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The use of insects as feed and food can be part of the solution towards a circular economy, in case the safety of insect products is assured. Black soldier fly larvae (BSFL, Hermetia illucens) can be reared on different waste streams. However, before BSFL can be legally reared on these streams, the safety of BSFL for feed and food should be assessed thoroughly. This study aimed to investigate several food safety aspects of BSFL grown on waste streams. Therefore, BSFL were reared for 7 days on substrate mixtures of waste streams with similar protein and moisture content. These waste streams included fast food waste (FF), mushroom stem (MS), pig manure solids (PS), poultry meal (PM) and slaughter waste (SW). The substrates, BSFL and the frass were analysed for the presence of metals and veterinary drugs. The substrates and BSFL were also analysed for presence of DNA of ruminant, pig and chicken. Some of the metals accumulated in BSFL, although the concentrations in BSFL (as these would be manufactured as feed) were below maximum limits for feed. Only traces of some of the analysed veterinary drugs were found in the BFSL and no accumulation thereof was observed. DNA of ruminant and pig was traced back in BSFL samples, however, chicken was not. A good understanding of the presence of food safety hazards and possible variance thereof in potential substrates, such as waste streams, and their possible residues in insects is necessary for implementation of this circular way of insect feeding in the food chain.

DNA extraction is at the forefront of further analytical measurements on DNA targets and affects the downstream results. This report from the European Network of GMO Laboratories (ENGL) provides guidance on the selection and use of fit-for-purpose DNA extraction methods. It focusses on DNA extraction in the context of official controls on the presence and content of genetically modified organisms in food and feed. It provides guidance on protocols and selection support systems, validation approaches, assessment of DNA quality parameters and examples of practical solutions derived from collective experiences. There are many variations on the theme of DNA extraction, but there is no single protocol that works adequately across all food and feed matrices. Before using a new method in the laboratory, or in case of modifications to a protocol, validation or verification is needed to show that a chosen method is fit for purpose for use in routine analysis. This guidance is aimed to help the DNA analysis laboratories in fulfilling the standardisation requirements and support their daily operations.

National Reference Laboratories (NRLs) are part of the system responsible for controlling and enforcing EU food and feed legislation. Wageningen Food Safety Research (WFSR) has been designated as NRL for thirteen areas of competence including milk and milk products, marine biotoxins, animal proteins in feedingstuffs, certain substances and residues thereof1, additives for use in animal nutrition (feed additives), genetically modified organisms in food and feed, pesticides, metals and nitrogenous substances in feed and food, mycotoxins and plant toxins in food and feed, processing contaminants, halogenated persistent organic pollutants in food and feed, food-borne viruses and water content of poultry. The tasks of WFSR’s NRLs depend on their respective fields of research. This annual report summarises all the activities carried out by WFSR’s NRLs in 2022. Staying updated on the latest developments is a crucial aspect of NRL’s responsibilities. The European Union Reference Laboratories (EURLs) arrange one or two workshops annually to facilitate this objective. Attendance at these workshops is mandatory for EURL-NRL members. In 2022, WFSR’s NRLs attended all the workshops. They actively participated in the EURL working groups to enhance their analytical methods. To assess the analytical capabilities of NRLs, EURLs conduct proficiency tests. Since the scope of these tests is sometimes limited, WFSR’s NRLs also participated in proficiency tests organised by other relevant organisations. The majority of the proficiency tests yielded satisfactory results, with only a few instances of ‘questionable’ or ‘unsatisfactorily’ outcomes. These were addressed through follow-up actions. To ensure the performance of Official Laboratories (OLs), their results in proficiency tests organised by WFSR’s NRLs were checked, or assurance samples were provided. Additionally, technical support related to their analyses was provided for some OLs. Furthermore, the scientific and technical backin

The current EU legislation on GMOs and GM food and feed requires analytical testing to support traceability of these products on the market. The European Network of GMO Laboratories has reviewed the implications of the analytical requirements when they are applied to plant products developed with the use of new genomic techniques, i.e. targeted mutagenesis and cisgenesis. This review concluded that analytical testing to support traceability is not considered feasible for all products obtained by targeted mutagenesis and cisgenesis, both due to technical restrictions and because of implementation issues.

