in the Finally, to quantify exposure, it environment. Exposure assessment is common to estimate an average

Chapter 7. Risk assessment and risk management of mycotoxins 109 The intake rate IR is the amount can also be used to assess the ef- the dose–response information from of the agent passing through the initial fectiveness of interventions to reduce the specific agent. Discussion of major intake barrier (mouth, nose, skin) into mycotoxin exposure. Measurement assumptions, scientific judgements, the body over a period of time. This of biomarkers related to mycotoxin and estimates of uncertainties can be measured in mg/day or L/day, exposure typically requires samples should also be part of the final risk for example. Then, the ADD is of either urine or serum. As described characterization. calculated as: in Chapter 6, the measurement of validated biomarkers for aflatoxin 2.4.1 Risk characterization of ADD = (E * IR)/(BW * AT), exposure in human populations has non-carcinogenic toxins greatly assisted epidemiological where BW is body weight and AT is studies. Validated biomarkers for Simplistically, determining whether the averaging time in days. LADD fumonisins and deoxynivalenol have an individual or a population may is calculated similarly; in this case, also been developed. However, a suffer a health risk from a hazard however, the exposure duration (used past occurrence of measurement relies on knowing whether their to calculate E) is the number of years of an unvalidated biomarker for average daily exposure is greater that an individual is exposed to a ochratoxin A resulted in inaccurate than the daily dose of the hazard carcinogen, and the averaging time representation of actual dietary that may cause adverse effects. To AT is the expected lifetime in years. intake (Gilbert et al., 2001). Moreover, recapitulate: the PMTDI is estimated collection of samples for measuring from dose–response assessments 2.3.2 Estimating human exposure biomarkers in human populations that determine a NOEL or BMDL of to mycotoxins may also pose cultural challenges. a particular hazardous agent. The Without accurate exposure data, PMTDI is obtained by dividing the Many difficulties have been en- quantitative risk assessments can NOEL or BMDL by uncertainty factors countered in estimating mycotoxin be limited, because exposure is a that take into account extrapolation exposure in human diets. Until major component of the calculations. from laboratory animals to humans recently, human exposure to Hence, it is important to ensure that and variation among humans. Thus, mycotoxins was measured almost measurements are carried out in the PMTDI is an estimate of a daily exclusively in one of two ways: by the most accurate and reliable way exposure level for humans, including questionnaires or food diaries relying possible. sensitive individuals, that is unlikely on recall of what and how much to cause adverse health effects. The had been eaten; or by food samples 2.4 Risk characterization ADD can be calculated based on human collected from populations, which exposure to the hazardous agent. ideally were representative of true Risk characterization, the final step If ADD > PMTDI, then the exposures. Both of these ways pose in risk assessment, combines the potential for health risk exists. One potential problems. Dietary recall is information on exposure with that way to express this is the hazard often inaccurate. It can be difficult in on toxicity and dose–response quotient (HQ) used by the United many cultures worldwide to take food assessment to determine whether States Environmental Protection samples without disturbing social an individual or a population is Agency. HQ is calculated as the ratio contexts, and measurement may experiencing a significant risk of of the ADD of a particular agent to the lead to abnormal eating behaviour illness or disease based on exposure PMTDI of that same agent: during recording. In addition, snack to a hazardous agent. It translates foods may be a significant source of the available data to describe this HQ = ADD/PMTDI. exposure to mycotoxins, but these significance to a broad audience. may not be recalled and may not be Risk characterization, like dose– If HQ > 1, then an individual or a measured (Hall and Wild, 1994). response assessment and exposure population may suffer a health risk In recent years, however, bio- assessment, is conducted differently due to their levels of exposure to the markers to assess mycotoxin for non-carcinogens and carcinogens. hazardous agent. If HQ < 1, then the exposure, internal dose, and bio- For toxic effects, the individual individual or population is unlikely logically effective dose have been or population ADD of an agent is to suffer a health risk from current developed and are increasingly being compared with the PMTDI of the agent. exposure levels to the agent. used to estimate human exposure For carcinogenic effects, the individual (Groopman et al., 2008). Biomarkers or population LADD is compared with

110 2.4.2 Risk characterization of quantitative cancer risk assessments consumption levels of maize and carcinogens can be conducted (WHO, 1998). Based groundnuts, and HBV prevalence. on the WOE of the effects of aflatoxins The cancer potencies of aflatoxin Rather than using a threshold, such in human, animal, and in vitro studies, for HBV-positive and HBV-negative as HQ in the evaluation of non- IARC has classified naturally occurring individuals were considered, together carcinogens, carcinogenic risk is mixtures of aflatoxins as Group 1, with uncertainties in all variables. Liu estimated for an individual or a carcinogenic to humans (IARC, 2002). and Wu estimated that of the 550 000 population over an expected lifetime. However, the carcinogenicity, or cancer to 600 000 new HCC cases worldwide Policy-makers may then determine potency, of aflatoxins differs in humans per year, about 25 200 to 155 000 may whether this expected lifetime with and without chronic hepatitis B be attributable to aflatoxin exposure. risk is acceptable or whether the virus (HBV) infection. The risk of HCC In other words, aflatoxin may play carcinogen should be regulated. The attributable to aflatoxins is up to 30- a causative role in 4.6–28.2% of all WOE, based on available studies as fold higher in populations chronically global HCC cases. Most cases occur described above, also factors into infected with HBV than in uninfected in sub-Saharan Africa, South-East policy decision-making about the populations (Groopman et al., 2008). Asia, and China, where populations carcinogenic agent in question. In 1998, the Joint FAO/WHO suffer from both high HBV prevalence To reiterate: the slope factor of Expert Committee on Food Additives and largely uncontrolled exposure to a carcinogen is derived by taking (JECFA) undertook an aflatoxin and aflatoxin in food. the slope of the linearized dose– HCC risk assessment to estimate the response curve. LADD is estimated impact on population HCC incidence 2.5.2 Fumonisins based on an exposure of an individual of moving from a hypothetical total to the carcinogenic agent. Then, aflatoxin standard of 20 μg/kg to a Compared with the risk assessment stricter standard of 10 μg/kg (WHO, of aflatoxins, risk assessments of Risk = LADD * slope factor, 1998; Henry et al., 1999). Assuming fumonisins and ochratoxin A for risk that all food that contained aflatoxin management are more complex where Risk is a unitless probability of levels higher than the standard would from the human health standpoint an individual developing cancer over be discarded and that enough maize because of the lack of convincing a lifetime (for example, 3 in a million, and nuts would remain to preserve evidence linking either mycotoxin or 3 × 10–6) from being exposed to the consumption patterns, JECFA de- to human disease (WHO, 2001b) carcinogenic agent. termined that HCC incidence would and, in the case of ochratoxin A, the decrease by about 300 cases per controversy over its mechanism of 2.5 Risk assessment of billion people per year if the stricter action (WHO, 2001b). mycotoxins aflatoxin standard were applied in Gelderblom et al. (2001) estimated countries with an HBV prevalence of the NOAEL for HCC induction by

A detailed discussion of the human 25%. However, in countries where fumonisin B1 in male rats at 0.8 mg/ health risks associated with af- the HBV prevalence was 1%, using kg bw/day, which translated to a TDI latoxins, fumonisins, ochratoxin A, the stricter aflatoxin standard would of 0.8 μg/kg bw/day when divided deoxynivalenol, and zearalenone is decrease HCC incidence by only 2 by a safety factor of 1000. The given in Chapter 6. In this section, we go cases per billion people per year. This justification for the large safety factor into more detail about risk assessment assessment associated HCC risk with is that although fumonisin does not of each of these mycotoxins for the particular doses of aflatoxin. However, directly damage DNA, it induces purposes of policy-making. these doses do not correspond to cancer in rats and mice (IARC, 2002) CHAPTER 7 CHAPTER actual doses in food in different parts of and is a cancer promoter (Carlson et 2.5.1 Aflatoxins the world, and two hypothetical values al., 2001; Gelderblom et al., 2002). for HBV prevalence were assumed: 1% Adopting such a standard, however, The evidence for aflatoxins causing and 25% (Liu and Wu, 2010). could seriously affect the food supply liver cancer (hepatocellular car- Liu and Wu (2010) estimated the and trade between producers and cinoma [HCC]) in humans has global burden of HCC induced by consumers (Steyn et al., 2008). been established from decades of aflatoxin by using the quantitative JECFA set and then retained a epidemiological research. These cancer risk assessment described PMTDI for fumonisins of 2 μg/kg bw/ studies have elucidated dose– above and collecting national data day, based on one evaluation of a response relationships from which on foodborne aflatoxin levels, NOAEL for nephrotoxicity in male

Chapter 7. Risk assessment and risk management of mycotoxins 111 rats and a safety factor of 100 (WHO, than did long-term effects such as 2001). IARC has categorized DON 2001b), and then more recently based carcinogenicity, so the previous PTWI, as Group 3, not classifiable as to its on a benchmark dose (BMDL10) that set on the basis of nephrotoxicity carcinogenicity to humans (IARC, causes megalocytic hepatocytes in before carcinogenicity was definitely 1993). A 2-year study of mice exposed male mice and a safety factor of 100 established, was retained (WHO, to DON suggested no carcinogenic (WHO, 2011). The safety factor of 100 2008). IARC has classified OTA as hazard. Although the weight of the was deemed appropriate because Group 2B, possibly carcinogenic to mice exposed to DON was lower than the mechanism of toxicity and humans (IARC, 1993). that of the controls, the difference was carcinogenicity did not involve direct The current average dietary ex- not considered biologically significant. damage to DNA. IARC has classified posure levels to OTA have been JECFA established a PTMDI for DON fumonisin B1 as Group 2B, possibly determined by JECFA to be 8–17 ng/ of 1 µg/kg bw/day, based on the NOEL carcinogenic to humans (IARC, 2002). kg bw/week (WHO, 2008). These (for decreased body weight at day levels are well below the PTWI; 500) in this mouse study of 100 µg/ 2.5.3 Ochratoxin A however, JECFA had previously kg bw/day and a safety factor of 100 determined (WHO, 2001b) that the (WHO, 2001a). The mechanism of carcinogenicity of 95th percentile for OTA consumption ochratoxin A (OTA) remains unclear. was about 84–92 ng/kg bw/week, 2.5.5 Zearalenone Six hypotheses about this mechanism approaching the PTWI. A recent were listed by JECFA at its most recent reappraisal of the risk associated The toxicity of zearalenone (ZEA) evaluation (WHO, 2008). Positive with OTA resulted in a reduced was evaluated by JECFA in 2000. results for genotoxicity were usually PTWI of 21 ng/kg bw/week, one fifth Reproductive and developmental obtained only at high OTA exposure of the JECFA estimate, based on effects, as well as estrogenic effects, levels and were usually indicative of applying an even larger uncertainty were found in a variety of animal oxidative damage. JECFA concluded factor to the study of Krogh et al. species, including rats, mink, and that a direct genotoxic mode of action (1974) evaluated by JECFA (Kuiper- pigs (WHO, 2000). There is, however, remains unconfirmed and that several Goodman et al., 2010). little evidence for acute toxicity or possible modes of non-genotoxic Dietary exposure is based pri- carcinogenicity. In humans, ZEA was action could be involved in the marily on data from Europe, where suspected to have caused premature formation of renal tumours induced processed cereal foods often show thelarche in girls, but the evidence by OTA. Non-carcinogenic effects high levels of contamination with was inconclusive and other causative may occur at lower levels that those OTA (Steyn et al., 2008; WHO, 2008). or contributing agents could not be inducing tumour formation, however. Contamination of cereals in Europe ruled out (WHO, 2000). IARC has On the basis of a study on damage is due to Penicillium verrucosum, categorized ZEA as Group 3, not to renal function in pigs (Krogh et which is a fungus of cool climates classifiable as to its carcinogenicity to al., 1974) and a safety factor of 500, and does not occur in the tropics. humans (IARC, 1993). Using a safety JECFA established a provisional Although sometimes contaminated factor of 80, JECFA set a PMTDI for tolerable weekly intake (PTWI) for by Aspergillus species capable of ZEA of 0.5 µg/kg bw/day, based on OTA of 112 ng/kg bw/week (WHO, producing OTA, cereals in tropical the NOEL (for reversible increase 1991). This finding was confirmed countries are not usually a major in length of estrous cycle) of 40 µg/ by JECFA in 1995, when the PTWI source. Coffee and cocoa are kg bw/day in a 15-day study in pigs was rounded off to 100 ng/kg bw/ potential sources of OTA exposure in (WHO, 2000). week (WHO, 1995). For the most tropical countries (WHO, 2008). recent evaluation, JECFA modelled 3. Risk management carcinogenicity data on OTA from rat 2.5.4 Deoxynivalenol bioassays performed by the United Risk assessments have confirmed States National Toxicology Program The toxicity of deoxynivalenol (DON) health risks to human populations (NTP, 1989). Six different models has been reviewed by WHO and worldwide from several mycotoxins were used on data on carcinoma in IARC, and risk assessments including in food, including aflatoxins, fu- the male rat kidney, the most sensitive toxicological reviews of DON have monisins, ochratoxin A, and de- sex, species, and target organ. Risk been published for Canada, the oxynivalenol. Although several assessment indicated that acute Nordic Council, the Netherlands, potential interventions exist by which toxicity occurred at lower levels and the European Union (WHO, to manage mycotoxin risks in food,

