Mycotoxins

BY D. S. P. PATTERSON

1 Introduction

It has long been known that certain moulds and larger fungi can be harmful to man and animals. Ergot and toadstool poisoning are obvious examples of the earliest known forms of fungal toxicity. But, by convention, toxins causing the latter form of poisoning are seldom termed mycotoxins. These then, are all formed by microscopic fungi or moulds, usually associated with foodcrops or animal feeds.

In the 1940’s and 1950’s certain fungal metabolites were found to possess useful properties and, particularly following the discovery of penicillin, many substances with antibiotic properties were isolated from laboratory cultures. All such substances were mycotoxins by definition but their unique property was their selective toxicity towards pathogenic micro-organisms while remaining relatively harmless to man and animals undergoing treatment. At about the same time it was also suspected that some diseases could be attributed to fungal toxins that were ingested involuntarily in food. Thus, in Russia, an epidemic of a fatal disease known as Alimentary Toxic Aleukia (ATA) swept through the Ukraine following the use of over-wintered wheat in local bread making and in the United States serious losses of livestock were caused by ‘mouldy feed toxicosis’. The former disaster was investigated by Soviet scientists but at that time little of this was known to the world at large, and in general, illnesses associated with mouldy food or feeds attracted so little scientific attention that in 1962 Forgacs and Carll described rnycotoxicoses as ‘neglected diseases’ and in a text-book of veterinary toxicology published at about that time Garner2 stated that ‘in only a few instances has it been shown that extracts from fungi are harmful’. The situation suddenly began to change when aflatoxin was discovered as the cause of ‘Turkey X disease’, an epidemic that had been responsible for the death of thousands of poultry in the UK during 1960. As soon as it was realized that this newly described toxin not only caused acute liver disease but induced hepatomas in the trout and laboratory animals, research on aflatoxin gathered momentum on both sides of the Atlantic. Today there is a vast literature

’ J. Forgacs and W. T. Carll. Ad!) Vef . Sci., 1962, 7, 273. * R. J . Garner, ‘Veterinary Toxicology’, 2nd Ed, Bailliere, Tindall and Cox, London, 196 1, p. 303.

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206 Environmental Chemistry

on the subject (see various reference works””) and even this is steadily being matched by research carried out on many more toxic metabolites of quite commonly occurring fungi.

It is not possible to comprehensively review the literature in a short report of this kind: instead, a broad overview of the subject has been attempted. This has entailed a degree of selection and simplification but sufficient references have been provided for the reader to obtain detail when this is required. It will be seen that mycotoxins are to some extent inescapable contaminants of many foods and animal feedstuffs and as many are at least potentially harmful to man and animals they are worthy of serious consideration. Besides, it will also be evident that in the study of mycotoxins there is enormous scope for the chemist with interests in the biosynthesis of natural products, trace analysis, metabolism, molecular pathology, and even chemical engineering.

2 Biogenesis of Mycotoxins

Growth of a fungus on cereal or other substrates does not necessarily denote the presence of my cotoxin because not all species and strains of fungi produce toxins and environmental conditions favouring optimal growth rarely coincide with those best suitable to toxin biosynthesis. Indeed, a number of physical, chemical, and biological factors’*# l 3 combine together to determine whether a particular mycotoxin is eventually produced on the substrate in question. He~se l t i ne ’~ has listed the following determinants; moisture (relative humidity in the field, drying or re-wetting after harvest), temperature, mechanical damage (bruising, insects, birds, etc.), blending with other grain, influence of ‘hot spots’, time interval before harvest and length of storage period, gaseous environment (especially carbon dioxide and oxygen), chemical composition of the substrate, mineral nutrition, chemical treatment of the crop, plant ‘stress’, invertebrate vectors, spore load, plant varietal differences, fungal strain differences, and the nature of the microbiological ecosystem. Consequently, although the presence of potentially toxigenic species of the fungus can be established by mycological examination in the laboratory, it can

L. A. Goldblatt (ed.), ‘Aflatoxin: Scientific Background, Control and Implications’, Academic Press, New York and London, 1969. A. Ciegler, S. Kadis, and S. J. Ajl (ed.), ‘Microbial Toxins’, Vol. VI, ‘Fungal Toxins‘. Academic Press, New York and London, 197 1. ’ S. Kadis. A. Ciegler. and S. J . Ajl (ed.), ‘Microbial Toxins’. Vol. VII, ‘Algal and Fungal Toxins’, Academic Press. New York and London. 1972. ‘ I. F. H. Purchase (ed.), ‘Mycotoxins’. Elsevier Scientific Publishing Co., Amsterdam, Oxford and

New York, 1974. ’ J . V . Rodricks (ed.), ‘Mycotoxins and other Fungal Related Food Problems’, Adr. Chem. Ser.,

American Chemical Society, Washington DC. 1976. No. 149. J. V . Rodricks, C . W. Hesseltine, and M. A. Mehlman (ed.), ‘Mycotoxins in Human and Animal Health’, Pathotox Publishers Inc., Park Forest South, Illinois, 1977.

9T. D. Wyllie and L. G . Morehouse (ed.), ‘Mycotoxic Fungi, Mycotoxins, Mycotoxicoses: an Encyclopedic Handbook’, 3 Vols. Marcel Dekker, New York and Basel, 1977 and 1978.

lo K. Uraguchi and K. Yamazaki (ed.), ‘Toxicology, Biochemistry and Pathology of Mycotoxins’, Kodansha Ltd.. Tokyo and John Wiley, New York. 1978.

IIJ. G. Heathcote and J. R. Hibbert. ‘Aflatoxins: Chemical and Biological Aspects’, Elsevier Scientific Publishing Co., Amsterdam, Oxford and New York. 1978.

’ * J . Lacey. S. T. Hill, and M. A. Edwards. Trop. Stored Prod. InJ, 1980. 39. 19. I ’ C. W. Hesseltine, i n ‘Mycotoxins and other Fungal Related Food Problems’, ed. J . V . Rodricks, A&.

Chem. Ser., American Chemical Society, Washington DC. 1976, No. 149, p. 1.

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Mycotoxins

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208 Environmental Chemistry

never provide certain evidence for the presence in food of the relevant mycotoxin. This can only be done by direct chemical analysis (see Section 4).

It is possible to distinguish two broad types of fungal metabolism, primary and secondary. Primary metabolism, while cells are still multiplying, involves aerobic and anerobic respiration, the energy-requiring processes of cell growth, main- tenance, and division, and the synthesis of structural and functional macro- molecules. Secondary metabolism, is a characteristic of the senescent organism and, for example, is observable in a laboratory culture following a period of exponential growth when there is no longer an increase in the biomass. At this stage different use is made of certain basic intermediate metabolites, such as acetate, malonate, mevalonate, pyruvate, and amino-acids, which are utilized in the synthesis of complex molecules having no obvious physiological function 1 4 . These ‘secondary metabolites’ constitute a chemically diverse group of compounds that can readily be classified into closely related families such as coumarins, flavonoids, ketides, macrolides, pyrroles, and terpenoids. Many are pigments and others possess antibiotic or toxic properties. It is the latter type of compound that concerns us at present and in Figure 1 is shown the broad relationship between primary and secondary metabolism and the origins of some mycotoxins.

Citrinin (see Figure 2) provides a simple example and is synthesized by the so-called polyketide pathway. which involves the head-to-tail condensation of 5 acetate units, cyclization, and the insertion of two methyl groups from methionine. Sterigmatocystin and aflatoxin arise by a fairly complex route but this too initially involves the formation of a polyketide chain. The isocoumarin moiety of the ochratoxin A molecule is formed from one acetate and 4 malonate units followed by chlorination and the subsequent condensation with phenylalanine. Many of the trichothecene mycotoxins (see Figure 2 for their general structure) are sesquiter- penes and are derived through the mevalonate pathway with the basic tricho- thacene skeleton being formed from farnesyl pyrophosphate.

An early catalogue of fungal metabolites was compiled by TurnerI5 in 1971 and biosynthetic pathways were considered in an even earlier symposium.16 Individual chapters of the various treatises 3-11 have subsequently provided detail of the pathways relating specifically to mycotoxins and an authoritative account of their biosynthesis has just (1 980) become available.”

A given mycotoxin often appears to be produced by a restricted number of fungal species but this is not generally so. For example. patulin, which at one time was tested clinically for its antibiotic potential. can be produced by at least 12 Penicillium species, three Aspergillus species, three Aspergillus species, one or more species of B-vssochlamys and perhaps other fungi.

l4 E. D. Weinburg, Perspect. B i d . Med., 1971, 14. 5 6 5 . l5 W. B. Turner, ‘Fungal Metabolites’, Academic Press. New York and London. 197 1 . I ‘ Z. Vanek and Z. Hostalek (ed.), ‘Riogenesis of Antibiotic Substances‘. Academic Press. New York

and London, 1965. P. S. Steyn (cd.). ‘The Biosynthesis of Mycotoxins. A Study in Secondary Metabolism’. Academic Press. New Yark, London, Toronto, Sydney and San Francisco. 1980.

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M y cotoxins 209

3 The Importance of Mycotoxins in the Environment

This can be assessed in at least two ways; first, by their frequency of detection in food and animal feeds and secondly, according to their toxicological potency.

Mycotoxins in Food and Feeds.-Fungal contamination of the basic food commodities can arise either when a plant is growing in the field or after harvest when stored. Manufactured or prepared food can also become contaminated during storage. For example, amongst other fungal species, living plants may be colonized by Fusarium graminearum (producing zearalenone) and Aspergillus fravus (aflatoxin), while stored material can support the growth of A . flavus and A . parasiticus (both producing aflatoxin), A . uersicolor (sterigmatocystin), A . ochraceus (ochratoxin A), and several Fusarium species (zearalenone and trichothecene mycotoxins).