WFSR voert jaarlijks duizenden analyses uit voor de Nederlandse Voedsel- en Warenautoriteit (NVWA) om te ondersteunen bij de handhaving op wettige verplichtingen op het gebied van voedsel, diervoer en consumentenveiligheid. In voorkomende gevallen wil de NVWA uitsluitsel hebben over de herkomst van mest. De vraag daarbij is of het aangetroffen DNA in het mestmonster overeenkomt met de opgegeven mestcode. Dit onderzoek is uitgevoerd om een beter inzicht te krijgen in de afbraak van het DNA van het brondier als functie van de bewaartemperatuur. Het onderzoek is uitgevoerd met dagverse feces afkomstig van varkens en koeien. In dit onderzoek is aangetoond dat DNA van het brondier nog tot en met 12 maanden aangetoond kan worden indien de mest is opgeslagen bij 4 °C of 20 °C, 6 maanden indien opgeslagen bij 55 °C en één week indien opgeslagen bij 70 °C.

National Reference Laboratories (NRLs) are part of the system responsible for controlling and enforcing the EU food and feed law. Wageningen Food Safety Research (WFSR) has been designated the NRL for thirteen subjects. The tasks of an NRL depend on its research fields. This report gives an overview of the activities performed by all of WFSR’s NRLs in 2021. These NRLs are for: milk and milk products, marine biotoxins,animal proteins, certain substances and residues thereof as laid down in Regulation (EU) 2017/625, additives for use in animal nutrition (feed additives), genetically modified organisms (GMOs) in food and feed,pesticides, metals and nitrogenous substances in feed and food, mycotoxins and plant toxins in food and feed, processing contaminants halogenated persistent organic pollutants in food and feed, food-borne viruses and water content of poultry.This report first gives an overview of relevant legislation and information on the networks of EURLs, NRLsand OLs. For every NRL, a description of all activities performed in the EURL-NRL network, such as participation in EURL-NRL workshops, working groups, and proficiency and comparative tests. This is followed by a description of the assistance provided to OLs, such as a quality check or advice. Finally, the scientific and technical support given to the competent authority is discussed. In some cases, contact with other NRLs is discussed. An important NRL task is to stay updated with current developments within its NRL domain. Every EURL organises one or two meetings (workshops) every year for that purpose. Participation in these EURL-NRL workshops is mandatory. In 2021, because of COVID-19, the workshops were almost all held online. NRLs of WFSR have attended all workshops and actively participated in EURL working groups to improve analytical methods.To test the analytical capabilities of NRLs, the EURLs organise proficiency tests. As the scope of the EURL proficiency tests is sometimes limited, the NRLs al

WFSR report 2021.014 | 9 Summary National Reference Laboratories (NRLs) are part of the system responsible for the control and enforcement of EU food and feed law. Wageningen Food Safety Research (WFSR) has been designated as the NRL for thirteen subjects. The tasks of a NRL depend on its research fields. This report gives an overview of the activities performed by all of NRLs of WFSR in 2020. These NRLs are for: milk and milk products, marine biotoxins, animal proteins, certain substances and residues thereof as laid down in Directive 96/23/EC, additives for use in animal nutrition (feed additives), genetically modified organisms (GMOs) in food and feed, pesticides, metals and nitrogenous substances in feed and food, mycotoxins and plant toxins in food and feed, processing contaminants, halogenated persistent organic pollutants in food and feed, food borne viruses and water content of poultry. This report first gives an overview of relevant legislation and information on the networks of EURLs, NRLs and OLs. For every NRL, a description is then given of all activities performed in the EURL-NRL network such as participation in EURL-NRL workshops, working groups, and proficiency and comparative tests. This is followed by a description of the assistance given to OLs in the form of quality control and/or advice. Finally, the scientific and technical support given to the competent authority is discussed. In some cases, the contact with other NRLs is discussed. An important NRL task is to stay up to date with current developments within its NRL domain. Every EURL organises one or two meetings (workshops) every year for that purpose. Participation in these EURL-NRL workshops is mandatory. In 2020, due to COVID-19, most of these workshops were online. All workshops have been attended by NRLs of WFSR. Additionally, the NRLs have actively participated in EURL working groups, to improve analytical methods. To test the analytical capabilities of NRLs, the EURLs organise profici

