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Mycotoxins are among the most important and highly prevalent nutritional contaminants worldwide. They are naturally produced as secondary metabolites of different fungal species, which can contaminate a wide range of agricultural products, especially cereal and grain-based food. Fusarium species are amongst the most prevalent fungal contaminants of grains, and produce the two most important classes of mycotoxins, trichothecenes (including deoxynivalenol (DON), T-2 toxin and HT-2 toxin) and zearalenones (ZEN). In humans and animals, exposure to Fusarium mycotoxins is associated with mutagenic, teratogenic, estrogenic, hemorrhagic, neurotoxic, hepatotoxic, and immunotoxic effects. Considering the widespread occurrence of these food contaminants and inevitable human exposure, this thesis aimed to investigate the adverse effects of exposure to commonly occurring Fusarium mycotoxins and potential mechanisms involved, with a special focus on DON, as this is the most prevalently occurring food contaminant worldwide. In this thesis, we showed that exposure to the major Fusarium mycotoxin DON, can adversely affect innate and adaptive immune responses, placental barrier, fetal survival and development. We further demonstrated that consuming DON-contaminated diets during pregnancy and lactation, induces long-term effects on the developing immune system of the offspring and shifts the balance in immune cell populations, hence increasing the susceptibility to allergic reactions and reducing immune responses to vaccination in later stages of life. Some specific non-digestible oligosaccharides (NDOs) have prebiotic and immune- modulating properties comparable to those observed for certain human milk oligosaccharides (HMOs). Specific structures of NDOs and HMOs are confirmed to have positive effects on improving adaptive immune responses. In this thesis we showed that nutritional supplementation with a specific NDO mixture rich in 3’-GL, can be beneficial in preventing or neutralising the adverse effects of DON on immune system. Thus, NDOs might be a suitable candidate as a supplement in infant formulae for boosting the immune system and preventing the harmful effects of early-life exposure to DON on the intestinal and immune systems.

An interlaboratory study was performed for the determination of phomopsin A in lupin seeds and lupin derived products. This was done in the frame of the 2nd Standardization Mandate (M/520) in the field of methods of analysis for mycotoxins in food, which had been issued by the European Commission in the framework of the Regulation EC 882/2004. The study was organized and carried out in accordance with collaborative study guidelines of AOAC. Test materials (seeds, flour, bakery product) were prepared by addition of naturally contaminated lupin seeds.

Ergot alkaloids are recognised as seriously toxic compounds, which caused a series of outbreaks in the past. In the EU, enforcement is implemented by visual detection and quantification of ergot sclerotia produced by moulds of the genus Claviceps. On behalf of the IAG section Feedstuff Microscopy, RIKILT organised a ring test for the visual detection of ergot sclerotia in two unground rye samples in September 2015. In this report the results from the ring test for ergot in rye 2015 are presented. The ring test ergot sclerotia in rye was designed to test the capability to visually detect sclerotia or parts thereof at relatively high levels. One sample was based on a level of approx. 400 ppm, and the second sample contained an amount of approx. 1000 ppm (EU legal limit for feeds and ingredients: 1000 ppm = 1 gram/kg = 0.1%). An amount of approx. 250 grams of rye grains was chosen as sample size. All samples were individually spiked. Thirty participants enrolled for the ring test. Participants were requested to report the number of recovered (fragments of) sclerotia and the total weight per sample. The percentage of recovery for every sample was calculated. A dedicated IAG method as well as other (lab internal) methods were allowed for application. Principally, methods are based on sieving (preferably with a mesh size of 0.5 mm), examination of every particle (grain) in the fraction with full grains or particles larger than 0.5 mm, selection of sclerotia fragments supported by documentation, and weighing the final selection of bodies. The average recovery for both samples was approx. 97%. All results except one were between the expected recovery limits (80 – 110 % w/w). Supporting data from an intralaboratory validation study of the IAG method showed trueness at different low spike levels between 98 and 105% w/w. Limit of detection was established at 7 ppm. It can be concluded that examination by visual detection of sclerotia is a valuable indicator of the expected presence of ergot alkaloids. The results of this study provides the data for a partial validation of the method of IAG for the examination of whole kernel cereal samples.