Some 30 mycotoxins have been found to be occasional contaminants of food or animal feed but they are not equally common. Indeed in the UK traces of only the following mycotoxins have so far been d e t e ~ t e d : ’ ~ - ~ ~ aflatoxins B,, B,, GI, G,, and M citrinin, ochratoxin A, patulin, sterigmatocystin, T2-toxin, vomitoxin, and zearalenone (Figure 2).

Toxicological Potencies of Mycotoxins.-LD,, values determined in the rat do not provide a very satisfactory means of assessing the relative importance of different mycotoxins as they generally fall within a fairly close range of 7-25 mg kg-’ body weight. Altogether different values may be obtained in other laboratory animals, although sex, strain, various dietary factors, and the route of administration also influence the determination of an LD,, value.

The only toxin that is evidently different from the others on this basis is zearalenone, which has been said to have an LD,, value in excess of 16 g kg-’ body weight 24 and, although this compound is usually termed a mycotoxin, it is therefore not an acute toxin at all. It is a potent oestrogen, however, particularly in the pig.25

When mycotoxins are considered as largely inevitable environmental con- taminants, perhaps their potential for causing chronic toxicity is a more important attribute. At least in man, single large dose acute toxicity is unlikely to occur because high toxin concentrations are often accompanied by macroscopic moulding of food which then has an unpleasant appearance and flavour. Therefore chronic exposure to very small amounts of mycotoxin would appear to present the greater risk to man. Animals also resist eating mouldy feed but not always to the

’’ B. J . Shreeve. D. S. P. Patterson, and B. A. Roberts, Vet. Rec.. 1975, 97, 275. ’’ D. S. P. Patterson. B. A. Roberts, B. J . Shreeve, A. E. Wrathall, and M . Gitter, Ann. Nutr. Aliment.,

2o D. S. P. Patterson, E. M. Glancy, and B. A. Roberts, Food Costnet. Toxicol., 1980, 18, 35. ” D. C. Hunt, L. A. Philp, and N. T. Crosby,Ana!,ist (London), 1979, 104, 1171. 2 2 A. E. Buckle, Proc. 2nd. int. Confr. Vet. Pharrnacol. Toxicol. Therap., 198 1, in press. 23 Anon., Food Surveillance Paper No. 4. ‘Survey of Mycotoxins in the United Kingdom’, H.M.S.O.,

24 D. E. Bailey. G. E. Cox, K. Morgareide. and J. Taylor. Toxicol. A p p l . Pharrnacol., 1976, 37, 144. 2 5 C. J. Mirocha and C. M. Christensen, in ‘Mycotoxins’, ed. I . F. H. Purchase, Elsevier Scientific

1977. 31. 643.

London. 1980.

Publishing Co.. Amsterdam, Oxford and New York, 1974, p. 129.

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2 10 Environmental Chemistry

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212 Environmental Chemistry

point of starvation and as compounded feeds are usually flavoured, the presence of trace amounts of mycotoxins does not usually diminish palatibility.

Numerous environmental chemicals including some mycotoxins have now been screened for embryotoxic, teratogenic, mutagenic, and carcinogenic properties. The pregnant rat, mouse, or guinea-pig are the usual laboratory test animals for embryotoxic and teratogenic effects.26 Mutagenicity is usually determined in an in vitro bacterial test system, with 27 or without 28 prior ‘activation’ by rodent-liver microsome preparations, and susceptible strains of the rat 29 or the rainbow trout 30

are used for carcinogenicity trials. Table 1 summarizes these biological effects of aflatoxins B and M,, ochratoxin A, patulin, sterigmatocystin, and T2-toxin. Ochratoxin A and T2-toxin are teratogenic and aflatoxin B, and patulin may be also. Aflatoxin B, and M, and sterigmatocystin behave as mutagens in one test system and patulin and sterigmatocystin in another. Where both biological activities have been tested for, mutagenicity seems to correlate with carcinogenic potential.

In unconfirmed reports, ochratoxin A31,32 has been shown to be carcinogenic and patulin induces tumours only at an intradermal injection site.32 The only other common toxins which are known to be carcinogenic are aflatoxin B,, aflatoxin MI, penicillic acid, and sterigmatocy~tin.~~ All except penicillic acid have been detected in food or animal feeds in the UK, but because of their more frequent detection in albeit low concentrations the aflatoxins would certainly appear to possess the greatest potential to harm animal and public health.

Table 1 Biological effects of some mycotoxins Mvcotoxin

Aflatoxin B, Aflatoxin M, Citrinin Ochratoxin A Patulin

Sterigmatocy stin T2-toxin

Zearalenone

Target organ Acute toxicity LD5, mg kg ‘

liver 0.36 (o ;d) liver 0.32 (0: d) kidney 35 (sc; rn) kidney, liver 2 0 - 2 2 (0: r) nervous system. 10 (sc: rn)

liver, kidney 120 (o:r) skin (contact). stomach. 4 (0 : r)

reproductive 16 000 (0; r)

visceral organs

bone marrow

system

Embryotoxic Teratogenic Mutagenic Carcinogenic

+ + + + nt nt + + + + + + * + +

- + -

- +

nt nt + + + + - f

+ - + nt

Adapted from, B. J. Wilson and A. W. Hayes, in ‘Toxicants Occurring Naturally in Foods’, National Academy of

Abbreviations: o = oral, sc = subcutaneous, d = duckling. m = mouse. r = rat, nt : not tezted Sciences, Washington DC. 1973, p. 372: A. W. Hayes, M.vcopa/ho/ogia. 1978. 65,29: Clin. Toxicol., 1980. 17,45

General Attributes of Mycotoxic Disease.-Mycotoxins are chemically diverse (see Figure 2) and their toxic effects are just as varied since they can interact with almost any organ or physiological system of the body (Table 1). Consequently the

26 A. W. Hayes, R. D. Hood, and H. L. Lee, Teratology, 1974,9,93. 27 B. N. Ames, W. E. Durston, E. Yamasaki, and F. D. Lee, Proc. Null. Acad. Sci. USA., 1973, 70,

28 Y. Ueno and K. Kubota, Cancer Res., 1976, 36,445. 2y International Agency for Research on Cancer, IARC Monographs on the Evaluation of Carcinogenic

Risk of Chemicals to Man, Vol. 10, ‘Some naturally occurring substances’, IARC, Lyon, 1976. R. 0. Sinnhuber, in ‘Mycotoxins in Human and Animal Health’, ed. J. V. Rodricks, C. W. Hesseltine, and M. A. Mehlman, Pathotox Publishers Inc., Park Forest South, Illinois. 1977, p. 7 3 1 .

228 1.

3 1 M. Kanisawa and S. Suzuki, Gann, 1978, 69,599. 32 M. Kanisawa and S. Suzuki, Abs. Annu. Meet. Am. SOC. Microbiol., 1979, Abs. 205. 3 3 F. Dickens and H. E. H. Jones, Br. J. Cancer, 1961, IS, 85.

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Mycotoxins 213

characteristics of mycotoxic disease can only be defined in the broadest of terms, although the cardinal feature of all such diseases is that they are food or feed related.

The following diagnostic criteria are based on those originally proposed by Forgacs and Carl1 and, essentially, the same criteria apply to mycotoxic disease of man: (1) the illness under investigation has no readily identifiable cause, (2) the condition is not transmissible, (3) syndromes are associated with particular consignments or batches of feed, (4) treatment with antibiotics or vitamins has little effect, and ( 5 ) outbreaks are often seasonal because weather conditions influence the growth of mould on harvested crops or stored feed.

Clinical Diseases of Farm Animals Caused by Mycotoxins.-Table 2, adapted from ref. 34, lists the clinical conditions of farm animals that have arisen naturally or have been produced experimentally following the ingestion of feeds con- taminated with certain mycotoxins. Here the list has been extended to include a few toxins that are so far unknown in the UK as natural contaminants, but which have been encountered in animal feeds in the United States and on the continent of Europe.

Whenever mycotoxicosis has been suspected in the field, it has always been difficult reliably to establish causal relationships between clinical disease and the presence of mycotoxins in animal feeds. This is partly because contamination is almost always patchy and truly representative samples can rarely have been obtained for chemical analysis. Another difficulty is that naturally moulded feeds are usually contaminated with several fungal species and while more than one may be toxigenic, the predominance of one toxin or the availability of analytical methods may in practice determine which mycotoxin is actually detected.

Human Mycotoxicosis.-Amongst the metabolic products of microscopic fungi, 4 toxins have been implicated in human disease; ergot, trichothecene mycotoxins (probably T2-toxin), aflatoxin B,, and ochratoxin A.

Ergotism in man is a well-known hazard of contaminated food35 and was the first such disease known to man, outbreaks in the Middle Ages being called ‘St. Anthony’s Fire’. The last outbreak of note36 probably attributable to ergot occurred in France in 195 1.

The war-time epidemic of ATA in Russia, mentioned briefly above, has been described in some detail by J ~ f f e . ~ ’ ? ~ * An authentic sample of crude toxin originally isolated from food implicated in the outbreaks of disease during the 1940’s has recently been found to contain 3 trichothecene mycotoxins (T2-toxin, neosolaniol, T2-tetraol) and ~ e a r a l e n o n e . ~ ~ Consequently, the presence in food of one or more of

34 B. J. Shreeve and D. S. P. Patterson, Vet. Rec., 1975, 97, 279. 35 S. J . van Rensburg and B. Altenkirk, in ‘Mycotoxins’, ed. I. F. H. Purchase, Elsevier Scientific

36 Anon., Lfe , 1951, 31. 25 (cited by B. J . Wilson and A. W. Hayes in ‘Toxicants Occurring Naturally

37 A. Z. Joffe, in ‘Microbial Toxins’, Vol. VII, ed. S. Kadis, A. Ciegler, and S. J . Ajl, Academic Press,

38 A. Z. Joffe, in ‘Mycotoxins’, ed. I. F. H. Purchase, Elsevier Scientific Publishing Co., Amsterdam,

3y C. J. Mirocha and S. V. Pathre, Appl . Microbiol., 1973. 26, 7 19.

