Occurrence of mycotoxins in feedstuffs of dairy cows and estimation of total dietary intakes.

A survey was conducted to determine the occurrence of mycotoxins in feedstuffs of dairy cows in the Netherlands and to estimate total dietary intakes of these compounds. Twenty-four dairy farms were visited twice and samples taken of all diet ingredients. Feed intake data were collected by means of questionnaires. A total of 169 feed samples were collected and analyzed for 20 mycotoxins using a liquid chromatography tandem mass spectrometry multimethod. Silage and compound feed were the main diet ingredients, representing on average 67 and 23% of dry matter intake, respectively. Deoxynivalenol (DON), zearalenone, roquefortine C, and mycophenolic acid were the mycotoxins with the highest incidence. The incidence of DON in silage, compound feed, and feed commodity samples was 38 to 54%. The incidence of zearalenone in silage, compound feed, and feed commodity samples was 17 to 38%. The DON and zearalenone had a low incidence in forage samples and were not detected in ensiled by-product samples. Roquefortine C and mycophenolic acid were only detected in silage and ensiled by-product samples (incidence 7 to 19%). Fumonisins B(1) and B(2) were detected in 2 compound feed samples and one feed commodity sample. Aflatoxins B(1), B(2), G(1), and G(2), ochratoxin A, T-2 and HT-2 toxin, 3-acetyl-DON, 15-acetyl-DON, diacetoxyscirpenol, sterigmatocystin, fusarenon-X, ergotamine, and penicillinic acid were not detected in any of the samples. Average concentrations of DON, zearalenone, roquefortine C, and mycophenolic acid in complete diets were 273, 28, 114, and 54 microg/kg, respectively. Maximum concentrations were 969, 203, 2,211, and 1,840 microg/kg, respectively. Calculated average daily intakes of these mycotoxins were 5.0, 0.5, 2.0, and 0.9 mg/animal, respectively, and maximum daily intakes 19.3, 3.5, 38.9, and 32.3 mg/animal, respectively. Corn silage was the major source of all 4 of these mycotoxins in the diet. Extremely high concentrations of roquefortine C and mycophenolic acid (up to 45 and 25 mg/kg, respectively) were detected in visibly molded areas in surface layers of corn silage. These areas appeared to be the main source of roquefortine C and mycophenolic acid in the diet. Because carry-over of DON, zearale-none, roquefortine C, and mycophenolic acid into milk is negligible, their occurrence in feedstuffs is not considered of significant concern with respect to the safety of dairy products for consumers. Potential implications for animal health are discussed.

Occurrence of mycotoxins in maize, grass and wheat silage for dairy cattle in the Netherlands.

The occurrence of mycotoxins in 140 maize silages, 120 grass silages and 30 wheat silages produced in the Netherlands between 2002 and 2004 was determined using a liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) multi-method. Deoxynivalenol (DON) was detected above the limit of quantification (LOQ) of 250 µg kg⁻¹ in 72% of maize and 10% of wheat silages. Average DON concentrations were 854 and 621 µg kg⁻¹, respectively, and maximum concentrations 3142 and 1165 µg kg⁻¹, respectively. Zearalenone was detected above the LOQ of 25 µg kg⁻¹ in 49% of maize and 6% of grass silages. Average zearalenone concentrations were 174 and 93 µg kg⁻¹, respectively, and maximum concentrations 943 and 308 µg kg⁻¹, respectively. The incidences and average concentrations of DON and zearalenone in maize silage were highest in 2004. The incidence of other mycotoxins was low: fumonisin B1 and 15-acetyl-DON were detected in 1.4 and 5% of maize silages, respectively, and roquefortin C in 0.8% of grass silages. None of the silages contained aflatoxins, ochratoxin A, T2-toxin, HT2-toxin, sterigmatocystin, diacetoxyscirpenol, fusarenon-X, ergotamine, penicillinic acid, or mycophenolic acid. This study demonstrates that maize silage is an important source of DON and zearalenone in the diet of dairy cattle. Since the carryover of these mycotoxins into milk is negligible, their occurrence in feed is not considered to be of significant concern with respect to the safety of dairy products for consumers. Potential implications for animal health are discussed.

