BACKGROUND:Narrow-leaved lupins (NLL, Lupinus angustifolius L.) is recognized as a climate-resilient protein crop but its use in food and feed is frequently limited by toxic quinolizidine alkaloids (QAs). The effect of intercropping with spring oat (Avena sativa L.) on grain QA content has not yet been quantified. RESULTS:In a 2-year field experiment, three NLL varieties (Lunabor, Probor, and Jowisz), grown as pure stands and in nine mixtures with the oat varieties Bison, Lion, and Troll were compared. Mixed cropping increased total grain QAs by between 16% and 46% relative to the respective pure stands. Absolute increases reached +168 mg kg-1 in Lunabor and +128 mg kg-1 in Probor, whereas Jowisz increased by only +76 mg kg-1. Among the mixtures, Jowisz-Bison exhibited the smallest increase (16%) and the lowest final QA content, whereas Lunabor-Troll showed the highest content. In mixed stands, both Lunabor and Probor exceeded the 500 mg kg-1 threshold, whereas Jowisz remained below this threshold. Profiles of the seven major QAs remained constant, with the exception of the 13-hydroxylupanine to lupanine ratio, which increased in the mixture. Year effects were not observed. CONCLUSION:Intercropping NLL with oat elevates the grain QA content to levels of toxicological relevance. The extent is variety-interaction dependent, presumably due to oat allelopathy. The evidence points to an indirect stress mechanism: allelopathic cues from the oat crop place NLLs under physiological stress, which in turn stimulates NLL to accumulate additional QAs in the grain. Additional mixed cropping experiments and breeding against QA accumulation in NLL grains should be pursued to understand and alleviate this issue. © 2026 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs) are phytotoxins that occur worldwide and are important contaminants of food, especially (herbal) teas. Their transfer rates to tea infusions strongly influence exposure estimates, but conflicting results have been obtained so far, making risk assessment uncertain. Therefore, infusions prepared under controlled conditions from artificially contaminated black and herbal tea and naturally PA-containing comfrey root (Symphytum officinale) were analyzed by UPLC-MS/MS. Brewing time, repeated infusion, pH and temperature of the infusion were evaluated. Infusions of black tea and herbal tea produced under standardized conditions resulted in PA/TA transfer rates between 30 % and 68 %, with an average of 49 %. Surprisingly, brewing time was not found to be a significant factor. Instead, the polarity of the compounds and the pH of the tea infusion due to the tea matrix were identified as main factors. Consequently, teas should be analyzed as infusion for realistic exposure estimates.

This Data Descriptor reports the submission of a High-Resolution Orbitrap Mass Spectral Library of Pyrrolizidine Alkaloids (PASL) to public repositories. The library contains 165 tandem mass spectra (MS/MS) from 84 pyrrolizidine alkaloid (PA) standards, along with 18 additional PAs manually annotated in crude plant extracts. This collection comprises most commercially available PAs and a unique selection of annotated (in crude extracts) and synthesized compounds. The PASL serves as a valuable resource for identifying PAs in complex mixtures without analytical standards and facilitates the annotation of novel PAs through molecular networking approach. To ensure high quality, the library was validated by dereplicating its spectra against the GNPS libraries and applying it to the annotation of two PA-producing plant species. The spectra of the PASL can be accessed through GNPS ( https://gnps.ucsd.edu/ProteoSAFe/gnpslibrary.jsp?library=PYRROLIZIDINE-ALKALOID-SPECTRAL-LIBRARY ), with accession numbers ranging from CCMSLIB00014205782 to CCMSLIB00014205946 . The PASL in .mgf format can be downloaded from GNPS under: https://external.gnps2.org/gnpslibrary . The PASL enables rapid and straightforward annotation of both known and novel PAs, accelerating research in food safety and related fields.

Alkaloids play an essential role in protecting plants against herbivores. Humans can also benefit from the pharmacological effects of these compounds. Plants produce an immense variety of structurally different alkaloids, including quinolizidine alkaloids, a group of bi-, tri-, and tetracyclic compounds produced by Lupinus species. Various lupin species produce different alkaloid profiles. To study the composition of quinolizidine alkaloids in lupin seeds, we collected 31 populations of two wild species native to Israel, L. pilosus and L. palaestinus, and analyzed their quinolizidine alkaloid contents. Our goal was to study the alkaloid profiles of these two wild species to better understand the challenges and prospective uses of wild lupins. We compared their profiles with those of other commercial and wild lupin species. To this end, a straightforward method for extracting alkaloids from seeds and determining the quinolizidine alkaloid profile by LC-MS/MS was developed and validated in-house. For the quantification of quinolizidine alkaloids, 15 analytical reference standards were used. We used GC-MS to verify and cross-reference the identity of certain alkaloids for which no analytical standards were available. The results enabled further exploration of quinolizidine alkaloid biosynthesis. We reviewed and re-analyzed the suggested quinolizidine alkaloid biosynthesis pathway, including the relationship between the amino acid precursor l-lysine and the different quinolizidine alkaloids occurring in seeds of lupin species. Revealing alkaloid compositions and highlighting some aspects of their formation pathway are important steps in evaluating the use of wild lupins as a novel legume crop.