112 control is very difficult in practice. At The risk of mycotoxin contam- dietary staple. There, populations may this time, no single strategy enables ination of commodities and foods is be exposed to potentially dangerous risk from mycotoxins to be eliminated greatest in LICs, where agricultural amounts of fumonisin from maize in any country. systems are often poorly equipped to even if that maize meets regulatory Mycotoxin risks in food can be handle food safety risks. Suboptimal standards. Table 7.2 highlights how managed either by governmental field practices and poor storage people who have a very high maize regulations or by agricultural and public conditions make the crops vulnerable intake can ingest dangerous levels health interventions. Governments to fungal infection and subsequent of fumonisin even if the maize itself can impose food safety standards mycotoxin accumulation. The prob- is considered relatively clean by that specify a maximum tolerable limit lem is exacerbated by the fact that regulatory standards, whereas people of a particular mycotoxin in human maize and groundnuts, two of the who consume very small amounts food. At the same time, agricultural food crops that are most susceptible of maize could ingest a much more and public health interventions can be to aflatoxin contamination, are staples contaminated commodity without adopted to reduce mycotoxin levels in the diets of many people worldwide, having significant fumonisin exposure in food or reduce the bioavailability of and thus aflatoxin exposure is higher (Gelderblom et al., 2008). mycotoxins. where dietary variety is difficult to Hence, regulatory standards Mycotoxin standards have reduced achieve (Shephard, 2008). In good for mycotoxins in food sometimes foodborne mycotoxin risk in developed seasons, subsistence farmers and have no impact, or even potentially countries because enforcement is local food traders may be able to adverse impacts, on human health strong and because technologies and avoid eating obviously mouldy maize in LICs (Wu, 2004; Shephard, 2008; methods exist to successfully reduce and groundnuts, but in drought Williams, 2008). Instead, the focus or remove mycotoxin contamination. seasons, or in situations of food for risk management should be Commodities with moderately exces- insecurity, often people have no on technologies and public health sive levels of mycotoxins are removed choice but to eat mouldy food or interventions to reduce mycotoxin from the food stream and used as starve (Wu and Khlangwiset, 2010a). risk, infrastructures to support these feeds for animals that tolerate higher Indeed, 125 people died due to acute technologies, and public education levels of mycotoxins, such as beef aflatoxicosis in rural Kenya in 2004 (Fig. 7.1). cattle, or as biofuels or fertilizers. when food insecurity, caused by a Mycotoxins can be managed at In low-income countries (LICs), variety of climatic and social factors, various points along the food production the situation is quite different. In led to widespread consumption of chain from the field to the plate. In many parts of the world, regulatory maize contaminated with high levels pre-harvest, or field, conditions, using standards for mycotoxins in food have of aflatoxins (Lewis et al., 2005). good agricultural practices, such as little or no impact on actually reducing One additional limitation of the choosing appropriate cultivars for the mycotoxin risk, for several reasons. setting of mycotoxin standards is geographical region, can reduce the First, many rural farmers engage that such standards are usually in risk of fungal infection and subsequent in subsistence farming, in which the form of an allowable mycotoxin mycotoxin accumulation. Post-harvest case food grown on farms is directly concentration in a particular food interventions involve careful sorting, consumed by the families without commodity, such as maize. Such a cleaning, drying, storage, transportation, ever undergoing a formal inspection standard does not take account of the and processing to reduce the risk of process for mycotoxins. Second, even fact that some populations consume further mycotoxin accumulation, or to if regulatory standards exist for certain much more of the food commodity lower mycotoxin levels directly. mycotoxins, there is often little to no than other populations in the world; Much of the work that has been CHAPTER 7 CHAPTER enforcement of these standards in hence, those populations could be done on dietary methods to reduce certain parts of the world. Third, if the much more exposed even if regulatory the bioavailability of mycotoxins regulatory standards are imposed by standards were enforced. has pertained specifically to afla- importing countries, farmers in LICs For example, regulations on toxin. However, one simple dietary may export their best quality foods and allowable fumonisin levels in maize intervention that applies to all keep the poorer quality for domestic may sufficiently protect populations mycotoxins is, where feasible, to use, inadvertently raising health risks that do not typically consume large consume less of the foodstuffs that related to mycotoxins in populations amounts of maize, but the allowable contain the mycotoxins. In the case of already vulnerable to disease because levels may be too high in many parts of aflatoxin, that would mean consuming of poverty (Wu, 2004). sub-Saharan Africa, where maize is a less maize and groundnuts if

Chapter 7. Risk assessment and risk management of mycotoxins 113 possible, in favour of other food Table 7.2. Probable daily intake of fumonisins (µg/kg bw/day) as a function of maize crops that have significantly lower intake and fumonisin contamination levels in food aflatoxin contamination, such as rice, a sorghum, or pearl millet. An example Fumonisin Maize intake (g/person [60 kg]/day) is the recent economic growth in level (mg/kg) China that has led to reduced maize 10 50 100 150 200 400 500 consumption, and hence reduced aflatoxin exposure (Wild and Gong, 0.2 0 0.2 0.3 0.5 0.7 1.4 1.7 2010). Where it is not easy to make such a dietary shift (e.g. where maize 0.5 0.1 0.4 0.8 1.3 1.7 3.4 4.2 and groundnuts have traditionally been staples), other dietary inter- 1 0.2 0.8 1.7 2.5 3.3 6.6 8.3 ventions may prove helpful. These interventions are described in greater 2 0.3 1.7 3.3 5.0 6.7 13.4 16.7 detail in Chapter 9. One public health intervention does not result in lower mycotoxin 3 0.5 2.5 5.0 7.5 10.0 20.0 25.0 levels but can reduce the adverse effects associated with HCC induced 4 0.7 3.3 6.7 10.0 13.3 26.6 33.3 by aflatoxin: vaccination against HBV. A regular practice now in developed a The shaded values are those intakes closest to the Joint FAO/WHO Expert Committee on Food Additives (JECFA) provisional maximum tolerable daily intake (PMTDI) of 2 µg/kg bw/day for fumonisin B1 (FB1), FB2, and FB3 alone countries, HBV vaccination in children or in combination. still requires wider implementation. Adapted, by permission of the publisher, from Gelderblom et al. (2008). Vaccinating children against HBV over the past three decades has Fig. 7.1. Mycotoxin control in developing countries. In the absence of enforcement resulted in significantly decreased of mycotoxin standards, technology, appropriate infrastructure, and public education are required to implement strategies that reduce mycotoxin risk. HBV infection rates in several countries and regions, including Europe (Williams et al., 1996, Bonanni et al., 2003); Taiwan, China (Chen et al., 1996); and Thailand (Jutavijittum et al., 2005). This vaccine may have significant impacts on HCC incidence, particularly in Africa and Asia, where the current prevalence of chronic HBV infection is relatively high. To date, a reduction in HCC incidence resulting from HBV vaccination has been demonstrated in children and adults in Taiwan, China, after vaccination was introduced in 1984 (Chang et al., 2009). By lowering the prevalence of chronic HBV infection, this vaccination should prevent the synergistic interaction between HBV and aflatoxin in inducing HCC (Khlangwiset and Wu, 2010; Liu and Wu, 2010).

114 4. Mycotoxin risk and public two crops susceptible to mycotoxin shown to be significantly correlated education contamination, are staples in the with farmers’ knowledge of aflatoxin diets of many people in Africa, risk (Jolly et al., 2006), whereas in There is no question that mycotoxins Asia, and Latin America. Therefore, Benin farmers’ knowledge of aflatoxin in food pose a significant public interventions should focus on risk has been shown to be correlated health risk in many parts of the world. reducing mycotoxin risk in simple and with the motivation to implement Risk assessments conducted using cost-effective ways. interventions to reduce aflatoxin human, animal, and in vitro studies It is important to remember, formation (Jolly et al., 2009). and exposure assessments have however, that public health inter- Education must take place on confirmed the reality of this global ventions must be readily accepted by at least three different levels. First, health problem. HCC is a leading their target populations to have any government policy-makers must cause of cancer deaths worldwide meaningful impact, and must have receive information about the burden (Ferlay et al., 2008), and an HCC risk financial and infrastructural support of mycotoxin-induced disease in their assessment reveals that aflatoxin to be feasible in the parts of the world countries – in terms of effects on both may play a causative role in more than where they are most needed (Wu public health and food markets – as one quarter of HCC cases (Liu and and Khlangwiset, 2010b). A critical well as information about possible Wu, 2010). Epidemiological studies component to implementing any or all interventions, their cost-effectiveness have also linked aflatoxin exposure of these methods is public education in reducing mycotoxins, and their with stunted growth in children, acute (Wild and Gong, 2010). Educational technical feasibility requirements. aflatoxicosis, and liver cirrhosis efforts should include not only how to Supplying the appropriate information and have suggested fumonisin use the intervention properly to achieve will be one step in sensitizing exposure as a possible risk factor for maximum benefit for mycotoxin risk governments to provide the finances oesophageal cancer and neural tube reduction but also why the interventions and other resources necessary to defects (Wild and Gong, 2010). are important from the perspectives of initiate the interventions. Second, In developed countries, mycotoxin public health and food markets, so that depending on the intervention exposure in the diet is controlled by users have incentives to continue with characteristics, the farmers, the good agricultural practices, good the interventions. consumers, or both these groups storage and processing, and control of Public and governmental ed- must receive education on why excess levels of the major mycotoxins ucation on mycotoxin risk is crucial mycotoxins are a concern and how by standards and enforced to provide incentives to adopt to implement the intervention in regulation. However, many countries interventions. Even if an intervention question. Third, international health where the risk is particularly high to reduce mycotoxin risk is cost and agricultural organizations must have neither the technologies nor the effective in terms of lives saved and be informed about the extent to which infrastructures to reduce mycotoxin improved quality of life (Wu and mycotoxins can affect both food exposure. Moreover, suboptimal Khlangwiset, 2010a), no incentive markets and public health. This will field practices and poor storage to implement it may exist unless provide incentives to aid countries in conditions make the crops vulnerable the effects of mycotoxins on public which foodborne mycotoxins are still to fungal infection and subsequent health and food markets are fully a significant public health risk. mycotoxin accumulation (Williams, understood. It is worth noting that in 2008). Maize and groundnuts, Ghana aflatoxin exposure has been CHAPTER 7 CHAPTER

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Chapter 7. Risk assessment and risk management of mycotoxins 117

chapter 8. Economics of mycotoxins: evaluating costs to society and cost-effectiveness of interventions

Summary is a lower risk of mycotoxins in the however, it is also important to food supply. Losses related to health assess the technical feasibility of The economic impacts of mycotoxins occur when mycotoxins are present interventions, particularly in low- to human society can be thought of in in food at levels that can cause income countries, where funds and two ways: (i) the direct market costs illness. In developed countries, such infrastructures are limited. associated with lost trade or reduced losses are often measured in terms revenues due to contaminated food or of cost of illness; around the world, 1. Introduction feed, and (ii) the human health losses such losses are more frequently from adverse effects associated measured in terms of disability- Two important topics are central to with mycotoxin consumption. adjusted life years (DALYs). It is also a discussion of the economics of Losses related to markets occur useful to assess the economics of mycotoxins: (i) the overall economic within systems in which mycotoxins interventions to reduce mycotoxins impact of mycotoxins on society, and are being monitored in the food and their attendant health effects; (ii) the benefits and costs of strategies and feed supply. Food that has the relative effectiveness of public to control mycotoxins in food. mycotoxin levels above a particular health interventions can be assessed A common misunderstanding maximum allowable level is either by estimating quality-adjusted life about the overall economic impact rejected outright for sale or sold years (QALYs) associated with each of mycotoxins on society is that only at a lower price for a different use. intervention. Cost-effectiveness as- market impacts – losses from food Such transactions can take place sessment can be conducted to lots rejected due to excessively high at local levels or at the level of compare the cost of implementing mycotoxin levels, as well as losses trade among countries. Sometimes the intervention with the resulting related to livestock and poultry – CHAPTER 8 CHAPTER this can result in heavy economic benefits, in terms of either improved are included in this estimation. In losses for food producers, but the markets or improved human health. estimating the economic impact of benefit of such monitoring systems Aside from cost-effectiveness, mycotoxins, human health impacts

Chapter 8. Economics of mycotoxins: 119 evaluating costs to society and cost-effectiveness of interventions matter just as much, if not more so, results should not be overinterpreted, to However, when the supply curve particularly in low-income countries avoid the danger of making long-term is shifted left, to S1 (as happens (LICs). Since the 1990s, the field of decisions based on analyses of current when food supply is decreased health economics has developed (limited) information. due to excessively high mycotoxin sufficiently that now improved levels), a new equilibrium is reached, methods exist to evaluate human 2. Market and trade impacts of represented by the intersection of the health impacts of diseases and mycotoxins demand curve with the new supply conditions, including those associated curve, S1. The reduced quantity of with mycotoxin exposure in food. To The primary way in which mycotoxins units of food sold, Q1, demands a derive an estimate for the total cost affect markets is to lower the value higher price per unit of food, P1. to society of mycotoxins in food and of the commodity being traded. The Thus, both producers and con- feed, both market impacts and health price paid for a particular lot of food or sumers bear costs associated with impacts must be included in the feed is reduced, or the lot is rejected mycotoxins. Producers sell less food calculation. entirely, or the lot must be treated and thus have reduced revenue, Strategies to control mycotoxin at additional cost before being sold and consumers must buy the food contamination should also be subject at a higher price. This can occur at a higher price. Specifically, the to economic analysis. Multiple strat- at multiple different levels of trade, decrease in food producers’ welfare egies have been developed to reduce from local all the way to international. due to mycotoxin contamination is mycotoxin risks before harvest (in Depending on the demands of the represented by the shaded area in the field), after harvest (in storage, buyer, the stakeholder group that Fig. 8.1. This area represents the transportation, or processing), in diets, bears the burden of mycotoxin cost difference between the producers’ and in clinical settings (see Chapter 9). can be individual farmers, handlers, initial welfare (area of triangle bounded

