Natural toxins: risks, regulations and the analytical situation in Europe

Transcription

1 Anal Bioanal Chem (2004) 378 : DOI /s REVIEW Hans P. van Egmond Natural toxins: risks, regulations and the analytical situation in Europe Received: 25 July 2003 / Revised: 15 October 2003 / Accepted: 27 October 2003 / Published online: 13 December 2003 Springer-Verlag 2003 H. P. van Egmond ( ) Laboratory for Food and Residue Analyses, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands hp.van.egmond@rivm.nl Abstract Natural toxins in food and feed are considered important food safety issues of growing concern, in particular mycotoxins, phycotoxins and plant toxins. Most scientific developments have occurred in the past few decades in the area of mycotoxins. Formal health risk assessments have been carried out by the Joint Expert Committee on Food Additives of the World Health Organization and the Food and Agriculture Organization. Limits and regulations for mycotoxins in food and feed have been established in many countries, including practically all European countries. An array of (formally validated) analytical methods and (certified) reference materials have become available. Several European research projects, funded by the European Commission and supported by the European Standardization Committee, have significantly contributed to this development. Quantitative methods of analysis for mycotoxins often make use of immunoaffinity cleanup with liquid chromatographic or gas chromatographic separation techniques in combination with various types of detectors, including mass spectroscopy. For screening purposes (bio)sensor-based techniques are among the promising newcomers. For the phycotoxins the situation is less advanced. Formal risk assessments by authoritative international bodies have not been carried out. Methods of analysis, formally validated according to internationally harmonized protocols, are scarce and animal testing still plays a key role in official methodology. The development of the analytical methodology is partly hampered by the limited availability of certain reliable calibrants and reference materials, although this situation is gradually improving. New regulations in the European Union have increased the pressure to develop and validate chemical methods of analysis. Joint efforts in the European context are now directed towards significantly improving this situation, and techniques such as liquid chromatography mass spectroscopy offer promise in this respect. Both the working group on biotoxins of the European Standardization Committee and the network of National Reference Laboratories for Marine Biotoxins have taken up responsibilities here. The plant toxins are a category of natural toxins, where the situation is the least developed with respect to regulations, validated methods of analysis and reference materials. Yet, their occurrence in a wide range of consumable plant species demands the attention of the analytical community. Keywords Natural toxin Mycotoxin Phycotoxin Plant toxin Analysis Reference material Introduction Over recent years there has been an increased scientific interest in natural toxins in food and feed. The occurrence of these harmful organic compounds of natural origin is of growing concern to human health. Some natural toxins are extremely potent acute toxins (e.g. botulin) or are strong carcinogens (e.g. aflatoxins). On the basis of their origin the natural toxins can be divided into five main categories: Mycotoxins: toxins produced by fungi. Most have relatively small molecular weights, and they are usually heat-stable. Mycotoxins that are significant in terms of toxicity and occurrence include the aflatoxins, ochratoxins, trichothecenes, patulin, fumonisins and zearalenone. Bacterial toxins: toxins produced by bacteria. Many bacterial toxins are proteins, which are not heat-stable. Some well-known bacterial toxins are botulin, Staphylococcus aureus enterotoxin and Bacillus cereus enterotoxin. Phycotoxins: toxins produced by algae that, through feed chains, end up in fishery products, such as shellfish. Some phycotoxins of significant human health

2 concern include the paralytic shellfish poisoning toxins, the diarrhoeic shellfish poisoning toxins and the amnesic shellfish poisoning toxins. Plant toxins (also named phytotoxins): toxins that are produced by edible plant species. Some play a role in the defence mechanism of plants against attacks of insects and fungi. Examples are the potato glycoalkaloids and toxins occurring in herbs, such as pyrrolizidine alkaloids, and anisatin in certain varieties of star anise. Zootoxins: toxins produced by animals, e.g. snakes, scorpions and certain frogs. Zootoxins are generally of lesser significance to human health by oral exposure, although exceptions occur, such as bufotoxin, a toxin excreted by Bufo marinus. Licking the head of this toad (a dangerous practice of drug addicts in the Netherlands) leads to hallucinations. The first three mentioned categories of natural toxins