In their natural habitat, insects may bioaccumulate toxins from plants for defence against predators. When insects are accidently raised on feed that is contaminated with toxins from co-harvested herbs, this may pose a health risk when used for human or animal consumption. Plant toxins of particular relevance are the pyrrolizidine alkaloids (PAs), which are genotoxic carcinogens produced by a wide variety of plant species and the tropane alkaloids (TAs) which are produced by a number of Solanaceae species. This study aimed to investigate the transfer of these plant toxins from substrates to black soldier fly larvae (BSFL) and lesser mealworm (LMW). PAs and the TAs atropine and scopolamine were added to insect substrate simulating the presence of different PA- or TA-containing herbs, and BSFL and LMW were grown on these substrates. Bioaccumulation from substrate to insects varied widely among the different plant toxins. Highest bioaccumulation was observed for the PAs europine, rinderine and echinatine. For most PAs and for atropine and scopolamine, bioaccumulation was very low. In the substrate, PA N-oxides were quickly converted to the corresponding tertiary amines. More research is needed to verify the findings of this study at larger scale, and to determine the potential role of the insect and/or substrate microbiome in metabolizing these toxins.

The European Commission requested EFSA to provide an update of the 2012 Scientific Opinion of the Panel on Contaminants in the Food Chain (CONTAM) on the risks for animal health related to the presence of ergot alkaloids (EAs) in feed. EAs are produced by several fungi of the Claviceps and Epichloë genera. This Opinion focussed on the 14 EAs produced by C. purpurea (ergocristine, ergotamine, ergocornine, α- and β-ergocryptine, ergometrine, ergosine and their corresponding 'inine' epimers). Effects observed with EAs from C. africana (mainly dihydroergosine) and Epichloë (ergovaline/-inine) were also evaluated. There is limited information on toxicokinetics in food and non-food producing animals. However, transfer from feed to food of animal origin is negligible. The major effects of EAs are related to vasoconstriction and are exaggerated during extreme temperatures. In addition, EAs cause a decrease in prolactin, resulting in a reduced milk production. Based on the sum of the EAs, the Panel considered the following as Reference Points (RPs) in complete feed for adverse animal health effects: for pigs and piglets 0.6 mg/kg, for chickens for fattening and hens 2.1 and 3.7 mg/kg, respectively, for ducks 0.2 mg/kg, bovines 0.1 mg/kg and sheep 0.3 mg/kg. A total of 19,023 analytical results on EAs (only from C. purpurea) in feed materials and compound feeds were available for the exposure assessment (1580 samples). Dietary exposure was assessed using two feeding scenarios (model diets and compound feeds). Risk characterisation was done for the animals for which an RP could be identified. The CONTAM Panel considers that, based on exposure from model diets, the presence of EAs in feed raises a health concern in piglets, pigs for fattening, sows and bovines, while for chickens for fattening, laying hens, ducks, ovines and caprines, the health concern related to EAs in feed is low.

Natural toxins (NTs) are poisonous secondary metabolites produced by living organisms developed to ward off predators. Especially low molecular weight NTs (MW<∼1 kDa), such as mycotoxins, phycotoxins, and plant toxins, are considered an important and growing food safety concern. Therefore, accurate risk assessment of food and feed for the presence of NTs is crucial. Currently, the analysis of NTs is predominantly performed with targeted high pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS) methods. Although these methods are highly sensitive and accurate, they are relatively expensive and time-consuming, while unknown or unexpected NTs will be missed. To overcome this, novel on-site screening methods and non-targeted HPLC high resolution mass spectrometry (HRMS) methods have been developed. On-site screening methods can give non-specialists the possibility for broad "scanning" of potential geographical regions of interest, while also providing sensitive and specific analysis at the point-of-need. Non-targeted chromatography-HRMS methods can detect unexpected as well as unknown NTs and their metabolites in a lab-based approach. The aim of this chapter is to provide an insight in the recent advances, challenges, and perspectives in the field of NTs analysis both from the on-site and the laboratory perspective.