Het is mogelijk vroegtijdig het optreden van mycotoxinen te voorspellen. Een model berekent – al tijdens de teelt – de verwachte contaminatie van tarwe met Deoxynivalenol bij de oogst. De voorspellingen kunnen helpen de voer- en voedselveiligheid verder te verbeteren

A total of 1 259 samples of cereal grains, cereal products, beer and nuts were analysed for the presence of the mycotoxin sterigmatocystin. Samples were mainly collected at processing plants, storage facilities, wholesale and retail between August 2013 and November 2014, in nine European countries (mostly Greece, Italy, the Netherlands and the United Kingdom (UK), additionally in Cyprus, Germany, Latvia, Lithuania, and Poland). The products originated from 27 European countries and 18 other countries (mostly rice and nuts). The samples comprised cereal grains (221 wheat, 35 rye, 33 maize, 59 barley, 51 oats, 2 spelt, 117 rice), grain milling products (125), pasta (115), bread/rolls (143), breakfast cereals/muesli (97), fine bakery ware (90), cereal-based infant food (54), beer (53), peanuts (28) and hazelnuts (36). All samples were analysed by methods based on liquid chromatography with tandem mass spectrometry (LC-MS/MS).

A total of 1 259 samples of cereal grains, cereal products, beer and nuts were analysed for the presence of the mycotoxin sterigmatocystin. Samples were mainly collected at processing plants, storage facilities, wholesale and retail between August 2013 and November 2014, in nine European countries (mostly Greece, Italy, the Netherlands and the United Kingdom (UK), additionally in Cyprus, Germany, Latvia, Lithuania, and Poland). The products originated from 27 European countries and 18 other countries (mostly rice and nuts). The samples comprised cereal grains (221 wheat, 35 rye, 33 maize, 59 barley, 51 oats, 2 spelt, 117 rice), grain milling products (125), pasta (115), bread/rolls (143), breakfast cereals/muesli (97), fine bakery ware (90), cereal-based infant food (54), beer (53), peanuts (28) and hazelnuts (36). All samples were analysed by methods based on liquid chromatography with tandem mass spectrometry (LC-MS/MS).

De belangrijkste factoren om de aanwezigheid van mycotoxinen in voedingsmiddelen te kunnen voorspellen zijn: weersomstandigheden (zoals temperatuur en luchtvochtigheid), landbouwkundige factoren (zoals gewasrotatie en grondbewerking) en factoren in de voedselketen (zoals het drogen van de gewassen en opslagcondities). Mycotoxinen zijn giftige chemische stoffen die door schimmels worden geproduceerd en in verschillende voedingsmiddelen terecht kunnen komen. Het Rijksinstituut voor Volksgezondheid en Milieu (RIVM) en Wageningen Universiteit en Researchcentrum (Wageningen UR) hebben een conceptmodel ontwikkeld waarmee de aanwezigheid van mycotoxinen op tarwe, mais en noten kan worden geschat. Om consumenten tegen mycotoxinen te kunnen beschermen is het noodzakelijk om een mogelijke besmetting met schimmels zo vroeg mogelijk te ontdekken. Overheidsorganisaties en het bedrijfsleven kunnen het model als hulpmiddel hierbij gebruiken. Bij de ontwikkeling van dit conceptmodel is onderzocht welke factoren binnen en buiten de voedselketen gebruikt kunnen worden als indicatoren voor de aanwezigheid van mycotoxinen op tarwe, mais en noten. Hiervoor zijn interviews en een workshop met deskundigen uit verschillende invalshoeken gehouden. Met deze inzichten is een concept 'stoplicht'-model ontwikkeld dat de mate van het risico op de aanwezigheid van mycotoxinen aangeeft door middel van een rood (hoog risico), geel (gemiddeld risico) of groen (laag risico) signaal. In een vervolgstudie zal het voorspellende vermogen van het conceptmodel verder worden onderzocht.

The aim of this survey was to provide data on the occurrence of mycotoxins and pesticide residues in straw and hay used as animal feed in the Netherlands.