Publishing Co., Amsterdam, Oxford and New York, 1974. p. 69.

in Foods’, Natl. Acad. Sci. USA, Washington DC, 1973, p. 372.)

New York and London, 1971, p. 139.

Oxford and New York, 1974. p. 229.

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Mycotoxins 215

these toxins, and particularly T2-toxin, may have been responsible for the outbreak of ATA.

In India40-43 and e l ~ e w h e r e ~ ~ . ~ ~ aflatoxin B , has been detected in food eaten by patients or their domestic pets suffering from acute liver failure, and there is some epidemiological evidence to suggest a link between the ingestion of aflatoxin- contaminated food and Reye’s syndrome (encephalopathy with fatty degeneration of the viscera) in ~ h i l d r e n ~ ~ - ~ ~ and primary liver-cell cancer in a d ~ l t s . ~ ~ , ~ ~

Ochratoxin A has been identified as one of the contributory causes of porcine nephropathy, mainly in Denmark.” A comparable illness of man in the Balkan

_ _

/ fungus

, / \

J* i toxlns produced in laborotory

cultures growing - stored cereals

crops and nuts I

I , / \ \ I

/ 4 I / * fungal extracts,

pure toxins / food for animal \ / human feed i / *

\ \

consumption

occupational exposure animals

*

waste

animal products (human food)

* =possible control points

Figure 3 Mycotoxins in the environment. Growing or harvested crops may be infected with a fungus and the toxin it produces passed directly or indirectly to man’s food. Pathways for occupational exposure to mycotoxins are also indicated

40 K. A. V. R. Krishnamachari, R. V. Bhat, V. Nagarajan, and T. B. G. Tilak, Lancet, 1975, ii, 1061. “ K. A. V. R. Krishnamachari, R. V. Bhat, V. Nagarajan, and T. B. G. Tilak, Indian J . Med. Res.,

1975,63, 1036. 4 2 B. N. Tandon, L. Krishnamurthy, A. Koshy, H. D. Tandon. V. Ramalingaswami, J. R. Bhandari,

M. M. Mathur, and P. D. Mathur, Gastroenterology, 1977, 72,488. 43 H. D. Tandon, B. N. Tandon, and V. Ramalingaswami,Arch. Pathol. Lab. Med., 1978, 102,372. 44 T. C. Campbell and L. Stoloff, J. Agric. Food Chem., 1974, 22, 1006. ” Anon., The Weekly Review (Nairobi), Nov. 10, 1978, p. 24; J. E. Price and R. Heinonen, Kenya Vet.,

46 R. C. Shank, C. H. Bourgeois, N. Keschamaras, and C. Chandavimold, Food Comet. Tuxicol.,

4’ I. Dvorackova, V . Kusak, D. Vesely, J. Vesela, and P. Nesdinol, Ann. Nutr. Aliment., 1977, 31.977. 48 G. R. Hogan, N. J . Ryan, and A. W. Hayes, Lancet, 1978, i, 56 I . 49 F. G. Peers and C. A. Linsell, Ann. Nutr. Aliment., 1977, 31, 1005. 50 World Health Organization, Environmental Health Criteria, No. I 1, ‘Mycotoxins’, W.H.O. Geneva,

J 1 P. Krogh in ‘Mycotoxins’, ed. I. F. H. Purchase, Elsevier Scientific Publishing Co., Amsterdam,

1978,2,45.

1971. 9, 501.

1979.

Oxford and New York. 1974, p. 419.

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216 Environmental Chemistyy

countries has also been tentatively attributed to this toxinS2 but the causes of this and other forms of mycotoxic n e p h r ~ p a t h y ~ ~ are probably complex.

A report published by the World Health OrganizationS0 considered the public health implications of mycotoxins in detail and the essential problem posed by mycotoxins as contaminants of the environment is illustrated diagramatically in Figure 3. It shows three routes by which man may become exposed to mycotoxins: (a) directly by eating cereals, nuts, and other vegetable products that are contaminated by toxigenic fungi, (b) indirectly by consuming meat, milk, and eggs that might contain residues of mycotoxins or their metabolites ‘carried over’ from contaminated animal feeds. (c) as an occupational hazard workers in the food and feed industries may inhale dust or otherwise be exposed to contaminated ingredients, and (d) certain laboratory workers may face similar hazards when they prepare or use crude and pure toxins prepared from fungal cultures.

4 Analysis of Mycotoxins

The Analytical Problem.-The simplest task for the analyst is to determine quantities of mycotoxins formed in laboratory cultures of specific toxigenic fungi. Concentrations of the toxin can be high (several mg g-I) and extracts in organic solvents are usually relatively free from interfering substances. The sensitivity of detection systems are often such that purification is unnecessary and the extract can be diluted before quantification.

The more usual problem facing the agricultural and food chemist is much more difficult. He is required to identify and determine trace amounts ( p g kg-’ ) of a particular toxin in one of a range of chemically complex matrices including cereals, nuts, pasture samples, hay, straw, silage, mixed animal feeds, processed and cooked food, meat. eggs, and milk. In some cases possible interfering substances may actually have been added. Thus mixed anim,al feeds contain added oil, vitamins, antibiotics, and antioxidants and human foods contain various preservatives as well as artifacts produced by cooking or processing. Just as specific methods are needed for the quantitative analysis of a given toxin, a specific extraction and purification procedure ideally is required for each type of sample. But, in practice, this is not possible and methods are usually applicable to a narrow range of similar foods or feeds.

The problem is further complicated because the analyst frequently does not know which, if any, mycotoxin is present in the suspect sample submitted to him. and this has led to the development of the multimycotoxin screening

5 2 P. Krogh. N. H. Axelsen. F. Elling, N. Gyrd-Hansen. B. Hald. J . Hyldgaard-Jensen. A. E. Larsen, A. Madsen, H. P. Mortensen, T. Mailer, 0. K. Petersen, U . Ravnskov, M. Rostgaard, and 0. Aalund, Acla. Palhol. Microbiol. Scatid., Secl. A , 1974. Suppl. 246, p. I .

53 G. C. Peristianis, P. K. C. Austwick. and K. L. Carter, Yirchows Archizl. R , 1978, 28. 321. 54 R. M. Eppley. J . Assoc. O f i Anal. Chem.. 1968. 5 1. 74. ’’ L. Stoloff. S. Nesheim, J . V . Rodricks, M. Stack, and A . D. Campbell. J. Assoc. Oif Anal. Chem.,

56 B. A. Roberts and D. S . P. Patterson, J . Assoc. OjJ Anal. Chcm.. 1975. 58, I 178. ’’ D. S. P. Patterson and B. A. Roberts. J . Assoc. OJK A n a l . Chcm.. 1979. 62, 1265. ’’ 6. G. E. Josefsson and T. E. Moller, J . Assoc. Of l A n a l . Cheni.. 1977.60. 1369. 5 9 A. Gimeno, J . Assoc. Off: Anal. Chetn.. 1979. 62. 579. 6o Y . E. Takeda. E. Isohata. R. Amano. and M. Uchiyarna. J . Assoc. Of l A n a l . Chem.. 1979. 62. 573.

1971. 54, 91.

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Mycotoxins 217

Table 3 Chemical analyses of mvcotoxins in food and feedstugs

(4) Final separation

( 5 ) Detection, quantitation

(6) Confirmation

Basic steps Brief description of general or alternative procedures mix, grind, sub-sample (general) agitate dry or wet sample with organic solvent (general)

precipitate impurities with salts of divalent metals column chromatography preliminary development of t.1.c. plate with diethyl ether dialysis t.1.c. (one- or two-dimensional) h.p.1.c. g.c. miniature multi-layer chromatography column (rapid

fluorescence (t.l.c.*, h.p.1.c.) absorbance (t.l.c.*, h.p.1.c.) visible reaction product (t.l.c.*) flame ionization (g.c., derivative) electron capture (g.c., derivative) co-chromatography (t.l.c., h.p.1.c.) derivative (t.l.c., h.p.1.c.) mass spectrometry biological test

(1) Sampling, sample preparation (2) Extraction (3) Purification (clean-up) liquid-liquid partition

screening method)

* Quantitation on t.1.c. is by densitometry or semi-quantitative visual comparison with standards

which is an analytical compromise. Such a method has as high as possible sensitivity for a small number of toxins that are thought likely to be present in food or animal feed of inferior quality and is applicable to as wide a range of commodities as possible. When the result of the 'screen' is positive it would be followed by a specific quantitative analysis.

Estimates of the concentrations of mycotoxins in food or feeds are markedly dependent upon the sample selected for chemical analysis. As already mentioned, moulding and the mycotoxin Contamination are generally uneven and, for example, in a 50 g sample of peanuts with a mean concentration of 30 pg aflatoxin B, kg-' it is quite conceivable that the entire content of 1.5 pg aflatoxin B, could be located in a single nut. Similarly, pockets of moulded animal feed may contain high concentrations of a toxin while the bulk of the feed is relatively uncontaminated. This uneven distribution of mycotoxins may well be of some significance in the epidemiology of mycotoxin-induced disease, but for regulatory and investigational purposes representative well-mixed samples of food or feed are required for chemical analysis.

The basic steps involved in mycotoxin analysis are summarized in Table 3.

Sampling Procedures.-Because of the very uneven distribution of mycotoxins in crops and stored commodities sampling is subject to considerable error. Thus, it has been shown6' that even when large samples (21.8 kg) were taken from consignments of peanuts with a mean aflatoxin concentration of 20 pg kg-', the coefficient of variation (CV) due to sampling was 60%. With its greatly reduced

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218 En u iron rn en t a I C h em is t ry

particle size the CV of a smaller sub-sample (1.1 kg) of ground peanuts was only 18% compared with 16% for the chemical analysis.

