Mycotoxins in Grains Harvested

The global frequency of mycotoxin contamination of feedstuffs and the severity of mycotoxicosis in livestock has increased in recent years. Wheat-based feed and food supply chains can be contaminated with mycotoxins produced by a variety of fungi, in particular by Fusarium species. Since wheat is an important source of energy in human nutrition and in nutrition of monogastric animals its quality is crucial. This paper gives an overview of the occurrence of 24 mycotoxins in feed grade wheat harvested in 2008 in three European countries: Czech Republic, Denmark and Hungary. DON was the most frequently observed mycotoxin and was found in 41% of samples tested (63, 14 and 86% for Czech Republic, Denmark and Hungary respectively) and the highest concentration of 8841 ppb was found in Czech Republic. Apart from DON, also nivalenol, 3- & 15- acetyldeoxynivalenol, fumonisins, zearalenone, HT-2 toxin, ochratoxin A and roquerfortine C were detected in wheat.

Introduction

Wheat-based feed and food supply chains can be contaminated with mycotoxins produced by a variety of fungi. There are >300 mycotoxins presently being isolated. The most frequently detected mycotoxins are aflatoxin, deoxynivalenol (DON), zearalenone, fumonisin, and T-2 toxin. The fungal growth can not only change the chemical and physical properties of feed but also the nutrient content of grains, which influences the nutritional value for farm animals. Since wheat is an important source of energy in human nutrition and in the nutrition of monogastric animals, its quality is crucial.

The global frequency of mycotoxin contamination of feedstuffs and the severity of mycotoxicosis in livestock and poultry increased in recent years. This may be due to the increased monitoring of feed materials and an increased awareness of the symptoms of mycotoxicosis by animal producers. Global climate change has also contributed to an increased frequency of mycotoxin contamination of feed grains. Additionally, the intensive international trading of feedstuffs increases the chance that compound feed may contain mixtures of different mycotoxins of various geographical origins. This can result in synergistic effects that increase the severity of mycotoxicosis in the animal.

The models for the prediction of the mycotoxin occurrence are based on statistical relations between the presence of the mycotoxin and geographically related factors.

Results of this study show the mycotoxin contamination of feed grade wheat harvested in 2008 in three EU countries with different agricultural practices.


Objective

The objective of the study was to evaluate the mycotoxin contamination of feed grade wheat harvested in 2008 in Czech Republic, Denmark and Hungary in order to estimate the potential risk of mycotoxicosis in livestock.

A total amount of 29 wheat samples were collected in three EU countries: 8 in Czech Republic, 14 in Denmark and 7 in Hungary. Samples were being collected in the period of July-October 2008 and stored frozen before analyses. All 29 samples were analysed in the laboratory of Food Analyses of Ghent University, Belgium on liquid chromatography tandem mass spectrometry (LC-MS/MS) - system with two stages of mass analysis. A total of 24 mycotoxins of Aspergillus, Penicillium, Fusarium and Alternaria moulds were identified quantitatively. Mycotoxin concentration was expressed in μg/kg (ppb) of wheat (natural moisture content). Limits of detection shown in the table 1 were specific for each mycotoxin


Table 1. Detection limits of mycotoxins on LC-MS/MS

 

A total of 29 multianalyses were conducted on wheat samples. Positive samples with mycotoxin level above detection limit contained mycotoxins of Fusarium and Penicillium origin such as nivalenol, DON, 3- & 15- acetyldeoxynivalenol, fumonisins, zearalenone, HT-2 toxin, ochratoxin A and roquefortine C.

Since mycotoxin contamination of wheat varied depending on the country of origin, the interpretation of the results is given for each of three countries separately (Figures 1-3).

In the Czech Republic 5 out of 8 wheat samples contained one or more mycotoxins produced by Fusarium species (Table 2). DON and its derivatives 3- and 15-ADON were present in all contaminated samples. The concentration of DON varied from 99 to 8841 ppb. Zearalenone was found in two samples at levels of 61 and 155 ppb. One sample contained 200 ppb of nivalenol.

Table 2.Average contamination of wheat samples harvested in Czech Republic in 2008 (ppb). 

Overall, the contamination of Czech wheat in 2008 was caused by Fusaruim mycotoxins. Despite the fact that the levels of DON and zearalenone were mostly in line with EU recommendation 2006/576/EC their levels could create problems when fed long time. More attention is required when this wheat is used in pig diets.