Maïskopbrand kan veel schade veroorzaken. Na constatering van de ziekte in Nederland is de vraag hoe de ziekte te bestrijden of beheerst kan worden. Op een zwaar besmet perceel in Megen is in 2012 een indicatief onderzoek gestart. De resultaten zijn aangevuld met informatie verkregen uit literatuuronderzoek.

Maïskopbrand kan veel schade veroorzaken. Na constatering van de ziekte in Nederland is de vraag hoe de ziekte te bestrijden of beheerst kan worden. Op een zwaar besmet perceel in Megen is in 2012 een indicatief onderzoek gestart. De resultaten zijn aangevuld met informatie verkregen uit literatuuronderzoek.

The aim of this survey was to provide data on the occurrence of mycotoxins and pesticide residues in straw and hay used as animal feed in the Netherlands.

This report of the Dutch National Reference Laboratory (NRL) for feed additives (RIKILT Wageningen UR) descreibes the activities employed in 2012. The main tasks of the NRL are: providing assistance to the European Union Reference Laboratory (EURL) on their request, as well as providing advice and support to the competent authority, which is the Dutch Ministry of Economic Affairs - Department 'Plantaardige Agroketens en Voedselkwaliteit'.

Keywords: informal seed systems, seed recycling, seed quality, germination, seed pathology, seed health, seed-borne diseases, mycotoxigenic fungi, Fusarium verticillioides, mycotoxins, Vigna unguiculata, Zea mays, Nigeria. Seed is a crucial input for agricultural production. Approximately 80% of the smallholder farmers in Africa depend for their seed on the informal seed system, consisting of farmers involved in selection, production and dissemination of seed. The lack of overhead, distribution and seed testing costs enables seed-producing farmers to offer seed for low prices, but seed quality is not always good. Seed-producing farmers multiply their seed on-farm without frequent seed renewal, referred to as seed recycling, which may lead to low seed quality. This research analysed the effect of seed recycling on physiological quality and seed health of cowpea and maize, and compared seed quality of the formal and informal seed system. We tested the physical and physiological quality of cowpea seeds produced by the formal and informal seed system. Five out of six foundation seed samples, 79 out of 81 samples of farmers’ seed, and six out of six seed company samples failed to meet standards for foundation and certified seeds of the National Agriculture Seed Council (NASC), the seed industry regulatory agency in Nigeria. No evidence was found for a negative effect of seed recycling on physiological quality of cowpea seeds. We analysed 45,500 cowpea seeds for seed-borne bacteria and fungi to compare the performance of formal and informal seed systems. All samples were heavily infected with seed-borne pathogens, including Fusarium oxysporum (69% of the samples) and Macrophomina phaseolina (76%). No evidence was found that seed recycling in the informal seed system did lead to increased levels of seed-borne pathogens. We also analysed seed quality of farmer-produced maize seed to compare it with the formal seed system. The seed company samples had significantly higher germination (99.3%) than farmer-produced seed (97.7%), but not a single sample passed the requirements for certified seed of the NASC. Twelve seed-borne pathogens were identified including Bipolaris maydis (found in 45% of the farmer-produced samples), Botryodiplodia theobromae (97%) and Fusarium verticillioides (100%). Seed recycling had no negative effect on the physiological quality or seed health of maize seed. We analysed formal and informal seed systems to assess the opportunities to prevent mycotoxigenic fungi infection in maize seeds. A range of control methods to avoid fungal infection and mycotoxin production is discussed in relation to three criteria for sustainable implementation in developing countries. An integrated approach is recommended, with special attention towards the local seed system. As an overall conclusion of the work it can be stated that the informal seed system did not underperform compared to the formal seed system for cowpea, but did underperform in relation to seed company samples of maize. There was no evidence that seed recycling reduces seed quality of cowpea and maize seed samples, so frequent seed renewal will not improve seed quality of the informal seed system. We recommend a new quality assurance system for the informal seed system based on seed quality testing by farmers themselves, without interference by government or external laboratories. Farmers publish their seed testing results on the bag, while buyers can retest the seed to verify the quality. Further research is required to develop and implement this system in different countries, agro-ecologies and crops, and to develop methods that enable farmers to test seed health quality themselves.

