Survey of ochratoxin A and deoxynivalenol in stored grains from the 1999 harvest in the UK

Three hundred and twenty samples from the 1999 UK harvest comprising wheat (201 samples), barley (106) and oats (13) were analysed for ochratoxin A and deoxynivalenol. A small number of organic samples was also obtained. Samples were collected from farms, central stores, mills, maltings and ports from across the UK from February to April 2000. Ochratoxin A and deoxynivalenol analysis was by affinity column clean up and high-performance liquid chromatography with fluorescence and ultraviolet light detection, respectively, with limits of detection of 0.2 and 20 μg kg^-1. The survey found ochratoxin A at below 5 μg kg^-1 in 97% of the samples indicating satisfactory storage conditions. The remaining 3% of the samples contained ochratoxin A at levels between 5.2 and 231 μg kg^-1, but none of these samples was intended for human consumption. Deoxynivalenol was detected in 88% of all samples, with 83% below 100 μg kg^-1; the maximum level was 600 μg kg^-1. Twenty samples containing deoxynivalenol at or above 150 μg kg^-1 by high-performance liquid chromatography were all confirmed by gas chromatography/mass spectrometry. Nivalenol was also detected by gas chromatography/mass spectrometry at levels of 50 μg kg^-1 or higher in 18 of 20 samples where deoxynivalenol was confirmed.     

Beauvericin and enniatins A,A1, B and B1 in Norwegian grain: a survey

Norwegian grain samples (73 oats, 75 barley, 80 wheat) from the 2000 to 2002 growing seasons were examined for contamination with five different enniatins and the association between the found concentrations and the prevalence or infection level with several common Fusarium species investigated. Enniatin B was the fungal metabolite with the highest prevalence (100%) and the highest maximum concentration (5800 μg/kg, wheat). The maximum concentration of all five enniatins together in a single sample was 7400 μg/kg (wheat). Enniatin concentrations were correlated with several independent variables, among them grain species. Beauvericin was only sporadically detected in barley and wheat and at concentrations just above the limit of detection of 3 μg/kg, while amounts up to 120 μg/kg were found in oats. The likelihood of detecting enniatin A1 as well as the concentrations of enniatins B and B1 could be mainly related to infection with Fusarium avenaceum/arthrosporioides, and the likelihood of detecting beauvericin could be related to infection with Fusarium poae. This survey indicates that the prevalence of enniatins A1, B and B1 in Norwegian grain is high, and that enniatin B concentrations of above 1000 μg/kg are common in barley and wheat.     

Moniliformin in Norwegian grain.

Norwegian grain samples (73 oats, 75 barley, 83 wheat) from the 2000-02 growing seasons were examined for contamination with moniliformin, and the association between the fungal metabolite and the number of kernels infected with common Fusaria was investigated. Before quantification of moniliformin using ion pairing reversed-phase high-performance liquid chromatography with diode array ultraviolet light detection, all samples were extracted using acetonitrile/water (84/16) and disposable strong anion exchange columns used for clean up. The limit of detection was 40 microg kg(-1). Moniliformin was found in 25, 32 and 76% of the barley, oats and wheat samples, respectively. The maximum concentrations of moniliformin in barley, oats and wheat were 380, 210 and 950 microg kg(-1), respectively. At the same time, the prevalence and infection level of the moniliformin-producing F. avenaceum/arthrosporioides was as high as 100 and >53% on average, respectively. Moniliformin concentrations were significantly correlated to the variables grain species, growing season and infection with F. avenaceum/arthrosporioides and F. culmorum. The survey indicates that the prevalence of moniliformin in Norwegian grain is high, especially in wheat. On the other hand, field conditions in Norway do not seem to favour contamination of grain with high levels of moniliformin.     

On ochratoxin A and fungal flora in Polish cereals from conventional and ecological farms. Part 2: occurrence of ochratoxin A and fungi in cereals in 1998.