Sample comminution for mycotoxin analysis: dry milling or slurry mixing?

A comparison was made between dry milling and slurry mixing as a comminuting step preceding mycotoxin analysis. Sample schemes of up to 30 kg are mandated by European Commission legislation. Cocoa, green coffee, almonds and pistachio samples of 10 kg were milled by a Romer analytical sampling mill and all three subsamples were analysed for aflatoxin B1 or ochratoxin A content. The homogenization process was evaluated in terms of the analytical results, coefficients of variation for different mills and particle size distributions. Coefficients of variation for the comminuting step were higher for dry milling than for slurry mixing. This difference was explained based on measured particle size distributions for both milling types. Measurements also showed slight differences in mycotoxin content of samples based on milling procedures. This might lead to lots being wrongly accepted or rejected based on an erroneous subsample result. It was concluded that sample comminution was best performed by slurry mixing, which produced smaller particles and, consequently, homogeneous samples with lowest coefficients of variation. Additional data are given on analytical results in 10-kg subsamples that originate from the aggregate 30-kg sample as described in Commission Directive 98/53/EC.

Novel sampling methods for the analysis of mycotoxins and the combination with spectroscopic methods for the rapid evaluation of deoxynivalenol contamination.

A novel non-destructive sampling approach is described for the identification of food matrices contaminated with deoxynivalenol and other mycotoxins. This technique is different from currently applied sampling procedures for this purpose and is based on the principle that surface material from the tested goods is collected on a filter and brought to chemical or spectroscopic analysis. This approach has been applied to several matrices and mycotoxins, with a focus on those mycotoxin-matrix combinations that are of main relevance due to current or future legislation. Tests were carried out with a facility that has been shown to be suitable to process large quantities of materials at points of transaction, such as harbour. Further experiments with a small sampling lance prototype showed that analytical results from the chemical analysis of the tested goods can be correlated with the results obtained with this novel sampling procedure.

Acrylamide exposure from foods of the Dutch population and an assessment of the consequent risks.

At the end of April 2002, the Swedish Food Administration reported the presence of acrylamide in heat treated food products. Acrylamide has been shown to be toxic and carcinogenic in animals, and has been classified by the WHO/IARC among others as 'probably carcinogenic for humans'. The purposes of this study were firstly to analyse acrylamide contents of the most important foods contributing to such exposure, secondly, to estimate the acrylamide exposure in a representative sample of the Dutch population, and thirdly to estimate the public health risks of this consumption. We analysed the acrylamide content of foods with an LC-MS-MS method. The results were then used to estimate the acrylamide exposure of consumers who participated in the National Food Consumption Survey (NFCS) in 1998 (n=6250). The exposure was estimated using the probabilistic approach for the total Dutch population and several age groups. For 344 food products, acrylamide amounts ranged from <30 to 3100 microg/kg. Foods with the highest mean acrylamide amounts were potato crisps (1249 microg/kg), chips (deep-fried) (351 microg/kg), cocktail snacks (1060 microg/kg), and gingerbread (890 microg/kg). The mean acrylamide exposure of the NFCS participants was 0.48 microg/kg bw/day. Risk of neurotoxicity is negligible. From exposure estimations it appears that the additional cancer risk might not be negligible.

Evaluation of effect of measures taken after a DON outbreak.

In April 1999 an amount of 2600 µg/kg DON was found in a sample breakfast cereals in the Netherlands. This event was the start of a lot of activities, which dealt with the prevention, control, health and consumer aspects of DON in food for human consumption. The Food Inspection Services started a monitoring program to measure DON in cereal products, flour and raw cereals. The National Institute of Public Health and the Environment, another part of the Ministry of Health in the Netherlands, was asked to carry out a risk analysis on DON. This was the basis for the Minister of Health to set an action limit for consumer products. She also informed Brussels and asked for a European limit. The Main Board on Agriculture set out to implement measures to be taken at harvesting, milling and bread baking industry. The Scientific Committee on Food of the EC expressed an opinion on DON in December 1999. Worldwide attention leads to discussion of a DON limit by JECFA in February 2001.In the period may 1999 until march 2002 a number of more than 1700 samples were analysed on DON. These originated from the cereal harvest of the years 1998 until 2001. The results showed a sharp decrease of DON content in samples of harvest 1999 when compared to 1998. This lower level was maintained in the 2000 and 2001 harvests. Apparently the measures taken to control the DON level succeeded to maintain values below the action limits. Despite these activities a smaller outbreak of DON appeared in 2001 in pasta products at a lower extent. This indicated that control should be done systematically, not sporadically, and at a European level, which is made possible since EC has set a limit in July 2000. Analytical results of the measurements are presented, together with the chronological order of the associated activities of national, EU and worldwide bodies on human health control. Special attention is paid to DON in bread, related to the level in flour.