If these mycotoxin control strategies processors, distributors, consumers, by P0, Q0, and the demand curve) and are to be adopted in the parts of the or government. their resulting welfare (area of triangle world where they are most needed, bounded by P1, Q1, and the demand then their expected benefits, or 2.1 Dynamics of market curve). The decrease in consumers’ effectiveness – in terms of both market supply and demand due to welfare because of mycotoxins and health outcomes – should exceed mycotoxin contamination is represented by the difference their costs. Moreover, their capital between their initial welfare (area costs should not be so high that LICs Microeconomic theory explains how of triangle bounded by P0, Q0, and would find it impossible to adopt the the overall market and trade costs of supply curve S0) and their resulting strategies. Low-tech strategies may mycotoxins can be evaluated. Simply, welfare (area of triangle bounded by prove the most economically feasible mycotoxin contamination decreases P1, Q1, and supply curve S1). option to control mycotoxins in LICs. the available supply of acceptable food In practical terms, producers of Finally, cultural acceptability of the to be sold or bought. Fig. 8.1 illustrates commodities vulnerable to mycotoxin interventions is crucial, to ensure the dynamics of supply and demand contamination may suffer market long-term adoption and effectiveness for food for human consumption when losses directly, if the buyers monitor in mycotoxin reduction. supply is decreased. The demand and enforce limits for mycotoxins. In addition to the overall economic curve represents the quantity of a Consumers suffer market losses impact of mycotoxins and the cost- particular food that consumers are indirectly, by facing a reduced supply of effectiveness of control strategies, willing to buy at a particular price. At the commodity and therefore a higher another important economic con- very high prices, less demand will exist price. (In contrast, consumers may sideration is the technical feasibility of for the food, whereas at lower prices, suffer health-related losses directly, as these strategies, which includes risk demand will be higher. The supply discussed in Section 3.) Often, this cost assessment of potential health and curves (labelled S0 and S1) represent to consumers is marginal, particularly environmental impacts. These issues the quantity of food that producers will in developed countries, where the are also discussed in this chapter. provide at different prices per unit of bulk of the cost of food is made up It is important to remember that food. Hence, the original equilibrium of food processing rather than the values for the different variables in of quantity of food supplied, Q0, and commodity itself. But in LICs, reducing economic models can change sub- price per unit of food, P0, is the the supply of food by removing heavily stantially with time. Hence, when the intersection of the demand curve contaminated commodities can result models are used at any point in time, the with the original supply curve, S0. in food shortage crises. Moreover, for

120 Fig. 8.1. The impact of a strict food quality standard on supply and subsequent price. may sell their crops to grain elevators Source: Wu (2008); reproduced with the permission of the publisher. (e.g. maize, wheat) or to shellers or other handlers (e.g. groundnuts, tree nuts), who may then further process the crop and sell it to animal operations or to food processors for human consumption. After processing, the food is sold to distributors, who then sell it to retail markets to be purchased by consumers. For the USA, mycotoxin action levels (for aflatoxin, specifically) or industry guidelines (for fumonisin and deoxynivalenol) set by the United States Food and Drug Administration are enforced at different levels, such as at grain elevators or by food handlers. Crops with the lowest mycotoxin levels can Fig. 8.2. Complexity of the value chain of maize in Kenya, and points at which be sold for human food or for feed to aflatoxin control strategies could be implemented. NCPB, National Cereals and the most sensitive animal species at Produce Board of Kenya. Source: Dr Jonathan Hellin, International Maize and Wheat Improvement Center (CIMMYT), personal communication. a higher price, whereas crops with higher mycotoxin levels can be sold for animal feed at a lower price or are rejected outright. Within each commodity, different stakeholder groups are affected differentially by mycotoxins. In the USA, producers of grain commodities generally bear the burden of costs related to mycotoxins, whereas shellers and handlers, not growers, bear the largest burden of mycotoxin-related costs associated with groundnuts and tree nuts (Wu et al., 2008). In other parts of the world, local commodity markets are far more complex. The value chain of maize in Kenya is illustrated in Fig. 8.2, to provide an example of trade of one commodity in one country. Maize growers (labelled in Fig. 8.2 as “Small- staple food items such as maize, the 2.2 Mycotoxin costs in local scale farmers”) may sell their maize price elasticity of demand is usually and regional markets to a wide variety of different local very low, which means that demand will maize traders, who may travel from not change significantly in response The complexity of local and re- household to household to purchase to a change in price; consumers will gional commodity markets varies maize. These traders, in turn, may sell purchase staples even if the price considerably among countries, and the maize to large-scale traders or to becomes much higher, because staple thus the extent to which mycotoxins a variety of millers. Each step further CHAPTER 8 CHAPTER foods are a necessity. may impose market costs varies. in the value chain of maize provides In the USA, for example, crop value different opportunities for both buyers chains are relatively simple. Farmers and sellers.

Chapter 8. Economics of mycotoxins: 121 evaluating costs to society and cost-effectiveness of interventions This complexity of the value for the country that imposes the Third, the cost of a rejected food chain of crops in Africa means that standard but also for countries that shipment is enormous (about $10 000 it can be very difficult to implement attempt to export foods there. per lot in transportation, storage, and mycotoxin control strategies that From an international trade dockage fees; Wu et al., 2008), even if would have widespread effects. standpoint, mycotoxin contamination the lot can be returned to the country The control strategies would have inflicts heavy economic burdens. attempting to export the food. to be implemented at multiple It reduces the price paid for crops These dilemmas led former United points, and these points would and can cause disposal of large Nations Secretary-General Kofi An- have to be coordinated. In the event amounts of food. In the USA, losses nan to recognize the magnitude of of an outbreak of mycotoxicosis, from mycotoxins – in the hundreds of the problem of setting appropriate interventions would need to be millions of US dollars annually – are aflatoxin standards worldwide. At the distributed, and communication usually associated with these market Third United Nations Conference on among different stakeholders is costs rather than with health effects the Least Developed Countries, held absolutely crucial. Depending on because enforcement of mycotoxin in Brussels in 2001, he commented, available communications infra- standards and pre-harvest and post- “The European regulation on afla- structures, implementing interven- harvest control methods have largely toxins costs Africa $670 million each tions may be a difficult task. eliminated harmful exposures in food year in exports. And what does it in the USA (Wu, 2004). achieve? It may possibly save the 2.3 Mycotoxin costs in In LICs, impacts of mycotoxins life of one citizen of the European international markets are far more severe. Many individuals Union every two years. Surely a more are not only malnourished but also reasonable balance can be found.” The issue of mycotoxin control is chronically exposed to high aflatoxin Annan had based his statement on becoming increasingly important levels in their diet, resulting in deaths a report of the Joint FAO/WHO Expert for LICs as international trade from aflatoxicosis, cancer, and other Committee on Food Additives (JECFA) becomes more prominent in a world conditions (Wild and Gong, 2010). LICs (WHO, 1998), which assessed of increasing demand for crops (to be often lack the resources, technology, the effect of aflatoxin regulations used for food, animal feed, or even and infrastructure necessary for on HCC incidence, depending on fuel). Hence, mycotoxin costs must routine tests of mycotoxin levels in HBV prevalence. JECFA developed also be considered in the context of food. Further complicating the problem two scenarios to determine the international trade. in the case of aflatoxin is that for a effect of moving from an enforced More than 100 countries have given level of aflatoxin exposure, cancer aflatoxin standard of 20 µg/kg to a established maximum tolerable levels risk may be more severe in LICs stricter standard of 10 µg/kg in two for aflatoxins in human food (FAO, than in developed countries because hypothetical countries: one with an 2004), whereas relatively few countries of higher prevalence of chronic HBV prevalence of 1% and another have established these levels for hepatitis B virus (HBV) infection, with an HBV prevalence of 25%. In the other mycotoxins such as fumonisin, which synergizes with aflatoxins to first country, tightening the aflatoxin ochratoxin A, and deoxynivalenol. significantly increase the risk of liver standard would yield a drop in the Table 8.1, which lists maximum cancer (hepatocellular carcinoma estimated population HCC incidence tolerated levels for foodborne [HCC]) (Groopman et al., 2008). by 2 cases per billion people per year. aflatoxins in selected countries and Globalization of trade has In the second country, tightening regions as of August 2010, shows exacerbated food losses due to the aflatoxin standard would yield that aflatoxin standards vary greatly mycotoxins in three ways. First, strict a drop in the estimated population among countries, even for a small mycotoxin standards mean that LICs HCC incidence of 300 cases per sample of countries; this difference will export their best quality foods and billion people per year. Hence, in may cause food trade barriers. keep more heavily contaminated foods high-income importing countries with Indeed, the Council for Agricultural for domestic consumption, resulting low HBV prevalence, tightening the Science and Technology states that in higher mycotoxin exposure in LICs aflatoxin standard would reduce HCC one key goal for the 21st century (Cardwell et al., 2001). Second, even incidence by an amount so small is to “develop uniform standards the best quality foods produced in as to be undetectable (Henry et al., and regulations for mycotoxin LICs may be rejected for export, 1999; Wu, 2004). contamination” (CAST, 2003). These resulting in millions of US dollars in Now that a global push exists standards have implications not just losses (Wu, 2004; Wu et al., 2008). to harmonize mycotoxin standards

122 Table 8.1. Maximum tolerated levels for aflatoxins in human food in selected countries where PDFi,j,k is the probability density and regionsa function of the percentage of crop i from country j having mycotoxin Country Total allowable level of total aflatoxins in levels at or lower than standard k, and or region human food (µg/kg) its integral over k is the cumulative

Australiab 5 or 15 distribution function. Then, export losses calculated for each country are China 20 summed across major food exporting countries to derive a total global c European Union 4, 10, or 15 burden of export loss at different mycotoxin standards. Guatemala 20

India 30 3. Health economic impacts of mycotoxins Kenya 20 Looking at the market and trade Taiwan, China 50 impacts of mycotoxins is only one USA 20 side of the story. The other important facet to consider is the public health a A more complete list can be found in FAO (2004). impact of setting different mycotoxin b The Australian standard for maximum allowable aflatoxins in groundnuts is 15 µg/kg, more permissive than those for other foods. standards worldwide, assuming c The European Union standard for maximum allowable aflatoxins is 4 µg/kg for cereals and all products derived from they could be enforced. The human cereals, except maize to be subjected to sorting, which has a standard of 10 µg/kg. The standard for groundnuts, diseases and conditions caused by almonds, hazelnuts, and pistachios “ready to eat” is 10 µg/kg, whereas the standard for groundnuts, almonds, hazel- nuts, and pistachios intended for further processing is 15 µg/kg. mycotoxin exposure must first be evaluated. Evaluating the human health (CAST, 2003), it is important to consider is the total export amount (in metric economic impacts of mycotoxins is on a global scale what the economic tons) of crop i from country j; and ri,j,k is crucial to understanding their total impacts would be of harmonizing the fraction of export volume of crop i economic impact because mycotoxins different standards, which range from from country j rejected at international primarily affect LICs, where trade- relatively strict to relatively permissive. mycotoxin standard k. related losses are not nearly as Wu (2004) provided a framework for A sensitivity analysis on k reveals prominent as adverse health effects assessing losses related to markets, how export losses for food crops from consuming food contaminated as outlined below. in a particular country change as a with mycotoxins. Subsistence farmers Given a particular internationally function of the harmonized standard and local food traders occasionally imposed mycotoxin standard, the total chosen. Values for ri,j,k are calculated have the luxury of discarding obviously national export loss of a particular food by fitting probability density functions mouldy food, but in conditions of crop can be calculated as the product PDFi,j,k, based on the relevant literature, drought or food insecurity, poor people of the price of the food crop per unit of concentrations of fumonisin and/ often have no choice but to eat the weight on the world market, the total or aflatoxin in crop i in country j. The contaminated food or starve. amount of that crop exported, and particular country j to study is chosen by Until recently, it was difficult to the fraction of the export crop that is looking at the most important exporting put an economic value on health rejected as a result of that mycotoxin countries of crop i. Cumulative effects. Fortunately, the field of health standard (Wu, 2004): distribution functions are estimated economics has made great strides from the probability density functions in the past two decades. Health

Export lossi,j,k = Pi * Wi,j * ri,j,k, of the percentage of the crop having economics strives to quantify health mycotoxin levels at or lower than a benefits and costs in such a way as where i is the crop (e.g. maize, given concentration. Then, the fraction to be comparable with monetary groundnuts); j is the country; k is the of export volume rejected at that benefits and costs. Otherwise, it can CHAPTER 8 CHAPTER international mycotoxin standard (e.g. concentration is: be difficult to understand how much a for fumonisin or aflatoxin); Pi is the world particular risk affects human society, price for food crop i per unit weight; Wi,j ri,j,k = 1 – ∫ PDFi,j,k dk, especially if death is not a significant

Chapter 8. Economics of mycotoxins: 123 evaluating costs to society and cost-effectiveness of interventions outcome, or, conversely, how much impacts are more difficult to evaluate. associated with the condition would a public health intervention benefits The burden of human diseases, also be included in this category. human society, if no direct market such as those caused by mycotoxin outcomes exist. Putting monetary consumption, can be calculated 3.2 Disability-adjusted life values on these health outcomes in two primary ways. The first is years helps decision-makers to understand cost of illness (COI), which is more how important a risky agent or a appropriate in developed countries The DALY, like COI, is a measure disease is, how useful an intervention because a large portion of the of the overall burden of disease. It might be, and how to compare the estimate is health-care cost. The extends the concept of potential years relative importance of risks and the second is disability-adjusted life of life lost due to premature mortality relative effectiveness of interventions years (DALYs), which is appropriate to include equivalent years of healthy (Wu and Khlangwiset, 2010a). for both developed and developing life lost in states of less than full health, Much of the literature in health countries. A third metric, quality- broadly termed disability (Havelaar, economics focuses on medical adjusted life years (QALYs), is more 2007; Wu and Khlangwiset, 2010a). treatments, with relatively few ap- often used to estimate the relative One DALY can be thought of as one plications in food and agriculture. effectiveness of different public lost year of healthy life. The total Some examples of health economic health interventions in improving DALYs associated with a particular assessments of food additives overall quality of life. disease are calculated as follows: include studies of the potential cost- effectiveness of transgenic golden rice 3.1 Cost of illness DALYs = YLL + YLD, in reducing vitamin A deficiency (Stein et al., 2008) and the cost-effectiveness For an individual or for a particular where YLL is the years of life lost of biofortifying foods in reducing population, COI caused by a disease due to premature mortality from the micronutrient deficiency (Meenakshi or condition is calculated as the sum of disease and YLD is the years lost et al., 2007). Havelaar (2007) provided three factors: direct health-care costs due to disability. YLD is estimated as a notable example of estimating (DHC), direct non-health-care costs the number of years lived with the the health costs of foodborne (DNHC), and indirect non-health-care disability multiplied by a weighting zoonoses, such as those caused costs (INHC): factor, between 0 and 1, that reflects by Campylobacter, Salmonella, or the severity of the disability. Cryptosporidium in Europe. COI = DHC + DNHC + INHC. The World Health Organization It is also important to consider (WHO), among other organizations, the health economic impacts of DHC are costs associated with has estimated DALYs for many mycotoxins, which can impose an medical services. These include gener- diseases and conditions in different enormous socioeconomic cost. As al practice consultations, consultations parts of the world. DALYs for any given stated above, in developed countries it with specialists, hospitalization, any disease are estimated separately for is relatively straightforward to estimate surgery or treatments required, drugs, high-income, middle-income, and the costs of mycotoxins because supplements (e.g. intravenous fluids), low-income countries. This strat- these costs are primarily related to and rehabilitation. DNHC are costs ification is based on assumptions markets. Commodities that contain associated with the disease that do not about how many years individuals will mycotoxins at levels exceeding relate to the medical system. These live with a disability in different parts regulatory guidelines for human food include travel costs to medical centres, of the world and what resources are or animal feed are discarded or sold costs of childcare, and co-payments available to alleviate disability (Wu and at a lower price for a different use by patients for medicines. Khlangwiset, 2010a). (Wu et al., 2008). One can estimate INHC are defined as the value the cost of mycotoxins to a particular of production lost to society due to 3.3 Quality-adjusted life years commodity group by estimating the disease or condition, as a result how much of the commodity must of temporary absence from work, The QALY is used to assess the be discarded or discounted due to permanent or long-term disability, or value for money of a medical or mycotoxin contamination. In LICs, premature mortality (Havelaar, 2007; public health intervention. It is based in contrast, health-related costs are Wu and Khlangwiset, 2010a). To the on the estimate of the number of usually much higher than market- extent that they can be evaluated, years of life that would be added by related costs, and health economic the costs of pain and of suffering the intervention, and hence is used