Over 200 samples of Polish cereal grain from the 1998 harvest obtained from conventional and ecological farms were investigated for the presence of ochratoxin A and for contamination by microscopic fungi. The frequency of contamination of rye and barley grains from conventional and ecological farms was similar in most cases; it varied from nearly 5 to 12%, respectively, for both types of farming. However, in samples from ecological farms, higher maximum concentrations of ochratoxin A were observed (35 micro g kg(-1), overall range 1.4-35.3 micro g kg(-1)) for both cereals rye and barley in comparison with rye and barley from conventional farms (maximum levels of 8.8 and 9.7 micro g kg(-1), respectively). However, wheat grain from the conventional farms showed ochratoxin A concentrations in a very wide range from 0.6 to 1024 micro g kg(-1) and the average frequency of contaminated samples was about 48%. In contrast, in wheat samples from ecological farming, the presence of ochratoxin A ranged from 0.8 to 1.6 micro g kg(-1) (mean 1.2 micro g kg(-1)) and the frequency of contamination was 23%. From samples containing detectable amounts of ochratoxin A, fungi producing ochratoxin A under laboratory conditions were isolated. They were classified as belonging to the species Penicillium cyclopium, P. viridicatum, Aspergillus ochraceus group, A. glaucus and A. versicolor. Penicillium strains-species known to be producers of ochratoxin A-were isolated from 71% of the samples; in 28% of samples, only Aspergillus strains (species known to be producers of this mycotoxin) were noted. These results have been compared with those obtained in 1997.     

Ochratoxin A contamination of cereal grain harvested in 2003 and 2004 years

The analysis of ochratoxin A--mycotoxin produced by widely distributed Aspergillus and Penicillium--of cereal grain harvested in 2003 - 2004 years were performed by immunoaffinity column clean-up and HPLC with fluorescence detection. This survey examined 282 samples of raw grain wheat, rye, barley and oat 13.8% of all samples contaminated by ochratoxin A in the range 0.2-33.3 mg/kg. Calculation made on the basis of the obtained means showed that the daily ochratoxin A intake of human from cereal grain were from 1.58 to 2.84 ng/kg b.w. Scientific Committee for Food of the European Commission suggested that it was prudent to reduce exposure to ochratoxin A as much as possible below 5 ng/kg bw/day. Codex Alimentarius and European Commission have established maximum permissible level of 5 mg/kg for ochratoxin A in raw cereal grains.     

Trichothecenes, ochratoxin A and zearalenone contamination and Fusarium infection in Finnish cereal samples in 1998

The occurrences and concentrations of trichothecenes, ochratoxin A and zearalenone in Finnish cereal samples are presented in this study. Furthermore, infections by moulds, especially Fusarium contamination of grains in the same samples, are reported. In total 68 cereal samples, including 43 rye, 4 wheat, 15 barley and 6 oats samples, were collected after a cool and very rainy growing season in 1998. A gas chromatograph combined with a mass spectrometric detector was used for determination of seven different trichothecenes. A high performance liquid chromatograph with a fluorescence detector was used for ochratoxin A and zearalenone determination. For the identification of moulds, the grain samples were incubated and the moulds were isolated and identified by microscopy. The analytical methods were validated for mycotoxin analysis and they were found to be adequately reliable and sensitive. Heavy rainfalls in the summer and autumn of 1998 caused abundant Fusarium mould infection in Finnish cereals, particularly in rye. Fusarium avenaceum was the most common Fusarium species found in cereals. However, the mycotoxin concentrations were very low and only deoxynivalenol, nivalenol and HT-2 toxin were detected. Deoxynivalenol was detected in 54 samples in the concentration range 5-111 µg/kg. Nivalenol and HT-2 toxin were detected in three and two samples, respectively, in the concentration range 10-20 µg/kg.     

Survey for aflatoxins, ochratoxin A, zearalenone and fumonisins in maize imported into the United Kingdom

This survey examined 140 samples of raw maize as received at ports or at major maize mills in the UK and 12 after initial cleaning. Samples were examined for aflatoxins B₁, B₂, G₁ and G₂, ochratoxin A, zearalenone and fumonisins B₁, B₂ and B₃ using fully validated analytical HPL C methods with detection limits of 0.1 mu g/kg for each aflatoxin and ochratoxin A, 4 mu g/ kg for zearalenone and 10 mu g/kg for each fumonisin. 95.0% and 92.1% of samples met the new EC statutory maximum permissible level for total aflatoxins and aflatoxin B₁ respectively. The maximum concentration of ochratoxin A found was 1.5 mu g/kg. Zearalenone and fumonisins were detected in almost every sample with 41.7% of maize containing more than 100 mu g/kg of zearalenone and 48% of samples containing more than 1000 mu g/kg total fumonisins. Initial cleaning of raw maize reduced aflatoxin concentrations by about 40% and total fumonisins by 32%.     

