Labinfo

LNRN A T I O N A L ER E F E R E N T I ELABORATORIA

L A B O R ATO I R E SN A T I O N A U XD E R E F E R E N C ENRLN A T I O N A L

R E F E R E N C ELABORATORIESNRL

Newsletter for the approved food safety laboratories

SEMIANNUAL NEWSLETTER - N° 11 FEBRUARY 2014

FASFCAC-Kruidtuin - Food Safety Center, Kruidtuinlaan 55, 1000 Brussels

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p. 4 Emerging diseases in forest ecosystems: challenges, prospects and role of NRL plant diseases

p. 7 Phycotoxins: an emerging problem for public health

p. 10 Diagnostic of brucellosis: difficulties and prospects

p. 12 A new type of multiplex assay to screen for GMO in food and feed samples developed within the ORIENT-EXPRESS project

p. 15 Susceptibility of co-contaminations of maize and its derived products in Belgium to fumonisins, fusaric acid and fusarin C

p. 18 ICP-MS: a promising tool in the detection and analysis of nanoparticles

p. 23 New understanding of ink components migrating from packaging material into foodstuffs

p. 27 Workshops & Symposia

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LabInfoNewsletter for the approved food safety laboratories

Editors’ groupDirk Courtheyn, Marnix De Gruyter, Mieke De Mits, Conny De Schepper, Alain Dubois, Marc Evrard, Geert Janssens, Alain Laure, Bert Vandenborre, Eva Wevers and Marie-Christine Wilem

Authors of this issueGeert De Poorter, Anne Chandelier, Philippe Delahaut, Mathieu Dubois, David Fretin, Sylvia Broeders, Sigrid De Keersmaecker, Nancy Roosens, Tangni Emmanuel K, Nadia Waegeneers, Benjamin Horemans, Hendrik De Ruyck and Jan De Block.

TranslationTranslation Service of the AgencyEditors’ group

Photographs and illustrationsSupplied by the laboratories

LayoutGert Van Kerckhove

Editor’s addressLabInfop.a. D. CourtheynFASFCAC-Kruidtuin – Food Safety Center4de verdieping, bureel K04/120218Kruidtuinlaan 551000 BrusselTel.: 02.211.87.33dirk.courtheyn@favv.be

LNRN A T I O N A L ER E F E R E N T I ELABORATORIA

L A B O R ATO I R E SN A T I O N A U XD E R E F E R E N C ENRLN A T I O N A L

R E F E R E N C ELABORATORIESNRL

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Dear reader,

Already one month into 2014, it seems that the economy is picking up again. The laboratory sector continues to strive towards increased efficiency and conver-gence. As I have pointed out before: small labs will disappear, increased specialization will gain ground, and increasingly heavy funds will be needed to develop a state-of-the-art laboratory. Our clients are also very demanding: they want increasingly shorter lead times, even lower detection limits (or maybe just the opposite!), even more components in the same run, faster service after results (clarification, advise, compulsory notification,...). These are all topics that are part and parcel of the daily management of a well-functioning and flexible laboratory. In this framework, the Laboratories Administration of the FASFC has granted its control program partim analyses 2014 via the formal procedure provided by the new law of 2006 concerning public procurement contracts, and certain procurements for public works, deliveries and services. This choice has made it possible to save several hundred thousands of Euros, as now more than ever, we have to pinch every penny before spending it. We attach great importance to maintaining cordial work relations with our partners who have subscribed to the framework agreement to work on clearly delineated assignments based on KPIs (Key Performance Indicators). The proportion of analyses conducted by our own staff with regard to those conducted by third parties fluctuates around 75/25. The outsourced package represents about €2 million, an amount not to be despised. I wish you a lot of reading pleasure with this 11th edition of Labinfo.

Geert De PoorterDirecteur-generaal Laboratoria

Editorial

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Emerging diseases in forest ecosystems: challenges, prospects and role of NRL plant diseases

Anne ChandelierWalloon centre of agronomic research, Life sciences Department, Biology of pest and biovigilance Unit (Centre wallon de Recherches agronomiques, Département Sciences du Vivant, Unité Biologie des Nuisibles et Biovigilance)

During the past 30 years, emerging diseases have been reported at an increasing rate in forest ecosystems in Europe. These diseases have economical (loss in value of forest products, loss of employment in the wood sector, financial loss for nurseries), ecological (loss of biodiversity, dysfunctional ecosystems) and societal (landscape alteration, disappearance of forest species valued by the public) impacts. The Dutch elm disease, caused by the fungus Ophiostoma novo-ulmi, the alder disease caused by Phytophthora alni (fig. 1a), the ash dieback caused by the fungus Hymenoscyphus pseudoalbidus (fig. 1b) or more recently the “Sudden Larch Death” caused by Phytoph-

thora ramorum are some examples of emerging diseases. Two of the causing agents (P. alni and H. pseudoalbidus) have already caused a lot of damage in forest ecosystems in Belgium.

What is the origin of an emerging disease?

Intensified international trade constitute the main introduction pathway of pathogenic organisms. Plants or parts of plants (seeds, cutting, living plants for nurseries), wood or wood packaging are potential ‘gateways’ for fungi, in-sects or nematodes in forests. The role of the public is also not to be put aside, whether by the direct introduction of plants or seeds brought back in the luggage and escaping border inspection or by the indirect introduction of pathogenic organisms polluting their cloths or present in soil sticking to their shoes. In parallel, climatic changes modify temperature and humidity conditions of a region making local host plants more susceptible to infections. Moreover, changed climatic conditions can also lead to the expansion of the distribution area of a pathogenic organism.

Although the causes of introduction are known (but sometimes difficult to control), the factors which contribute to the establishment and subsequent dispersal of a pathogenic exotic agent are hard to control because of the complexity of invasions. While the directive 2000/29/EC (interceptions of non-conforming products) has shown to be useful for regulated pathogens, it has no impact on emerging agents because the diseases they cause are not foreseeable. Moreover, most of the time, they do not cause major plant health problems in their place of origin (due to the co-evolution of host plants and the pathogenic agent). This means that the emerging organ-isms escape, at least initially, plant health inspections. Finally, it is also important to notice that emerging diseases which occur in natural environments are very difficult to control by emergency phytosanitary measures because they spread rapidly.

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Fig. 1. Symptoms caused by emerging diseases in Belgian forests. A: Alder (Alnus glutinosa) disease caused by Phy-

tophthora alni – symptoms on the trunk and crown dieback; B : ash (Fraxinus excelsior) dieback caused by Hymenoscyphus pseudoalbidus - symptoms in the wood and crown dieback (photos, A. Chandelier)

How to respond to these new phytosanitary threats?