124 to rank the relative effectiveness of specific human health end-points are 4. Evaluating total economic different interventions for a particular difficult to attribute quantitatively or even impacts of mycotoxins condition. Every year of “perfect qualitatively to a particular mycotoxin. health” is assigned a value of 1, For example, fumonisins have been If both the market and the health whereas a year not lived in perfect associated with oesophageal cancer economic impacts of mycotoxins can health is assigned a value between and neural tube defects in humans, be estimated, the cost-effectiveness 0 and 1 that reflects the quality of but these associations are not clearly of different interventions to reduce life (similar to the weighting factor for established and there are no human mycotoxin risk can then be assessed. YLD in DALYs). dose–response data, i.e. doses of Various studies have attempted to QALYs are calculated as follows. fumonisins causing particular levels of quantify the potential market losses Individuals with a serious, life- disease incidence, by which to perform associated with mycotoxins in crops. threatening condition (as is often a reliable quantitative risk assessment. In the USA, Vardon et al. (2003) the case with excessive exposure Further complicating the issue is that estimated the total annual losses to aflatoxin) can receive a standard each mycotoxin may have multiple due to three mycotoxins – aflatoxin, treatment (which, in LICs, may be no health end-points, including cancer, fumonisin, and deoxynivalenol – to treatment at all) that will allow them acute toxicity, and immunomodulation. reach as high as US$ 1 billion. Almost to live for X more years with a quality Even if quantitative relationships could all of this loss was borne by maize, of life of A. However, if they receive a be established for each end-point, groundnut, and wheat growers. new treatment instead, they will live an analyst would need to ensure However, a small portion of this loss for Y more years with a quality of life that every possible human health was estimated to be suffered by of B. The difference between the new outcome of a mycotoxin was included livestock producers due to adverse and the standard treatment in terms of in the calculation, to derive an accurate animal health effects. QALYs gained is health economic estimate. In three Asian countries – Thai- With aflatoxins, more progress land, Indonesia, and the Philippines – QALYs gained = Y * B – X * A. has been made in quantifying the the total estimated annual loss due to link between exposure and disease aflatoxin was about 1 billion Australian To assess the relative effective- incidence. Aflatoxins cause a multi- dollars (Lubulwa and Davis, 1994). ness of an intervention, the cost of the tude of conditions, including acute This loss was a combination of new treatment must also be taken into aflatoxicosis and HCC, and are market impacts, through rejected account. The difference in treatment believed to contribute to immu- lots with excessively high mycotoxin costs divided by the QALYs gained is nosuppression and stunted growth in levels, and adverse health effects – used to estimate the cost per QALY, children. In an aflatoxicosis outbreak specifically the impacts of HCC in i.e. how much would need to be spent in Kenya in 2004 (Strosnider et al., these populations. to provide one additional QALY. 2006), it was possible to estimate the Wu (2004) estimated the market total number of cases, the number impacts to the world’s top maize- 3.4 Challenges to evaluating of deaths, and the concentrations of exporting and groundnut-exporting health economic impacts of aflatoxins in the contaminated maize countries and regions of conforming mycotoxins that caused the toxicoses. Decades to hypothetical harmonized standards of work have likewise established for fumonisin in maize and aflatoxin To use either the cost of illness dose–response relationships between in groundnuts. If the current United or the DALYs method to calculate aflatoxins and HCC in HBV-positive States Food and Drug Administration socioeconomic costs, one must first and HBV-negative individuals, from (FDA) total fumonisin guideline of identify human health end-points, i.e. which aflatoxin cancer potency fac- 2 mg/kg were adopted worldwide, diseases or conditions, to assess. tors can be derived for quantitative the total annual maize export losses Otherwise, it is impossible to gather cancer risk assessment (WHO, 1998). for the USA, Argentina, and China data on the necessary factors to The immunosuppressive effects of would be US$ 100 million, whereas assess the economic impact: mortality aflatoxins cannot yet be quantified in if a fumonisin standard of 0.5 mg/ and morbidity, incidence, duration, and humans, but limited quantitative data kg were adopted worldwide, those severity associated with the disease. are available to assess the link between total annual losses would increase to CHAPTER 8 CHAPTER The challenge in calculating the aflatoxin exposure and stunted growth US$ 300 million. If the current FDA socioeconomic costs of mycotoxins is in children (Gong et al., 2002). total aflatoxin action level of 20 μg/kg that, with the exception of aflatoxins, were adopted worldwide, total annual

Chapter 8. Economics of mycotoxins: 125 evaluating costs to society and cost-effectiveness of interventions groundnut export losses for the USA, levels in food. Dietary and clinical where Y is the crop yield per hectare, Argentina, China, and Africa would be interventions can be considered P is the price differential for high- US$ 92 million, whereas if an aflatoxin secondary interventions. They cannot quality use (low mycotoxin levels standard of 4 μg/kg were adopted reduce actual mycotoxin levels in required) versus other uses, and R worldwide, those total annual losses food, but they can reduce mycotoxin- is the percentage of the crop with would increase to US$ 450 million. related illness either by reducing mycotoxin levels above the limit for Liu and Wu (2010) and Liu et al. the bioavailability of mycotoxins high-quality use. (2012) estimated the global burden (e.g. through enterosorption) or by This cost C is compared with of aflatoxin-related HCC using two ameliorating damage induced by the benefits and costs of applying a different approaches: quantitative mycotoxins (e.g. through inducing mycotoxin control method. The net cancer risk assessment and pop- phase 2 enzymes that detoxify benefit per hectare, B, of applying ulation attributable risk (PAR), metabolites of mycotoxins). These the intervention can be expressed as respectively. The quantitative cancer control strategies are described in risk assessment methodology (Liu and greater detail in Chapters 7 and 9. B = (E * C) – A, Wu, 2010), relying on dietary surveys In developed countries, it is and cancer potency factors, yielded relatively straightforward to estimate where E is the percentage efficacy an estimate of 5–28% of total global the cost-effectiveness of controlling in reducing mycotoxins to levels that HCC cases attributable to aflatoxin. mycotoxins because the costs and allow growers a premium, C is the Similarly, the PAR approach (Liu et benefits are primarily market-related cost per hectare associated with al., 2012), making use of a systematic (Wu et al., 2008). The cost of a mycotoxin contamination (as shown review and meta-analysis of human particular mycotoxin to a particular in the previous equation), and A is the biomarker studies on aflatoxin-related commodity group is calculated total cost of purchasing and applying cancer, yielded an estimate of 21– by assessing how much of the the mycotoxin control strategy. 24% of global HCC cases attributable commodity must be discarded or In LICs, however, interventions to to aflatoxin. Because the total number discounted due to contamination. reduce mycotoxins have both market of new HCC cases worldwide is Then, measuring the benefit accrued and human health importance. hundreds of thousands each year and from a particular intervention requires How can the cost-effectiveness of each HCC case is associated with 13 estimating how much levels of the a health intervention be determined DALYs (Wu and Khlangwiset, 2010a), mycotoxin are reduced as a result if no direct market benefits aflatoxin-related HCC alone may of the intervention and how much exist? The WHO Commission on cause > 2 million DALYs each year. more of the commodity can thus be Macroeconomics and Health has sold. The difference between the total provided the following guideline for 5. Assessing cost- market value of the commodity with thresholds of cost-effectiveness effectiveness of interventions and without the intervention is a rough (WHO, 2001). An intervention is to control mycotoxins estimate of the cost-effectiveness of considered very cost effective if that intervention. the monetary amount spent on the Multiple public health interventions To calculate the cost of mycotoxin intervention per DALY saved is less exist by which to control mycotoxins contamination in developed countries, than the per capita gross domestic or their burden in the human body. three market economic factors need product (GDP) of the country in Interventions to reduce illness induced to be considered: the expected cost of which the intervention is applied. by mycotoxins can be roughly grouped mycotoxin contamination to growers An intervention is considered into three categories: agricultural, or handlers in the absence of any moderately cost effective if the dietary, and clinical. Agricultural interventions, the cost of purchasing monetary amount spent on the interventions are methods or and applying an intervention, and the intervention per DALY saved is less technologies that can be applied expected net benefit of applying the than 3 times the per capita GDP. An either in the field (pre-harvest) or in intervention in terms of mycotoxin intervention is considered not cost drying, storage, and transportation reduction. The cost per hectare, effective if the monetary amount (post-harvest) to reduce mycotoxin C, of mycotoxin contamination to spent on the intervention per DALY levels in food. Agricultural inter- growers in the absence of agricultural saved is greater than 3 times the per ventions can thus be considered interventions can be expressed as capita GDP. primary interventions because they directly reduce mycotoxin C = Y * P * R,

126 As described in Section 3.4, the Fig. 8.3. Framework for assessing the technical feasibility of public health health effects of the mycotoxin in interventions. Source: Wu and Khlangwiset (2010b); reproduced with the permission question must be identified to estimate of the publisher. DALYs for this cost-effectiveness calculation. DALYs for any given disease are estimated separately for high-income, middle-income, and low-income countries. The DALYs estimate for each kind of country is based on assumptions about how many years individuals will live with a disability in different parts of the world and what resources are available to alleviate disability. This WHO guideline is not without controversy. First, the cut-off of 3 times the per capita GDP for the cost- effectiveness of an intervention is a debatable metric. Second, using the average GDP in the estimation can vention? Is the intervention culturally and temporally, to allow maximum be controversial in itself, especially appropriate and easily adopted by the effectiveness of the intervention for countries in which income is target population? If an intervention to in a target population. To achieve distributed bimodally rather than reduce mycotoxins fails in any or all of large-scale implementation, the normally (i.e. average GDP can be these points, then it is not likely to be following characteristics of the meaningless in a country with very adopted on the large scale, no matter basic intervention would influence rich versus very poor populations how cost effective it may be. feasibility: (i) the stability of the and no middle class). Third, DALYs A conceptual framework has been product, including its usable lifetime can be a controversial measure, developed for evaluating the technical and its risk of degradation or particularly in selecting a weighting complexity – and hence the feasibil- destruction; (ii) the degree to which factor associated with each illness or ity – of public health interventions for the intervention can be standardized condition. Finally, cost-effectiveness is LICs with limited resources (Gericke for production and sale; (iii) the but one component of the feasibility of et al., 2005). The framework has four safety profile of the intervention, aflatoxin reduction strategies in LICs. relevant dimensions (see Fig. 8.3): in terms of both adverse health Many other factors are important, intervention characteristics, delivery and environmental effects and risk including the technical feasibility of the characteristics, government capacity, associated with inappropriate use; intervention, which is described next. and usage characteristics. Each of and (iv) the ease of storage and these is discussed below. transportation of the intervention. 6. Technical feasibility of In considering safety, it is important interventions to control 6.1 Intervention to recognize that implementing certain mycotoxins characteristics mycotoxin control interventions in LICs may result in health or environmental The cost-effectiveness of a myco- What aspects of the intervention risks that would be less likely to occur toxin control strategy is not enough to itself make it more or less feasible in developed countries. Items to be ensure its successful adoption in the for large-scale adoption? Gericke considered include occupational haz- parts of the world where it is needed. et al. (2005) pointed out that one of ards associated with producing or More questions must be addressed. the most important aspects of the implementing the intervention, health Does the strategy entail countervailing feasibility of an intervention, once risks to workers, quality control of health or environmental risks? What it has been proven to have some production and application, immune would the delivery mechanism be, and level of efficacy in a field or clinical status of the target population, local CHAPTER 8 CHAPTER would local infrastructures support trial, is that it has the potential to ecologies, and potential side-effects that mechanism? Do governmental be implemented on a much larger of dietary interventions (Wu and regulations inhibit or promote the inter- scale. This is crucial both spatially Khlangwiset, 2010b).