Deoxynivalenol and other Fusarium toxins in wheat and rye flours on the Danish market

Information on the contamination of Danish cereals and cereal products with Fusarium toxins is limited and the last survey is from 1984/1985. In the present study, the occurrence of deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin, T-2 toxin and zearalenone (ZON) was investigated in flour of common wheat, durum wheat and rye. The samples were collected from 1998 to 2001 from both mills and the retail market in Denmark. A total of 190 flour samples were analysed for DON and NIV and about 60 samples for HT-2, T-2 toxin and ZON. DON was most frequently detected with an incidence rate of 78% over all samples for all years. The contamination level varied considerably from year to year, and for wheat and rye the highest incidence and DON concentrations were found in samples from the 1998 harvest. There were regular and heavy rainfalls in Denmark during the flowering period of the crops that year, and DON was found in all samples, with mean concentrations in wheat and rye flour of 191 μg kg^-1 (n =14) and 99 μg kg^-1 (n =16), respectively. Comparison of data from each harvest year showed higher contents of DON in samples of wheat (range 20-527 μg kg^-1) than in rye (20-257 μg kg^-1). Contents of NIV, HT-2 toxin and ZON in samples of wheat and rye were generally low, and even in positive samples the contents were close to the detection limit of the methods. The T-2 toxin was detected in only a few of the wheat samples and in low amounts. However, the toxin was found in about 50% of the rye samples collected during 1998-2000, with a mean content of 49 μg kg^-1 (n =25). Durum wheat flour showed the highest DON contamination level, and all samples (n =33) collected during 2000 and 2001 contained DON with means and medians above 1100 μg kg^-1. Over 70% of the samples contained more than 500 μg kg^-1 DON, and the highest observed concentration was 2591 μg kg^-1. The concentration of T-2 toxin in durum wheat flour was also high with five of the 10 analysed samples containing more than 100 g kg^-1.     

Mycotoxins in South African traditionally brewed beers

Traditionally brewed alcoholic beverages are regularly consumed by most ethnic black South Africans. Maize and barley, both of which are used for producing locally brewed alcoholic beer, are frequently contaminated by mycotoxin-producing moulds. The study was undertaken to investigate whether these toxins are present in raw grains and the traditional beers imbibed by the local black African population. It was established that the raw ingredients (sorghum, sorghum malt grains, maize grits), commercially produced traditional beers (Utshwala and Utshwala special) and home-brewed beers (Umqombotha, Isiqatha, Imfulamfula) were contaminated by bacteria and fungi (both yeasts and moulds). The contaminating moulds were isolated and identified. The contaminated samples were analysed for aflatoxins B 1 , B 2 , G 1 and G 2 , zearalenone, citrinin, deoxynivalenol, and ochratoxin A using a multi-mycotoxin thin-layer chromatography screening method and the toxins were quantified by high-performance liquid chromatography. Grain samples were infected by Aspergillus flavus , A. alliaceus , A. clavatus , Penicillium spp., Rhizopus spp. and Mucor spp. Sorghum malt grain samples contained the toxin zearalenone. No mycotoxin-producing fungi were present in the fermented beers but two of six commercial beer samples contained aflatoxins (200 and 400 μgl -1 ) and 45% (13 of 29) of the home-brewed beers had zearalenone (range 2.6-426 μg l -1 ) and/or ochratoxin A (3-2340 μg l -1 ).     