In September 2022, the 3rd International Workshop on pyrrolizidine alkaloids (PAs) and related phytotoxins was held on-line, entitled 'Toxins in botanical drugs and plant-derived food and feed - from science to regulation'. The workshop focused on new findings about the occurrence, exposure, toxicity, and risk assessment of PAs. In addition, new scientific results related to the risk assessment of alkenylbenzenes, a distinct class of herbal constituents, were presented. The presence of PAs and alkenylbenzenes in plant-derived food, feed, and herbal medicines has raised health concerns with respect to their acute and chronic toxicity but mainly related to the genotoxic and carcinogenic properties of several congeners. The compounds are natural constituents of a variety of plant families and species widely used in medicinal, food, and feed products. Their individual occurrence, levels, and toxic properties, together with the broad range of congeners present in nature, represent a striking challenge to modern toxicology. This review tries to provide an overview of the current knowledge on these compounds and indicates needs and perspectives for future research.

Botanical preparations, such as essential oils are increasingly used as feed additives to improve the feed quality and animal health while reducing antibiotic use. Regarding safe usage of these preparations for both humans and relevant animal species (e.g., food-producing animals and pets), the toxicity of their constituents requires to be characterised. Particularly, p -allylalkoxybenzene derivatives, that are present in certain botanical preparations, are known to be both genotoxic and carcinogenic and raise health concerns. However, little is known about species differences in sensitivity to the biological effects of these compounds, nor on the transfer from feed to food. Moreover, toxicokinetic properties and resulting toxicity of these compounds can be influenced by other substances present in the botanical preparations. Narrowing this knowledge gap, a NAM-based approach was carried out for five representative p -allylalkoxybenzenes: elemicin, estragole, methyleugenol, myristicin and safrole for six model species: cat, chicken, cow, human, pig and rat. The aim was to generate in vitro kinetic data from liver S9 incubations to explore potential species differences in bioactivation of p -allylalkoxybenzenes combined with PBK modelling. The influence of terpenoids on the p -allylalkoxybenzene-bioactivation was investigated in parallel. In vitro bioactivation was observed in all species for phase I and phase II metabolism at the species and compound-specific level. The presence of terpenoids revealed to be of minor influence on the p -allylalkoxybenzene-bioactivation. The PBK model simulations for two p -allylalkoxybenzenes revealed that all species have a relatively higher formation of the 1’-sulfooxy metabolite compared to that in rats (differences exceed the default uncertainty factor of 4). PBK model simulations of excretion/deposition of the p -allylalkoxybenzenes and their related 1’-hydroxy metabolite indicated limited transfer into milk, eggs and edible tissues. This case study demonstrates the applicability of NAMs to identify species differences in the metabolism of natural compounds from botanical preparations in support of risk assessment.

Abstract The European Commission asked EFSA for a scientific opinion on the risks for human health of the presence of grayanotoxins (GTXs) in ‘certain honey’ from Ericaceae plants. The risk assessment included all structurally related grayananes occurring with GTXs in ‘certain’ honey. Oral exposure is associated with acute intoxication in humans. Acute symptoms affect the muscles, nervous and cardiovascular systems. These may lead to complete atrioventricular block, convulsions, mental confusion, agitation, syncope and respiratory depression. For acute effects, the CONTAM Panel derived a reference point (RP) of 15.3 μg/kg body weight for the sum of GTX I and III based on a BMDL10 for reduced heart rate in rats. A similar relative potency was considered for GTX I. Without chronic toxicity studies, an RP for long‐term effects could not be derived. There is evidence for genotoxicity in mice exposed to GTX III or honey containing GTX I and III, showing increased levels of chromosomal damage. The mechanism of genotoxicity is unknown. Without representative occurrence data for the sum of GTX I and III and consumption data from Ericaceae honey, acute dietary exposure was estimated based on selected concentrations for GTX I and III reflecting concentrations measured in ‘certain’ honeys. Applying a margin of exposure (MOE) approach, the estimated MOEs raised health concerns for acute toxicity. The Panel calculated the highest concentrations for GTX I and III below which no acute effects would be expected following ‘certain honey’ consumption. The Panel is 75% or more certain that the calculated highest concentration of 0.05 mg for the sum of GTX I and III per kg honey is protective for all age groups regarding acute intoxications. This value does not consider other grayananes in ‘certain honey’ and does not cover the identified genotoxicity.