There were 14 wheat samples collected in Denmark during the harvest season in 2008. Five samples contained a single mycotoxin, either DON or HT-2 or roquefortine C at relatively low levels (Table 3).

Table 3. Average contamination of wheat samples harvested in Denmark in 2008 (ppb). 

Generally, wheat harvested in Denmark in 2008 had an insignificant mycotoxin contamination.

Out of 7 wheat samples from Hungary, 6 contained one or more mycotoxins produced by Fusarium or Penicillium moulds (Table 4). One sample contained fumonisins B₁ and B₂. Two samples were positive for ochratoxin A. DON and its derivatives 3- and 15-ADON were found in 5 samples. The concentration of DON varied from 154 up to 2113 ppb.

 

In general, mycotoxin levels in Hungarian wheat were not critical except for DON contamination. Use of wheat harvested in 2008 is potentially unsafe for pig producers.

F. graminearum can produce a variety of mycotoxins: deoxynivalenol, zearalenone, 3-ADON, 15-ADON, diacetyldeoxynivalenol, nivalenol, T-2, neosolaniol, and diacetoxyscirpenol. 15-ADON was found in two samples from Czech Republic and in 3 samples from Hungary. 3-ADON accompanied 15-ADON in 3 wheat samples with maximum concentrations of 67±26 and 100±38 ppb respectively. Potentially 3-ADON and 15-ADON can be converted into DON during storage and feed processing. Therefore the DON levels can be higher in wheat where 3-ADON and 15-ADON are present. Both 3- and 15-ADON are equivalently or less toxic than DON (Pestka, 2007). Similar findings were reported by Swamy et al. (2004) who suggested that other co-occurring Fusarium toxins such as fusaric acid, 15-ADON and zearalenone could impact DON's effects.

Overall, 41% of examined samples were positive for DON - one of the major contaminants of feedingstuffs worldwide. DON was the common toxin in all studied countries with the highest level of 8841 ppb detected in wheat from Czech Republic. Fusarium graminearum is the parent fungi of DON. Wheat and barley are the most commonly effected grain crops but the same fungus does infect corn too. Fusarium toxins in general and in particular zearalenone and DON cause major problems in the worldwide livestock production. Especially pigs react most sensitively to feedstuffs contaminated with these mycotoxins. The most common effects of prolonged dietary exposure of experimental animals to DON are decreased weight gain, anorexia, decreased nutritional efficiency and altered immune function (Pestka, 2007). The officially published values for critical concentrations of DON in complete diets for farm animals are based on literature data, which were not always consistent for these values. In practice adverse effects are reported at dietary DON concentrations and concentrations below the orientation values (Döll et al. 2002).

Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum. They occur worldwide and are found predominantly in maize and in maize-based animal feeds. However, we found fumonisin B₁ and fumonisin B₂ in one wheat sample collected in Hungary. Of the fumonisins, fumonisin B₁ is the most common and the most thoroughly studied. Fumonisin B₁ causes equine leukoencephalomalacia (ELEM) and porcine pulmonary edema (PPE), diseases long associated with the consumption of mouldy feed by horses and pigs, respectively. Fumonisin B₁ is toxic to the liver in all species and the kidney in a range of laboratory and farm animal species, causing apoptosis in the affected tissues. Fumonisins inhibit ceramide synthase in all species including laboratory and farm animals and disrupt sphingolipid metabolism, a process underlying the mechanism of toxicity and pathogenesis of fumonisin-related diseases. The USFDA has set guidances for fumonisin concentrations in animal feeds that range from 1 to 50 ppm in the formulated rations depending upon the animal species. The European Union Commission has recommended guidance levels for fumonisins B₁ plus B₂ in feed materials and formulated feedstuffs. The levels also vary according to species and range from 5 ppm for horses, pigs, rabbits and pet animals to 50 ppm for adult ruminants and mink. Awareness of fumonisin-related animal diseases, monitoring feed and feed components, and adherence to guidance recommendations are important for reducing fumonisin-induced diseases in agriculturally important species (Voss et al., 2007).

HT-2 was detected in two wheat samples from Denmark at 27±10 and 29±11 ppb. Survey data from Nordic countries have indicated that levels of HT-2 and T-2 toxins have increased since last years. F. langsethiae has been iassociated with the production of HT-2 and usually occurs more frequently and at higher concentrations on oats followed by barley and wheat (Edwards, 2008).