Chapter 8. Economics of mycotoxins: 127 evaluating costs to society and cost-effectiveness of interventions 6.2 Delivery characteristics be adopted broadly in a region. If, challenges in implementing the for example, regulations have been intervention – challenges in terms of How will a particular intervention enacted against genetically modified human resource needs; equipment, be delivered to a target population? organisms, certain food additives, or technology, and transportation require- First, the target population must be certain chemical or microbial agents, ments; financial aid; and user adoption identified, which can be a challenge then particular interventions related to constraints. Feasibility analyses can in countries where food is grown agriculture and food safety cannot be indicate research and development in smallholdings. Communication implemented on a scale that achieves priorities to increase the likelihood of of a need to those able to respond widespread public health benefits adopting interventions that can improve may pose difficulties. In addition, (Wu and Khlangwiset, 2010b). public health and market outcomes there are requirements for facilities, (Wu and Khlangwiset, 2010b). transportation, human resources, and 6.4 Usage characteristics communication (Gericke et al., 2005). 7. Conclusions Proper facilities are necessary to store Generally, the more readily a target and to administer the intervention and population can use or adopt an For developed countries, it is relatively must be distributed widely enough intervention, the more likely it is to be easy to estimate the cost to human within the target population so that most adopted with a frequency that actually society of mycotoxins as well as to people have reasonably easy access makes a difference to public health assess the cost-effectiveness and to the intervention. Transportation (i.e. long-term use) and the more likely technical feasibility of interventions may necessitate specific infrastructure people are to adopt it (i.e. breadth of to control them. This is because the (e.g. cold storage in vehicles for use). Gericke et al. (2005) identified costs of mycotoxins and the benefits vaccines, and power to maintain cold three crucial dimensions of usage: (i) of interventions are largely restricted temperatures). Transportation issues the ease of usage; (ii) the pre-existing to the marketplace; human health may also make a significant difference demand for the intervention; and (iii) effects can largely be considered in cost if the intervention needs to the risk of diminished effectiveness negligible. Moreover, in developed be imported rather than produced and efficiency because of illicit trade countries, the feasibility of mycotoxin locally. Human resources and activities, such as counterfeit products. control becomes an issue only if the communication are crucial when the Ease of usage includes the need for intervention is extremely expensive, public, or any subgroup thereof (such consumer information and education or in which case another, less expensive as farmers or food storage handlers), training on how to use the mycotoxin intervention is usually available. must be educated on proper use of the control strategy effectively and safely. For LICs, including the impact of intervention and why it is important for If no pre-existing demand exists for mycotoxins on human health results health and economic reasons. the intervention, adoption might be in much more complicated economic more difficult and would require more analyses. Health economic tools from 6.3 Government capacity time. Finally, illicit trade activities can the past two decades have improved pose dangers, especially in the case of the ability to place monetized values on How would national or local gov- dietary interventions. human health effects. This, in turn, aids ernments either support or inhibit Understanding constraints on the cost-effectiveness analysis because adoption of the intervention? Govern- feasibility of mycotoxin control inter- the monetary cost of interventions mental financial support and other ventions helps scientists and policy- can be compared with the human resource support, such as staff makers to think beyond efficacy, health benefits of implementing the support and outreach activities, would and even beyond material costs. For interventions. Evaluating the technical be crucial for at least the start-up interventions to succeed in LICs, feasibility of interventions is still phase of an intervention. Moreover, governments, scientists, international complex because the many impeding governmental regulations can deter- organizations, farmers, and consumers factors and external risks and benefits mine whether an intervention can must work collaboratively to overcome need to be considered.

128 References

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Chapter 8. Economics of mycotoxins: 129 evaluating costs to society and cost-effectiveness of interventions

chapter 9. Practical approaches to control mycotoxins

Summary of resistant plant cultivars, good 1. Introduction agricultural practice, and biocontrol Control strategies to minimize using non-toxigenic strains. Post- This chapter provides detailed mycotoxin levels in food comprise harvest interventions include the information about interventions and several broad categories, including removal of infected and/or insect- practices that can help to reduce good agricultural practice, good damaged food components by mycotoxin risks in a variety of settings: manufacturing practice, and hazard sorting, maintaining correct drying before harvest (in the field), after analysis and critical control point and storage conditions, and chemical harvest (in storage, transportation, principles. In general, intervention deactivation such as nixtamaliza- or processing), and in diets. The strategies include pre-harvest, post- tion. Dietary interventions include sections are organized by mycotoxin harvest, and dietary approaches, reducing mycotoxin bioavailability and the interventions that have been depending on the specific myco- or modulating metabolism in ways developed to control them, as follows: toxins and the food commodity that reduce the harmful effects of aflatoxins, fumonisins, ochratoxin A, likely to be contaminated. This reactive metabolites. Cost-effective and deoxynivalenol and zearalenone chapter describes practical inter- and simple intervention methods, (treated together because they are ventions, which are arranged by predominantly at the population level, produced by the same fungi). the major groups of mycotoxins and should be emphasized in developing For each intervention, information are described according to their countries, where resources are limited about each of the following aspects is stage of development, efficacy, and sophisticated technologies are listed. geographical regions in which lacking. Description. A description of the they have been tested or applied, intervention. simplicity or complexity, and breadth Stage of development. How well of usefulness. Typical pre-harvest developed and tested is the interventions include the breeding intervention in terms of controlling

Chapter 9. Practical approaches to control mycotoxins 131 CHAPTER 9 CHAPTER the mycotoxin? Has it undergone categories of interventions apply for control over conditions. In subtropical testing under laboratory, field, or epi- general control strategies across and temperate regions, the weather demiological conditions? How broad multiple mycotoxins. is usually drier at harvest time and is the current adoption? field drying is effective. In tropical Efficacy. By how much can the 1.1 Good agricultural practice countries, groundnuts are frequently intervention reduce mycotoxin risk, harvested and left to dry in stacks in compared with conditions where Good agricultural practice (GAP) the field, or are separated from plants there is no intervention? involves good farm management. at harvest and dried on the ground, Geographical regions. Where has Many definitions exist, depending on some form of matting, or on plastic this intervention been tested or adopt- on local conditions, but in general sheets. Maize is sometimes shelled ed around the world? GAP means maintaining healthy wet and then dried mechanically Simplicity/complexity. How simple or crops and sustainable agriculture by, by middlemen. Storage practices complex is the intervention to among other activities: (i) planting in developed countries normally implement? Is it so complex that with optimal row and seed spacing prevent development of mycotoxins individuals must have advanced for local conditions, especially water after drying. However, less than ideal education and training, or can it be availability, to reduce plant stress; (ii) storage conditions in LICs may permit made sufficiently simple for farmers maintaining adequate water supplies, increases in moisture content, leading or even the general public to adopt by irrigation where practicable; (iii) to increases in production of aflatoxins effectively? reducing erosion by contouring, or ochratoxin A. Storage conditions Population/individual. Is the inter- ditching, or hedging; (iv) controlling can be improved by using dry, well- vention tailored, or can it be tailored, weeds, and mulching crops to reduce ventilated rooms with protection to a population-level approach (e.g. moisture stress; (v) controlling insects from sunlight (to prevent moisture so that a government can broadly that damage developing grains or migration) and control of insects and implement the intervention), or does nuts and permit entry of the fungi rodents. This is true not just for maize, the intervention rely on individuals for that produce mycotoxins; (vi) rotating cereals, and groundnuts (the major implementation? crops to reduce insect infestation sources of mycotoxin exposure for Useful in emergencies. Is this and fungal infection, which are humans) but also for tree nuts such as intervention suitable for use in a time exacerbated by monoculture; (vii) pistachios, for which there have been of emergency, e.g. when mycotoxin applying fertilizers at appropriate dramatic improvements in aflatoxin levels are known to be high in times and concentrations, to benefit reduction in Iran over the past decade available foods, or in the event of the crop but limit run-off of nutrients due to improved drying and storage acute poisoning? such as nitrogen and phosphorus; conditions (Wu, 2008). Locality of resources. Can the (viii) harvesting crops at or before full For further information about intervention be manufactured using maturity, because overmature crops GAP, see FAO (2002). local resources and drawing on are liable to increased risk from insect local expertise, or does it require damage and water stress and hence 1.2 Good manufacturing practice importation of resources and/or mycotoxin production; (ix) drying trained operators? crops rapidly and completely, as soon Good manufacturing practice (GMP) Accessibility. Is the intervention as possible after harvest; and (x) involves a wide range of prac- applicable in low-income countries maintaining good storage conditions tices that maintain the quality of (LICs) and among subsistence on the farm (storage facilities should foods, in developed countries often farmers with respect to access, cost, be soundly constructed to prevent through legislation. In the context of and feasibility? water ingress, with raised floors to mycotoxins, GMP includes practices References. For further reading prevent moisture migration from soil; that prevent fungal growth and on the topic. properly dried crops should be stored hence reduce mycotoxin formation Before individual interventions for in closely woven sacks that permit air and that reduce or remove mycotoxin each mycotoxin are discussed, three exchange; and rodents and insects contamination in crops after harvest- broad categories of interventions should be controlled). ing and drying. On the farm, this are described: good agricultural Drying of crops is a critical most commonly involves removal of practice, good manufacturing prac- process in reducing development of defects, including immature nuts or tice, and the hazard analysis and mycotoxins. Grains and nuts are often grains and also weed seeds, sticks, critical control point system. These dried in the field, with consequent poor stones, earth, husks, and so on,

132 by hand sorting, winnowing, and Codex Alimentarius Commission HACCP control for mycotoxins gravity separation or other methods. (Codex Alimentarius Commission, is an integrated approach (Bryden, Sorting out obviously mouldy nuts or 1995), the Food and Agriculture 2009), which includes GAP and GMP kernels by hand and eye has proven Organization of the United Nations (described above) as complementary a particularly effective method of (FAO) has outlined the following approaches (Aldred et al., 2004). removing a large proportion of the seven principles of HACCP for food In developed countries, several mycotoxin contamination in the safety (FAO, 2001). First, identify HACCP programmes have been food (Turner et al., 2005; Van der potential hazards associated with developed for aflatoxin in a variety of Westhuizen et al., 2011). Practices food production at all stages, assess commodities as well as for ochratoxin downstream, involving middlemen, the likelihood of hazard occurrence, A in coffee; these programmes rely cooperatives, and factories, depend and identify preventive measures for on rapid diagnostic tools to monitor on the crop and the mycotoxin. These control. Second, determine points, fungal occurrences and application include practices such as extrusion procedures, and operational steps of methods to quantify mycotoxins. (Bullerman and Bianchini, 2007) and that can be controlled to eliminate, In LICs, some HACCP processes nixtamalization (described in more or reduce the likelihood of, hazards. may not yet be technically and eco- detail in Section 2.2.2), both of which These are the critical control points nomically feasible (Bryden, 2009), have been shown to reduce levels of (CCPs). Third, establish critical limits necessitating other strategies to multiple mycotoxins in food. that must be met to ensure that CCPs reduce mycotoxins. However, it can are under control. Fourth, establish be useful to adopt HACCP principles 1.3 The hazard analysis and a system to monitor control of CCPs as a way of thinking, regardless of critical control point system by scheduled testing or observations. economic and technical constraints. Fifth, establish corrective actions A 1999 conference on mycotoxins The hazard analysis and critical when monitoring indicates that certain emphasized that GAP and GMP control point (HACCP) system CCPs are not under control. Sixth, overlap and are prerequisites for for food safety management in- establish procedures for verification HACCP. HACCP will ensure and volves controlling critical points in to confirm that the HACCP system improve food quality in a controlled food handling (FAO, 2001) and is is working effectively. Seventh, environment; hence, such systems important in managing the problem of establish documentation concerning must be kept simple, practical, and mycotoxins in the food supply (Bryden, all procedures and records appro- understandable for those who use 2009; Chulze, 2010). Adopting priate to these principles and their them to reduce mycotoxin risk (FAO/ guidelines of the United Nations application. WHO/UNEP, 1999).

Table 9.1. Risk management strategies for major mycotoxins in pre-harvest, post-harvest, and dietary settings

Mycotoxins

Setting Deoxynivalenol Aflatoxins Fumonisins Ochratoxin A and zearalenone

Pre-harvest GAP GAP GAP GAP Developing drought-resistant Ensuring that cultivars are Timely harvesting Breeding for host plant cultivars adapted to local environments resistance Biocontrol Breeding for insect resistance Using cultivars that mature Forecasting aflatoxin formation Transgenic Bt maize over a range of dates Timely harvesting Forecasting fumonisin Transgenic Bt maize formation Using fungicides at anthesis Timely harvesting or silking Forecasting toxin formation

Post-harvest GMP, HACCP, sorting, drying, GMP, HACCP, sorting, drying, GMP, HACCP GMP, HACCP, sorting, drying nixtamalization washing, nixtamalization

Dietary Enterosorbents (e.g. organic clays) Chlorophyll and chlorophyllin

GAP, good agricultural practice; GMP, good manufacturing practice; HACCP, hazard analysis and critical control point.

Chapter 9. Practical approaches to control mycotoxins 133 CHAPTER 9 CHAPTER Table 9.2. Likelihood of mycotoxin contamination in major commodities and current stage of development of potential or actual interventions

Commodity

Maize Groundnuts Tree nuts Small grains Others

Mycotoxin

Aflatoxins X X X Figs, copra, spices, cottonseed Fumonisins X Sorghum, millet, soybeans, asparagus

Ochratoxin A X X Dried vine fruits, wine, coffee, cocoa, chocolate Deoxynivalenol X X and zearalenone

Relevant intervention

GAP Practice Practice Practice Practice Practice

GMP Practice Practice Practice Practice Practice HACCP Practice Practice Practice Practice Practice

Biocontrol via Pilot Practice Pilot Practice (cottonseed) non-toxigenic strains

Fungicides Practice Practice Plant breeding Practice Pilot Pilot Practice (conventional and transgenic) Sorting Practice Practice Practice Practice Practice

Nixtamalization Practice

Enterosorbents Practice (animal feeds Practice (animal feeds Practice (animal feeds Practice (animal feeds in the USA) in the USA) in the USA) in the USA) Chlorophyllin Promising; needs Promising; needs Promising; needs Promising; needs more research more research more research more research Dietary Promising; needs Promising; needs Promising; needs Promising; needs chemoprevention more research more research more research more research

GAP, good agricultural practice; GMP, good manufacturing practice; HACCP, hazard analysis and critical control point; Pilot, studied or tested in experimental studies or on a pilot scale, but not in commercial use; Practice, currently being used by growers or producers to control a particular mycotoxin.