Contents of Fusarium toxins in Finnish and imported grains and feeds

The aim of the present study was to investigate the levels of trichothecenes in representative samples of Finnish and imported cereals and feeds from the 1987 and 1988 crops. Trichothecene concentrations were determined in samples as trimethylsilylether derivatives by a gas chromatograph equipped with a mass selective detector (GC-MSD) using selected ion monitoring (SIM) which proved to be a reliable and sufficiently sensitive technique. Representative samples of Finnish and imported oats, wheat, barley, rye, maize gluten, soy granules, rapeseed, turnip rapeseed, fish meal, poultry feed and pig feed were analysed employing 19-nortestosterone as an internal standard. Almost all grain and feed samples contained deoxynivalenol (DON) from 7 to 300 micrograms/kg and smaller amounts (13-120 micrograms/kg) of 3-acetyldeoxynivalenol (3-AcDON). The most toxic trichothecenes, T-2, HT-2 toxins and nivalenol (NIV) and also zeralenone (ZEN) were found at low concentrations in some samples. Six lots of oats containing 1.3-2.6 mg/kg of DON and 0.2-0.6 mg/kg of 3-AcDON were found in the Finnish grain samples. Silo samples of almost all imported grains contained DON but at levels below 50 micrograms/kg. Imported grains contained no other trichothecenes. DON, 3-AcDON and ZEN were found in imported feedstuffs at low concentrations.     

A SURVEY OF OCHRATOXIN A IN WHEAT AND BARLEY IN INDIA

Ochratoxin A (OTA), a secondary metabolite produced by the species of Aspergillus and Penicillium has been found to be nephrotoxic, immunosuppressive and teratogenic in many species and is a possible human carcinogen. Its prevalence in cereals, namely wheat and barley, has been reported from many European countries. In this study, 126 samples of wheat and barley collected from different regions in India were analyzed by high‐performance liquid chromatography for the presence of OTA. The highest OTA content determined in one sample of wheat was 2.3 µg/kg. None of the 126 samples of wheat and barley contained more than 5 µg/kg. Wheat (82.6%) and barley (90%) samples contained less than 0.1 µg/kg, which is the limit of quantification of OTA in this study. The maximum limit of OTA for wheat, barley and rye that is being debated at Codex Alimentarius Commission meetings is 5 µg/kg. In an attempt to determine if there was any correlation between the ochratoxigenic mold mycoflora and the ochratoxin content in the samples analyzed, enumeration of the molds that belonged to Aspergillus and Penicillium species was determined using general mycological media. The aflatoxigenic and ochratoxigenic mycoflora was also enumerated using selective agar media. It was observed that more aflatoxigenic molds were recovered in both wheat and barley samples. None of the species of Penicillium detected from the samples was found to be ochratoxigenic. However, Aspergillus species belonging to section Nigri, Aspergillus niger group, also an ochratoxin producer, were recovered from the samples.     

Analysis of ochratoxin A in pig kidney and rye flour using liquid chromatography tandem mass spectrometry (LC/MS/MS)

A liquid chromatography electrospray tandem mass spectrometric (LC/MS/MS) method is described for analysis and confirmation of ochratoxin A in pig kidney and rye flour using derivatization of ochratoxin A to the methyl ester. Ochratoxin A methyl(d₃)ester is used as internal standard. The method works well, the detection limit is 0.02 μ g/kg and the repeatability (coefficient of variation) is between 6% and 16% in the contamination range 0.5 to 8 μ g/kg.     

On ochratoxin A and fungal flora in Polish cereals from conventional and ecological farms - Part 1: occurrence of ochratoxin A and fungi in cereals in 1997.

Over 200 samples of Polish cereal grain from the 1997 harvest obtained from conventional and ecological farms were tested for the presence of ochratoxin A as well as for contamination by microscopic fungi. Ochratoxin A contamination of rye from ecological farms was over six times more frequent than that from conventional cultivation. The ochratoxin A content in wheat and barley samples from ecological farms was also higher. No wheat sample from conventional farms contained the mycotoxin. In the group of ecological farms, there were differences in the percentage of cereal samples containing ochratoxin A. The ochratoxin A levels ranged from 0.2 to 57 microg kg(-1). The mean concentration of ochratoxin A in investigated cereal grain was 5.7 microg kg(-1). From samples containing detectable amounts of ochratoxin A, fungi producing ochratoxin A under laboratory conditions were isolated. They were classified as belonging to the species Penicillium cyclopium, P. viridicatum, P. chrysogenum and also Aspergillus alliaceus, A. versicolor, A. glaucus and A. flavus. Penicillium strains - producers of ochratoxin A - were isolated from 93% of the samples; in 7% of samples, only Aspergillus strains producing this mycotoxin were noted. Rye samples mainly from one farm with an ecological type of cultivation and from one conventional farm were contaminated with both Aspergillus and Penicillium mycotoxigenic strains.     