The mathematical and statistical t h e o r i e ~ ~ l - ~ ~ applicable to mycotoxin con- tamination have been developed by several investigators and in the case of aflatoxin contamination samples procedures have been devised6’ so as to give optimum protection to the consumer and the US Peanut Industry alike. Practical sampling plans concerned with mycotoxin surveys have also been r e ~ i e w e d . ~ ~ , ~ ~

In the UK, legally valid analytical figures for aflatoxin and certain other contaminants of agricultural commodities may be obtained only when sampling procedures laid down in the appropriate Fertilisers and Feeding Stuffs Regula- tions66 have been followed. For example, when applied to a consignment of 100 packages of feed in the form of a meal, this involves taking a similar quantity from 10 or, in general, the square root of the number of packages. The individual samples are thoroughly mixed in a manner described and by a process of ‘quartering’ reduced to a sub-sample of 1-2 kg. Before analysis, it is finally ground in a suitable mill.

Representative samples of fruit, meat, and eggs can be obtained by homo- genizing appropriate quantities in a food blender, whereas already homogenous foods like milk and fruit juice can be taken directly for analysis without preparatory treatment. Preparation of samples is discussed in detail by Jones,67 in the official methods of analysis of the Association of Official Analytical Chemists,68 and in a review by Schuller et

Analytical Methods.-Chemical analysis, bioassay, and radioimmunoassay have all been used for the detection and measurement of mycotoxins in food and feeds. Details of only one bioassay are included in the AOAC Official Methods of Analysis6* and this is for aflatoxin using the chick embryo as the test system. Possibly, the brine shrimp larva ( A rtemia salina) test 69 has found wider application as a general screen for mycotoxins but it is subject to interference from other toxic extractants including fatty acids.70 Radioimmunoassay systems assay procedures have been developed for the a f l a t o x i n ~ , ~ ~ ochratoxin A,72 and T 2 - t o ~ i n ~ ~ but it is almost impossible to devise an assay that is sufficiently specific for a single member of a group of closely related mycotoxins (e.g., the aflatoxins) and the sensitivity of

T. B. Whitaker, Pure Appl. Chem., 1977, 49, 1709. G. Berry and N. E. Day, Am. J . Epidemiol., 1973, 97, 160. W. F. Kwolek and E. B. Lillehoj, J. Assoc. Ofl Anal. Chem., 1976,59, 787.

64 P. L. Schuller, W. Horwitz, and L. Stoloff, J . Assoc. Off Anal. Chem., 1976, 59, 13 15. 6J N. D. Davis, J. W. Dickens, R. L. Freie, P. B. Hamilton, 0. L. Shotwell, T. D. Wyllie, and J. F.

Fulkerson, J. Assoc. Of l Anal. Chem., 1980, 63, 95. 66 Anon., Fertilisers and Feeding Stuffs Regulations, Statutory Instrument No. 1521, 1973, H.M.S.O.,

London. 67 B. D. Jones, ‘Methods of Aflatoxin Analysis’, Report G70, Tropical Products Institute, London,

1972. Association of Official Analytical Chemists, ‘Official Methods of Analysis’, 13th Edn., Chapter 26 ‘Natural Poisons’ (issued separately), 1980, AOAC Washington DC.

69 J. Harwig and P. M. Scott, Appl. Microbiol., 1971, 21, 1011. lo R. F. Curtis, D. T. Coxon, and G. Levett, Food Cosmef. Toxicol., 1974, 12,233. ” G. Yang, S. Nesheim, J. Benavides, I . Ueno, A. D. Campbell, and A. Pohland, Zenfralbl. Bakf. , 1980,

” F. S. Chu, F. C. C. Chang, and R. D. Hinsdall, Appl. Enuiron. Microbiol., 1976, 31, 831. 73 F. S. Chu, S. Grossman, R.-D. Wei, and C. J. Mirocha, Appl. Enoiron. Microbiol., 1979, 37. 104.

Suppl. 8, 329.

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M y cot oxins 219

these assays are little different from t.1.c. or h.p.1.c. methods. Official quantitative AOAC methods (as at 1980; ref. 68) still involve t.l.c., although the current trend in mycotoxin methodology is to use h.p.1.c. instead. A case can be made for the use of both, the one technique complementing the other.

Only collaboratively tested methods are recommended by the AOAC and at present these are limited to the following mycotoxins in food and animal feed; aflatoxins B,, B,, G,, G,, and M,, ochratoxin A, patulin, and sterigmatocystin.68 In the UK the Tropical Products Institute was first to issue a handbook of recommended methods6’ for aflatoxin and a method for aflatoxin B,74 used throughout the European Economic Community (E.E.C.) is laid down by the current Fertilisers and Feeding Stuffs Regulations. This has an analytical limit of about 10 ,ug aflatoxin B, kg-’, employs t.l.c., and is very similar to the ‘CB’ method.68 In these procedures separate extraction and clean-up steps are generally recommended for the specific toxins and different types of food and feedstuff. Determination by t.1.c. is by visual or densitometric comparison with standard amounts of pure toxin applied to the same chromatoplate as the extract. This is done either directly after evaporating the developing solvent or subsequently after reaction with a spray or dip reagent. Plates are viewed or scanned under visible or U.V. illumination. As shown in Table 4, of the commonly occurring mycotoxins, aflatoxin B , is most appropriately determined by t.l.c., sub-nanogram amounts being readily detected.

Confirmation of the identity of a mycotoxin usually involves co-chromatography (the standard toxin is mixed with the unknown on the chromatoplate prior to t.1.c.) in several developing solvents and derivitization followed by t.1.c. with the pure toxin treated ~imilarly.~*.’~

Table 4 Minimum quantities ofpure mycotoxins detected on t.1.c. Mycotoxin and how visualized Quantity detected Moles detected relative

(ng) to aflatoxin B I

Nativefluorescence in U.V. light Aflatoxin B, Citrinin Zearalenone

Spray reagent producing or increasing fluorescence in u. v. light

T2-toxin Ochratoxin A Sterigmatocystin Diacetox y scirpenol

Spray reagent producing a visible spot

Patulin Penitrem A

0.4 10 20

10 20 20 50

20 1000

1 3 1 49

16 39 48

108

1 00 1215

Data from, B. A. Roberts and D. S . P. Patterson, J. Assoc. Off Anal . Chem., 1975, 58, I178

74 Anon., Fertilisers and Feeding Stuffs (Amendment) Regulations, Statutory Instrument No. 840,

l5 B. A. Roberts and D. S . P. Patterson, Proc. 2nd Meet. Mvcotoxins Animal Disease, Aberdeen, 1976, 1976, H.M.S.O., London.

p. 42.

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220 Environmental Chemistry

A group of 30 to 40 mycotoxins formed by Fusariurn and certain other fungal species, known collectively as the trichothecene mycotoxins, possess few exploit- able chemical properties and, indeed, the sole functional groups of many members consist of an isolated double bond and an epoxide (see Figure 2). They do not fluoresce or absorb U.V. light to any significant extent and on t.1.c. milligram amounts of untreated trichothecene would be undetectable. However, they are converted to unknown but highly fluorescent derivatives by strong acids and the epoxide reagent of Hammock et al.,76 4-(p-nitrobenzyl)pyridine, permits the detection of about 200 pg T2-toxin kg-I animal feed77 and provides some specificity. Suitable extracts of contaminated feed may also be assayed biologically making use of the fact that about 0.5 pg of diacetoxyscirpenol and T2-toxin produce an erythematous skin reaction in the guinea-pig. This permits the detection of trichothecene mycotoxins at concentrations of about 100 pg kg-I of feedstuff.78

Gas chromatography-mass spectrometry (g.c.-m.s. with selected ion moni- toring) is a powerful tool for the identification and quantification of my cot ox in^^^ but this technique is currently the preserve of the specialist laboratory. As an example of its application to the analysis of trichothecenes in agricultural commodities, it has been recorded** that, although t.1.c. and dermal bioassay failed to detect the presence of epoxide-containing skin irritants in appropriate feedstuff extracts, g.c.-m.s. confidently revealed the presence of 10 pg T2-toxin kg-' of barley.

Fast semi-quantitative methods have a special place in mycotoxin analysis as the suitability of consignments of agricultural commodities often need to be decided quickly. Peanuts (groundnuts) or corn (maize) kernels contaminated with aflatoxin fluoresce under U.V. light, partly due to the intrinsic fluorescence of aflatoxin B, and partly due to the fluorescence of other fungal metabolites, notably kojic acid [5-hydroxy 2-(hydroxymethyl)4 pyrone1.80 It has been found empirically that there is a good correlation between bright greenish yellow (BGY) fluorescence and aflatoxin contamination," and it has therefore been possible to devise electronic sorting procedures 82 to separate substandard kernels mechanically.

Another screening method for aflatoxin c o n t a r n i n a t i ~ n ~ ~ * ' ~ depends upon the fact that crude organic extracts of corn fluoresce when treated with an iodine reagent. This is slightly more elaborate than the BGY test but it is claimed to be more specific.

Increased specificity and semi-quantitative assessment can be achieved with various 'mini-column' methods, some of which have been tested collaboratively and

76 L. G. Hammock, B. D. Hammock, and J . E. Casia, Bull. Environ. Contam. Toxicol., 1974, 12, 759. 7 7 S. Takitani, Y. Asabe, T. Kato, M. Suzuki, and Y. Ueno,J. Chromatogr., 1979, 172, 3 5 5 . '' D. S. P. Patterson, B. J. Shreeve, and B. A. Roberts, Zentalbl. Bakt., 1980, Suppl. 8, 321. 7y C. J . Mirocha, S. V. Pathre, and C . M. Christensen in 'Mycotoxic Fungi, Mycotoxins, Mycotoxicoses:

an Encyclopedic Handbook', Vol. I, ed. T. D. Wyllie and L. G. Morehouse, Marcel Dekker Inc., New York and Basel. 1977, p. 365.

E. B. Lillehoj and C. W. Hesseltine, in 'Mycotoxins in Human and Animal Health', ed. J. V. Rodricks, C. W. Hesseltine, and M. A. Mehlman, Pathotox Publishers Inc., Park Forest South, Illinois, 1977, p. 107. L. J. Ashworth, J. L. McMeans, J . L. Pyle, C. M. Brown, J. W. Osgood, and R. Ponton, Phytoparho1og.v. 1968, 58, 102.