are in fact bio-contaminants. They are produced by microorganisms and may contaminate food and food products. This is not the case with the last two categories, which are inherent components of plants or animals. This article will be restricted to the mycotoxins, the phycotoxins and the plant toxins. These three categories of chemical hazards are characterized by the World Health Organization (WHO) as significant sources of food-borne illnesses [1]. Of these categories, most research has been carried out on the mycotoxins, which are currently a major food safety issue. Problems with mycotoxins are more severe than with the other categories of toxins, due to the widespread consumption of goods being contaminated. Various networks of scientists are active, and the European Commission (EC) is a major sponsor of European mycotoxin research projects. Research on phycotoxins (in particular the marine biotoxins) is of growing significance in Europe where problems regularly lead to closures of shellfish harvesting areas. Also here networking exists in Europe, and EC-funded research activities take place, but to a lesser extent than for the mycotoxins. Investigations on plant toxins still seem to have a lower priority in European food safety research activities, and scientific networking is in its infancy here. Mycotoxins Risks and regulations Mycotoxins are secondary metabolites of fungi, which are capable of producing acute toxic, carcinogenic, mutagenic, teratogenic, immunotoxic and oestrogenic effects on animals at the level of exposure. Biological conversion products of mycotoxins are also referred to as mycotoxins. Toxic syndromes resulting from the intake of mycotoxins by man and animals are known to as mycotoxicoses. For the mycotoxins currently considered most significant (aflatoxins, ochratoxin A, patulin, fumonisins, zearalenone and some trichothecenes including deoxynivalenol), the Joint Expert Committee on Food Additives (JECFA), 1153 a scientific advisory body of the WHO and the Food and Agriculture Organization (FAO), has recently evaluated their hazard [2, 3]. JECFA provides a mechanism for assessing the toxicity of additives, veterinary drug residues and contaminants. Safety evaluation of contaminants incorporates various steps in a formal health risk assessment approach. The 56th session of JECFA was specifically directed to mycotoxins. The report addressed several concerns about each mycotoxin: explanation of the mycotoxin, absorption through excretion, toxicological studies and final evaluation. It is interesting to note that, along with the mycotoxin evaluations, JECFA put forward general considerations on analytical methods, sampling, associated intake issues and control [2, 3]. In particular, good surveillance data should be generated using validated analytical methods and reference materials to ensure reliable results. Methods used should be validated through collaborative studies of analytical performance; however, the committee recognized that it may not always be possible to use a validated method due to expense or an official method being irreconcilable to a particular toxin matrix combination. The committee also stressed the importance of laboratories participating in interlaboratory comparison studies to ensure analytical quality assurance. In Europe various organizations are involved with risk assessment of mycotoxins: Both the Scientific Committee on Food (SCF) and the Scientific Committee on Animal Nutrition (SCAN) of the EC have regularly expressed scientific opinions about risks associated with the occurrence of mycotoxins in food or animal feed, respectively. For example, SCAN recently updated its opinion on undesirable substances in animal feed [4], which included several mycotoxins for which the EC might establish new regulations in the near future. The newly formed Scientific Panel on Contaminants in the Food Chain of the recently established European Food Safety Authority (EFSA) is specifically charged (among other issues) with mycotoxins. Efforts to assess exposure, one of the main ingredients of risk assessment, are undertaken within SCOOP (SCOOP: Scientific Cooperation on Questions relating to Food) projects, funded by the EC. SCOOP projects on mycotoxins are targeted to make the best estimates of intake of these toxins by EU inhabitants. In the 1990s these activities resulted in a report on the exposure assessment of aflatoxins [5]; later SCOOP reports for several other mycotoxins followed (for ochratoxin A, patulin, trichothecenes, fumonisins and zearalenone) [6, 7, 8]. SCOOP data were used by the SCF for its evaluation and advisory work on the risks to public health arising from dietary exposure to certain mycotoxins. Some projects in the EC s fifth Framework Programme are directed towards determining the risk of certain mycotoxins (e.g. the ongoing project Ochratoxin A risk assessment: mechanisms of ochratoxin A-induced carcinogenicity as a basis for an improved risk assessment, supported by the Quality of Life Programme [9]).