The tables organize several key in this chapter because it does not Table 9.2 provides information characteristics of the interventions control mycotoxin levels directly. about which food commodities are presented here. Table 9.1 sum- Likewise, food replacement – sourcing likely to be contaminated by which marizes the information on applicable clean food from another region to mycotoxins, and whether particular interventions by mycotoxin and by type a region suffering high foodborne interventions are common agricultural of intervention, i.e. pre-harvest, post- mycotoxin contamination – is useful practice or have only been tested in pilot harvest, or dietary. Although hepatitis in emergency situations; however, it is experiments. Table 9.3 describes usage B virus vaccination, described in also not included in the table because characteristics of each intervention: the Chapter 7, is a clinical intervention that it does not directly reduce mycotoxin local availability of resources needed may reduce the potency of aflatoxin in levels in the original food supply. to develop it, the technical simplicity causing liver cancer, it is not included of implementing the intervention,

134 Table 9.3. Usage characteristics of interventions: local availability of intervention materials, ease of implementation, usefulness in emergencies, and whether the intervention is applicable at the population or individual level

Usage characteristic Intervention Intervention materials Implementation Population or individual Useful in emergencies available locally technically simple level

GAP Yes Yes No I, P

GMP Yes Yes No P

HACCP Yes Yes No P

Biocontrol via non- Yes (fungal strains and Yes (after development) No P toxigenic strains substrate)

Fungicides Yes Yes No P

Plant breeding No No No P

Sorting Yes Yes Yes I, P

Nixtamalization Yes Yes Yes I

Enterosorbents No No Promising; needs more I, P research Chlorophyllin No No Promising; needs more I research

Dietary chemoprevention No No Needs more research I

GAP, good agricultural practice; GMP, good manufacturing practice; HACCP, hazard analysis and critical control point; I, individual; P, population. its usefulness in emergencies, and aflatoxin production have been addition, breeding for characteristics whether the intervention is applicable identified, so that genomics as a tool for to reduce environmental stressors on at the population or individual level. understanding aflatoxin biosynthesis maize has shown some efficacy in has gained much ground (Yu et al., reducing aflatoxin. 2. Aflatoxins 2008). It is hoped that these findings Efficacy. Suggestive evidence of may have practical application in the efficacy. 2.1 Pre-harvest interventions future; for now, they have improved Geographical regions. Tested in our understanding of the regulation the USA for maize. Several centres 2.1.1 Conventional breeding for and biosynthesis of aflatoxins. Also, of the Consultative Group on In- host plant resistance recent proteomic studies involving ternational Agricultural Research the generation of > 20 000 expressed (CGIAR) have conducted research. Description. Breeding methods have sequence tags from developing Simplicity/complexity. High degree been explored to improve resistance groundnut plants under drought of complexity. Other strategies, such as to drought, insect herbivory, or other stress have yielded several proteins giving germ plasm of improved open- environmental stressors that would potentially associated with resistance to pollinating lines of maize to indigenous predispose groundnuts and maize aflatoxin production (Wang et al., 2010). farmers, have been proposed and to pre-harvest formation of aflatoxin. Stagef o development. Pilot. Much possibly deserve reconsideration. This has included work on identifying research has been conducted on Population/individual. Population. resistant crop lines and identifying resistance, but potentially resistant Useful in emergencies. No. biochemical and genetic resistance varieties lack other characteristics Locality of resources. Local crop markers in crops. Sequencing of the necessary for commercial application. lines should preferably be identified for Aspergillus flavus genome has been For groundnuts, characteristics that suitability for each geographical region. completed. Genes that potentially confer drought tolerance may reduce Accessibility. Likely to be acces- encode for enzymes involved in pre-harvest aflatoxin accumulation. In sible if appropriate hybrids are devel-

Chapter 9. Practical approaches to control mycotoxins 135 CHAPTER 9 CHAPTER oped for different regions of the world Population/individual. Individu- Accessibility. High in geographical and prices are established that are al in current commercial applica- regions where predictive models affordable for small-scale farmers. tions; possibility for population-level have been developed and access References. Gorman and Kang approach. to computers is readily available to (1991), Brown et al. (2001), Cleveland Useful in emergencies. No. growers. Low in other regions. et al. (2003), Maupin et al. (2003), Locality of resources. Appropriate References. Henderson et al. Menkir et al. (2006), Chen et al. fungal strains must be sourced locally (2000), Chauhan et al. (2010), DEEDI (2007), Yu et al. (2008), Arunyanark et for each region where the process (2010). al. (2010), Girdthai et al. (2010), Wang is used. Different substrates may be et al. (2010), Warburton et al. (2011). more economical in various regions; 2.2 Post-harvest interventions economic barriers for subsistence 2.1.2 Biocontrol farmers. 2.2.1 Sorting, drying, and storage Accessibility. Unlikely to be Description. Biocontrol of aflatoxins highly accessible, unless local soil Description. Post-harvest control relies on competitive exclusion. High samples are tested and facilities methods are based on GMP. Some numbers of spores of a non-toxigenic are built and maintained to develop specific methods that apply to aflatoxins strain of A. flavus (or, less commonly, biocontrol strains appropriate for the follow: community-based approaches A. parasiticus) are introduced into the geographical region. and industrial sorting methods. soil where crops are being grown, References. Dorner et al. (1999), At the level of communities, basic where they compete with existing Bandyopadhyay et al. (2005), Pitt and visual hand sorting can remove a toxin-producing spores for sites on Hocking (2006), Cotty et al. (2007), large proportion of nuts or kernels that the developing crop, thus reducing Dorner and Horn (2007), Atehnkeng are significantly contaminated with aflatoxin production. Usually the et al. (2008). aflatoxins. Additional community- selected strain is introduced to the based methods to keep aflatoxin field on a carrier substrate that permits 2.1.3 Forecasting levels low in post-harvest settings growth of the fungus with consequent include proper drying, storage in production of high numbers of spores. Description. AfloMan is a forecasting bags that allow for air circulation, Stage of development. Commer- system for the formation of aflatoxin and use of well-ventilated storage cial in the USA, and pilot. in groundnuts, in use in Queensland, facilities that control for pests. Efficacy. Depending on climatic Australia. Industrial sorting methods vary factors that affect biocontrol agent Stage of development. Commercial. with the crop. growth, and concentration of toxigenic Efficacy. The model accounts Maize. Primary sorting of maize spores in a given field, aflatoxin for up to 95% of the variation in kernels is by examination using an reductions may range widely, from aflatoxin accumulation in groundnut ultraviolet (UV) light (365 nm) after no reduction at all to high levels crops at harvest. As with all models cracking; grains containing appre- (0–80%). of this type, it is highly dependent on ciable aflatoxin fluoresce, enabling Geographical regions. Used com- the reliability of weather and crop sorting of lots. Fluorescence is due mercially in cottonseed and groundnut development data. Similar systems to plant peroxidase enzyme reacting fields in the USA. Tested in pilot have been explored in the USA, but with kojic acid produced by A. flavus. studies on maize in the USA, Nigeria, not beyond the conceptual stage. Some A. flavus strains do not produce Kenya, and Thailand. Tested in pilot Geographical regions. South Bur- kojic acid and therefore do not studies on tree nuts in the USA. nett region of Queensland, Australia. cause fluorescence. Further, tropical Simplicity/complexity. Selecting Simplicity/complexity. Requires temperatures induce isomerization of and maintaining non-toxigenic strains access to reliable climatic data kojic acid, preventing the fluorescence is a specialist undertaking; producing and sophisticated mathematical reaction, so sorting under UV light is substrates for field inoculation modelling, but AfloMan is used daily ineffective in the tropics. requires a feed mill or similar factory by South Burnett groundnut growers Groundnuts. When fungi invade with suitable protection for workers via the Internet. groundnuts, enzymatic changes because A. flavus is a known human Population/individual. Population. cause nut discolouration; thus, pathogen. Application of the product Useful in emergencies. No. sorting out discoloured nuts also onto fields can be carried out by Locality of resources. Not applicable. removes those that contain aflatoxin. trained farmers. Colour sorting should be followed

136 by aflatoxin assays. UV sorting countries use the control measures reducing concentrations of both of groundnuts is possible using a described above. aflatoxins and fumonisins (which are somewhat higher wavelength. In Efficacy. A pilot test in Guinea of a described in Section 3). severe circumstances, groundnuts post-harvest intervention package for Stage of development. This pro- are blanched and roasted, which groundnuts (including education on cess has been used for centuries increases discolouration, enabling how to sort and dry nuts, natural fibre to produce masa (a dough made more effective colour sorting, which drying mats and storage bags, wooden from ground maize), by indigenous again should be followed by aflatoxin pallets on which to store groundnut populations in regions throughout Latin assays. Blanched and roasted nuts bags, and pesticide for storage floors) America, especially Mexico and Central are susceptible to oxidative rancidity achieved a 70% reduction in aflatoxin America, where maize is produced. and thus should be packed under levels in groundnuts after 5 months Efficacy. Under laboratory and nitrogen in gas-tight packaging. of storage compared with untreated commercial conditions, nixtamalization Pistachios. Pistachios have groundnuts, and a 57% mean re- by the traditional process can reduce been very difficult to sort by UV or duction in aflatoxin–albumin adduct aflatoxin levels by up to 90%. discolouration and have traditionally levels in individuals who implemented Geographical regions. Mexico, Cen- not been sorted. However, recent the post-harvest intervention package tral America. studies have shown that sorting by compared with controls. Simplicity/complexity. Simple pro- fluorescence and discolouration Geographical regions. Control cess; adequate clean water is required. may be potentially useful. Aflatoxin measures outlined above are of uni- Population/individual. Individual; in pistachios results from shells versal applicability. possibility for population-level approach. of nuts opening before the nut is Simplicity/complexity. Control mea- Useful in emergencies. Yes. dry, permitting ingress of A. flavus sures vary in complexity, but many Locality of resources. The activated spores. Because shell opening is a can be applied in all regions. lime used in nixtamalization is widely desirable characteristic, control has Population/individual. Individual; available. However, this process involved the development of cultivars possibility for population-level approach. appears to be culturally acceptable only with later shell opening. Useful in emergencies. No. in the Americas. Water can sometimes Almonds. Aflatoxin in almonds is Locality of resources. Most ele- be difficult to obtain, which can cause usually caused by insect damage, so ments of post-harvest control methods problems if washing is inadequate after control relates to insect control. UV can be obtained or manufactured alkaline steeping. In addition, use of light can be used for sorting almonds locally. polluted water for steeping can present containing aflatoxins. Accessibility. Community-based a different set of risks. Brazil nuts. Brazil nuts are infected approaches are generally accessible Accessibility. Likely to be accessible by A. flavus when allowed to remain among subsistence farmers, but in geographical regions where this for extended periods on the forest education is necessary. Industrial practice is accepted and where clean floor before harvest (by picking them sorting approaches are accessible in water is readily accessible. up). Infection by A. nomius, which developed countries on the scale of References. Torres et al. (2001), also produces aflatoxins, apparently large commercial farming. Elias-Orozco et al. (2002), De La Campa occurs before harvest. No control References. Pearson and et al. (2004), Méndez-Albores et al. measures, other than aflatoxin Slaughter (1996), Hadavi (2005), (2004), Bullerman and Bianchini (2007). assays, exist for Brazil nuts at this Turner et al. (2005), Kabak et al. time. Some studies have suggested (2006), De Mello and Scussel (2007), 2.3 Dietary interventions that sorting by size or physical Magan and Aldred (2007), Wagacha appearance may be useful, but more and Muthomi (2008), Pacheco and 2.3.1 NovaSil clay research is needed. Scussel (2009), Khlangwiset and Wu Figs. Aflatoxin in figs results (2010), Pacheco et al. (2010). Description. NovaSil is a dioctahedral from A. flavus infection carried by smectite clay that can bind aflatoxin insects during pollination. Control is 2.2.2 Nixtamalization in the gastrointestinal tract and aid by examination of individual dried or in its elimination. NovaSil can be fresh fruit under UV light to detect the Description. Alkaline cooking of included in food or feed or taken presence of aflatoxin. maize in a solution of ash, lime, or separately during mealtimes. Stage of development. Nut- other materials containing inorganic Stage of development. Commer- producing regions of developed calcium. This process is useful for cial in animal feed; pilot in humans.

Chapter 9. Practical approaches to control mycotoxins 137 CHAPTER 9 CHAPTER Efficacy. In a pilot study in humans Stage of development. Pilot; Lactic acid bacteria from fermented in Ghana, after 3 months of treatment, clinical trials. vegetables, fruits, and dairy products

NovaSil clay added to diets achieved Efficacy. A clinical trial in hu- have the ability to bind aflatoxin B1 in a 59% reduction in aflatoxin M1 levels, mans in Qidong, China, achieved laboratory tests; this has not yet been and a 25% reduction in aflatoxin– a 55% reduction in aflatoxin-N7- tested in animals or humans. albumin adduct levels, in treated in- guanine levels in treated individuals Geographical regions. China dividuals compared with controls. compared with controls. (green tea polyphenols, sulforaphane). Geographical regions. USA (as Geographical regions. Clinical Simplicity/complexity. These in- an anticaking agent in animal feed), trials have been carried out in the terventions would be simple in the Ghana (humans). USA and China. parts of the world where the foods Simplicity/complexity. Currently, Simplicity/complexity. The inter- or drinks containing these dietary NovaSil clay comes only from one mine vention was administered as a constituents are already common in in the USA, so although the material chemopreventive pill, which requires diets. They could be complex where is cheap, importation costs must be regular and continued administration. this is not the case already. Optimized considered. Inclusion in the diet, in Population/individual. Individual. consumption patterns to modify bread or in maize or groundnut meal, Useful in emergencies. Promis- aflatoxin metabolism over extended should be reasonably simple. Studies ing; needs more research for effects periods would need to be developed. indicate some limitations, including the in situations of very high exposure to Population/individual. Individual; risk of vitamin and mineral binding in a aflatoxin in vivo. possibility for population-level approach. nutritionally compromised population. Locality of resources. Depends Useful in emergencies. No. Further, distribution under government on availability of the chemopreven- Locality of resources. Common. supervision will be essential because tive pills. Accessibility. Readily accessible imitation clay materials, similar in Accessibility. Will depend on the in developed countries where these appearance to NovaSil but without the availability of the medication and cost. dietary constituents are affordable; potential benefit, are readily available. References. Dashwood et al. less accessible in LICs, where diets Population/individual. Individual; (1998), Egner et al. (2001), Simonich are much less varied. possibility for population-level approach. et al. (2007, 2008), Groopman et al. References. Haskard et al. (2000), Useful in emergencies. Promising; (2008), Jubert et al. (2009). Fujiki et al. (2002), Kensler et al. (2005), needs more research. Yates and Kensler (2007), Hernandez- Locality of resources. So far, 2.3.3 Naturally occurring dietary Mendoza et al. (2009), Gao et al. (2010), NovaSil clay has been mined only in constituents Gross-Steinmeyer et al. (2010). the USA; would have to be exported to countries in need. Description. Green tea polyphenols, 3. Fumonisins Accessibility. Will depend on cul- sulforaphane derived from cruciferous tural acceptance and cost. vegetables, and lactic acid bacteria. 3.1 Pre-harvest interventions References. Pimpukdee et al. Stage of development. Pilot. (2004), Afriyie-Gyawu et al. (2008), Efficacy. In rat studies, green 3.1.1 Breeding for host plant Phillips et al. (2008), Wang et al. tea polyphenols have been shown resistance (2008). to inhibit initiation of liver cancer induced by aflatoxin. In humans, Description. Breeding and selection 2.3.2 Chlorophyll and inverse associations between green methods have been used for centuries chlorophyllin tea consumption and overall cancer to improve maize resistance to risk have been observed. Sulforaph- fungal and insect infection or other Description. Chlorophyll and its ane, metabolized from glucoraphanin environmental stressors, stressors derivative chlorophyllin, which in cruciferous vegetables such as that have been discovered recently are natural constituents of green broccoli and cabbage, induces phase to predispose plants to fumonisin vegetables, can sequester aflatoxin 2 enzymes such as the glutathione-S- contamination. This has included work in the gastrointestinal tract and transferases that prevent DNA damage on developing resistant inbred crop impede its absorption. In addition, induced by aflatoxin. In human studies, lines and identifying biochemical and these compounds may have enzyme- those individuals who converted more genetic resistance markers in crops. inducing properties that contribute glucoraphanin to sulforaphane had Stage of development. It is reliably to mechanisms of detoxification. lower aflatoxin-N7-guanine levels. known that improved insect and