At the European level, the scientific community is involved in different projects notably FORTHREAT (1) or ISEFOR (2). Several options are proposed to limit the development of emerging diseases of tree species (Stenlid and al. 2011) (3):• to intensify monitoring in Europe;• to introduce Early Warning Systems for pathogenic organisms and especially spore traps (fig. 2);• to introduce ‘sentinel’ plants (i.e. forest species found in Europe) in different regions of the world to evaluate

their behaviour in new environment;• to adapt plant health legislation (better implementation of existing norms, reinforcement of phytosanitary

analysis in developing countries from which the plants come from, phytosanitary risk assessment by using PRA (« Pest Risk Analysis »).

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Fig. 2. Installation of spore traps in forest to monitor airborne inoculum of pathogenic fungi (photo, A. Chandelier)

What does the NRL “plant diseases” do?

In Belgium, the NRL “plant diseases” participates to studies on forest pathogens in several nationally and inter-nationally financed research projects. By its participation to different European meetings dealing with regulated pathogens (especially meetings of the European and Mediterranean Plant Protection Organisation, EPPO), it informs regional and federal political authorities about potential new phytosanitary threat to forest environment. Ultimately, it also advises the FASFC on diseases for which trainings dealing with symptoms identification could be organised. Moreover, as the reference laboratory of the Walloon Observatory of Forest Health for fungal diseases, the laboratory of mycology of the CRAW carry out phytosanitary survey in forests and organizes trainings on dis-ease identification for forest managers.

Bibliography:

1. European network on emerging diseases and threats through invasive alien species in forest ecosystems (FORTHREATS, FP6, 2007-2009)

2. Increasing Sustainability of European forests : modelling for security against invasive pests and pathogens under climate changes (ISEFOR, FP7, 2011-2013)

3. Stenlid J, Oliva J, Boberg JB, Hopkins AJM (2011). Emerging diseases in European forest ecosystems and responses in society. Forests 2, 486-504

a.chandelier@cra.wallonie.be

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Phycotoxins: an emerging problem for public health

Philippe Delahaut and Mathieu DuboisCER Group - Health Department, Rue du Point du Jour 8, 6900 Marloie, Belgium

Introduction

During these last years the increased blooming of potentially toxic algae causes a safety problem to drinking water and fishery products, especially shellfish.

Phycotoxins are secondary metabolites, synthesized by dinoflagellates and diatoms. These toxins can be toxic for the marine fauna or for consumers via shellfish or fish. Usually these toxins are classified by their clinical signs observed during intoxication.

Main phycotoxins

The Diarrheic Shellfish poison (DSP) comprises several compounds, of which okadaic acid is the best known. The presence and toxicity of the poison is subject to seasonal and geographical variations.Several toxins (DTXs, PTXs, YTXs, etc) can cause these digestive problems, which can lead to confusion with viral or bacteriological infections. During the sixties, gastro-enteritis was mentioned for the first time, in the Netherlands, after consumption of mussels. The clinical signs appear on average 4 hours after the ingestion of contaminated shellfish. These signs are diarrhea, vomiting and abdominal pain. These symptoms disappear after 3 days without after effects.

The Paralytic Shellfish poison (PSP) forms a family of approximately twenty chemically related molecules of which saxitoxin represents the main poison. Between 1689 and 1962, numerous cases of intoxications were reported of which several had a fatal ending. Most of them were counted in temperate zones with predominance on the northern pacific coast of the American continent. Afterwards European countries, the South American continent and Asia have also described cases of intoxication via mussels or shellfish.The signs of intoxication appear very fast after the ingestion and the seriousness depends on the ingested dose and the individual sensitivity.In most of the cases, the recovery is completed in several days, but in the most serious cases consumers can die following a respiratory paralysis. The symptoms are mainly nervous, type paraesthesia. Unfortunately there exists until these days no antidote.

The first cases of Amnesic Shellfish Poison (ASP) broke out at the end of 1978, after the consumption of mussels harvested in the estuary of Prince Edward Island in Canada. The identified phycotoxin was domoic acid. From then on, this toxin has been identified in several places, and in countries with surveillance variable quantities of domoic acid were demonstrated in shellfish.

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Short time after ingestion, patients show classical signs of a food poisoning (vomiting, abdominal cramps, diar-rhea,...), then approximately 2 days later neurological disorders appear (cephalea, memory disorders, confusion, coma,...). Usually, recovery happens in between 1 to 4 days, but cases are known were the victim died of severe convulsions.After analysis, a relation between the observed disorders and the ingested dose was found.

Besides these best known 3 groups, there exist toxins like brevetoxins, ciguatoxins and more recently azaspiracids, spirolids and gymnodinium. The symptoms are mainly of digestive or neurological order.

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Method of detection

The difficult nature of analyzing phycotoxins is mainly due to the very complex chemical structure of the toxic molecules. This structure makes the chemical synthesis nearly impossible. The only way to obtain them in a lim-ited quantity is to try to produce them in vitro from toxic algae cultures, which represents an uncertain and very tedious task. This difficulty limits the access to reference materials needed for analysis.

In Belgium, the FASFC carries out inspections on different types of shellfish and especially on mussels. For the de-tection of domoic acid, HPLC-UV is used which has a limit of quantification of 0.8 mg/kg. Two official methods for the PSP group exist, a bioassay on mice and an HPLC with fluorescence detection. In the future, an LC-MS method will be used for molecules of this group. This technique is already used in routine for okadaic acid and related compounds.

Conclusion

The field of phycotoxins is expanding a lot. New toxic species, phycotoxins and contaminated zones are regularly discovered. Because a simple elimination is impossible, the scientific research tries to predict the phenomena of blooming of toxigenic phytoplankton species.

In parallel, the development of purification methods of these toxins has led to the development of new biological and physiochemical detection methods. The detection and the quantification of molecules are confronted with the availability of toxin standards. The bioassays are not sensitive enough, contrary to physicochemical methods which are too specific.

infolnr@cergroupe.be

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Diagnostic of brucellosis: difficulties and prospectsDavid FRETINUnit « Bacterial Zoonoses of Livestock », Operational Direction Bacterial Diseases, CODA-CERVA, Groeselenberg 99, 1180 Brussels

Since 2003, Belgium is (European decision 2003/467/EC) officially Bovine Brucellosis-free. To hold its officially free status, a surveillance program was introduced. This program is based on a bacteriological diagnostic of abortions, a random winter screening of cattle herds and a serological diagnostic of purchases (if these are applied accord-ing to the epidemiological situation) and cattle imports from countries which are not officially Bovine Brucellosis-free. This surveillance program, in particular the abortion protocol introduced by ARSIA and DGZ and financed by the FASFC, allowed it to detect new outbreaks of Bovine Brucellosis in 2010, 2012 and 2013. The causes of return of Bovine Brucellosis in our country are until now not discovered.The surveillance program and the follow-up of 2010, 2012 and 2013 breakouts have made the problem of sero-logical diagnostic of Brucellosis, that is to say false positive serological reactions, stand out. These reactions are in particular problematic in countries where the disease’s prevalence is low or zero.