3 1154 ILSI-Europe, the European branch of the International Life Sciences Institute (a non-profit-making, worldwide foundation established to advance the understanding of scientific issues relating to nutrition, food safety, toxicology, risk assessment and the environment), has a working group on natural toxins that organizes international symposia on mycotoxins of European concern, e.g. on ochratoxin A in 1996 and on trichothecenes in The knowledge that mycotoxins can have serious adverse effects on man and animals has led many countries to establish regulations which limit the presence of these compounds in food and feed. To date, there are more than 100 countries in the world which have specific limits for mycotoxins in foodstuffs and feedstuffs. The total population in these countries represents approximately 90% of the world s inhabitants [10]. In Europe, practically all nations were known to have specific mycotoxin regulations in Compared to the other continents, Europe has the most extensive and detailed regulations for mycotoxins in food and feed (see Figs. 1 and 2). In the EU, now consisting of 15 countries but soon to be expanded with another ten countries, harmonized regulations or detailed guidelines exist for aflatoxins in various foodstuffs, aflatoxin M 1 in milk, ochratoxin A in cereals and dried vine fruits, patulin in several fruit products, deoxynivalenol in cereals and cereal products and for aflatoxin B 1 in various feedstuffs [11]. In the future (2004 and following years) a significant further expansion of EU-harmonized mycotoxin regulations is expected both for foods and feeds, and limits will be lowered for some mycotoxins. For foods this concerns aflatoxin B 1, ochratoxin A and deoxynivalenol (DON) in infant formulae and follow-up formulae, ochratoxin A in coffee, wine, beer, spices, grape juice, cocoa and cocoa products, and several Fusarium-produced mycotoxins, i.e. trichothecenes (nivalenol, T-2 and HT-2 toxins, in addition to DON), fumonisins and zearalenone in cereal-based foodstuffs. For feeds, specific limits are to be expected for ergot alkaloids, zearalenone, trichothecenes and ochratoxin A, and possibly fumonisins. The developments described above, and in particular those on limits and regulations, have dictated and stimulated the analytical community to develop, validate and standardize suitable analytical methodology, to develop (certified) reference materials and to establish analytical Fig. 1 Mycotoxins regulated in food in Europe (situation 2003). AFT: total aflatoxins B 1, B 2, G 1 and G 2 ; AFB1: aflatoxin B 1 ; AFB1/G1: aflatoxins B 1 and G 1 ; AFM1: aflatoxin M 1 ; OTA: ochratoxin A; PAT: patulin; DON: deoxynivalenol; T-2: T-2 toxin; DAS: diacetoxyscirpenol; ZEN: zearalenone; FUM 1/2: fumonisins B 1 and B 2 ; FUM 1: fumonisin B 1 ; STE: sterigmatocystin Fig. 2 Mycotoxins regulated in feed in Europe (situation 2003). Abbreviations: see Fig. 1

4 quality assurance programmes such as international proficiency tests. Analytical methods and reference materials Current analytical methods for mycotoxins are based on chemical procedures. Test portions are usually extracted with (combinations of) organic solvents and water. Purification of the extracts to remove lipids and other substances is often done by leading the extracts through chromatography columns or pre-packed cartridges. The latter are commercially available with many types of adsorbents and in many formats, to suit the needs of the analyst. A purification technique, which is widely applied now in mycotoxin methodology, is based on immunoaffinity cleanup of extracts. Immunoaffinity columns are composed of monoclonal antibodies, specific for the toxin of interest, which are immobilized on Sepharose and packed into small columns. These immunoaffinity columns have sufficient capacity to clean up heavily contaminated samples, and they usually efficiently remove compounds that could interfere in the determination, because the antibodies specifically recognize the toxin of interest. The purification techniques described can be incorporated in fully automated sample preparation systems that take the sample from the extraction stage through to completion of liquid chromatography (LC) in an unattended mode of action. In addition to LC, separation techniques such as thin-layer chromatography (TLC), gas chromatography (GC) and enzyme-linked immunosorbent assay (ELISA) are among the ones most often used in mycotoxin methodology. Several immunoaffinity-based mycotoxin methods have been formally validated in the last five years in European collaborative studies for various mycotoxin/matrix combinations. These studies were conducted in agreement with the protocol for the design, conduct and interpretation of method-performance studies (the IUPAC/AOAC/ ISO harmonized protocol ) [12], and most of them have also been approved by AOAC International, which has made them widely acceptable as tools for regulatory analysis. Many of the European methods were standardized by working group 5 Biotoxins of