Keywords: informal seed systems, seed recycling, seed quality, germination, seed pathology, seed health, seed-borne diseases, mycotoxigenic fungi, Fusarium verticillioides, mycotoxins, Vigna unguiculata, Zea mays, Nigeria. Seed is a crucial input for agricultural production. Approximately 80% of the smallholder farmers in Africa depend for their seed on the informal seed system, consisting of farmers involved in selection, production and dissemination of seed. The lack of overhead, distribution and seed testing costs enables seed-producing farmers to offer seed for low prices, but seed quality is not always good. Seed-producing farmers multiply their seed on-farm without frequent seed renewal, referred to as seed recycling, which may lead to low seed quality. This research analysed the effect of seed recycling on physiological quality and seed health of cowpea and maize, and compared seed quality of the formal and informal seed system. We tested the physical and physiological quality of cowpea seeds produced by the formal and informal seed system. Five out of six foundation seed samples, 79 out of 81 samples of farmers’ seed, and six out of six seed company samples failed to meet standards for foundation and certified seeds of the National Agriculture Seed Council (NASC), the seed industry regulatory agency in Nigeria. No evidence was found for a negative effect of seed recycling on physiological quality of cowpea seeds. We analysed 45,500 cowpea seeds for seed-borne bacteria and fungi to compare the performance of formal and informal seed systems. All samples were heavily infected with seed-borne pathogens, including Fusarium oxysporum (69% of the samples) and Macrophomina phaseolina (76%). No evidence was found that seed recycling in the informal seed system did lead to increased levels of seed-borne pathogens. We also analysed seed quality of farmer-produced maize seed to compare it with the formal seed system. The seed company samples had significantly higher germination (99.3%) than farmer-produced seed (97.7%), but not a single sample passed the requirements for certified seed of the NASC. Twelve seed-borne pathogens were identified including Bipolaris maydis (found in 45% of the farmer-produced samples), Botryodiplodia theobromae (97%) and Fusarium verticillioides (100%). Seed recycling had no negative effect on the physiological quality or seed health of maize seed. We analysed formal and informal seed systems to assess the opportunities to prevent mycotoxigenic fungi infection in maize seeds. A range of control methods to avoid fungal infection and mycotoxin production is discussed in relation to three criteria for sustainable implementation in developing countries. An integrated approach is recommended, with special attention towards the local seed system. As an overall conclusion of the work it can be stated that the informal seed system did not underperform compared to the formal seed system for cowpea, but did underperform in relation to seed company samples of maize. There was no evidence that seed recycling reduces seed quality of cowpea and maize seed samples, so frequent seed renewal will not improve seed quality of the informal seed system. We recommend a new quality assurance system for the informal seed system based on seed quality testing by farmers themselves, without interference by government or external laboratories. Farmers publish their seed testing results on the bag, while buyers can retest the seed to verify the quality. Further research is required to develop and implement this system in different countries, agro-ecologies and crops, and to develop methods that enable farmers to test seed health quality themselves.

This report of the Dutch National Reference Laboratory (NRL) for feed additives (RIKILT Wageningen UR) descreibes the activities employed in 2012. The main tasks of the NRL are: providing assistance to the European Union Reference Laboratory (EURL) on their request, as well as providing advice and support to the competent authority, which is the Dutch Ministry of Economic Affairs - Department 'Plantaardige Agroketens en Voedselkwaliteit'.

Op verzoek van de directie RIVM werd een inventarisatie gemaakt van het gewenste RIVM-onderzoek naar natuurlijke toxinen in de komende jaren. Dit omvat hoofdzakelijk analytisch en toxicologisch onderzoek van en naar mycotoxinen, marine toxinen en fytotoxinen. De veelheid aan mogelijke onderwerpen voor onderzoek maakt een prioriteitsafweging noodzakelijk. Een voorstel daartoe is aan het eind van deze nota opgenomen.

The data about the possible toxicity of natural toxins indicate that many of these toxins are immunotoxic. This was found in various animal species and in man. The effects on the immune system often involve clinical implications, i.e. reduced resistance towards infections. For many of the natural toxins, however, investigations on the immunotoxic effects are far from complete. The relation of the observed effects on the immune system to other toxic effects is often not clear. On several occasions effects on one or more immunological parameters have been described, whereas the significance for the ultimate functionality remains unclear. This means that the knowledge about the potential immunotoxicity is fragmentary for the majority of the natural toxins. The knowledge on the toxicological profile of many less well investigated natural toxins would be desirable. In particular attention should be given to the immune system as a possible target for toxicity. The mycotoxins Fumonisin B1 and Ochratoxin A should be considered for further immune toxicity studies, in view of the toxicological significance, the gaps in the knowledge and the possibilities to have enough material available for (immune-)toxicity studies.