False positive serological reaction

The serological diagnostic of Brucellosis is based on the detection of antibodies against bacterial lipopolysac-charides. Lipopolysaccharides (LPS) are complex sugars which are found in Gram-negative bacteria. The composi-tion and the structure of this sugar vary depending on the bacterial species. This sugar forms a protective barrier around the bacteria, so LPS are a major target of the immune system. The problem within the context of Brucel-losis diagnostics is that the LPS of Brucella has a similar structure as the LPS of other bacteria, and in particular Yersinia enterocolitica O9 causes a problem. This bacteria is a common Enterobacteriaceae for cattle, but it is very weakly pathogenic. Experimental infections on cattle carried out by CERVA show this phenomenon of serological cross reaction well. Y. enterocolitica O9 infected cattle show a serological positive response in diagnostic tests of Brucellosis.Until now, no scientific research has made it possible to find a solution to this major problem of diagnostic.

Bacteriology of Brucellosis

Due to the lack of a serological test which can certify the status of an animal, isolating the bacteria remains the gold standard. The transmission of the disease on cattle is visible at the abortion at the end of gestation. During abortion, a big quantity of germs are set free, up to 1 billion of germs per gram aborted material. In consequence, it is possible to see the germ under the optical microscope, especially after Stamp coloring (figure 1). This coloring doesn’t allow to distinguish between Brucella sp, Chlamydophila abortus and Coxiella burnetii, all infectious agents which can lead to an abortion. The culturing of aborted material on selective medium during 10 days allows revealing Brucella. Complementary tests of cultures with different colorants, agglutination and molecular biology help typing the bacteria. The bacteriological diagnostic on slaughtered animals will be more delicate because outside of gestation time, Brucella abortus has no organ of predilection.

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Figure 1: Optical microscopy with 500 times magnification of a Stamp’s coloring of a Brucella sp. culture

Challenge for the future

For the labs and the research team, two points must be the aim of all attentions. Firstly, the focus on serological tests that reduce or eliminate wrongly positive reactions, and secondly, the focus on molecular tests that allow fast detection and typing of the bacteria. A molecular characterization is essential to identify a relation between two or more strains and possibly the source of infection.

david.fretin@coda-cerva.be

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A new type of multiplex assay to screen for GMO in food and feed samples developed within the ORIENT-EXPRESS project

Sylvia Broeders, Sigrid De Keersmaecker and Nancy RoosensWIV-ISP, Unit Platform Biotechnology and Molecular Biology (PBB)

CONTEXT AND AIM

Commercialization of Genetically Modified Organisms (GMO) is strictly regulated in Europe and detection makes up an integrated part of these regulations (1, 2). To date, real-time PCR is the method of choice in GMO detection. Methods for quantification are validated and published by the European Union Reference Laboratory (EU-RL) and further implemented by the GMO detection laboratories. These event-specific methods are also used to identify unequivocally the GMO in food/feed samples. However, with the increasing number of Genetically Modified (GM) events being put on the market (3), the one-by-one approach for identification of GMO is no longer realistic. Con-sequently, many enforcement laboratories have established a screening tool as the first step in GMO analysis. This tool does not only permit to narrow down the number of GM events to be identified but allows also to presume the presence of events that are unauthorized (UGM) in Europe. Indeed, when a GM element is detected in a sam-ple and cannot be covered by an authorized event, it may be suspected to be part of a UGM.

In the near future, the diversity of the taxonomy (taxon host plants) and biotechnology (new traits) of the GM events as well as the possibility of having a UGM present in a sample will increase (4). Consequently, it will be nec-essary for enforcement laboratories to enlarge their panel of screening methods and the screening tool will not be time and cost effective anymore. Many laboratories are therefore evolving in the direction of using methods that target different elements simultaneously, i.e. multiplex methods. However, multiplex detection by real-time PCR has some limitations such as the number of fluorescent dyes that can be used and the appearance of false positives (5). In addition, it should be guaranteed that the sensitivity and efficiency are equal to those obtained for the individual simplex methods and that no primer dimer formation will occur. There is thus a need for new nucleic acid-based methods allowing reliable multiplex detection without loss of specificity and sensitivity. Such a high-throughput strategy will reduce costs and time of analysis.

The project ORIENT-EXPRESS, financed by the WIV-ISP, aims at developing such a tool based on the xMAP technol-ogy (6). This bead-based multiplex technology (Figure 1) allows to detect up to 500 targets in a sample in a single run.

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Figure 1: Principle of the xMAP technology applied to GMO detection (based on 6)The xMAP technology is based on the use of beads (microspheres), each set having a unique spectral ad-dress (i.e. a different shade of red). Each bead set can be coupled with a reagent (here: a specific oligonu-cleotide) specific to a particular bioassay, allowing the capture and detection of a specific analyte (here: plant/GM DNA, labelled in green during sample preparation and hybridised to the oligo). Multiplexing is achieved by combining different bead sets to analyse one sample. Inside the Luminex analyzer, a light source excites the internal dyes allowing to identify each microsphere particle by measuring the red fluorescence signal emitted by the bead. In addition, the presence of the bound element is detected by measuring the green fluorescence signal of the hybridised target.

To date the WIV-ISP GMOlab (coordinator of the Belgian NRL-GMO) has developed and validated 17 GMO screen-ing methods using the SYBR®Green real-time PCR technology (7). These methods are currently used in the routine GMO analysis of food and feed samples, performed for the Federal Agency for the Safety of the Food Chain (FASFC), under ISO17025 accreditation. In the ORIENT-EXPRESS project, it will in the first place be investigated which approach can be used to transfer these real-time PCR methods into the xMAP technology allowing them to be tested simultaneously in a single well. A comparison will be made between the real-time PCR and the xMAP methods to permit evaluating the performance of the newly developed assays. Important in this context are parameters such as specificity (i.e. does the method exclusively detects the targeted sequence? is there cross-reactivity with other species/targets?), sensitivity (i.e. is detection/identification of low levels of the analyte still possible?) and repeatability (i.e. are similar results obtained under repeatability conditions?).

 

 

 

 

 

 

Figure 1: Principle of the xMAP technology applied to GMO detection (based on 6) The xMAP technology is based on the use of beads (microspheres), each set having a unique spectral address (i.e. a different shade of red). Each bead set can be coupled with a reagent (here: a specific oligonucleotide) specific to a particular bioassay, allowing the capture and detection of a specific analyte (here: plant/GM DNA, labelled in green during sample preparation and hybridised to the oligo). Multiplexing is achieved by combining different bead sets to analyse one sample. Inside the Luminex analyzer, a light source excites the internal dyes allowing to identify each microsphere particle by measuring the red fluorescence signal emitted by the bead. In addition, the presence of the bound element is detected by measuring the green fluorescence signal of the hybridised target.