Nivalenol was found in one sample from Czech Republic at a level of 200±63 ppb. This mycotoxin is produced mainly by the Fusarium nivale fungi. Studies have shown a low occurrence rare; it has only been found in a few samples of barley, wheat, wheat flour and rice. Preliminary testing shows that nivalenol is 10 times more potent than DON. If the advisory level for DON in wheat is used as a guide for toxicity, nivalenol would have an advisory level of only 0.8 ppm. In a variety of in vitro systems, T-2 consistently has been the most toxic, followed by nivalenol and then deoxynivalenol (Ueno, 1987).

Ochratoxin A was present in two samples from Hungary. The ochratoxin A contamination was low but occurred simultaneously with DON contamination. Ochratoxins were initially associated with Aspergillus ochraceus but are produced primarily by Penicillium verrucosum. Ochratoxin A can cause listlessness, huddling, diarrhea, tremors, and other neural abnormalities in poultry and has been associated with kidney disease in swine in Scandinavia and northern Europe.

Roquerfortine C is very typical for the geographical region of the UK, Ireland and Scandinavia and frequently found in grass silages. One sample from Denmark contained a low level of 2.5±0.9 ppb of roquefortine C. Nine mycotoxins, potentially threatening to human and animal health mycotoxins: citreoviridin, citrinin, cyclopiazonic acid, ochratoxin A, patulin, penitrem A, PR toxin, roquefortine C, and secalonic acid D are produced by 17 Penicillium species. Roquefortine C is a neurotoxin and it has been suggested to have a link to paralytic effects and hemorrhagic syndromes in animals.

Zearalenone is known to exhibit estrogenic properties and is therefore associated with reproductive disorders. Moreover, it has also been shown to be hepatotoxic, haematotoxic, immunotoxic and genotoxic. Zearalenone is a mycotoxin produced mainly by fungi belonging to the genus Fusarium. The biotransformation for zearalenone in animals involves the formation of two metabolites α-zearalenol and β-zearalenol which are subsequently conjugated with glucuronic acid. Zearalenone is commonly found on several foods and feeds in Europe, Africa, Asia, America and Oceania (Zinedine et al., 2007). We found 155 ppb of zearalenone in one Czech wheat sample, which was also contaminated with nivalenol, DON, 3- and 15-ADON at relatively high levels.

Mycotoxins are ubiquitous and toxic. They globally present a potential danger for animal and human health when absorbed in high amounts or over a long period of exposure.

Generally, wheat harvested in 2008 seems to be contaminated mainly with Fusarium mycotoxins. Based on the toxicological evaluation of certain mycotoxins (deoxynivalenol, T-2-toxin, HT-2-toxin, zearalenone, fumonisin B₁+B₂, aflatoxins, and ochratoxin A) the European Union has set (or will set in the near future) a maximum permitted level of mycotoxins in foods and feeds. However, as far as the emerging mycotoxins (nivalenol, roquerfortine C, 3- and 15-ADON, etc.) are concerned, maximum tolerable levels are not expected to be proposed in the near future. This is principally due to the lack of data about their occurrence, contamination level and toxicity. In order to assess the risk of these mycotoxins, regular surveillance is a prerequisite to understand their significance as natural contaminants in human and animal nutrition, and in consequence sensitive, repeatable, and reliable analytical methods such as LC-MS/MS are needed to detect these mycotoxins.

In order to prevent the possible increase of mycotoxin levels during storage, the control of mould growth and mycotoxin production is very important. Control of mould growth in feeds can be accomplished by keeping moisture levels low and equipment clean and by using mould inhibitors as a part of Good Storage Practices (Commission Recommendation 2006/583/EC). Grains and other dry feedstuffs should be stored at a moisture level of less than 14% and/or with the use of chemical mould inhibitors to prevent mould growth.

Binding agents such as mineral clay products, for example, bentonites, zeolites and aluminosilicates, have been found to be effective in binding/adsorbing mycotoxins (Ramos et al., 1996). The molecular surfaces of these additives attract the polar functional groups of mycotoxins and trap them against its surface. This mechanism isolates the mycotoxin from the digestive process and thereby prevents absorption by the animal.

This work has been supported by Petr Hurka and the team (Kemin Central Europe), Torben Petersen (Kemin Scandinavia) and Georgia Kolbert and the team (Kemin Hungary) who obtained all samples.

Analyses were conducted in collaboration with Ghent University, Laboratory of Food Analyses