Results material A (wheat)Annex 10 Results material B

A proficiency test (PT) for the quantitative analysis of pyrrolizidine alkaloids (PAs) in the food matrices black tea and the culinary herb marjoram was organised by the European Union Reference Laboratory for mycotoxins & plant toxins in food and feed (EURL-MP) between December 2021 and March 2022. This PT was carried out by Wageningen Food Safety Research (WFSR) under accreditation (R013, Dutch Accreditation Council RvA, ISO/IEC 17043:2010. In December 2020 Commission Regulation (EU) 2020/2040 on maximum levels of PAs in certain foodstuffs was published and this has come into effect from July 1, 2022. The primary goal of this PT was to assess the proficiency of the National Reference Laboratories for mycotoxins & plant toxins in food and feed (NRLs) with respect to the quantitative determination of the 35 PAs mentioned in Commission Regulation (EU) 2020/2040.

The occurrence of tropane alkaloids (TAs), toxic plant metabolites, in food in Europe was studied to identify those TAs in food most relevant for human health. Information was extracted from the literature and the 2016 study from the European Food Safety Authority. Calystegines were identified as being inherent TAs in foods common in Europe, such as Solanum tuberosum (potato), S. melongena (eggplant, aubergine), Capsicum annuum (bell pepper) and Brassica oleracea (broccoli, Brussels sprouts). In addition, some low-molecular-weight tropanes and Convolvulaceae-type TAs were found inherent to bell pepper. On the other hand, atropine, scopolamine, convolvine, pseudotropine and tropine were identified as emerging TAs resulting from the presence of associated weeds in food. The most relevant food products in this respect are unprocessed and processed cereal-based foods for infants, young children or adults, dry (herbal) teas and canned or frozen vegetables. Overall, the occurrence data on both inherent as well as on associated TAs in foods are still scarce, highlighting the need for monitoring data. It also indicates the urge for food safety authorities to work with farmers, plant breeders and food business operators to prevent the spreading of invasive weeds and to increase awareness.

Pyrrolizidine alkaloids (PAs) are noted for their hepatotoxic, genotoxic, and carcinogenic effects in animals and humans following metabolic activation in the liver. In this study, herbal supplements sold in Ghana for sexual improvement were analysed for the presence of 64 PAs using LC-MS/MS analysis. Up to 17 different PAs were identified in 19 out of the 37 samples analysed. The sum of PAs in samples ranged from 5 to 3204 & mu;g kg(-1). Since the PA content in the herbal medicinal preparations was generally lower than in honey samples, their presence was mainly attributed to cross-contamination. The observed levels would result in estimated daily intakes from 0.01 to 12 & mu;g per day or 0.0002 to 0.2 & mu;g kg(-1) bw day(-1) for a person weighing 70 kg. The margins of exposure ranged from 1200 to 1,400,000 with eight samples showing values below 10,000, thus indicating a health concern.

Pyrrolizidine alkaloids (PAs) are produced by various plant species and have been detected as contaminants in food and feed. Monitoring programmes should include PAs that are present in relevant matrices and that exhibit a high toxic potential. The aim of the present study was to use a bioassay-directed analysis approach to identify relevant PAs not yet included in monitoring programmes. To that end, extracts of Heliotropium europaeum and H. popovii were prepared and analysed with LC–MS/MS for the presence of 35 PAs included in monitoring programmes, as well as for genotoxic activity in the HepaRG/γH2AX assay. Europine, heliotrine and lasiocarpine were found to be the most abundant PAs. The extracts showed a higher γH2AX activity than related artificial mixtures of quantified known PAs, which might point to the presence of unknown toxic PAs. The H. europaeum extract was fractionated and γH2AX activities of individual fractions were determined. Fractions were further analysed applying LC–Orbitrap-MS analysis and Compound Discoverer software, identifying various candidate PAs responsible for the non-explained genotoxic activity. Altogether, the results obtained show that bioassay-directed analysis allows identification of candidate PAs that can be included in monitoring programmes.

Plants produce many secondary metabolites showing considerable inter- and intraspecific diversity of concentration and composition as a strategy to cope with environmental stresses. The evolution of plant defenses against herbivores and pathogens can be unraveled by understanding the mechanisms underlying chemical diversity. Pyrrolizidine alkaloids are a class of secondary metabolites with high diversity. We performed a qualitative and quantitative analysis of 80 pyrrolizidine alkaloids with liquid chromatography-tandem mass spectrometry of leaves from 17Jacobaeaspecies including one to three populations per species with 4-10 individuals per population grown under controlled conditions in a climate chamber. We observed large inter- and intraspecific variation in pyrrolizidine alkaloid concentration and composition, which were both species-specific. Furthermore, we sequenced 11 plastid and three nuclear regions to reconstruct the phylogeny of the 17Jacobaeaspecies. Ancestral state reconstruction at the species level showed mainly random distributions of individual pyrrolizidine alkaloids. We found little evidence for phylogenetic signals, as nine out of 80 pyrrolizidine alkaloids showed a significant phylogenetic signal for Pagel's lambda statistics only, whereas no significance was detected for Blomberg'sKmeasure. We speculate that this high pyrrolizidine alkaloid diversity is the result of the upregulation and downregulation of specific pyrrolizidine alkaloids depending on ecological needs rather than gains and losses of particular pyrrolizidine alkaloid biosynthesis genes during evolution.