To date the WIV-ISP GMOlab (coordinator of the Belgian NRL-GMO) has developed and validated 17 GMO screening methods using the SYBR®Green real-time PCR technology (7). These methods are currently used in the routine GMO analysis of food and feed samples, performed for the Federal Agency for the Safety of the Food Chain (FASFC), under ISO17025 accreditation. In the ORIENT-EXPRESS project, it will in the first place be investigated which approach can be used to transfer these real-time PCR methods into the xMAP technology allowing them to be tested simultaneously in a single well. A comparison will be made between the real-time PCR and the xMAP methods to permit evaluating the performance of the newly developed assays. Important in this context are parameters such as specificity (i.e. does the method exclusively detects the targeted sequence? is there cross-reactivity with other species/targets?), sensitivity (i.e. is detection/identification of low levels of the analyte still possible?) and repeatability (i.e. are similar results obtained under repeatability conditions?).

OTHER APPLICATIONS

The approach described for GMO will be extended within ORIENT-EXPRESS to gastro-intestinal food-borne pathogens and respiratory viruses in collaboration with the respective National Reference Laboratories and Centres (NRL/NRC) at the WIV-ISP. The implemented technology will allow in this case a rapid identification of the pathogenic organism(s) present in biological/food samples in case of crisis and permit a rapid orientation of the sample(s) towards the specific NRL/NRC for confirmation and further analysis. This will result in a fast diagnosis and thus in a more efficient treatment and rapid containment of possible spreading of the infectious agent(s).

Bead (labelled in a shade of red) coupled with GM element-specific oligo

GM element-specific DNA amplicon (labelled in green during sample preparation)

+

Label interrogated with green light source

Label interrogated with red light source

Identify bound amplicon: GM element was detected

Identify bead: GM element was targeted

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OTHER APPLICATIONS

The approach described for GMO will be extended within ORIENT-EXPRESS to gastro-intestinal food-borne pathogens and respiratory viruses in collaboration with the respective National Reference Laboratories and Centres (NRL/NRC) at the WIV-ISP. The implemented technology will allow in this case a rapid identification of the pathogenic organism(s) present in biological/food samples in case of crisis and permit a rapid orientation of the sample(s) towards the specific NRL/NRC for confirmation and further analysis. This will result in a fast diagnosis and thus in a more efficient treatment and rapid containment of possible spreading of the infectious agent(s).

Hereto, an inventory will be made of the organisms most frequently encountered in the analysed samples by the respective NRL/NRC. Additionally, the sequences to be targeted and the real-time PCR methods already existing and possible to be transferred to the xMAP technology will be listed. The in-house developed xMAP methods will also be compared with commercialized kits. The use of multiple targets for each organism can also permit the detection of emerging organisms which may no longer be detected by the single-target method due to changes in the targeted sequence.

The project will deliver a rational, modular high-throughput tool for the identification and orientation of patho-gens and GMO in times of incidence and surveillance. It will in this way contribute to the preparedness of the institute and of enforcement laboratories to handle future outbreaks in a time and cost effective manner and play an important role in the public health sector.

REFERENCES

(1) Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. Official Journal of the European Union, L 268, p 1-23

(2) Regulation (EC) No 1830/2003 of the European Parliament and of the Council of 22 September 2003 con-cerning the traceability and labelling of genetically modified organisms and the traceability of food and feed products produced from genetically modified organisms and amending Directive 2001/18/EC. Official Journal of the European Union, L 268, p 24-28

(3) James C (2012) Global status of Commercialized Biotech/GM Crops: 2012. Executive summary. ISAAA brief, No 44

(4) Stein, A. & Rodriguez-Cerezo, E. (2009). The global pipeline of new GM crops. Implications of asynchroneous approval for international trade. EU23846-EN.

(5) M. Querci, M. Van den Bulcke, J. ˇZel, G. Van Den Eede, and H. Broll, “New approaches in GMO detection,” Ana-lytical and Bioanalytical Chemistry, vol. 396, no. 6, pp. 1991–2002, 2010.

(6) http://www.luminexcorp.com/TechnologiesScience/xMAPTechnology/

(7) Broeders S, Papazova N, Van den Bulcke M, Roosens N. Development of a molecular platform for GMO detection in food and feed on the basis of ‘‘combinatory qPCR’’ technology. In Polymerase Chain Reaction; Rodríguez PH, Ramirez APG (eds); InTech, Rijeka (2012): Chapter 18:363-404.

nancy.roosens@wiv-isp.be

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Susceptibility of co-contaminations of maize and its derived products in Belgium to fumonisins, fusaric acid and fusarin C

Tangni Emmanuel KVeterinary and Agrochemical Research Centre (CODA-CERVA)Operational Department Chemical Safety of the Food Chain Unit Toxins and Natural Substances, National Reference Laboratory Mycotoxins Leuvensesteenweg 17, 3080 Tervuren, Belgium

In 2012, the seeded maize surfaces in Belgium represented 173540 ha maize forage and 72025 ha grain maize (www.statbel.fgov.be), destined for the internal market. The major problem of this crop remains the attack on the field by different species of Fusarium (F. crookwellense, F. culmorum, F. equiseti, F. graminearum, F. oxysporum, F. poae,

F. proliferatum, F. sambucinum, F. solani, F. sporotrichoïdes, F. subglutinans, F. verticillioides). Recently, a new species, F. temperatum, related to F. subglutinans, has been identified on maize in Belgium. According to the intensity of the infection, the yield losses go up to 15% for maize forage and 30 % for grain maize. These fungi are not only phytopathogenic but can also cause serious problems for the consumer of maize and derived products, because of their highly mycotoxinogenic character. Indeed, the strains of Fusarium produce a large variety of toxins such as fumonisine, zearalenone, trichothecenes (deoxynivalenol, nivalenol, toxins T2 and HT2) who resist against several methods of processing and for which maximum permissible limits have been fixed by the authorities of the European Commission (EC/856/2005, EC/576/2006, EC/1126/2007) and in other countries (ftp://ftp.fao.org/do-crep/fao/007/y5499f/y5499f00.pdf ).