138 drought tolerance results in reduced Geographical regions. USA, Can- implement; education can improve con- risk for fumonisin accumulation. ada, Argentina, Brazil, Uruguay, fidence in the results. This approach is being applied to South Africa, Honduras, Philippines, Population/individual. Individual; the extent that hybrids suitable Hungary. possibility for population-level approach. for particular areas have become Simplicity/complexity. Although Useful in emergencies. Yes. available. the commercial breeding process Locality of resources. These Efficacy. In years of high insect involves significant expertise and post-harvest control methods can be pressure and drought, such resistant expense at first, Bt seeds can be carried out locally. hybrids can increase the percentage disseminated as readily as other types Accessibility. Generally accessible of the crop suitable for human of seeds, within the area of adaptation. techniques worldwide. consumption. Population/individual. Individual; References. Desjardins et al. Geographical regions. USA, Europe. possibility for population-level ap- (2000), Pearson et al. (2004; 2010), Simplicity/complexity. Although proach in countries with commercial Fandohan et al. (2005), Afolabi et al. the commercial breeding process agriculture. (2006), Kimanya et al. (2009a, 2009b), involves significant expertise and Useful in emergencies. No. Van der Westhuizen et al. (2010, 2011). expense at first, seeds resistant to Locality of resources. Highly vari- fungal development and fumonisin able; requires reliance on biotech- 3.2.2 Nixtamalization formation can be disseminated as nology companies to permit small- readily as other types of seeds, scale farmers access to seed at Description. Alkaline cooking of maize within the area of adaptation. prices under fair conditions. in a solution of ash, lime, or other Population/individual. Population. Accessibility. High in developed materials containing inorganic calcium. Useful in emergencies. No. countries that have permitted Bt maize Stage of development. This process Locality of resources. Local in- planting and commercialization; low has been used for centuries to produce bred maize lines can be identified for elsewhere worldwide. masa, by indigenous populations in suitability to the geographical region; References. Munkvold et al. regions throughout Latin America, questions of access and affordability (1999), Bakan et al. (2002), Hammond especially Mexico and Central America, for LICs. et al. (2004), de la Campa et al. where maize is produced. Accessibility. Likely to be widely (2005), Papst et al. (2005), Wu (2007), Efficacy. Nixtamalization can re- accessible after local varieties with Folcher et al. (2010). duce fumonisin B1 levels in fried tortilla improved traits are developed. chips by up to 80%. References. Miller (2001), Clements 3.2 Post-harvest interventions Geographical regions. All maize- et al. (2004), Afolabi et al. (2007), Henry growing areas in Latin America. et al. (2009), Loeffler et al. (2010), 3.2.1 Sorting and washing Simplicity/complexity. Simple pro- Parsons and Munkvold (2010). cess; adequate clean water is required. Description. Hand sorting of obvi- Population/individual. Individual; 3.1.2 Transgenic Bt maize ously contaminated kernels of home- possibility for population-level approach. grown maize, and washing before Useful in emergencies. Yes. Description. Transgenic Bt maize consumption. Locality of resources. The activated contains a gene from the soil Stage of development. Traditional lime used in nixtamalization is very bacterium Bacillus thuringiensis hand and eye sorting methods are widely available. However, this process that results in the accumulation of well developed; optical sorting using appears to be culturally acceptable only proteins toxic to key insect pests of two wavelengths is possible but in the Americas. Water can sometimes maize. Insect damage predisposes requires expensive equipment. be difficult to obtain, which can cause maize to fumonisin contamination by Efficacy. Sorting and washing problems if washing is inadequate after facilitating fungal infection. maize kernels can reduce fumonisin alkaline steeping. In addition, use of Stage of development. Commercial. contamination by > 84% in maize polluted water for steeping can present Efficacy. Depending on the sever- grains and by > 60% in maize porridge. a different set of risks. ity of insect infestation in a given Geographical regions. Traditional Accessibility. Likely to be accessible year, fumonisin reductions afforded sorting methods are used in many in geographical regions where this by Bt maize can greatly increase the maize-producing regions. practice is accepted and where clean percentage of the crop acceptable Simplicity/complexity. Sorting and water is readily accessible. for human consumption. washing techniques are simple to

Chapter 9. Practical approaches to control mycotoxins 139 CHAPTER 9 CHAPTER References. Dombrink-Kurtzman OTA production in small grains is 5. Deoxynivalenol and et al. (2000), Voss et al. (2001, not a problem in warm temperate or zearalenone 2009), Palencia et al. (2003), De La tropical crops. OTA occurs in maize Campa et al. (2004), Bullerman and but appears to be a minor problem 5.1 Pre-harvest interventions Bianchini (2007), Torres et al. (2007), compared with aflatoxins, fumonisins, Burns et al. (2008). or deoxynivalenol. 5.1.1 Breeding for host plant Dried vine fruits and wines. As resistance in small grains 4. Ochratoxin A noted above, infection in grapes by A. carbonarius and A. niger may occur Description. Breeding methods have 4.1 Pre-harvest interventions before harvest. In winemaking, fungal been explored to improve host plant growth and toxin production cease resistance either to fungal infection Little evidence exists that any of the when fermentation commences. or to other environmental stressors important fungi producing ochratoxin Wine-making reduces the level of that would predispose the plants A (OTA) invade crops before harvest, OTA in wine by up to 80%. In grape to accumulation of deoxynivalenol so plant breeding is of little value in drying, mechanical damage during (DON) and, secondarily, zearalenone the control of OTA formation. The harvest and drying pretreatments (ZEA). This has included work on one exception is that the invasion of increases the possibility of infection identifying resistant cultivars and grapes by Aspergillus carbonarius (and of grapes by A. carbonarius and A. identifying biochemical and genetic A. niger) takes place before harvest. niger, so rapid drying will reduce resistance markers in cultivars. The evidence is that these fungi OTA formation. Some regions (notably Germany cannot infect intact grapes, so entry Coffee. In coffee, OTA infection and Ontario, Canada) have stringent is dependent on infection by fungal occurs immediately after harvest, registration rules that place specific pathogens such as powdery mildews when green coffee cherries are weight on eliminating the worst or Rhizopus stolonifer, mechanical handled, hulled, and dried. The cultivars in trials each year. damage, or skin splitting due to rain. process is often slow because in Stage of development. Commercial. Some cultivars are more susceptible many regions coffee is grown under Efficacy. Efficacy varies, depend- to skin splitting, so plant breeding is of climatic conditions that favour mist ing on climatic conditions by year value in that area. and rain at harvest time. Poor storage and on agronomic factors, including Pre-harvest control of OTA produc- is also a documented factor causing the topology of the field. Current tion in grapes, and hence dried vine increases in OTA levels. Partial efforts are focusing on developing fruits and wine, is based on limiting control can be achieved during the high-yielding cultivars with good damage by the powdery mildews (by early stages of manufacture, where quality characteristics and moderate fungicidal spray programmes) and defective cherries, which frequently resistance to Fusarium head blight by Rhizopus (by a defoliant spray to contain most OTA in a sample, and DON production. increase exposure of grapes to sunlight, are sorted out. Roasting of coffee Geographical regions. Cultivars which limits growth of Rhizopus). reduces OTA levels by amounts that with reasonable efficacy are in use in vary with the severity of the roasting the USA, Canada, and Europe. 4.2 Post-harvest interventions process, e.g. from a 50% reduction Simplicity/complexity. Although the for a light roast (12 minutes at 180 °C) breeding process involves significant Fungi producing OTA invade crops to a > 90% reduction for a dark roast expertise and expense at first, after harvest. Types of control vary (8 minutes at 240 °C). resistant seeds can be disseminated with the crop. Cocoa and chocolate. OTA occurs as readily as other types of seeds, Small grains. OTA is produced in cocoa, and hence in chocolate, within the area of adaptation. after harvest, during drying in small but levels are usually low. Rapid and Because small grains are open- grain cereal crops (wheat and adequate drying of cocoa beans is the pollinated, seeds can be saved from barley) in cool temperate zones, important control step. year to year. by Penicillium verrucosum. The References. Taniwaki et al. (2003), Population/individual. Population. problem is widespread, and the Leong et al. (2006), Copetti et al. Useful in emergencies. No. only effective control measure (2010), Ferraz et al. (2010). Locality of resources. Local lines consists of rapid drying, where this can be identified for suitability to is possible. Because P. verrucosum the geographical region through does not grow in warmer climates, the International Maize and Wheat

140 Improvement Center (CIMMYT) shut- 5.1.3 Transgenic Bt maize during anthesis, the optimal time for tle breeding program. F. graminearum infection and DON Accessibility. Likely to be widely Description. Transgenic Bt maize accumulation. accessible after local varieties with contains a gene from the soil bacterium Stage of development. Commercial. improved traits are developed. Bacillus thuringiensis that encodes for Efficacy. Varies depending on References. Mesterhazy et al. proteins toxic to key insect pests of fungal infection risk by year. (1999), Boutigny et al. (2008), Foroud maize. Insect damage predisposes Geographical regions. Worldwide. and Eudes (2009), Snijders (2004), maize to DON and ZEA contamination Simplicity/complexity. Simple to Müller et al. (2010). by facilitating fungal infection. implement, although basic education Stage of development. Commercial. is required. 5.1.2 Breeding for host plant Efficacy. Reductions of DON Population/individual. Individual; resistance in maize levels by up to 59% and significantly possibility for population-level approach. lower ZEA levels in animal feed Useful in emergencies. No. Description. Breeding methods made from Bt maize compared with Locality of resources. Widely avail- have been explored to improve host maize lacking the Bt gene. able in developed countries, much plant resistance either to fungal Geographical regions. USA, less so in LICs; cost implications for infection or to other environmental Canada, Argentina, Brazil, Uruguay, subsistence farmers. stressors that would predispose the South Africa, Honduras, the Philip- Accessibility. Accessible in devel- plants to accumulation of DON and, pines, Hungary, China. oped countries, much less so in LICs. secondarily, ZEA. This has included Simplicity/complexity. Although References. Hollingsworth et al. work on identifying resistant cultivars the transgenic breeding process (2008), Odenbach et al. (2008), Paul and identifying biochemical and involves substantial initial expertise et al. (2008), Zhang et al. (2009). genetic resistance markers in hybrids. and expense, Bt seeds, once Some regions (e.g. Ontario, Canada) developed, can be disseminated as 5.1.5 Forecasting have stringent registration rules that easily as other types of seeds. place specific weight on eliminating Population/individual. Individual; Description. The most evolved mod- the worst hybrids in trials each year. possibility for population-level approach. els for forecasting the formation of Stage of development. Commercial. Useful in emergencies. No. DON in small grains were developed Efficacy. Moderate resistance Locality of resources. Not local; in Ontario, Canada. Only preliminary has been achieved. requires reliance on biotechnology work has been reported on a model Geographical regions. Cultivars companies to permit small-scale for DON in maize. with moderate resistance are in use farmers access to seed at prices under Stage of development. Commer- in the USA, Canada, and Europe. fair conditions. cial in Ontario, Canada, and in Europe. Simplicity/complexity. Although Accessibility. High in developed Efficacy. Analyses of data from the breeding process involves signifi- countries that have permitted Bt maize produced a model with initial cant expertise and expense at first, maize planting and commercializa- validation, i.e. data for enough resistant seeds can be disseminated tion; low elsewhere worldwide. years of variation in two countries to as readily as other types of seeds, References. Munkvold et al. explain 70% of variation in fumonisin within the area of adaptation. (1999), Aulrich et al. (2001), Magg accumulation. A different model, Population/individual. Population. et al. (2002), Schaafsma (2002), Wu developed in Italy, has had only Useful in emergencies. No. (2007), Burachik (2010), Folcher et al. limited validation thus far. Locality of resources. Local in- (2010), Loeffler et al. (2010). Geographical regions. These mod- bred varieties can be identified els have been applied in Uruguay, in for suitability to the geographical 5.1.4 Fungicides France, and (since 2009) throughout region through the CIMMYT shuttle Europe. breeding program. Description. Chemicals that control Simplicity/complexity. These mod- Accessibility. Likely to be widely fungal species; azoles, commonly els are used to enable management accessible after local varieties with used to control Fusarium head blight, decisions, e.g. harvest time, whether improved traits are developed. inhibit sterol biosynthesis in Fusarium foliar fungicides would be useful in References. Boutigny et al. species. However, an important a given year, and crop segregation. (2008), Loeffler et al. (2010) and issue for high efficacy of fungicides These models must be tested and references therein. is the correct timing of application validated in many areas, not only to

Chapter 9. Practical approaches to control mycotoxins 141 CHAPTER 9 CHAPTER make the models more widely useful Accessibility. High in regions of 5.2 Post-harvest interventions but also to test their robustness to the world for which forecasting models climatic variation. As with all predictive have been developed (Canada, Because Fusarium species do models, these require reliable climatic Europe); low elsewhere worldwide. not grow at water activities below and agronomic data and some so- References. de la Campa et about 0.90, DON and ZEA are not phisticated mathematics. al. (2005), Hooker and Schaafsma produced in grains that have been Population/individual. Population. (2005), Schaafsma and Hooker even partially dried. Post-harvest Useful in emergencies. No. (2007), Maiorano et al. (2009), Müller treatments are not applicable Locality of resources. To date, et al. (2010), Van der Fels-Klerx et al. to these mycotoxins in wheat or Canada and Europe. (2010). maize.