Non-destructive automated sampling of mycotoxins in bulk food and feed - A new tool for required harmonization.

Mycotoxins contamination is highly non-uniformly distributed as is well recog-nized by the EC, by not only setting legal limits in a series of commodities, but also schedule a sampling plan that takes this heterogeneity into account. In practice however, it turns out that it is very difficult to carry out this sampling plan in a harmonised way. Applying the sampling plan to a container filled with pallets of bags (i.e. with nuts or coffee beans) varies from very laborious to almost impossible. The presented non-destructive automated method to sample bulk food could help to overcome these practical problems and to enforcing of EC directives. It is derived from a tested and approved technology for detection of illicit substances in security applications. It has capability to collect and iden-tify ultra trace contaminants, i.e. from a fingerprint of chemical substance in a bulk of goods, a cargo pallet load (~ 1000 kg) with boxes and commodities.The technology, patented for explosives detection, uses physical and chemistry processes for excitation and remote rapid enhanced release of contaminant residues, vapours and particulate, of the inner/outer surfaces of inspected bulk and collect them on selective probes. The process is automated, takes only 10 minutes, is non-destructive and the bulk itself remains unharmed. The system design is based on applicable international regulations for shipped cargo hand-ling and transportation by road, sea and air. After this process the pallet can be loaded on a truck, ship or plane. Analysis can be carried out before the cargo leaves the place of shipping. The potent application of this technology for myco-toxins detection, has been demonstrated by preliminary feasibility experiments. Aflatoxins were detected in pistachios and ochratoxin A in green coffee beans bulk. Both commodities were naturally contaminated, priory found and confirm-ed by common methods as used at routine inspections. Once the contaminants are extracted from a bulk shipment, an appropriate existing analytical method, i.e. a CEN method, can be used to measure the mycotoxins.The system, routinely in use for explosives detection, was able to screen bulk food and feed for mycotoxins, through non-destructive automated sampling of a whole batch/lot/sublot of commodities. The opportunity to sample a whole bulk would provide more effective tools for inspection at seaports, production facili-ties and distri-bution points. It will advance the current process of myco-toxins check because: (i) Checks will be automated and harmonized, (ii) Checks will be non-destructive, (iii) Checks will be faster and allow a greater amount of bulk commodities to be inspected and (iv) The ability to check, with automated equipment, larger portions of lots of a shipment will increase the probability to detect the heterogeneous mycotoxins contamination in bulk foods. The poster provides some results of feasibility experiments indicating the capability of this technology for inspection of commodities bulks for the detection of mycotoxins, at legal limits, in naturally contaminated food.

Determination of aflatoxin B1 in pistachio kernels and shells.

A method was developed for accurate measurement of aflatoxin B1 in the edible portion of pistachio nuts. Twenty-nine samples of kernels with and without their shells were slurried with a Mega Ultra Turrax. A subsample of the homogenate was extracted with water-methanol, defatted with petroleum ether, purified with a silica solid-phase extraction column, and redissolved in methanol. After separation on an octadecyl column and postcolumn reaction with on-line electrochemically generated bromine, the aflatoxin B1 derivative was detected fluorometrically. The shells contained less than 1% of the aflatoxin B1 found in the edible kernel, and they accounted for 41.7-46.8% of the weight of the whole pistachio. These observations indicate it is possible to analyze an entire sample, up to 25 kg, as a whole and still be able to judge whether it meets the legal tolerance limit of 5 micrograms aflatoxin B1/kg edible part, as set by the Dutch Food Act.