These regulated toxins remain largely under control by means of surveillance programs of their occurrence in food and possibly by corrective actions. On the contrary, few information is gathered on the not yet regulated fu-sarine C, which represents real health risks because it has mutagen and immunosuppressive activities comparable to aflatoxins B1 and sterigmatocystin. Fusarin C is classified by the International Agency for Research on Cancer (IARC) as a probable carcinogenic agent. The absence of standards on the market and the instability of fusarin C in calibration solutions can justify the non determination of its presence in foodstuffs. Moreover, fusaric acid which synergizes the toxicity of some trichothecenes is also not targeted. In this context, it is important to establish an analytical procedure to identify these new emerging chemical risks in order to evaluate its scale. In general, the national reference laboratories (NRLs) and the official control laboratories use reference methods based in majori-ty on HPLC for their control program. At present, a reliable and sensitive method based on liquid chromatography linked with mass spectrometry (LC-MS/MS) has been developed by the NRL for the simultaneous determination of fusarin C, fusaric acid and fumonisines B1 (FB1), FB2 and FB3 in samples of maize inoculated with strains of F.

graminearum, F. venenatum and F. verticillioides, which have been isolated on plants of maize in Belgium previously. This development has started with a synthesis step of Fusarin C, needed to identify and quantify toxins. Fusarin C has been identified, isolated, purified by HPLC-DAD and characterized by LC/MS-MS from a liquid culture of F. graminearum. The analytical method is based on a rapid extraction commonly referred to as ‘QuEChERS’, followed by a short time detection (< 8 min). This method allowed it to evaluate the capacity to produce 11 strains of F. verticillioides which have been collected in Belgium and have been inoculated onto sterilized grain maize (figure 1).

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Fig. 1.: All the strains produce the 5 mycotoxins in a different way, targeted concentrations can reach 441 mg/kg, fusaric acid, 74 mg/kg of fusarin C, 1301 mg/kg of FB1, 367 mg/kg of FB2 and 753 mg/kg of FB3 (Table 1).

Table 1:Concentrations (mg/kg) of 5 mycotoxins produced by 11 strains of F. verticillioides on grains of sterilized maize.

NoStrains

Fumonisins (mg/kg) Fusarine C (mg/kg)

Fusaric acid (mg/kg)B1 B2 B3

1 1301.3 367.2 504.8 19.7 9.5

2 382.4 71.7 165.8 12.5 0.7

3 134.6 12.5 83.4 32.6 24.8

4 49.5 14.8 24.3 27.6 132.3

5 96.3 17.5 14.2 43.2 441.2

6 94.8 22.1 48.0 17.5 154.1

7 967.6 268.4 753.3 20.3 30.8

8 637.8 122.2 562.6 11.3 9.8

9 697.0 105.7 324.1 11.2 2.8

10 310.2 38.9 173.9 74.3 77.3

11 863.9 119.5 340.2 49.6 7.8

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These values let foreshadow a potential risk that all these mycotoxins are present in food produced from cultivars susceptible to be naturally infested by these fungi under favorable climatic conditions beneficial to their sprea-ding. At the same time, products made from contaminated material are susceptible to contamination with these toxins, as it was proven in Germany through a study that reveals the presence of fusarin C in samples of maize grains and processing products such as popcorn, polenta and cornflakes. Other mycotoxins such as enniatine, beauvericin, fusarenon X and nivalenol can also be concerned according to the fungal species. It is necessary to adjust the analytical method according to the specificity of each implied matrix. We urgently need to take precau-tionary measures all the long of the food chain (from field to plate) to maintain the contamination at an achieva-ble level, the lowest possible.

As a consequence, the European regulations setting the maximum acceptable levels individually for each of its contaminants in food have to be revised taking into account natural inseparable interactions and the fragility of some consumers, in particular children with a low body weight.

The ‘multi-mycotoxins’ control analyses will allow it to provide an explanation and a realistic control strategy of contaminants for a greater efficiency of food programs. The resort to liquid chromatography linked with mass spectrometry is highly recommended because of the simplicity of the used extraction and the multi-detection of mycotoxins with a higher sensitivity.

emtan@coda-cerva.be

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ICP-MS: a promising tool in the detection and analysis of nanoparticles

Nadia WaegeneersUnit “Trace Elements”Operational Department Chemical Safety of the Food ChainVeterinary and Agrochemical Research Centre (CODA-CERVA) Leuvensesteenweg 17, 3080 Tervuren, Belgium

Introduction

In October 2011 the European Commission published its recommendation on a common definition of the term “nanomaterial” for regulatory purposes. According to this recommendation, a nanomaterial is a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external diameters is in the size range 1 nm – 100 nm. In this definition, a particle, aggregate and agglomerate are defined as follows:1) “particle” means a minute piece of matter with defined physical boundaries2) “aggregate” means a particle comprising of strongly bound or fused particles3) “agglomerate” means a collection of weakly bound particles or aggregates where the resulting external

surface area is similar to the sum of the surface areas of the individual components

In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the number size distribution threshold of 50% may be replaced by a threshold between 1 and 50%. Furthermore, fullerenes, grapheme flakes and single wall carbon nanotubes with one or more external dimensions below 1 nm should be considered as nanomaterials.

This definition was developed specifically for use in the regulatory field. Hence, through measurements it should be verified whether a material meets the definition of a nanomaterial. Especially the type of the constituent parti-cles (their chemical composition), their external size and the median value of the particle size distribution have to be determined. Methods for measuring the size and/or number of nanoparticles can be grouped as follows:1) “ensemble methods” like dynamic light scattering (DLS), measure large numbers of particles simultaneously

and report intensity-weighted particle sizes 2) “counting methods” like particle tracking analysis (PTA), electron microscopy (EM) and atomic force micros-

copy (AFM), study particle by particle3) “fractionation methods” like field-flow fractionation (FFF), hydrodynamic chromatography (HDC) and cen-

trifugal liquid sedimentation (CLS), separate samples into monodisperse fractions prior to quantifying the particles. Fractionation methods often have to be coupled to a suitable detector system.

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There is currently not a single technique able to measure satisfactorily and routinely the chemical composition, the size and the particle size distribution simultaneously. Furthermore, most of the available techniques are inade-quate for the study of nanoparticles in complex systems such as food. One particular challenge is distinguishing nanoparticles from other constituents of the matrix such as carbon-rich substances and debris. Another problem is that method detection limits are for many techniques higher than the expected exposure concentrations.

In practice the only method that is technically capable to count and size particles in both free and agglomera-ted states is transmission electron microscopy. However, sample preparation artifacts, the high particle count required, and unsuitability for high-throughput applications limit the applicability of this technique for numerous routine measurements.

Because of its elemental specificity, excellent resolution and low detection limit, inductively coupled plasma – mass spectrometry (ICP-MS) is becoming a very promising detection method for inorganic nanomaterials. ICP-MS can be coupled to fractionation methods such as FFF and HDC, or used as a stand-alone technique when opera-ted in single particle mode (SP-ICP-MS).