CEN Technical Committee 275: Horizontal methods for food [CEN= Comité Européan de Normalisation (European Standardization Committee), the European equivalent of ISO (International Standardization Organization)]. They are now available as reference methods. The CEN work was and is done in response to the needs of the EC to make enforcement possible of the new or proposed EU-harmonized regulations (with tolerance limits that are among the lowest in the world). CEN has also produced a document that provides criteria for the performance of mycotoxin methods [13]. This document gives information concerning the performance of analytical methods to determine mycotoxins in certain matrices that can be expected from experienced analytical laboratories. Some of these CEN criteria are reflected as method-performance requirements in official EU legislation, e.g. on aflatoxins and ochratoxin A [14, 15] The foreseen expansion in the European mycotoxin regulatory area (see previous paragraph) demands ongoing activities of European research groups to develop and validate suitable methodology. Alternative methods dedicated to special needs, e.g. the procurement of precise estimates of exposure of certain groups of the population (e.g. babies), and low-cost methods widely applicable for rapid screening at an early stage in the food/feed chain (e.g. based on TLC or ELISA), will be needed to supplement the reference methods mentioned above. There exist interesting newer developments in mycotoxin methodology, for which the application would need to be further explored, tested for performance and compared with the existing conventional methods. These newer methods include the powerful LC MS( MS) (mass spectroscopy) and GC MS benchtop techniques, capillary zone electrophoresis, cell tests, biosensors, MIP (molecularly imprinted polymers), fluorescence polarization immunoassays, dipstick assays and non-invasive methods based on infrared and acoustic techniques. Worthwhile to read are the yearly published reports of the General Referee for Mycotoxins of AOAC International, e.g. the one published in 2003 [16]. These reports provide insight into the analytical developments in the mycotoxin area and contain many references for further reading. LC MS MS is rapidly gaining ground in routine laboratories as a method which enables the simultaneous detection of several mycotoxins in one run. One of the most recent developments includes the application of a multitoxin immunoaffinity column, which allows the determination of the aflatoxins, deoxynivalenol, ochratoxin A, zearalenone and the fumonisins in one run [17]. An example of a very sensitive GC MS method is the one recently developed in our laboratory for the determination of deoxynivalenol (DON) and other trichothecenes in toddler food. The method allows one to estimate the real exposure of toddlers (age category 9 months to 2 years) to trichothecenes, by analysing samples of 24-h duplicate diets of toddlers. The method has a limit of detection for DON of ~0.3 µg/kg, which has been shown to be sufficiently low to find the first few dozen samples positive for DON [18]. This newly developed method has shown itself to be useful for the detection of trichothecenes at very low levels, but quantification of DON at these low, (sub-)µg/kg levels is required only for special studies, because (proposed) legal limits for DON are usually in the range µg/kg [11]. For rapid screening of cereals for DON other, much faster, methods are to be preferred, e.g. ELISAs or methods based on biosensors. An overview of ELISAs for mycotoxins is available through the website of the European EMAN project (EMAN=European Mycotoxin Awareness Network) [19]. An example of a recently developed biosensor-based method is the one by Tüdos et al. [20] for the selective and quantitative determination of DON. This biosensor assay is of the surface plasmon resonance (SPR) type, where DON conjugate is immobilized on the sensor. The analysis results of the optimized biosensor assay and an (in-house validated) LC MS MS method, practiced by the Netherlands Food

5 1156 Inspection Service (Amsterdam, the Netherlands)(see also [17]), were compared for naturally contaminated wheat samples with DON levels ranging from µg/kg. The results indicated good agreement between the two techniques, especially for wet-ground samples. Low-cost biosensor-based methods are currently developed for aflatoxin B 1 and ochratoxin A (target costs: approx. 