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The data about the possible toxicity of natural toxins indicate that many of these toxins are immunotoxic. This was found in various animal species and in man. The effects on the immune system often involve clinical implications, i.e. reduced resistance towards infections. For many of the natural toxins, however, investigations on the immunotoxic effects are far from complete. The relation of the observed effects on the immune system to other toxic effects is often not clear. On several occasions effects on one or more immunological parameters have been described, whereas the significance for the ultimate functionality remains unclear. This means that the knowledge about the potential immunotoxicity is fragmentary for the majority of the natural toxins. The knowledge on the toxicological profile of many less well investigated natural toxins would be desirable. In particular attention should be given to the immune system as a possible target for toxicity. The mycotoxins Fumonisin B1 and Ochratoxin A should be considered for further immune toxicity studies, in view of the toxicological significance, the gaps in the knowledge and the possibilities to have enough material available for (immune-)toxicity studies.

Op verzoek van de directie RIVM werd een inventarisatie gemaakt van het gewenste RIVM-onderzoek naar natuurlijke toxinen in de komende jaren. Dit omvat hoofdzakelijk analytisch en toxicologisch onderzoek van en naar mycotoxinen, marine toxinen en fytotoxinen. De veelheid aan mogelijke onderwerpen voor onderzoek maakt een prioriteitsafweging noodzakelijk. Een voorstel daartoe is aan het eind van deze nota opgenomen.

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Ergot alkaloids are toxins produced by members of the fungal family of Clavicipitaceae represented among others by Claviceps purpurea, C. paspali, and C. fusiformis, occurring predominantly on rye, wheat and barley, but also on rice, maize and sorghum, oats and millet.

Feed materials were analysed for the mycotoxins deoxynivalenol, ochratoxin A, zearalenone, aflatoxin B1, fumonisin B1 and B2, and HT-2- and T-2-toxins. In this report trends in the average content during the period 2001-2009 are reported for these mycotoxins. Monitoring data from the National Feed monitoring program and from the Commodity Board Animal Feed are used for this study.

Feed materials were analysed for the mycotoxins deoxynivalenol, ochratoxin A, zearalenone, aflatoxin B1, fumonisin B1 and B2, and HT-2- and T-2-toxins. In this report trends in the average content during the period 2001-2009 are reported for these mycotoxins. Monitoring data from the National Feed monitoring program and from the Commodity Board Animal Feed are used for this study.

Ergot alkaloids are toxins produced by members of the fungal family of Clavicipitaceae represented among others by Claviceps purpurea, C. paspali, and C. fusiformis, occurring predominantly on rye, wheat and barley, but also on rice, maize and sorghum, oats and millet.

Bestrijding van Fusarium in granen kan alleen als tijdens de bloei wordt gespoten, blijkt keer op keer uit PPO-onderzoek. 100 procent bestrijding kan niet.

In dit rapport wordt de monsternameproblematiek toegespitst op de analyse van mycotoxines en dioxinen in diervoedergrondstoffen. Eerst wordt in hoofdstuk 2 de Europese regelgeving betreffende monstername van diervoeders uiteengezet. In hoofdstuk 3 wordt de homogeniteit gedefinieerd en in hoofdstuk 4 wordt ingegaan op de onzekerheid in het eindresultaat. In hoofdstuk 5 worden relevante parameters voor beperking van de monsternameonzekerheid geïdentificeerd en worden methodes ter bepaling van de monsternameonzekerheid bediscussieerd. Op basis hiervan worden in hoofdstuk 6 conclusies getrokken en aanbevelingen gedaan voor vervolgonderzoek en een praktisch toepasbare strategie voor de controle van diervoedergrondstoffen op mycotoxines en dioxinen die moet leiden tot een betere interpretatie van analyseresultaten.