8o D. 1. Fennell, R. J. Bothast, E. B. Lillehoj, and R. E. Peterson, Cereal Chem., 1973, 50,404.

8 3 N. D. Davis and U. L. Diener, J . Appl. Biochem., 1979, I . 115. 84 N. D. Davis and U. L. Diener, J. Appl. Biochem.. 1979, 1. 123.

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My cotoxins 22 1

recommended in the book of AOAC official methods. Extraction and column chromatography steps are scaled down and by using layers of appropriate absorbents aflatoxin is trapped as a concentrated band on the minature column. Appropriate measurement is made by viewing in U.V. light and comparing with columns containing standard amounts of pure toxin. A similar method is available for ochratoxin A.68 Two recent developments have sped up and increased the reliability of quantitative t.1.c. analysis. First, column chromatography can be performed in a fraction of the usual time if commercially available chro- matography cartridges attached to 25 ml glass syringes are used in place of conventional chromatography columns. As in the mini-column methods, small volumes of food or feedstuff extract are required and with the use of smaller quantities of eluting solvent, the analyst is exposed to less toxic solvent vapour. Secondly, many interfering substances can be distinguished from aflatoxin and other mycotoxins by using 2-dimensional t.l.c., which can now be carried out quickly and precisely with small (10 x 10 cm2) aluminium backed ready-spread silica chromatographic plates. With this technique as little as 0.01 pg aflatoxin B , kg-' has been detected in compounded animal feeds.85 Progressively smaller amounts of many mycotoxins are being measured and, for example, with laser fluorimetry as an h.p.1.c. detection system concentrations measured in pg kg-I would seem to be a possibility.

However, little regard is paid to the need for high levels of sophistication. A brief consideration of sterigmatocystin will serve as an example. This is a biosynthetic precursor of aflatoxin B, but has an oral LD,, in the rat some 30 times larger.86p87 It is found less often as a contaminant of animal feeds, is 125 times less effective than aflatoxin B in initiating bile-duct hyperplasia in a standard duckling bioassay and some 250 times less effective as a liver carcinogen.88 Analytical limits for the measurement of aflatoxin B , in most food and animal feedstuffs is about 10 pg kg-' 67 and from the above comparisons of toxicological potential it can be argued that the need is to detect 300-2500 pg sterigmatocystin kg-I, whereas several methods are currently available for the determination of sterigmatocystin in various foods with detection of 5-50 pg kg-',89790 As in other fields of environmental toxicology trace mycotoxin analysis tends to become an end in itself.

5 Occurrence in Food and Animal Feed

Contamination Resulting from Direct Fungal Attack.-The natural moulding of food has been discussed in an earlier section and Table 5 lists various foods that have been shown at one time or another to contain traces of mycotoxins. Cereals, nuts, and foods manufactured from them are especially susceptible. The same commodities form the basis of many animal feedstuffs, and from Table 6 it will be

'' B. A Roberts, E. M. Glancy, and D. S. P. Patterson. J . Assoc. Off Anal. Chem.. 1981. 64, 961. 86 K. J. van der Watt, in 'Mycotoxins', ed. I . F. H. Purchase. Elsevier Scientific Publishing Co..

Amsterdam, Oxford, and New York, 1974, p. 369. E. B. Lillehoj and A. Ciegler, Mycopath. Mvcol. Appl., 1968, 35. 373. F. Dickens, H. E. H. Jones, and H. B. Waynforth, Br.J . Cancer. 1966,20, 134.

89 G. M. Shannon and 0. L. Shotwell, J . Assoc. Off Anal. Chem., 1976.59.963. 90 H. P. van Egmond, W. E. Paulsch, E. Deijill, and P. Schuller, .I. Assoc. Of Anal. Chem., 1980, 63,

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222 Eni7ironmental Chemistry

Table 5 The natural occurrence of mvcotoxins as contaminants of food and animal feedingstufls

Peanuts, groundnut meal Brazil nuts, pistachio nuts, pecans, almonds,

Cotton seed Copra Various seed oils (unrefined only) Sorghum Barley Corn/Maize Oats RY e Rice Wheat Soya bean White, navy, field beans Coffee beans Peppers, spices Dried figs Apples, apple juice, pears, peaches, apricots,

bananas, pineapple, grapes Dairy products: milk, cheese Pig meat Meat products: sausages Mixed animal feeds H aY Silage

walnuts, filberts

- E

a L - x .-. z 3 s + +

+ + + + + +

+ + +

+ +

+

+ +

+

+

+ + +

+

+ +? +

+

Data from, L. Stoloff, in 'Mycotoxins and Other Fungal Related Food Problems', ed. J. V. Rodricks, Adv. Chem. Ser., American Chemical Society, Washington D C , 1976, No. 149, p. 23; H. K. Frank, Ann. Nutr. Aliment., 1977. 31, 459; D. S. P. Patterson, B. A. Roberts, B. J. Shreeve, A. E. Wrathall, and M. Gitter, Ann. Nutr. Aliment., 1977, 31, 643; Anon., Food Surveillance Paper No. 4, 'Survey of Mycotoxins in the United Kingdom', H.M.S.O., London, 1980

Table 6 Mycotoxins in home grown cereals and animal feeds (based on observa- tions made during 1966- 1979)

Torn/ No.

No. somples contaminated with mycoroxin"

AJlaloxin B , Citrinin Ochrnloxin A Sterigmarocystin Zearalenone

Home grown cereals 523 3 I 1 67 17 8 Compound animal feeds 8 12 47 27 -

Groundnut mealh 31 30 - -

Detection limits 0.2 20 5 10 4 0

-

- -

~

(pg kg-')''

'Some samples were contaminated with more than one toxin: ' a component of compound animal feeds;

Data from. A. E. Buckle, Pror. 2nd In!. Cortfr. Vet. Pharmrrcol. Toxicvl. Therap.. 1981. in press: D. S. P. Patterson approximate figures: limits vary with the type of feed analvsed

and B. A. Roberts. Vet. Key., 1080. 107. 240

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M-vcotoxins 223

seen that in the UK aflatoxin has recently been a common contaminant of groundnut meal, and ochratoxin A is an occasional contaminant of barley. Other my cotoxins have been found less frequently.

Indirect Contamination of Food.-Mycotoxins present in animal feeds may appear as residues in the tissues and products of farm animals but, in general, concentrations are many times lower than in the feed. Indeed, farm animals behave as very effective filters for feed-borne mycotoxins. Aflatoxin will be used to illustrate this general principle of mycotoxin ‘carry-over’.

So small is the degree of residue accumulation that with rations containing 300-500 pg aflatoxin B, kg--’ it has been found that pigs had to be fed this diet for 17 weeks or longer before measurable amounts of aflatoxin B , could be found in the meat.91 Furthermore, the toxic diet induced such an erratic effect on the liver, that by no means all were condemned at routine meat inspection and of those that were acceptable to the Meat Inspector not all were found to contain aflatoxin B , on chemical analysis. In this experiment, the levels of aflatoxin were far in excess of those permitted by current UK Fertilisers and Feeding Stuffs regulation^.^^

The proportion of dietary aflatoxin B, ‘carried-over’ into meat and liver of cattle has been estimated to be about 0.1 and 0.3%, respectivelyY2 with similar quantities of aflatoxin M I detectable. Only with aflatoxin B , at dietary concentrations of at least 1-2 pg g-l could residues be detected that were in excess of West German tolerances for food (10 pg total aflatoxins kg-’, 5 pg aflatoxin B, kg-l).

The hen seems to be an even better filter for dietary aflatoxin than large farm animals. Thus aflatoxin residues have been only detectable in hens eggs when dietary levels of 3-4 pg aflatoxin B, g-’ (white and brown laying birds) were reached and corresponding maximum concentrations in the eggs were only 0.4 and 0.2 pg kg-1.93

Aflatoxin is readily transferred to cows’ milk predominantly in the form of aflatoxin M, and it has been shown that its concentration in milk is directly related to the daily dietary intake of aflatoxin B,.94-96 Levels of milk contamination can be predicted from a detailed knowledge of dietary aflatoxin intakes, but alternatively it has been shown that the concentration of aflatoxin M, in milk is very approximately a 300th of the concentration of aflatoxin B, in a dairy ration.97 This approximation takes no account of breed differences, stages of lactation, milk yield, etc., but it has been used98 to demonstrate that with current regulations governing aflatoxin B , contamination in animal feeds,74 the maximum expected concentration of aflatoxin M, in milk would be around 0.1 pg I-,,

9 * P. Krogh, B. Hald, E. Hasselager, A. Madsen, H. P. Mortensen, A. E. Larsen, and A. D. Campbell,

’* R. Loetzsch and L. Leistner, Chem. Abstr., 1975, 85, 158 076; Proc. In( . Conf. Mycotoxins, Munich,

93 R. Loetzsch and L. Leistner, Fleischwirtschafi, 1976, 56, 1777. 94 I . F. H. Purchase, Food Cosmet. Toxicol., 1972, 10 ,53 1. 95 F. Kiermeier, Pure Appl. Chem., 1973, 35 , 271. y6 D. S. P. Patterson, in ‘Mycotoxic Fungi, Mycotoxins, Mycotoxicoses: an Encyclopedic Handbook’,

Vol. 1, ed. T. D. Wyllie and L. G. Morehouse, Marcel Dekker Inc., New York and Basel, 1977, p. 159.

97J. V. Rodricks and L. Stoloff, in ‘Mycotoxins in Human and Animal Health’, ed. J. V. Rodricks, C. W. Hesseltine, and M. A. Mehlman. Pathotox Publishers Inc., Park Forest South, Illinois, 1977, p. 67.

Pure Appl. Chem., 1973,35,275.

1978, p. 32.

y8 B. J. Shreeve, D. S. P. Patterson, and B. A. Roberts. Food Cosmef. Toxicol., 1979, 17, 15 1.