Fusariotoxins in Russian Federation 2005–2010 grain harvests

Monitoring results of food grain contamination with fusariotoxins–deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FB1&FB2), T-2 and HT-2 toxins–are presented. Harvests of 2005–2010 in different regions of Russia were investigated. The occurrence of DON in wheat was 8%, barley 9%, oats 4%, rye 2% and maize 2%. The highest frequency of ZEN contamination was found in oats, the lowest in wheat. Calculated average daily intake of DON varied from 0.066 to 0.096 µg/kg body weight, the highest being found in the Southern region, but substantially lower than the provisional maximum tolerable daily intake. The results of enzyme-linked immunosorbent assay and high-performance liquid chromatography–mass spectrometry analysis demonstrated the presence of T-2 toxin in 14% and HT-2 toxin in 17% of all samples. The maximum level of T-2 toxin was exceeded only in one sample of barley. Relatively high frequency and levels of FB1&FB2 contamination were found in maize.     

Natural co-occurrence of deoxynivalenol and fumonisins B1 and B2 in Italian marketed foodstuffs

A survey was performed to obtain the frequency and levels of contamination by deoxynivalenol (DON) and fumonisins B₁ and B₂ (FB1, FB2) mycotoxins in Italian marketed foods. Of 202 samples investigated, including raw materials and processed cereal foods (bread, pasta, breakfast cereals, biscuits, baby and infant foods), 84% were contaminated with DON at levels from 0.007 to 0.930 μg g^-1 (median 0.065 μg g^-1); 26% contained FB1 ranging from 0.010 to 2.870 μg g^-1 (0.070 μg g^-1); 35% contained FB2 at 0.010-0.790 μg g^-1 (0.080 μg g^-1). The highest levels of DON and FB1 were detected in raw cereals and wholemeal flours. The highest levels of FB2 were detected in durum wheat pasta. A widespread DON contamination was found in baby and infant foods at levels varying from 0.007 to 0.166 μg g^-1.     

Natural occurrence of scirpentriol in cereals infected by Fusarium species.

Wheat, barley and oat grain samples naturally contaminated with Fusarium spp. were analysed for the presence of scirpentriol (STO). This toxin was detected in 1, 37 and 8% of 248 wheat, 32 barley and 99 oat grain samples, respectively, and the maximum concentration was 83 microg x kg(-1). Samples of wheat and oat grain with visible scab symptoms were also analysed, and STO (mean level 255 microg x kg(-1)) was detected only in oat samples infected with F. sporotrichioides and F. poae as the dominant species. We analysed 15 barley samples that were subdivided based on seed size into fractions of <2.5 and > 2.5 mm in diameter. The smaller kernels contained an average 94% of the STO in the samples (in kernel fraction > 2.5 mm 28 microg x kg(-1), <2.5 mm 297 microg x kg(-1)). In oats, STO levels were highest in the chaff, lower in the stalk's apical internode and lowest in the grain.     

Ochratoxin A in cereal-based baby foods: occurrence and safety evaluation

Ochratoxin A is a typical cereal contaminant with strong nephrotoxic activity. To estimate the quantity of ochratoxin A that can be taken in by a child in the weaning period, several samples of cereal-based baby foods were analysed. Although most samples analysed contained ochratoxin A in undetectable amounts or below the Italian legal limit of 0.5 μg kg -1 , some irregular products were found. In particular, the analyses of the 119 batches (338 samples) of baby foods considered indicated that: 20 batches (16.8%) contained detectable quantities of ochratoxin A and four of these (3.4% of the total) contained ochratoxin A above the Italian permitted value. All samples coming from agricultural practices based on integrated pest management contained undetectable amounts of ochratoxin A, while ~5% of batches coming from conventional and organic agricultural practices were above the legal limit. On the basis of the established provisional tolerable weekly intake (PTWI), there is no significant toxicological risk for a child who occasionally consumes a formula with ochratoxin concentration slightly above the permitted level. However, stricter controls have to be applied to reject the batches containing irregular concentrations of ochratoxin A.     