Field flow fractionation

Field flow fractionation is a fractionation or separation technique: it separates particles based on their hydrody-namic size. The separation process is similar to chromatography, except that the separation is based on physical forces as opposed to chemical interaction. The sample, which has to be a suspension of particles, is pumped through a narrow channel in a laminar flow, which means that the fluid in the center moves faster than the fluid at the edges of the channel (Figure 1). A ‘field’ is applied perpendicular to this flow, which is in most cases a second flow (flow field flow fractionation – F4), but this field can also be electric, magnetic, thermal, etc., which pushes the particles to the edge of the channel, the accumulation wall, where they move slower. Diffusion associated with Brownian motion tends to counteract this motion. Particles with a smaller mean hydrodynamic diameter, which have higher diffusion rates, tend to reach an equilibrium position closer to the center of the channel, where they move faster. The two effects result in a separation between big and small particles. It is possible to transform the retention time to a hydrodynamic diameter, requiring either calibration with particle size standards or theoretical calculations using the physical properties of the medium, the particles and the channel parameters. Calibration with particle size standards is only reliable if the particles that have to be measured, have the same properties as the calibration standards. Quantification of the amount of particles depends on the sort of detector used and its calibration. In the case of inorganic particles, the method can be combined on-line with ICP-MS. The resultant hyphenation of FFF-ICP-MS provides nanoparticle sizing, detection and compositional analysis capabilities at the parts per billion level, which is critical to environmental and toxicological investigations of nanomaterials.

FFF exploits a rather complex system, where interactions between particles, the carrier liquid and the channel membrane must be considered. Highlights of the FFF-ICP-MS technique are the capability to detect very small particles (circa 1 nm) over a wide size range (10- to 20-fold) with superb resolution (10 nm), and its multi-element capabilities (i.e. it is suitable for use with mixed nanoparticle systems). The size range and separation capability can be altered by varying flow rates and operation conditions. However, significant experience is required to deve-lop methods for FFF. As a separation technique, FFF is well suited to deal with polydispersity (i.e. the presence of particles of different sizes in one sample), but it does not distinguish between primary particles, aggregates and agglomerates. These need to be broken up to obtain information on the primary particles. Presuming all particles

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in the sample are of regular (spherical) shape and have equal interaction with the membrane, the interpretation of the elution profile is rather straightforward. On the other hand, particles build up at the lower channel wall in the course of an analysis, resulting in poor recoveries and limiting the number of samples that can be analysed in one run. Furthermore, FFF is prone to interference of large particles (> 1 µm), hence sample preparation is required for most samples. The preparation methods depend on both the character of the matrix and the properties of the nanoparticles, so development of suitable sample preparation techniques plays a major role in FFF analysis. With more studies being published, researchers will have a better understanding of how to minimize the problems associated with FFF, and it will only be a matter of time before FFF-ICP-MS becomes a routine analytical technique for the measurement of inorganic nanoparticles in a variety of samples.

Figure 1. Schematic view of a field flow fractionation channel showing smaller particles moving closer to the cen-ter of the channel in a higher velocity zone and thus eluting before the larger ones.

Hydrodynamic chromatography

Hydrodynamic chromatography is not a sizing method as such but also a separation method. The nanoparticle suspension flows along a column packed with non-porous beads, which build up flow channels or capillaries. In the narrow conduits, larger nanoparticles are transported faster than the smaller ones, as they cannot get as close to the slow-flow regions near the non-porous beads, resulting in separation between particles according to size. Similar to FFF, the time from sample introduction to arrival at the detector can be calibrated for apparent (equiva-lent spherical) particle size, and coupled to ICP-MS it allows the simultaneous analysis of most of the commonly used inorganic nanoparticles in a single run.

In general, the method has rather poor separation power, i.e. unless particle sizes vary widely, they will leave the instrument as one broad ‘peak’. Furthermore, currently only one column is available for HDC. On the other hand, investment costs are much lower than for FFF, sample analysis time is less than 10 minutes per sample, and sepa-ration over a wide range (5-300 nm) is possible. As hydrodynamic chromatography separates particles indepen-dent of their density, gold nanoparticles can function as universal size calibration standards. Another attractive feature is its minimum requirement for sample pretreatment.

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Given the poor separation power, HDC is not suitable for measuring nanoparticles according to the definition recommendation. However, the method is useful to separate the nanoparticles in question from other material components and can therefore play an important role in the determination of nanoparticles in finalized products.

The quantitative aspects of both FFF-ICP-MS and HDC-ICP-MS are not yet fully addressed, as quantitative ionic standards are not suitable for use with FFF or the HDC column. This might be achieved using on-channel/column and post-channel/column calibration with standards of different sizes and concentrations of the nanoparticle of interest, but for many nanoparticles these standards are not yet available. Even though hyphenated methods like HDC-ICP-MS and FFF-ICP-MS are suitable for providing both metal content and size distribution information, they cannot provide particle number concentration information, neither can they distinguish between a high concen-tration of nanoparticles of a certain size and containing a small fraction of a given metal, from a low concentration of nanoparticles of the same size with a large fraction of the metal. To address this drawback the use of SP-ICP-MS online with HDC or FFF is currently being investigated.

Single particle ICP-MS

Single particle ICP-MS is a new technique that relies on the extremely sensitive elemental detection capability of ICP-MS. The liquid sample is transformed into an aerosol, which is then transported into a plasma of very high temperature (10000 K), where the particles are atomized and to some extent ionized. Each plume of ions en-ters the mass spectrometer over a period of approximately 0.5 ms. Single particle ICP-MS hence splits the total observation time into thousands of very small time windows, called “dwell times” of 10 ms and below. By injecting sufficiently diluted samples, each discrete ion plume is originating from a single particle and gives rise to a single signal peak, the intensity of which is proportional to the mass of the particle. Assuming a certain particle shape, the particle size can be calculated. Ideally the method is combined with imaging methods to gain information on the shape to assume during size calculation. As the technique measures the total mass during a time window, it cannot distinguish between particles, aggregates and agglomerates. On the other hand the intensity readings can be collected as a function of time, which makes it both a counting and a sizing technique and allows particle size distribution calculations. The technique requires little sample preparation, even for complex matrices, and lit-tle additional method development for a given matrix and/or analyte. It can differentiate the particle of interest of other incidental particles of the same size, but different composition, by its elemental specificity. The size resoluti-on is about 10 nm. Distinguishing dissolved from nanoparticulate constituents of a given metal is another distinct advantage of SP-ICP-MS, as is the low sample analysis time (1-3 min per sample). Furthermore, as SP-ICP-MS uses a relatively standard laboratory instrument and no additional equipment, laboratories would not incur extra costs in performing this type of analysis.

However, the required dilution might change particle properties (e.g. due to dissolution). Furthermore, the tech-nique is highly dependent on the signal-to-noise ratio of a given ICP-MS, which may significantly hinder analysis of smaller sized nanoparticles. The lowest particle sizes detectable are between 10 and 20 nm and depending on the chemical composition of the particle, which may exclude it as a viable option to be used for measurements in the regulatory field. However, efforts are underway to deconvolute smaller sized nanoparticles from background intensities.