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224 Environmental Chemistry

a figure which compared very favourably with observed v a l ~ e s ~ ~ , ~ ~ ~ ~ ~ and is much lower than the tolerances for aflatoxin B, in human food of about 5 pg kg-I set by law in certain Western countries.99

With the food industry’s increasing awareness of the mycotoxin problem, the introduction of tolerances and our natural reluctance to eat obviously mouldy food, there is only a slight risk of acute aflatoxin poisoning due to high levels of aflatoxin in nuts, nut products, and similar foods. The indirect contamination of milk is different in that milk in one form or another is such a constant part of man’s diet, especially that of infants. And, although levels of aflatoxin M, in milk are low (sub-micrograms l-’), it obviously constitutes a significant form of chronic exposure. There is an epidemiologically demonstrable association between dietary aflatoxin and the risk of developing liver cancer49~so~100 and so chronic human exposure should be kept to an absolute minimum.50 In the case of milk this has been done by reducing levels of aflatoxin B, in the rations of dairy COWS'^*^^ and also by choosing ‘action levels’ for aflatoxin M , in milk (e.g., 0.5 ,ug 1-’ in the United States).

6 Metabolism and Mode of Action of Mycotoxins

Twenty years after its discovery, the relationship between aflatoxin metabolism and its toxic action is still being explored and in the case of the other toxins known occasionally to be present in animal feedstuffs in the UK very little is known of this aspect of metabolism. Several reviewers93. lo*-’ l o have considered the relationships between the metabolism of aflatoxin, sterigmatocystin, ochratoxin A, and Fusarium toxins (trichothecenes and zearalenone), their toxicology, their mode of action, and their potential for accumulating toxic residues in animal tissues. A brief account of this topic follows and, although some recent or important references are listed, many more referring to original work are to be found in the reviews cited above. Metabolic pathways for 10 mycotoxins have been summarized in Table 7.

Metabolic Activation and Detoxification.-Like other xenobiotics, mycotoxins may be metabolized to physiologically inactive or more readily excreted molecules, usually by the hepatic mixed-function oxidases, in a process of detoxification. Alternatively they may be ‘activated’ by conversion to highly reactive derivatives,

99 P. Krogh, Pure Appl. Chem., 1977.49. 17 19. loo F. G. Peers. Zentralbl. Bakt., 1980, Suppl. 8. 279.

lo‘ D. S. P. Patterson, Food Cosmet. Toxicol., 1973. 11. 287. lo’ D. S. P. Patterson, Cah. Nutr. Dietet.. 1976. Suppl. 2. 71. I o 4 D. S. P. Patterson, Pure Appl. Chem., 1977, 49. 1723. lo‘ D. S. P. Patterson, in ‘Mycotoxins and Liver Injury’. ed. T. F. Slater. Academic Press, London and

I”‘ D. S. P. Patterson, in ‘Natural Toxins’, ed. D. Eaker and T. Wadstrom. Pergamon Press, Oxford and

lo’ Y. Ueno, Proc. Jpn. Assoc. Mycotoxicol., 1976. No. 314, 25. lo* Y. Ueno, Pure Appl. Chem., 1977,49, 1737. j o y D. P. H. Hsieh. Z. A. Wong, J . J. Wong, C. Michas, and R. H. Ruebner, in ‘Mycotoxins in Human

and Animal Health’, ed. J. V. Rodricks, C. W. Hesseltine. and M. A. Mehlman, Pathotox Publishers Jnc., Park Forest South. Illinois, 1977. p. 37.

’ l o T. C. Campbell. in ‘Mycotoxins in Human and Animal Health’, ed. J. V . Rodricks. C. W. Hesseltine, and M. A. Mehlman, Pathotox Publishers Inc.. Park Forest South, Illinois. 1977. p. 687.

Anon., Food Chem. News, 1977, 19.38.

New York, 1978, p. 403.

New York. 1980. p. 681.

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Tabl

e 7

Met

abol

ism

of m

ycot

oxin

s: d

etox

ijica

tion

and

activ

atio

n in

the

liver

Myc

otox

in

(for

stru

ctur

al fo

rmul

a,

see

Figu

re 2

)

Afla

toxi

n B

,

Afla

toxi

n B,

Afla

toxi

n G

, A

flato

xin

G,

Afla

toxi

n M

, St

erig

mat

ocy

stin

Och

rato

xin

A

Patu

lin

T-2

toxi

n

Zear

alen

one

Det

oxiJ

catio

n pa

th w

avs"

4-hy

drox

y la

tion

+ a

flato

xin

M,

0-de

met

hyla

tion

-+ af

lato

xin

P,

22-h

ydro

xyla

tion

(cyc

lope

nten

one

-+ af

lato

xin Q,

conj

ugat

ion

of a

flato

xins

M,,

P,,

Q,

conj

ugat

ion

of 2

,3-o

xide

cy

clop

ente

none

redu

ctio

n (c

ytos

ol

and

mic

roso

mal

deh

ydro

gena

se)

- as

for a

flato

xin

B, e

xcep

t for

act

ivat

ion

reac

tion

? 2,

3-de

hydr

ogen

atio

n -+ af

lato

xin

B,

- as

for a

flato

xin

B, e

xcep

t for

reac

tions

invo

lvin

g th

e cy

clop

ente

none

moi

ety

- se

e re

mar

ks u

nder

afla

toxi

ns B

, an

d G

, - as

for a

flat

oxin

B, e

xcep

t for

4-h

ydro

xyla

tion

0-de

met

hyla

tion

-+ de

met

hyl s

teri

gmat

ocys

tin

conj

ugat

ion

-+ gl

ucur

onid

e hy

drol

ysis

(car

boxy

pept

idas

e A

) -+ ph

enyl

alan

ine

+ di

hydr

oiso

-

h ydr

ox yl

atio

n -+

4-

hydr

oxyo

chra

toxi

n A

no

ne d

efin

ed

hydr

olys

is (p

-ace

tyl)

+

HT2

-tox

in (P

-hyd

roxy

l)

conj

ugat

ion

(cyt

osol

tran

sfer

ase)

-+ gl

utat

hion

e co

njug

ate

NA

DP/

NA

DP

linke

d de

hydr

ogen

ases

-+

se

vera

l red

uced

form

s of

ring)

-+

co

rres

pond

ing

gluc

uron

ides

+

glu

tath

ione

conj

ugat

e +

afla

toxi

col (

reve

rsib

le re

actio

n: a

lso

afla

toxi

n Q, -+

afla

toxi

col H

,)

coum

aric

aci

d (o

chra

toxi

n a)

(cyt

osol

enz

ymes

) ? c

onju

gatio

n ze

aral

enon

e re

actio

ns

Activ

atio

n re

actio

na

z

oxid

atio

n +

2,3

-oxi

de (

chro

nic

effe

cts)

1

dihy

drod

iol (

? ac

ute

toxi

city

)

none

pos

sibl

e

- as

for a

flato

xin

B,

none

pos

sibl

e - as

for a

flato

xin

BIb

ox

idat

ion +

2,3

-oxi

deb

none

kno

wn

none

kno

wn

none

kno

wn

zear

alen

ol m

ay b

e an

'act

ive'

met

abol

ite

rnic

roso

mal

enz

ymes

exc

ept

whe

re s

tate

d;

by a

nalo

gy w

ith a

flat

oxin

B,

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B1

Dih

ydro

diol

DN

A o

dduc

t 0 a] a1 B2

2.3

oxid

.

Figu

re 4

Pat

hway

sfor

the

met

abol

ism

of

afat

oxin

B,

in t

he li

ver.

AJa

toxi

n 2,

s-ox

ide

isfo

rmed

by

mic

roso

rnal

enz

ymes

(1)

. It

alk

jdat

es D

NA

(2)

. m

ay b

e co

njug

ated

with

glu

tath

ione

or

met

abol

ized

to

the

2,3-

dihy

drod

iol (

3). A

t phy

siol

ogic

al p

H th

e di

hydr

odio

l re

arra

nges

to a

dia

ldeh

yde,

whi

ch fo

rms

Schi

ff ba

ses

with

pri

mar

y am

ino-

grou

ps o

f pro

tein

mol

ecul

es (4). A

flato

xin

B,,

(her

niac

etal

), pro

duce

d by

rea

ctin

g af

lato

xin

B wi

th d

ilute

min

eral

ac

id (

9, is

a

mod

el c

ompo

und

(6)

(see

als

o Fi

gure

6).

Hyd

roxy

late

d m

etab

olite

s (7

-9)

are

also

for

med

, bv

mix

edfu

ncti

on o

xida

ses.

Oth

er

hydr

oxyl

ated

met

abol

ites

(afla

toxi

col a

nd t

he a

nalo

gue,

HI)

form

reve

rsib

ly in

rea

ctio

ns c

atal

ped

by N

ADP-

depe

nden

t cyt

opla

smic

and

mic

roso

mal

de

hydr

ogen

ases

. Afla

toxi

n B,

may

be

conv

erte

d to

afla

toxi

n B

, (1

l), b

ut is o

ther

wis

e m

etab

oliz

ed b

ypat

hway

s 7-

10

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it4-vcotoxins 227

which then interact with functional macromolecules (often DNA) at specific locations in the target organ. On the basis of our present knowledge it is believed that only mutagenic and carcinogenic mycotoxins undergo metabolic activation as well as detoxification (see Table 7, adapted from ref. 105).

The precise outcome of ingesting my cotoxin is highly species dependent because comparative studies show that metabolic pathways are developed to varying extent in different animal species.