Concentration of plant secondary metabolites (SMs) show seasonal variations. However, it is still not well understood how these abiotic and biotic factors influence the seasonal variations of SMs. In addition, it is of interest to know if and how SMs are reallocated to the different plant organs, in particular whether SMs are reallocated to the remaining tissues when biomass is lost, e.g., during winter. Here we used Jacobaea vulgaris, Jacobaea aquatica, two F1 and four F2 hybrids that differed in their pyrrolizidine alkaloids (PAs) bouquet as a study system. A series of clones of these genotypes were investigated during their vegetative stage spanning 14 months in a semi-natural environment. We found that the total PA concentration in roots and shoots showed a gradual increase until the spring of the second year, whereafter it dropped substantially in shoots. The variation in PA composition due to seasonal changes was significant but relatively small. Senecionine-like PAs were the dominant PAs in roots, while jacobine-/erucifoline-like PAs were dominant in shoots. The variation of PA concentration was significantly correlated with temperature, day length, and plant age. A correlation analysis showed that PAs were not reallocated when biomass was lost in winter. Overall, our study showed that PA composition of each genotype changed over seasons in a different manner but seasonal variation did not overrule the differences in PA composition among genotypes.

This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAs pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.

A proficiency test (PT) for the determination of the tropane alkaloids (TAs) atropine and scopolaminein buckwheat flour and maize flour was organised by the European Union Reference Laboratory for mycotoxins & plant toxins (EURLMP) between August and October 2020. This PT was carried out by Wageningen Food Safety Research (WFSR) in accordance with ISO/IEC 17043 (R013). The measurandlevels were targeted to provide insight on the measurement capabilities of the EU Member States’National Reference Laboratories (NRLs) at concentrations corresponding to the levels of TAs in processed cereal-based foods and baby foods for infants and young children, containing millet, sorghum, buckwheat or their derived products as regulated by Commission Regulation (EU) 2016/239. In addition to this food product, a maize sample containing a higher level of TAs was included in this PT since an amendment to the legislation is foreseen.The participants were asked to quantify atropine and scopolamine in two materials and to report the compounds individually as well as the sum value. The participants performance was assessed as z-score in both materials for the individual TAs (maximum score 4 out of 4) and for the sum of the two TAs in one sample (maximum score 2 out of 2).Thirty-eight participants, of which 29 NRLs for mycotoxins and/or plant toxins in food and feed (from 22 EU Member States plus Iceland and Norway) and 9 Official Laboratories (8 from 4 EU Member States and Switzerland) participated in the PT.Two materials, buckwheat flour (material A) and maize flour (material B), were prepared containing atropine and scopolamine. Levels were artificially increased by spiking with atropine and scopolamine standard solutions. Both materials were sufficiently homogeneous and stable during the PT. Each participant received one test sample of each material.For the identification and quantification of atropine and scopolamine 34 participants used liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS), one participant used LC single quadrupole MS, two participants used LC high resolution mass spectrometry (HRMS) and one participant provided no information. In this PT the robust mean was used as consensus value. The consensus value based on the participants’ results was used as the assigned value. The assigned values of atropine and scopolamine in material A were, respectively, 1.15 and 1.16 μg/kg and in material B, respectively, 15.3 and 52.7 μg/kg. Obtained interlaboratory reproducibility (RSDR) ranged from 14% to 26%. The RSDR were for all TAs below the target standard deviation, except for scopolamine (26%) in material A. For the sum of TAs (atropine and scopolamine) the RSDR was 20% and 17% for material A and B, respectively. The proficiency of the participants was assessed through z-scores, calculated using the assigned values and a relative target standard deviation of 25%. All participants submitted results for atropine and scopolamine. One participant analysed only material A. For both materials (A and B) 87% of the results for atropine and scopolamine were rated with satisfactory z-scores (|z|≤ 2), 6% of the results fell into the questionable range with 2<|z|<3 and 7% of the results fell into the unsatisfactory range with |z|≥ 3. Twenty-six participants achieved optimal performance for both materials by detecting both TAs with the correct quantification and the absence of false negative results. In this PT, two false negatives were reported.