15 per analysis) within the ongoing EC CRAFT project MycoSense [21]. Many analytical methods for mycotoxins have been validated through collaborative studies, and a relevant overview was recently published [22]. However, the availability of modern and reliable methods of analysis for mycotoxins is no guarantee of accurate results. Proficiency tests for aflatoxins and ochratoxin A, organized by the International Agency for Research on Cancer in the past, have shown that wide scatter of analytical results is of little comfort to those who must either pay for the measurements, or base potentially important decisions upon them [23]. Analytical quality assurance (AQA) is needed to demonstrate acceptable analytical performance. For AQA purposes (certified) reference materials for mycotoxins have been developed in EC programmes and proficiency tests for mycotoxins are regularly organized. The (certified) reference materials have been developed since the 1980s under the auspices of the BCR Programme (BCR: Bureau Communautaire de Référence, or in English, Community Bureau of Reference), later known as the Standards, Measurements and Testing (SMT) Programme. Currently available BCR reference materials include aflatoxin M 1 in milk powder, aflatoxin M 1 calibrant, total aflatoxins in peanut butter, aflatoxin B 1 in peanut meal, aflatoxin B 1 in feedstuff, ochratoxin A in wheat, DON in maize and wheat, zearalenone in maize and zearalenone calibrant. A project is ongoing on the development of trichothecene calibrants. The mycotoxin reference materials are currently available from the EC s Joint Research Centre, Institute for Reference Materials and Measurements, Geel, Belgium (see Certified reference materials are relatively expensive, due to the huge amount of work involved with their development, and supplies are limited. Therefore laboratories are advised to develop their own in-house reference materials (homogeneous and stable) for routine use, the toxin content of which should be established on the basis of the certified materials. Besides the application of (certified) reference materials, regular participation in interlaboratory comparisons, such as proficiency testing schemes, is becoming increasingly important as part of AQA measures. A number of proficiency testing schemes for mycotoxins exist at the international level, including those organized by the Food Analysis Performance Assessment Scheme (FAPAS), operated from Europe by the Central Science Laboratory in the UK, and those organized by the American Oil Chemists Society (AOCS), operated from the USA. The experiences of FAPAS with mycotoxin analysis were recently published [24]. Phycotoxins Risks and regulations Phycotoxins are toxic components produced by unicellular microalgae. They may enter fishery products through feed chains in the aquatic environment. Phycotoxins may demonstrate various (mostly acute) toxic effects. Human intoxications caused by phycotoxins occur worldwide through consumption of marine fishery products in which the toxins have accumulated. Toxins of freshwater algae have induced intoxications in farm animals and they can threaten human health, e.g. through drinking water or consumption of freshwater algae. Among the most important marine phycotoxins are the shellfish poisons and the ciguatera toxins. The latter accumulate in finned fish. According to their toxic effects, the shellfish poisons are usually divided into four groups: the paralytic shellfish poisoning (PSP) toxins, the diarrhoeic shellfish poisoning (DSP) toxins, the amnesic shellfish poisoning (ASP) toxins, and the neurotoxic shellfish poisoning (NSP) toxins. Contrary to the mycotoxins, for which chronic effects are of particular concern, the danger of phycotoxins is mainly through their acute effects. Because toxin production and accumulation occur only under certain environmental conditions that stimulate algal blooms, marine fishery products may become incidentally contaminated with (relatively) high concentrations. At present the risk assessment of phycotoxins has not been performed in a straightforward way. Risk assessments have often been based on pragmatic decisions taken during intoxication events with risk management mixed into the process, complicating the procedure. The few data that exist about human toxicity of phycotoxins leave the impression that the margin of safety between exposure at a regulatory limit and human effect levels is often rather small, especially for PSP and DSP toxins [25]. Until now formal risk assessments, e.g. by JECFA, have not been carried out. However, such activities would be highly recommended to get insight into the real margins of safety, although it is questionable whether enough reliable toxicological data exist, particularly on repeated exposure. If adequate scientific (toxicological, epidemiological and occurrence) data were available, risk assessment would need to be performed by applying generally accepted safety or uncertainty factors. The fact that marine phycotoxins pose a threat to human health led a number of countries to establish regulations to control the presence of these toxins in seafood. Several dozens of countries have specific limits for marine phycotoxins in shellfish and fish [26]. Also, for microcystins (toxins produced by freshwater algae) a guideline limit was released by the WHO for drinking water [27]. In the EU the following harmonized limits and analytical requirements exist for ASP toxins [28], DSP toxins and related compounds [29], PSP toxins [30] and ciguatera toxins [31]: For ASP: limit 20 mg/kg; an LC UV method has to be used.