Background: DNA metabarcoding, which involves Next-Generation Sequencing (NGS) of DNA barcodes, holds great promise for species identification in complex samples such as food supplements and Traditional Medicines (TMs). Such method would aid CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for wildlife forensic species identification and to evaluate the applicability and reproducibility of the this approach across different laboratories. Results: The DNA metabarcoding method developed in this study makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa, and that facilitate the identification of plant and animal species in highly processed samples containing degraded DNA. The DNA metabarcoding method was developed on the basis of NGS data generated for 15 well-defined experimental mixtures using Illumina MiSeq technology, for which a bioinformatics pipeline with user-friendly web interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. Conclusion: The advanced, multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES species. The method provides improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to enhanced quality assurance., Background: DNA metabarcoding, which involves Next-Generation Sequencing (NGS) of DNA barcodes, holds great promise for species identification in complex samples such as food supplements and Traditional Medicines (TMs). Such method would aid CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for wildlife forensic species identification and to evaluate the applicability and reproducibility of the this approach across different laboratories. Results: The DNA metabarcoding method developed in this study makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa, and that facilitate the identification of plant and animal species in highly processed samples containing degraded DNA. The DNA metabarcoding method was developed on the basis of NGS data generated for 15 well-defined experimental mixtures using Illumina MiSeq technology, for which a bioinformatics pipeline with user-friendly web interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. Conclusion: The advanced, multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES species. The method provides improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to enhanced quality assurance.

The annual ring test for the detection of animal proteins in animal feed of the IAG - International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy was organized by RIKILT - Wageningen UR, The Netherlands. The aim of the ring study was to provide the participants information on the performance of the local implementation of the detection method for their local quality systems. A further aim was to gather information about the application of the microscopic method. The current 2016 version of the IAG ring test for animal proteins facilitated the full scenario with the methods for microscopy and PCR as published in Regulation (EC) 51/2013 amending Annex VI of Regulation (EC) 152/2009 together with accompanying SOPs. All four samples were based on an artificial feed mimicking a formulation for ruminant feed. Two samples were labelled as fish feed (B and D), which was effectuated by adding 2% of a general fish meal. Adulteration was achieved by adding 0.1% pig MBM (B), 0.1% ruminant MBM (D) and a combination of 0.1% ruminant MBM and 0.1% fish meal (C). This combination of different spikes allowed the diverse application of the detection methods. Forty eight participants enrolled for the ring test, of which 45 submitted microscopic results. Of these, 20 participants applied the combination of microscopic and PCR analysis. Three participants submitted exclusively PCR results.

Background With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study. Results Of the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands). Conclusions From the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the interlaboratory exchange study it

The authenticity of food is of increasing importance for producers, retailers and consumers. All groups benefit from the correct labelling of the contents of food products. Producers and retailers want to guarantee the origin of their products and check for adulteration with cheaper or inferior ingredients. Consumers are also more demanding about the origin of their food for various socioeconomic reasons. In contrast to this increasing demand, correct labelling has become much more complex because of global transportation networks of raw materials and processed food products. Within the European integrated research project ‘Tracing the origin of food’ (TRACE), a DNA-based multiplex detection tool was developed—the padlock probe ligation and microarray detection (PPLMD) tool. In this paper, this method is extended to a 15-plex traceability tool with a focus on products of commercial importance such as the emmer wheat Farro della Garfagnana (FdG) and Basmati rice. The specificity of 14 plant-related padlock probes was determined and initially validated in mixtures comprising seven or nine plant species/varieties. One nucleotide difference in target sequence was sufficient for the distinction between the presence or absence of a specific target. At least 5% FdG or Basmati rice was detected in mixtures with cheaper bread wheat or non-fragrant rice, respectively. The results suggested that even lower levels of (un-)intentional adulteration could be detected. PPLMD has been shown to be a useful tool for the detection of fraudulent/intentional admixtures in premium foods and is ready for the monitoring of correct labelling of premium foods worldwide.

Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples.

Background To maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs), but only to a limited extent for EU-non-authorised GMOs (NAGs). In the last decade the diversity of genetically modified (GM) ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products. Results In this paper we present an innovative method for detecting (approved) GMOs as well as the potential presence of NAGs in complex DNA samples containing different crop species. An optimised protocol has been developed for padlock probe ligation in combination with microarray detection (PPLMD) that can easily be scaled up. Linear padlock probes targeted against GMO-events, -elements and -species have been developed that can hybridise to their genomic target DNA and are visualised using microarray hybridisation. In a tenplex PPLMD experiment, different genomic targets in Roundup-Ready soya, MON1445 cotton and Bt176 maize were detected down to at least 1%. In single experiments, the targets were detected down to 0.1%, i.e. comparable to standard qPCR. Conclusion Compared to currently available methods this is a significant step forward towards multiplex detection in complex raw materials and derived products. It is shown that the PPLMD approach is suitable for large-scale detection of GMOs in real-life samples and provides the possibility to detect and/or identify NAGs that would otherwise remain undetected