142 References

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146 Subject index

Acceptance sampling plans Alimentary toxic aleukia (ATA) Biomarkers 48–49 2, 14 60, 61, 64, 66, 72, 74–76, 88, Aflatoxicosis Alternaria arborescens 96, 97, 110 2, 89–90 19–20 of exposure to aflatoxins, 72,

Aflatoxin B1 Analytical methods 88, 89, 91 chemical and physical 54–58 of exposure to fumonisins, 72, properties of –, 31–32 in developed countries, 54 74, 93

Aflatoxin B2 in developing countries, 54–55 Brazil nuts chemical and physical in rural areas, 55–56 7, 8 properties of –, 31–32 Animal Breeding for host plant resistance

Aflatoxin G1 contributing factors to – 19, 135–136, 138, 140–141 chemical and physical diseases, 60–63 Cancer potency factor properties of –, 31–32 disease outbreaks, 60–63 108

Aflatoxin G2 diseases, 60–63, 72–74 Carcinogenic effects chemical and physical performance problems, 71, 73, 108–109 properties of –, 31–32 75, 77, 78 Carcinogenicity

Aflatoxin M1 production problems, 61 of aflatoxins, 88–89, 90 chemical and physical reproductive effects, 63, 71, 77 of deoxynivalenol, 97–98 properties of –, 31–32 Antibody-based methods of fumonisins, 93 testing for –, 40–41 56 of ochratoxin A, 96–97 Aflatoxins Aspergillus of zearalenone, 99 4–8, 31–32, 62, 88–92, 111 3–12 Case–control studies carcinogenicity of –, 88–89, 90 carbonarius, 8, 10–12 106, 107 effects in farm animals of –, flavus, 4–12 Cashew nuts 71–72, 78 japonicus, 11 7 formation of –, 6–8 niger, 8, 10–12, 19–20 Cattle human health effects of –, ochraceus, 8–12 60, 61, 63, 70, 71, 76–77, 78, 79 89–92 parasiticus, 4–12 Cereals interventions to control –, Assessment 14 135–139 dose–response –, 107–109 Chickpeas maximum tolerated levels of –, exposure –, 109–110 10 122–123 risk –, 106–112 Child growth impairment mechanisms of action of –, 71, Average daily dose (ADD) 91 88–89 109–110 Chlorophyll occupational exposure to –, Beans 138 91–92 10 Chlorophyllin risk assessment of –, 111 Benchmark dose 138 testing for –, 40 108 Chromatographic methods toxicokinetics and metabolism Biocontrol 54–58 of –, 64 136

Subject index 147 Cirrhosis Deoxynivalenol Ergot alkaloids 90 34, 62–63, 97–98, 112 23–24, 36, 63 Citreoviridin carcinogenicity of –, 97–98 chemical and physical 3 chemical and physical properties of –, 36 Citrinin properties of –, 34 effects in farm animals of –, 3 effects in farm animals of –, 76–77, 79 Classification of agents as to 75–76, 79 poisoning, 63 carcinogenicity in humans formation of –, 20–22 toxicokinetics and metabolism 95, 109, 111–112 human health effects of –, of –, 70–71 Claviceps 97–98 Ergotamine 4, 23–24 interventions to control –, 36 purpurea, 23–24 140–142 Ergotism Clinical signs in farm animals mechanisms of action of –, 75, 2, 61, 76 71–77 97 Erosion Coconuts occupational exposure to –, 98 132 7 risk assessment of –, 112 Estrogenic effects Coefficient of variation (CV) testing for –, 41–42 60, 63, 76, 79 41 toxicokinetics and metabolism Eurotium Coffee beans of –, 68–69 4 10, 140 Dietary interventions Export loss Cohort studies 137–138 123 106 Disability-adjusted life years (DALYs) Exposure assessment Commodities at risk 124 109–110 6–8 Dose–response assessment F-2 toxin Consumer risk 107–109 63 49–50 Drought Feasibility of interventions Contributing factors 18–19 127–128 60–63 Drying Feed refusal Control strategies 10, 132, 136–137 60, 62–63, 75, 79 131–142 Economic impacts of mycotoxins Fertilizers Cost-effectiveness of interventions 125–126 132 126–127 Economics of mycotoxins Fescue foot Cost of illness (COI) 119 –128 63, 77 124 Emericella Field outbreaks Costs of mycotoxins 4 59–63, 72 in international markets, Environmental stressors Figs 122–123 135 8, 12 in local and regional markets, Enzyme-linked immunosorbent Fish 121–122 assay (ELISA) 7, 10 to society, 120–126 41– 42, 54, 56 Fluorescence polarization (FP) Cottonseed Epidemics immunoassay 7 2 55 Cyclopiazonic acid Equine leukoencephalomalacia Fluorometric methods 3 (ELEM) 42, 54 Decision trees 60, 62, 72–74 Forecasting 23 136, 141

Fumonisin B1 chemical and physical properties of –, 32–34

Fumonisin B2 chemical and physical properties of –, 32–34

148 Fumonisins Haemorrhagic syndrome Interventions 32–34, 62, 92–94, 111–112 61 126–128, 131–142 carcinogenicity of –, 93 Harvesting cost-effectiveness of –, effects in farm animals of –, 132 126–127 72–74, 78 Hazard dietary –, 137–138 formation of –, 19–20 analysis and critical control point feasibility of –, 127–128 human health effects of –, (HACCP) system, 133–135 post-harvest –, 139–140, 142 93–94 identification, 106–107 pre-harvest –, 135–136, 138, interventions to control –, quotient (HQ), 110 –111 140–142 138–139 Hazelnuts public health –, 126–128 mechanisms of action of –, 74, 7 Joint FAO/WHO Expert Committee 92–93 Health economic impacts of on Food Additives (JECFA) probable daily intake of –, mycotoxins 66, 92, 111–112, 122 113 –114 123–125 Kidney disease risk assessment of –, 111–112 Hepatocellular carcinoma (HCC) 61, 96 testing for –, 41 88, 90, 93, 111 Lactic acid bacteria toxicokinetics and metabolism Hepatotoxicity 138–139 of –, 65–67 71 Laminitis Fungicides High-performance liquid 63, 77 141–142 chromatography (HPLC) Lateral flow devices Fusarium 54 54 3–4, 14–22 Horses Lifetime average daily dose (LADD) crookwellense, 21 62, 63, 66, 72–74, 77, 78, 79 109–110 culmorum, 21 Human health effects Lipid metabolism graminearum, 20–22 87–104 74 proliferatum, 16–19 of aflatoxins, 89–92 Liver cancer subglutinans, 18 of deoxynivalenol, 97–98 88, 90, 93, 111 verticillioides, 16–19 of fumonisins, 93–94 Lowest-observed-effect level (LOEL) Fusarium of ochratoxin A, 96–97 108 head blight, 21–22 of zearalenone, 98–99 Maize kernel rot, 18–19 Human health losses 7, 17–19, 62, 72, 88, 89–90, 93 Gibberella moniliformis 123–125 bag stack of –, 42 16 Immunoaffinity columns (IACs) crib of – cobs, 42 Glutathione-S-transferases 54 transgenic –, 139, 141 138 Immunological methods Market Good agricultural practice (GAP) 54–58 costs of mycotoxins, 121–122 132 Immunomodulation impacts of mycotoxins, 120–123 Good manufacturing practice (GMP) 90–91 Matrix 132–133 Individual-level intervention 54 Grain and groundnut dusts 132–142 effects, 56 occupational exposure to –, 99 Insects Meat Grains 18–19, 132 10, 14 21–22, 140 Interlaboratory collaborative studies Mechanisms of action Grapes 54 of aflatoxins, 71, 88–89 11–12 International Agency for Research on of deoxynivalenol, 75, 97 Green tea polyphenols Cancer (IARC) of fumonisins, 92–93 138–139 90, 93, 95, 109, 111–112 of ochratoxin A, 94–96 Gross domestic product (GDP) International trade of zearalenone, 98

126–127 122–123 Median effective dose (ED50) Groundnuts 108

7, 43, 49, 88 Median lethal dose (LD50) 89, 108

Subject index 149 Metabolism Nivalenol Penicillium 63–71, 72, 76 34–35 3–4, 12–14 Metabolites chemical and physical expansum, 3 68 properties of –, 34–35 verrucosum, 8, 12–14 Methods formation of –, 20–22 Penitrem A analytical –, 54–58 Nixtamalization 3 antibody-based –, 56 137–138, 139 Phase 2 enzymes chromatographic –, 54–58 No-observed-effect level (NOEL) 138 fluorometric –, 42, 54 108 Phomopsin immunological –, 54–58 NovaSil clay 3 Mouldy corn poisoning 137 Phomopsis leptostromiformis 62 Occupational exposure 3 Mycotoxic nephropathy to aflatoxins, 91–92 Pigs 62, 78 to deoxynivalenol, 98 60–63, 64, 65, 66–71, 72, 73, Mycotoxicoses to grain and groundnut dusts, 74, 75, 76, 78–79 60–63 99 Pistachio nuts Mycotoxigenic fungi to ochratoxin A, 96–97 7–8, 10 2–3 Ochratoxicosis Pithomyces chartarum Mycotoxins 62 3 analysis of –, 53–58 Ochratoxin A Plant chemical and physical 8–14, 32–34, 62, 94–97, 112 breeding, 135–136, 138, properties of –, 31–38 carcinogenicity of –, 96–97 140–141 control of –, 113–114, 131–142 chemical and physical stress, 132 costs in international markets, properties of –, 32–34 Population attributable risk (PAR) 122–123 effects in farm animals of –, 126 costs in local and regional 74–75, 78 Population-level intervention markets, 121–122 formation of –, 8–14 132–142 costs of – to society, 120–126 human health effects of –, Porcine pulmonary oedema (PPE) definition of –, 2 96–97 60, 72–74 economic impacts of –, interventions to control –, 140 Post-harvest interventions 125–126 mechanisms of action of –, 136–140, 142 effects in animals of –, 59–86 94–96 Poultry fungi producing –, 2–3 occupational exposure to –, 61, 62, 64, 67, 68, 69, 70, 71, health economic impacts of –, 96–97 72, 73, 74, 78, 79 123–125 risk assessment of –, 112 Precision spotters human health effects of –, testing for –, 41 55 87–89 toxicokinetics and metabolism Pre-harvest interventions market impacts of –, 120–123 of –, 67–68 135–136, 138, 140–142 standards for –, 122–123 Odds ratio (OR) Probable daily intake (PDI) testing for –, 40–42 106 92, 113 –114 toxicological effects of –, 71–77 Oesophageal cancer Producer risk trade impacts of –, 120–123 93–94 49–50 Neosartorya Oilseeds Product monitoring 4 7 39–40 Nephrotoxicity Operating characteristic (OC) curve Provisional maximum tolerable daily 68, 74 49 intake (PMTDI) Neural tube defects Optical readers 92, 108 93–94 55 Public Neurological effects Patulin education, 114 –115 63 3 health interventions, 126–128 Pecan nuts Quality-adjusted life years (QALYs) 10 124–125

150 Rabbits Slope factor Trade 66, 67, 68, 74, 78 108 barriers, 122 Rd-toxin Sorting impacts of mycotoxins, 62 133, 136–137, 139 120–123 Red mould poisoning Species sensitivity Transgenic maize 62 60, 61, 77–79 139, 141 Reference dose (RfD) Spices Transportation 108 7 128 Regulations Sporidesmin Trichothecenes 48–50, 128 3 3, 21–22 Relative risk (RR) Stachybotryotoxicosis Turkey “X” disease 106 2 62 Reproductive effects in animals Storage Ultraviolet light 63, 71, 77 8, 132, 136–137 136 Resources Structure–activity relationship (SAR) Value chain of crops 132–142 models 121–122 Risk 107 Vomitoxin consumer –, 49–50 Subsistence farming 62 producer –, 49–50 47–50 Walnuts Risk assessment Sulforaphane 7 106 –112, 126 138 Washing of mycotoxins, 111–112 Summer slump 139 Risk characterization 63, 77 Water supplies 110 –111 Supply and demand 132 of carcinogens, 111 120–121 Weeds of non-carcinogenic toxins, Surveillance 132 110 –111 sampling plans, 43–45 Weight of evidence (WOE) Risk management studies, 42–48 108 112–114 Target organ Welfare strategies, 131–142 61 120 Rubratoxin A Taxonomy Wheat 3 3–4 21–22, 41–43, 97–98, 140 Sample of Aspergillus species, 4–12 Wines preparation, 50 of Fusarium species, 14–22 11–12, 140 preparation variance, 40 of Penicillium species, 12–14 Zearalenone size, 43–48 Tenuazonic acid 35, 63, 98–99, 112 storage, 50 3 carcinogenicity of –, 99 variation, 40 Testing for mycotoxins chemical and physical Sampling 40–42 properties of –, 35 48–50 Thin-layer chromatography (TLC) effects in farm animals of –, acceptance –, 48–49 54, 55–56 76, 79 in subsistence farming areas, Tolerable daily intake (TDI) formation of –, 20–22 47–50 108 human health effects of –, methods, 43–48 Toxicokinetics 98–99 of processed commodities, 43 59, 63–71 interventions to control –, of whole kernels, 43 Toxicological effects 140–142 plans, 42–48 71–77 mechanisms of action of –, 98 variability, 40–42 Toxicology risk assessment of –, 112 variance, 40 60 toxicokinetics and metabolism Sheep of –, 69–70 63, 64, 65, 67, 68, 69, 70, 77, 78, 79

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