6 For PSP: limit 80 µg/100 g; a biological assay has to be used. A chemical assay is allowed for saxitoxin, but if the results are challenged, the bioassay is the reference method. For DSP and related compounds: various limits are applied: Okadaic acid+dynophysis toxins+pectenotoxins: 160 µg okadaic acid equivalents/kg Yessotoxins: 1 mg yessotoxin equivalents/kg Azaspiracids: 160 µg/kg A biological method is to be used, (bio)chemical alternatives are allowed, if formally validated. For ciguatera toxins: placing on the market of fishery products containing ciguatera toxins is not allowed, no information about analytical methods is given. As for the mycotoxins, the problems caused by phycotoxins and the existence of specific regulations has led to the development of analytical methodology and (certified) reference materials. The progress has been relatively slow, however, due to the chronic lack of sufficiently pure materials which can be used as calibrants. Also new phycotoxins are regularly discovered, e.g. during intoxication events, which require the immediate attention of the scientific community, without the possibility that extensive analytical experience can be built up and reliable reference materials can be developed. Analytical developments and reference materials 1157 A variety of analytical techniques are available for the detection and determination of marine biotoxins, but animal tests still play a major role, in particular in the control of PSP and DSP toxin events. In the EU, a mouse bioassay is the reference method for PSP determination, and a mouse or rat assay is to be used for the determination of toxins belonging to the DSP complex. For PSP, a chemical method for saxitoxin may be used in addition to the mouse assay according to the relevant EU directive [30], but a problem here is that the PSP complex comprises about 20 different toxins, and in European waters saxitoxin is often not the prevalent PSP toxin. If the results are challenged, the mouse bioassay has to be used, but a serious limitation here is that this test is formally not allowed in several European countries, for animal welfare reasons. The mouse assay requires the injection of shellfish extract by the intraperitoneal route. The shellfish extract is crude and animals suffer severely, as the PSP toxins act as neuromuscular blocking agents, causing animal death in 5 7 min. In the mouse assay applied to detect toxins of the DSP complex, extracts are also injected intraperitoneally, and survival time is checked from 24 to 48 h. In this mouse assay all DSP components are likely to be detected, including those DSP toxins which do not cause diarrhoea (pectenotoxins and yessotoxins) and have an unknown toxicity for humans. Also other unknown toxin groups exhibiting ichthyotoxic and haemolytic properties may cause mortality of mice in this bioassay. The major disadvantages of this assay are the lack of specificity (no differentiation between the various components of DSP toxins, whereas in EU legislation specific limits have been set for the various toxins, see risks and regulations), the subjectivity of death time of the animals and the maintaining and killing of laboratory animals. In addition, this assay is time consuming and expensive, may give false positives because of interferences by other lipids (e.g. free fatty acids) and shows variable results between wholebody and hepatopancreas extracts [25]. In the EU a rat bioassay is also officially allowed to be used. This assay is based on diarrhoea induction in rats [32]. The (starved) animals are fed with suspect shellfish tissue (mixed into the diet) and observed during 16 h for signs of diarrhoea, consistency of the faeces and food refusal. The method is at best semi-quantitative and does not detect pectenotoxins and yessotoxins. The test is still used routinely in the Netherlands [25], as it is less animal-unfriendly than the mouse assay. The in vivo tests can in principle be replaced by in vitro tests, provided that there is analytical proof through convincing validation studies that these alternatives (e.g. physicochemical, functional or immunochemical methods) fulfil reasonable performance criteria. In the EU, Commission Decision 2002/225/EC (which lays down maximum levels for specific marine toxins within the DSP complex and related compounds) [29] permits the use of such alternatives, if these are connected with a validation of the methods following an internationally accepted protocol. At the time of writing such validated methodology hardly exists. This is partly due to the fact that, at present, reference standards and reference materials are not available or are in short supply for all the individual components of the DSP toxin groups. In addition, the conducting of a formal interlaboratory validation study is accompanied by a considerable investment of manpower, time and money. In an attempt to stimulate the development of accepted alternatives to the in vivo assays for PSP and DSP toxins currently practised in Europe, a recommendation with supportive and convincing documentation has recently been issued by representatives of governmental institutions of Germany, the UK and the Netherlands to the members of the ECVAM (European Centre for the Validation of Alternative Methods) Scientific Advisory Committee (ESAC) [33]. This has had a positive influence on the contents of the call for project proposals in the 6 th EU Framework Programme for Research and Technological Developments on the activities of the EU National Reference Laboratories for Marine Biotoxins (see below). Which possibilities, alternative to rodent tests, do exist to determine marine biotoxins and which European organizations can play a significant role in the further development and validation of these methods? Various methods, based on different principles, have been explored and published. These include techniques such as LC with UV and fluorescence detectors, LC MS, capillary zone electrophoresis, capillary electrochromatography, enzyme activity tests, receptor binding assays, cell toxicity tests, solid-