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Figure 2. A) Time scan of intensity readings during single particle ICP-MS; B) Histogram of the particle size distribu-tion after single particle ICP-MS analysis.

Conclusions

The problem of the detection and analysis of nanomaterials in complex matrices is only starting to be addressed. The analysis is challenging because appropriate and fit-for-purpose methods, i.e. suitable, robust, standardized and of reasonable cost, are not yet available. The use of ICP-MS, either hyphenated or stand-alone, provides a number of nanoparticle properties that are specifically relevant to environmental and toxicological studies and in the regulatory field, such as size, concentration and associated dissolved constituents. However, each technique has specific strengths, which prove valuable for defining a given set of nanoparticle characteristics, as well as limitations that are inherent to each technique.

nadia.waegeneers@coda-cerva.be

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New understanding of ink components migrating from packaging material into foodstuffsBenjamin Horemans, Jan De Block and Hendrik De RuyckInstituut voor Landbouw- en Visserijonderzoek / Institute for Agricultural and Fisheries ResearchEenheid Technologie en Voeding - Voedselveiligheid / Technology and Food Science Unit - Food Safety; Brusselses-teenweg 370 - 9090 Melle

Food packaging is important and for food producers the visual aspect of packaging material is a very powerful medium, so that consumers and competitors consider a brand name as an established value. In the process, the chosen varieties of ink play a crucial role. The choice may not only be based on marketing strategy, but the poten-tial health risks as a result of the migration issues of the ink components have to be taken into account. Printing inks used in food packaging materials contain substances that are able to migrate, such as raw materials and by-products of hardeners and solvents. So far, the knowledge of migration of specific components from printing inks is limited, therefore public health risk cannot be well assessed.

The issue of migration of components from inks and glues originated in 2005 when 2-isopropylthioxanton (ITX) threw the packaging and food industry into commotion. ITX was found in undesirable high concentrations in milk powder for babies and chocolate milk powder. Later on it appeared that the commonly used photoinitiator origi-nated from the used packaging. In 2009, the photoinitiators benzophenone and 4-methylbenzophenone were found in cereals and milk, increasingly alarming migration issues. Next to primary migrant substances, secondary products can also be formed due to a reaction in the foodstuff. These secondary products can also be harmful and they could possibly have an effect on food too, such as odor, taste, etc. Problems with bisphenol A diglycidyl ether (BADGE), used in epoxy coatings of tins, clarified this additional aspect. Moreover, it has recently been revealed that BADGE is not only unstable in acetic acid, but shows certain reactivity towards food components such as peptides and amino acids. This reveals that characterizing secondary migration products is at least as important as the migration issue of primary migrant substances.

In order to gain a larger understanding concerning the stability of migrating components from printing inks of packaging materials to food, an investigation of the FPS at ILVO-T&V is taking place together with the research group NutriFOODchem of the UGent. First of all, an inventory was made of components which can migrate from printing inks and of which could be derived, based on their chemical properties, that they are reactive/unstable in the official food simulants water, acetic acid and ethanol with or without the use of light. Harmful components shown in simulants indicate a potentially food safety risk in foodstuffs. A priorities list was composed in consulta-tion with some industrial stakeholders. Components of the following 9 different classes have been put forward: nitrocellulose, acrylates, phthalates, isocyanates, phosphates, adipates, citrates, high boiling point solvents and photoinitiators. Because of the limited research time, acrylates, phthalates and nitrocellulose are the compounds of interest in this research.

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Acylates are energy hardening monomers present as such during the printing process and they polymerize dur-ing ink drying. Sometimes photoinitiators are added to initiate polymerization. A considerable amount of these acrylates can still be present in the final print. Phthalates on the contrary are added to ink as an additive to affect the physical properties. These are plasticizers having an impact on rheological and elastic properties of inks. The stability of bis(2-ethylhexyl)phthalate and dodecyl acrylate has been investigated in the official food simulants 3% aqueous acetic acid (pH = 2.5), ethanol and modified polyphenylene oxide (MPPO). Both components are stable in 95% ethanol. They did not undergo any thermal degradation or oxidation at high temperatures on MPPO. On the contrary, phthalate as well as acrylate appear to hydrolyze in a 3% aqueous amino acid solution. Degradation curves showed pseudo first order kinetics. The activation energy of the phthalate was 96 ± 24 kJ mol-1 (k = 0.09 ± 0.02 and 8.1 ± 0.3 μs-1, at 20 and 60°C respectively). The activation energy for the hydrolysis of acrylate took about half the energy (41 ± 4 kJ mol-1) with k = 14.5 ± 0.5 and 162 ± 2 μs-1, at 20 and 60°C respectively.

Sometimes nitrocellulose is used as a polymer in the binding agent of solvent-based inks. At increased tempera-ture, it causes the formation of nitrogen oxides (NOx). Strictly speaking, these gases cannot be considered as migrants, but they can cause reactions in the foodstuff. It is possible that NOx react with amines in food and form carcinogenic nitrosamines. In a preliminary experiment, it has been revealed that NOx can be released significant-ly from nitrocellulose printing inks heated to 85°C and that the release increases exponentially at a temperature rise. This experiment also confirmed the hypothesis that the released NOx is capable to form the carcinogenic N-nitroso-di-n-butylamine out of the equivalent amine. Heating up ready-to-eat meals in printed packaging materi-als is a potential risk for nitrosamines to be formed in our food. In order to evaluate these findings quantitatively, an analytical procedure has been developed based on a solid phase extraction (SPE), followed by gas chromatog-raphy-mass spectrometry (GC-MS; Figure 1). Nitrosamines have been extracted from an aqueous solution using SPE and the extract has been evaporated using mild concentration techniques. Analysis by GC-MS is carried out by programmed temperature vaporization (PTV) and large volume injection (LVI). Figure 2 displays the result of a GC-MS analysis of a 20 µg/ml nitrosamine standard mixture. Combined with SPE and evaporation, the method is capable of detecting nitrosamine concentrations on sub-ppb level with a better precision than 5% and a recovery of about 70%, depending on the component.