Less Fusarium infestation and mycotoxin contamination in organic than in conventional cereals

A total of 602 samples of cereals, consisting of organically and conventionally produced barley, oats and wheat, were collected at harvest during 2002–2004 in Norway. Organic and conventional cereals were sampled in comparable numbers regarding cereal species, localisation and harvest time, and analysed for Fusarium mould and mycotoxins. Fusarium infestation and mycotoxin content were dependent on cereal species and varied year-by-year. However, in all cereal species, Fusarium infestation and levels of important mycotoxins were significantly lower when grown organically than conventionally. Concerning the most toxic trichothecenes, HT-2 and T-2 toxin, lower concentrations were found in organic oats and barley. Wheat was not contaminated by HT-2 and T-2, but lower concentrations of deoxynivalenol (DON) and moniliformin (MON) were found when organically produced. For mycotoxins considered to constitute the main risk to humans and animals in Norwegian cereals, i.e. HT-2 in oats and DON in oats and wheat, the median figures (mean levels in brackets) were as follows: HT-2 in organic and conventional oats were <20 (80) and 62 (117) µg/kg, DON in organic and conventional oats were 24 (114) and 36 (426) µg/kg, and DON in organic and conventional wheat were 29 (86) and 51 (170) µg/kg, respectively. Concentrations of HT-2 and T-2 in the samples were strongly correlated (r = 0.94). Other mycotoxins did not show a significant correlation to each other. Both HT-2 and T-2 concentrations were significantly correlated with infestation of F. langsethiae (r = 0.65 and r = 0.60, respectively). Concentrations of DON were significantly correlated with F. graminearum infestation (r = 0.61). Furthermore, nivalenol (NIV) was significantly correlated with infestation of F. poae (r = 0.55) and MON with F. avenaceum (r = 0.37). As lower Fusarium infestation and mycotoxin levels were found in organic cereals, factors related to agricultural practice may reduce the risk of contamination with Fusarium mycotoxins. Studies of these issues will be presented separately.     

Performance of new clean-up column for the determination of ochratoxin A in cereals and foodstuffs by HPLC-FLD

The performance of the newly developed Mycosep® 229 Ochra and Multisep® 229 Ochra clean-up columns for ochratoxin A (OTA) determination was evaluated. OTA was subsequently analysed using RP-HPLC with fluorescence detection. Recoveries for frequently contaminated commodities, like cereals, red wine, raisins and green coffee, were estimated. The recoveries obtained for the Mycosep 229 Ochra column were in the range from 87.9 ± 12.5% (n = 6) for wheat to 99.4 ± 2.7% (n = 24) for raisins. For Multisep 229 Ochra, recoveries from 76.5 ± 8.0% (n = 6) for barley to 86.4 ± 1.4% (n = 24) for raisins were achieved. Limits of detection for all matrices investigated (maize, wheat, rice, barley, raisins, green coffee beans, red wine) were in the range 0.4-2.4 μg kg^-1. The trueness of the method was tested using a certified reference material.     

A comparison of contents of group A and B trichothecenes and microbial counts in different cereal species

Concentrations of trichothecenes and the amounts of microbial biomass were compared in grain of bread wheat, durum wheat, triticale, rye, oat and barley. Grain samples came from lines regionalised in Poland grown under identical climatic and agricultural conditions in 2007. Among the six analysed cereals, the highest mean concentration of toxic metabolites of 151.89?µg/kg was found for grain of Triticum durum, whereas the lowest was for barley grain (25.56?µg/kg). The highest contamination with microscopic fungi was recorded in case of barley and rice grain (mean concentrations of ergosterol were 12.53?mg/kg and 11.24?mg/kg, respectively). In case of the analysed cereals, the total microbial biomass expressed in the amount of ATP turned out to be the highest (4.7?×?10⁵ relative light units [RLU]) for rye and oat (3.2?×?10⁵ RLU). The results of the applied classical discrimination analysis indicate a significant diversification of species in terms of all the 11 analysed metabolites.