7 1158 phase immunobead assays and biosensors. LC MS is currently one of the most interesting techniques which has applications in the determination of several of the shellfish toxins, e.g. the PSP, for which a new type of LC column (hydrophilic interaction) is used [34]. A detailed overview of the various approaches that are possible for the determination of the different shellfish poisoning toxins and the ciguatera toxins, with their advantages and disadvantages, is given in the draft FAO Food and Nutrition Paper Marine biotoxins a review [25]. The number of methods that have been validated through formal collaborative studies is rather scarce though, for reasons explained above. This makes it difficult to give an opinion about the practical usefulness of many of these procedures. In Europe, two networks of scientists are worth mentioning in respect of joint efforts to advance the development of analytical methods [35]. Firstly there is the network of EU National Reference Laboratories (NRL) for Marine Biotoxins, coordinated by the Community Reference Laboratory (CRL) for Marine Biotoxins in Vigo, created in Secondly there is working group 5 Biotoxins of CEN Technical Committee 275, also active in the area of mycotoxins (see previous section on mycotoxins), which took the phycotoxins subject on board in The work done by this CEN working group follows the same pattern as described for the mycotoxins. Typical examples of CRL-coordinated activities carried out in the last few years within the NRL network include the conduct of collaborative studies of methods to determine ASP and DSP toxins (ongoing in 2003), and intercalibration studies on DSP [36] and PSP toxins (ongoing in 2003). A typical example of an NRL-coordinated activity is the organization of a series of proficiency tests among Dutch laboratories involved in analysis of marine biotoxins [37].The interaction between members of the EU NRL network takes place through annual meetings and by means of working groups (WGs) hosted by the European Commission. The outcomes of some of the WGs in the way of conclusions and/or recommendations have been used as pre-normative studies to elaborate new regulations. The increasing demand for quality assurance has led several organizations to undertake proficiency tests, in addition to those incidentally undertaken as described above. There are now two European organizations starting to undertake proficiency tests for shellfish toxins on a regular scale: FAPAS (see section on mycotoxins) and Quasimeme, operated from the FRS Marine Laboratory in Aberdeen, UK. Both FAPAS and Quasimeme have existed for a long time, but their activities in the marine biotoxin area are just starting for PSP (FAPAS) [38] and ASP (Quasimeme). It is expected that these programmes will expand their activities in the future. Similarly as for the mycotoxins, efforts have been undertaken to develop (certified) reference materials for phycotoxins. In 2003, certified calibrants and some reference matrices were commercially available for domoic acid (ASP toxin), and for some of the PSP and DSP toxins, from the National Research Council (NRC) Canada (see In Europe efforts undertaken by the EC s SMT programme have led to lyophilized mussel reference materials with certified mass fractions of some of the toxicologically most significant PSP toxins [39], which are stored at the IRMM in Geel, but their distribution is severely hampered by the fact that saxitoxin is a schedule 1 chemical in the list of the Chemical Weapon Convention. For the other shellfish toxins no European activities to develop certified reference materials are currently undertaken or foreseen. Despite the positive developments with the activities undertaken by the NRC, the availability of reference materials for phycotoxins remains limited. This has an impact not only for the analytical activities on phycotoxins, but it is also a limiting factor in the development of reliable risk assessments for these compounds. Plant toxins Risks and regulations Inherent plant toxins are naturally occurring components in plants that are toxic and/or have negative effects on the bioavailability of nutrients. Toxins in the latter category are often referred to as antinutritional factors or antinutrients. Some plant toxins exhibit chronic effects, others acute effects, or both. Some toxins produced by the plant play a role in the defence system against bacteria, fungi, insects, or other threats. In general, the lack of essential data on the toxicological properties and occurrence of inherent plant toxins does not allow for an adequate safety evaluation. Some important staple foods form inherent toxins, such as potatoes that contain the glycoalkaloids α-solanine and α-chaconine. Because of the virtual absence of chronic toxicity data, an adequate no-observed-adverseeffect level (NOAEL) for potato glycoalkaloids has not been assessed, and a tolerable daily intake (TDI) for humans could not be determined by JECFA [40]. The committee was only able to recognize that the development of empirical data to support a safe level would require considerable effort. Another emerging food safety problem is the toxins occurring in herbs. Herbs are increasingly used, and newer ways for the consumer to order herbs, such as through the Internet, make it difficult for food inspection services to keep control over the situation. Examples of toxins occurring in herbs are anisatin in star anise and the large group of the pyrrolizidine alkaloids. In the Netherlands in late 2001, a few dozen people got severely ill due to the consumption of a blended herbal tea, in which star anise was mixed. Instead of the Chinese variety (Illicium verum, containing the desired substance trans-anethole), the Japanese variety (Illicium anisatum, containing anisatin, a sesquiterpene dilactone) was accidentally blended into the tea. All victims survived. As a consequence, the EC established a decision in 2002 on the import of star anise from Third World countries [41], which should avoid the mistakes described.