Figure 1: Gas chromatography coupled to mass spectrometry (GC-MS) detection

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NDMA: N-nitrosodimethylamineNEMA: N-nitrosoethylmethylamineNDEA: N-nitrosodiethylamineNDPA: N-nitrosodi-n-propylamineNPYR: N-nitrosopyrrolidineNPIP: N-nitrosopiperidineNDBA: N-nitrosodi-n-butylamine

Figure 2: Extracted ion chromatogram (EIC) for target ions in a 20 ppb nitrosamine standard

In order to have an experimental approach of the above-mentioned hypothesis, packaging material and food or a food simulant have to be brought into contact as if it were a real packaged food product. A migration cell has been chosen to approach reality as good as possible. This cell is composed of two plates of stainless steel covered with a packaging material of choice. Both plates are clipped onto both sides of a metal ring (Figure 3). A liquid simulant fills the space between the plates and the ring, creating close contact with the packaging material. In order to follow up nitrosation during the heating of nitrocellulose ink, relevant amines are dissolved in water and buffered at an optimal pH. The migration cell is covered by a film homogeneously printed with nitrocellulose ink. This cell is filled with a buffered amino solution and heated during a few hours. After this process and a successive cooling down step, the quantity of nitrosamines in the cell solution is analyzed using the above-mentioned SPE-GC-MS determination method.

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Figure 3: Experimental migration cell

The purpose of this research project is to obtain a full picture of kinetics and of the circumstances during the formation of nitrosamines out of printed food packages. At present experiments are carried out. There is strong evidence that nitrosamines indeed can be formed during heating of ready-to-eat meals in packaging materials with nitrocellulose ink prints. In order to be able to evaluate the importance for public health, exposure by this process must be compared with other ways of exposure in our environment. However, more experimental data are needed to prove this. In any case, the packaging sector is looking for a solution to eliminate the current uncer-tainty of using nitrocellulose in food contact materials.

benjamin.horemans@ilvo.vlaanderen.be

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The trainings for the approved laboratories organized by the FASFC in co-operation with the National Reference Laboratories are available on the website of the FASFC

(www.favv.be > Business Sectors > Laboratories > Trainings).

The schedule is updated regularly, it is therefore recommended to check the website from time to time.

Other interesting workshops and symposia are mentioned below.

Date Subject Place More information (website)

9-12.02.2014 Translational Cereal Genomics

Bundesamtsgebäude Radetzkystrasse, Hintere Zollamtsstrasse 1, 1031 Vienna, Austria

http://www.lisavienna.at/en/events/translational- cereal-genomics

12-15.02.2014 Plant Transformation Technologies III

Bundesamtsgebäude Radetzkystrasse, Hintere Zollamtsstrasse 1, 1031 Vienna, Austria

http://www.lisavienna.at/en/events/plant-transformation-technologies-iii

16-19.02.2014 Plant Gene Discovery & “Omics” Technologies

Bundesamtsgebäude Radetzkystrasse, Hintere Zollamtsstrasse 1, 1031 Vienna, Austria

http://www.lisavienna.at/en/events/plant-gene- discovery-omics-technologies

19-22.02.2014 Applied Vegetables Genomics

Bundesamtsgebäude Radetzkystrasse, Hintere Zollamtsstrasse 1, 1031 Vienna, Austria

http://www.lisavienna.at/en/events/applied-vegetables-genomics

26-28/02/2014 Global Food Safety Conference

California, USA http://tcgffoodsafety.com/pro/fiche/quest.jsp;jsessionid=HXnhMppwo7YQHFAu9sW5llkM.gl3

3-4.03.2014 IDF Symposium on Microstructure of Dairy Products

Melbourne, Australia http://dairyscienceconf.com/

6-7.03.2014 IDF Symposium on Science and Technology of Fermented Milk

Melbourne, Australia http://dairyscienceconf.com/

5.03-6.03.2014 12th International Fresenius Conference Food Safety and Dietary Risk Assessment

Mainz, Germany http://www.akademie-fresenius.com/konferenz/output.php?thema=3&kurs=415

27-29.03.2014 “Innovation for the Management of Echinococcosis”

Besançon, France http://extranet.insight-outside.fr/upload/compte487/File/ImE-2014%20Symposium%20-%20Besancon,%20France%20-%20March%202014.pdf en http://imes2014.scientific-event.com/

Workshops & Symposia

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31.03-2.04.2014 The RME Conference Series – 9th conference Food Feed Water Analysis: innovations and break-throughs!

The Netherlands http://www.bastiaanse-communication.com/rme2014/

7.04-8.04.2014 9th International Fresenius Conference Contaminants and Residues in Food

Mainz, Germany http://www.akademie-fresenius.com/konferenz/output.php?thema=3&kurs=428

7-9.05.2014 IAFP’s European Sympo-sium on Food Safety

Budapest, Hungary http://www.foodprotection.org/europeansymposium/

15-20.05.2014 IDF/ISO Analytical Week 2014

Berlin, Germany http://www.fil-idf.org/Public/SiteEventType.php?ID=23123http://www.idf-iso-analytical-week.org/

19.05-20.05.2014 International Fresenius Conference Nanotechnology in Food

19.05.2014 bis 20.05.2014 in Mainz (near Frankfurt)/Germany

http://www.akademie-fresenius.com/konferenz/output.php?thema=3&kurs=425

20.05.2014 66th International Symposium on Crop Protection

Ghent, Belgium http://www.iscp.ugent.be/

2-5.06.2014 7th International Symposium on Hormone and Veterinary Drug Residue Analysis

Ghent, Belgium www.vdra.ugent.be

8-13.06.2014 13th International Conference on Plant Pathogenic Bacteria (ICPPB)

Shanghai Jiao Tong University, Shanghai, China

http://www.icppb2014.org/

31.08-05.09.2014 The 34th International Symposium on Halogenated Persistent Organic Pollutants – Dioxin 2014

Madrid, Spain http://www.dioxin2014.org/

7-10.09.2014 128th AOAC Annual Meeting & Exposition

Boca Raton Resort & Club501 East Camino RealBoca Raton, Florida 33432 USA

http://www.aoac.org/imis15_prod/AOAC/Meetings_Events/14AM_Annual_Meeting/AOAC_ Member/Meetings___Events/14AM/Annual_Meeting.aspx?hkey=4cd073ff-a131-49e4-8e19-97230bbb0d82

18-19.09.2014 19th Conference on Food Microbiology

Brussels, Belgium Organized by the Belgian Society for Food Microbiology vzw/aslb (BSFM)www.bsfm.be

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29.09-1.10.2014 3rd International Conference on Responsible Use of Antibiotic in Animals

Amsterdam, the Nether-lands

http://www.bastiaanse-communication.com/html/ upcoming.html

27-31.10.2014 IDF World Dairy Summit 2014

Tel Aviv, Israel http://www.idfwds2014.com/

3-7.11.2014 Management of Microbiological Hazards in Foods (15th edition)

Wageningen, The Nether-lands

Organised in cooperation with: European Chair in Food Safety Microbiologyhttp://www.vlaggraduateschool.nl/eduvlco.html

10-12.11.2014 The World Mycotoxin Forum8th Conference

Vienna, Austria http://www.bastiaanse-communication.com/html/ upcoming.html

10.11.2014 The Plant Toxin Forum Vienna, Austria http://www.bastiaanse-communication.com/html/ upcoming.html

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