Reactive Toxin Molecules.-AJatoxin and Related Compounds. The metabolism of aflatoxin B , is summarized in Figure 4. Metabolic activation for aflatoxin B, has been inferred from the fact that this toxin is a potent carcinogen and that it becomes a bacterial mutagen in vitro only after prior incubation with a preparation with liver microsomes. By analogy with the carcinogenic polycyclic hydrocarbons, it was therefore proposed that the mutagenic and presumably the carcinogenic properties of aflatoxin B, involved its prior conversion to the 2,3-oxide after which it exerted these effects by alkylating DNA molecules of bacterial or liver cells. There is experimental evidence for the covalent attachment of labelled aflatoxin molecules to DNA in vitro and in vivo. Thus, aflatoxin 2,3-dihydrodiol, presumably a metabolite of the 2,3-oxide, has been isolated from liver microsome preparations incubated with aflatoxin and DNA and from liver tissues of dosed experimental animals (see Figure 5). Synthetic aflatoxin 2,3-dichloride has been used as a model compound for the epoxide, which is probably very reactive, may have only a transient existence, and has not yet been isolated or detected directly.

The 2,3-vinyl ether double bond also occurs in the molecular structures of aflatoxin GI and M, and sterigmatocystin besides several other related compounds and metabolic activation similar to that described for aflatoxin B, is at least

Aflotoxin B , dlhydrodiol + D N A

1lT-p ' o

Aflotoxin B , 2.3 oxide

/ <flNH2 - t - DN A \

other products

Figure 5 Aflatoxin B , 2,3-oxide (the activated form of aflatoxin, shown as its partial structure) alkylates DNA to form a N1-guanyl derivative. On acid hydrolysis or spontaneously in the liver, the DNA adduct breaks down to form ajlatoxin B, 2,3-dihydrodiol and DNA. Other less important reactions give rise to aguanic DNA amongst other products (after Tzu-chien V. Wang, and P. Cerutti, Biochemistry, 1980, 19, 1962). Formation of the dihydrodiol is a well developed merabolic pathway in livers of animal species that are particularly susceptible to acute ajlatoxin poisoning (G. E. Neal. D. J. Judah, F. Stirpe, and D. S. P. Patterson, Toxicol. Appl. Pharrnacol., 198 1, 58,43 1)

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OH

C

amin

o ac

ids

at pH

74

or

abov

e an

d pr

otei

ns

HO

Sch

iff b

ase

ofla

toxm

her

niac

etal

or

BzO

(A,,,,,,,

36

3m

)

OH

C

reso

nanc

e fo

rms

of

Bza

phen

okik

ion

( Ama

x ap

prox

. 400 rw

n )

Figu

re 6

Rea

rran

gem

ent of

the

afla

toxi

n B,

, m

olec

ule

and

its r

eact

ion

with

am

ino-

acid

s (a

fter

A.

E.

Pohl

and,

M.

E.

Cus

hmac

, an

d P.

J.

And

rello

s, J.

Ass

oc.

Ofl.

Ana

l. C

hem

., 19

68, 5

1,9

07).

Afi’

atox

in B

, 2,3-dihydrodiol is

belie

ved

to re

act s

imila

rly

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My cotoxins 229

theoretically possible. The fact that aflatoxin G, and M I and sterigmatocystin are reactive in the Ames test for bacterial mutagens'' supports the proposition that these toxins are also activated to the respective 2,3-oxides (Table 8).

The conversion by the liver of a high proportion of incoming aflatoxin B, molecules to its 2,3-dihydrodiol appears to be a characteristic of animal species that are highly susceptible to acute aflatoxin poisoning and it is probable that in this form it interacts with key enzymes leading to hepatocellular necrosis. Thus, at physiological pH, the hemiacetal rearranges to the resonant hybrids of a dialdehydic phenolate ion when it can react with amino-acids and proteins including enzymes to form Schiff bases (see Figure 6). In particular, it has recently been shown that aflatoxin 2,3-dihydrodiol inhibits protein synthesis in uitro. l1

Aflatoxin B2a, the hemiacetal of aflatoxin B,, is formed by the acid-catalysed addition of water across the isolated vinyl ether double bond and with a structure closely related to that of the 2,3-dihydrodiol (see Figures 4 and 6) its chemical properties are also similar. In particular, it also undergoes rearrangement to the corresponding dialdehyde at pH 7 and above, has an u.v.-absorption spectrum identical to that of the dihydrodio1112 and is thought to react with primary amines and proteins to form Schiff bases. For this reason, in the early 1970's it had been incorrectly concluded from in vitro metabolism studies that the hemiacetal was a major metabolic product of certain animal species'02 and that its role in the pathogenesis of acute liver necrosis was that now ascribed to the dihydrodiol."' Only recently has this rather confusing aspect of aflatoxin metabolism been resolved owing to the discovery that tris[ (hydroxymethy1)aminomethanel derivatives of the two compounds could be separated readily by an h.p.1.c. m e t h ~ d . ~ ~ ' * ~ ~ ~

The unchanged aflatoxin B molecule is probably also intrinsically reactive. Thus, probably by virtue of its coumarin-type structure it inhibits the synthesis of vitamin K-dependent blood-coagulation factors, and in addition it behaves as a pseudo- steroid reacting with steroid-binding sites on the hepatic endoplasmic reticulum and the soluble 17-hydroxysteroid dehydrogenase of the liver cytoplasm.

Ochratoxin A . The two known routes for the metabolism of ochratoxin A (see Table 7 ) lead to a loss of toxicity and it is therefore probable that the unchanged toxin is the reactive molecular species. Other than determining its propensity for protein binding, the mode of action at the molecular level has not been studied although it is known that the site of toxin action in the kidney is remarkably specific, being confined to the proximal tubules.

T2-toxin and Related Trichothecenes. Little is known of the mode of action of the trichothecene mycotoxins in vivo although T2-toxin is an inhibitor of protein synthesis in vitro and its ingestion causes a sub-optimal production of blood coagulation factors in avian liver. Based on the clinical course of ATA and a similar disease experimentally reproduced in the cat, it is believed to have an affinity for the bone marrow. Trichothecenes applied topically to the skin cause a severe necrotic reaction but when the 12,13-epoxide group (Figure 2) is selectively destroyed (e.g.,

G. E. Neal, D. J . Judah, F. Stirpe, and D. S. P. Patterson, Toxicol. Appl . Pharmacol., 198 1, 58,43 1 I l l

'Iz D. H. Swenson, J. A. Miller, and E. C. Miller, Biochem. Biophys. Res. Cornrnun., 1973,53, 1260. ' I 3 G. E. Neal and P. J. Colley, Biochern. J . , 1978, 174,839.

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230 E nu iro nmenta 1 C hem is try

by reduction with lithum aluminium hydride) they are no longer reactive. It is for this reason that it is believed that the epoxide group of the unchanged molecule is the probable reactive site.

Zearalenone. Assayed biologically in immature female mice this mycotoxin and its reduction products are oestrogenic but much less so than oestradiol or the synthetic diethylstilboestrol. One such product, zearalanol (9,lO double bond and the 6-0x0 group of the undecenyl ring are reduced; see Figure 2) is a permitted growth promotant for livestock.

a-Hydroxysteroid dehydrogenase, an enzyme contained in the cytosol of hepatic cells, is able to catalyse the reduction of zearalenone to the corresponding alcohol, zearalenol (only the 6-0x0 group reduced) and other active metabolite^.^^^^"^ Thus, with a steroid-metabolizing pathway implicated, it is possible that zearalenone interacts with target cells (e.g., in the uterus) in that form.

Metabolism and Toxic Residues.-Residues of mycotoxins appear in animal tissues and their products but, although a steady state is reached in a few days, at least in the case of aflatoxin they do not seem to accumulate. From Table 8 it can be seen that all the metabolites of aflatoxin B , are less toxic than the parent toxin measured in terms of LD,, values for day-old ducklings or chick embryos. Several of these metabolites retain the important 2,3-vinyl ether grouping, however, and are therefore potentially carcinogenic. But aflatoxin M the only metabolite so far tested is still somewhat less potent than aflatoxin B , . The activated forms of

Table 8 Acute toxicities of aflatoxin B , and its metabolites

AJatoxin

Bl

MI PI Q l

Aflatoxicol Aflatoxicol H I

B,, (hemiacetal)

2,3-oxide (epoxide)

Type of metabolite (parent toxin)

formed by liver microsomal enzymes

reduction products of microsomal and cytosol deh ydrogenases

dihydrodiol

by liver microsomal enzymes

model compound for the

'activated metabolite' formed

Toxicity relative to BIa 100 72b

v. low" 5 d

6e v. lowf

v. IOWK

presumably a transient metabolite

Calculated from toxicity data given in the respective references; * LD,, determined in day-old duckling: value for aflatoxin B , about 0.3 mg kg-I body wt, C. W. Holzapfel, P. S. Steyn, and I . F. H. Purchase, Tetrahedron Lett., 1966, 2799; 'Chick embryo LD,,, L. Stoloff, M. J . Verrett, J . Dantzman, and E. F. Reynoldo. Toxicol. Appl . Pharmacol.. 1972. 23. 528: Chick embryo LD,,, D. P. H. Hsieh, A. S. Salhab, J. J. Wong, and S. L. Yang, Toxicol. Appl . Pharmacol.. 1974, 30, 231;

Bile-duct hyperplasia assay, day-old duckling, R. W. Detroy and C. W. Hesseltine, Nature (London), 1968. 219, 967: /Chick embryo LD,,, A. S. Salhab and D. P. H. Hsieh, Res. Commun. Chem. Pathol. Pharmacol., 1975, 10, 419; a Bile-duct hyperplasia assay, day-old duckling, E. B. Lillehoj and A. Ciegler. Appl . Microbiol.. 1969, 17. 5 16

' I 4 F. Tashiro. Y. Kawabata, M . Naoi. and Y. Ueno. Zentralbl. Bakt.. 1980, Suppl. 8. 3 1 I . ' I 5 M. Olsen, H. Pettersson. and K.-H. Kiessling. Toxicon, 1979. Suppl. I , 134.

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Mycotoxins 23 1

aflatoxin do not present a residue problem at all as they are incapable of further activity after interaction with protein or DNA. Consequently, apart from the unchanged aflatoxin B , , the sole problem would appear to arise from residues of aflatoxin M , in the tissues and milk of farm animals. Similar considerations would apply to other mycotoxins and their metabolites but relevant data is scarce.