8 The pyrrolizidine alkaloids form a group of 200 different compounds, occurring in herbs, herbal teas and comfrey. They are a possible under-recognized cause of liver cancer. They occur at levels that are too low to produce acute liver damage but high enough to be of concern as a possible long-term cause of cirrhosis and liver failure, especially in children [42, 43]. Some toxicity data are available, but further studies on the exposure and toxicity of many pyrrolizidine alkaloids are required to make a meaningful risk assessment possible. Incidentally, European countries (e.g. Germany and the Netherlands) have established regulations for pyrrolizidine alkaloids in herbs, but the limits differ significantly. Contrary to the mycotoxins and phycotoxins, there are no efforts yet within the EU to harmonize the regulatory area, a situation which is not only the case for the pyrrolizidine alkaloids, but also general for the other plant toxins. This European situation is not exceptional. Only few countries in the world have regulations for certain inherent plant toxins. The fact that the new EFSA Scientific Panel for Contaminants in the Food Chain has specifically named the area of natural toxicants as one of its fields of attention, may point at a growing interest in the EU. The above-mentioned groups of toxins are just examples of plant toxins. There are many more plant toxins of concern, but these cannot all be discussed here. Analytical developments and reference materials Inherent plant toxins are quite variable in their chemical structures and physicochemical behaviour, so there exist a variety of analytical chemical approaches to determine them. Compared to biocontaminants, such as mycotoxins, the development of analytical methodology for the plant toxins has been a rather neglected area, and validated methods of analysis are rather scarce. One of the few exceptions is an LC UV method for potato glycoalkaloids, which was validated in an AOAC collaborative study [44]. The lack of formally validated methods is partly due to the fact that only few countries have official regulations for inherent plant toxins, so that there is not much legal pressure to have adequate analytical methods available. In the future, growing interest in the food safety aspects of plant resistance may lead to the development of regulations for certain plant toxins and, inevitably, to the further development of analytical methods to make enforcement of the regulations possible. The methods that are applied in practice to determine plant toxins are often based on chromatographic procedures, such as LC in combination with UV and MS detectors, GC in combination with FID and MS detectors, TLC, immunochemical procedures and, incidentally, NMR. An example of a GC FID method is that of Wiedenfeld [45], used for the determination of pyrrolizidine alkaloids such as senecionine and senkirkine in herbal juices. In the method an extract is purified over a Chromabonddiol column, and then separated over a diphenyldimethylpolysiloxane capillary GC column. Detection takes place with a flame 1159 ionization detector. The method is able to determine senkirkine and senecionine at 0.1 and 0.02 µg/ml juice, respectively. This limit of determination may not be low enough in European situations where legal limits have been set at very low levels, such as in the Netherlands (1 ng/g in herbs). The methodology may need to be further refined, formally validated and standardized before it can become a generalized tool in regulatory analysis. The chronic lack of reliable analytical standards and reference materials has contributed to a situation where the progress in plant toxin analytical methodology is slow. A favourable exception in this regard is the glucosinolates. Glucosinolates, toxins mainly occurring in the family of the Crucifera, are among the very few plant toxins for which the analytical methodology is rather advanced, and for which BCR certified reference materials have become available [46], distributed through the IRMM (see also irmm.jrc.be). Another factor hampering analytical progress is the lack of a European network of food analysts to specifically deal with plant toxins. This situation was recognized by the CEN working group 5 Biotoxins of CEN Technical Committee 275. This working group, very active and successful in the areas of mycotoxins and phycotoxins (see previous sections), has recently decided to include the item plant toxins in their work programme. Despite this welcome development, it will probably take several more years before the first CEN-standardized method for plant toxins will become available. 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