7 Control of Mycotoxins in the Food Chain

General.-Four types of control have received attention in recent years:’ 1 6 - ’ l 9 (1) prevention of fungal infection in the crops, (2) prevention of moulding during storage, (3) selection of shipments with minimal mycotoxin contamination, and (4) decontamination of commodities containing mycotoxins (see Figure 3).

Control of Fungal Infection.-For many years aflatoxin contamination was thought to result from bad storage. This is still true but it has gradually become apparent that pre-harvest contamination also occurs. In corn and peanuts, for example, fungal infection of this kind may be largely unavoidable. Early studies of the post-harvest problems centred on the control of the environment and the use of antifungal agents during storage. But emphasis is now placed on good farm practice generally.l18 This includes insect control 120 because damage caused by insects is a major predisposing factor, the prompt harvesting of crops at maturity, and the use of cultivars of food plants’21*’22 that tend to resist insect damage and fungal infection. It is also sensible to segregate evidently mouldy crops to avoid the further spread of contamination during storage.

Control by Selection.-Mycotoxin contamination commonly occurs in food or feed commodities imported from tropical or sub-tropical countries and whenever a choice can be made it is clearly desirable to use shipments with little or no contamination. Selection can be voluntary as for example where compounders of animal feeds follow an established ‘code of practice’123 or adhere to official guideline^'.'^^ It can also be compulsory and in many countries the use of

I I 6 L. A. Goldblatt and F. G. Dollear, in ‘Mycotoxins in Human and Animal Health’, ed. J. V. Rodricks. C . W. Hesseltine, and M. A. Mehlman, Pathotox Publishers Inc.. Park Forest South. Illinois, 1977, p. 139.

11’ L. A. Goldblatt and F. G . Dollear, Pure Appl. Chem., 1977, 49, 1759. 118 L. A. Goldblatt and F. G. Dollear, in ‘Interactions of Mycotoxins in Animal Production’. Proc. Symp.

13 July 1978 Michigan State Univ., National Academy Sciences, Washington DC. 1979, p. 167. ‘I9 K. Aibara and N. Yano, in ‘Mycotoxins in Human and Animal Health’, ed. J. V. Rodricks, C . W.

Hesseltine. and M. A. Mehlman, Pathotox Publishers Inc., Park Forest South, Illinois, 1977. p. 151. I 2 O E. B. Lillehoj, W. F. Kwolek, A. Manwiller, J . A. D u Rant, J . C. La Prade. E. S. Horner, J. Reid, and

M. S. Zuber, Crop Sci., 1976. 16,483. A. C. Mixon, in ‘Mycotoxins in Human and Animal Health’, ed. J . V. Rodricks, C. W. Hesseltine, and M. A. Mehlman, Pathotox Publishers Inc., Park Forest South, Illinois, 1977, p. 163.

122 M. S. Zuber, in ‘Mycotoxins in Human and Animal Health’, ed. J. V. Rodricks, C. W. Hesseltine, and M. A. Mehlman, Pathotox Publishers Inc., Park Forest South, Illinois, 1977, p. 173.

1 2 3 Anon., Report, Interdepartmental Working Party ‘Toxicity associated with certain samples of groundnuts’, 1962, p. 21 (cited by P. K. C . Austwick, p. 296 in Vol. 2, ref. 9).

124 J . V. Rodricks and H. R. Roberts. in ’Mycotoxins in Human and Animal Health’. ed. J . V. Rodricks, C. W. Hesseltine. and M. A. Mehlman, Pathotox Publishers Inc.. Park Forest South. Illinois. 1977, p. 753.

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23 2 Environmental Chemistry

aflatoxin-contaminated food or feed commodities is restricted by statutory regulation^.^^ 3”

Effective selection is necessarily dependent on reliable chemical analysis and methods of sampling, screening, and rapid aflatoxin or ochratoxin analysis have already been referred to (see also ref. 125).

Detoxification of Aflatoxin.-Heat treatment and various reactions of aflatoxin B , with common laboratory reagents have been studied with a view to developing methods for the decontamination of aflatoxin contaminated commodities. Thus, aflatoxin B, has been found to be stable to heat in the dry state up to its melting point (268-269 OC),ll* but in the presence of moisture the lactone moiety of the molecule probably opens and the o-coumaric acid so formed may subsequently undergo decarboxylation.’26 Hydrolysis of the lactone ring occurs in alkaline solution but aflatoxin B , reforms on a~idif icat i0n. l~~ At elevated temperatures (about 100 O C) irreversible changes occur with ring-opening, decarboxylation, and loss of the single methoxy group from the aromatic ring.12* Similar reactions occur with ammonia (gaseous or aqueous) and certain amines to form products with much lower toxicity compared with the parent toxin. 129 ,130

In the presence of mineral acid, water is added across the biologically active vinyl ether double bond of aflatoxins B, or G, to form the corresponding relatively non-toxic hemiacetals, B,, and H2,.1314 1 3 * Destruction of aflatoxin is achieved by the addition of various oxidizing agents and for example, sodium hypochlorite is widely used to decontaminate laboratory glassware used for aflatoxin or other mycotoxin analyses. l 3

Aflatoxins B, and G, are also degraded by U.V. radiation134 and this is readily observed when the toxins are absorbed onto the silica of a t.1.c. plate. The hemiacetal or their alkoxy-derivatives are formed when aflatoxins B , and G, are irradiated in phosphate buffer or in alcoholic s o l ~ t i o n . ~ ~ ~ - ~ ~ ~ Dry films of aflatoxin B, are resistant to gamma radiation but destruction has been observed when the toxin was dissolved in chloroform and particularly in water.13*

‘I5 E. B. Lillehoj. in ‘Interactions of Mycotoxins in Animal Production’. Proc. Symp. 13 July 1978.

I26 T. J. Coomes, P. C. Crowther, A. J. Feuell. and B. J. Francis, Nature (London). 1966, 209,406. 12’ J. de Iongh. R. K. Beerthuis, R. 0. Vles, C. B. Barrett, and W. 0. Ord. Biochim. Biophjs. Acta, 1962,

1 2 ’ F. Kiermeier and L. Ruffer, Z . Lebensm. (Inters. Forsch., 1974, 155, 129. Iz9 R. F. Vesonder. A. C. Beckwith, A. Ciegler, and R. J. Dimler, J . Agric. Food Chem.. 1975, 23. 242. I 3 O L. S. Lee, J. B. Stanley. A. F. Cucullu. W. A. Pons, and L. A. Goldblatt, J . Assoc. Of( Anal. Chem.,

1 3 ’ A. E. Pohland, M. E. Cushrnac, and P. J. Andrellos, J . Assoc. 08 Anal. Chem., 1968, 51.907. 1 3 * A. Ciegler and R. E. Peterson, Appl. Microbial.. 1968, 16,665. 13’ L. Stoloff and W. Trager, J . Assoc. Off Anal. Chem., 1965.48.681. 134 P. J. Andrellos, A. C . Beckwith. and R. M. Eppley, J . Assoc. Off Anal. Chem., 1967. 50, 346. 135 D. A. Lillard and R. S. Lantin, J . Assoc. Off: Anal. Chem., 1970, 53. 1060. 1 3 6 A. C. Waiss and M. Wiley. J. Chem. Soc., Chem. Commun.. 1969. 5 12. 1 3 ’ R.-D. Wei and F. S. Chu. J . Assoc. Off Anal. Chem.. 1973. 56. 1425.

Michigan State Univ., National Academy of Sciences, Washington DC, 1979, p. 139.

65, 548.

1974, 57,626.

H. K. Frank and T. Grunewald, Food Irrad., 1970, 11, 15.

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My cotoxins 233

Promising Decontamination Processes.-Aflatoxin residues are reduced con- siderably by roasting peanuts,’39 pecans,*4o corn (maize) and oil-seed residues14* and the toxin is removed from vegetable oils during normal refining processes since it is concentrated in the soap

More than 60 chemical treatments have been screened for decontaminating animal feeds mainly involving alkalis. 144 Ammonia gas provides the most promising treatment as indicated by chemical analysis and livestock feeding trials with ammoniated corn, cotton seed, and groundnut meals.11s,145, 146 Accordingly, ammoniation plants are beginning to operate commercially in some parts of the world.

Mycotoxins other than Aflatoxia-As sterigmatocystin is structurally closely related to aflatoxin it is therefore likely to respond to similar detoxification procedures. Although other mycotoxins are destroyed by strong oxidizing agents there have been no comparable decontamination studies. Good husbandry and careful storage of food or feed crops are the two basic means available for the control of mycotoxins generally.

139 L. S. Lee, A. F. Cucullu, A. 0. Franz. and W. A. Pons, J . Agric. Food Chem., 1969, 17.451. I 4 O F. E. Escher, P. E. Koehler, and J. C. Ayres, J. Food Sci., 1973, 38, 889. 14‘ H. F. Conway, R. A. Anderson, and E. B. Bagley, Cereal Chem.. 1978. 5 5 . 1 15. 1 4 * G. E. Mann, L. P. Codifer, and F. G. Dollear. J. Agric. Food Chem., 1967. 15. 1090. 14’ W. A. Parker and D. Melnick, J. Am. Oil Chem. SOC., 1966. 43,635. 144 G. E. Mann, L. P. Codifer, H. K. Gardiner. S. P. Koltun. and F. G. Dollear, J. Am. Oil Chem. SOC.,

145 H. K. Gardiner, S. P. Koltun, F. G. Dollear, and E. T. Rayner, J . Am. Oil Chem. Soc., 1971. 48, 70. 146 A. C. Key1 and W. P. Norred. in ‘Interactions of Mycotoxins in Animal Production’, Proc. Symp. 13

July 1978, Michigan State Univ., National Academy of Sciences. Washington DC, 1979, p. 185.

1970, 47, 173.

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