Scientific Opinion on marine biotoxins in shellfish Palytoxin group 1

Transcription

1 SCIENTIFIC OPINION Scientific Opinion on marine biotoxins in shellfish Palytoxin group 1 EFSA Panel on Contaminants in the Food Chain (CONTAM) 2, 3 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT The EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) assessed the risks to human health related to the presence of palytoxin (PlTX)-group toxins in shellfish. PlTX-group toxins have mainly been detected in soft corals of the genus Palythoa and in algae of the genus Ostreopsis. Blooms of Ostreopsis spp. have recently been reported in some European countries. Occurrence of Ostreopsis spp. may result in contamination of shellfish intended for human consumption. Currently there are no regulations on PlTX-group toxins in shellfish, either in the European Union (EU), or in other regions of the world. The toxicological database of PlTX-group toxins is limited, comprising only acute toxicity studies for PlTX and ostreocin-d via several routes of administration in various animal species. The oral route was least sensitive. Acute toxicity and deaths have been reported from human outbreaks, but there are no reliable quantitative data on acute toxicity in humans. In view of the acute toxicity and the lack of chronic toxicity data for PlTX-group toxins, the CONTAM Panel was only able to derive an oral acute reference dose (ARfD) of 0.2 µg/kg b.w. for the sum of PlTX and its analogue ostreocin-d. In order for a 60 kg adult to avoid exceeding the ARfD a 400 g portion of shellfish meat should not contain more than 12 µg of the sum of PlTX and ostreocin-d, corresponding to 30 µg/kg shellfish meat. The mouse bioassay (MBA) has been used to detect PlTX-group toxins, but cell based assays have been developed as alternative. However, positive results require confirmation by chemical methods. High performance liquid chromatography-fluorescence detection (HPLC-FLD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods can be valuable tools for the determination, but method optimisation and validation as well as the development of certified reference materials and standards are necessary. KEY WORDS Marine biotoxins, palytoxin (PlTX)-toxin group toxins, shellfish, mussels, sea urchins, mouse bioassay (MBA), acute reference dose, portion size, methods of analysis, human health, risk assessment. 1 On request from the European Commission, Question No EFSA-Q G, adopted on 26 November Panel members: Jan Alexander, Diane Benford, Alan Boobis, Sandra Ceccatelli, Jean-Pierre Cravedi, Alessandro Di Domenico, Daniel Doerge, Eugenia Dogliotti, Lutz Edler, Peter Farmer, Metka Filipič, Johanna Fink-Gremmels, Peter Fürst, Thierry Guerin, Helle Katrine Knutsen, Miroslav Machala, Antonio Mutti, Josef Schlatter, Rolaf van Leeuwen and Philippe Verger. Correspondence: contam@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on marine biotoxins for the preparation of this opinion: Jan Alexander, Diane Benford, Luis Botana, Peter Fürst, Gerhard Heinemeyer, Philipp Hess, Angelika Preiss-Weigert, Gian Paolo Rossini, Hans van Egmond, Rolaf van Leeuwen and Philippe Verger, and EFSA s staff Mari Eskola and Francesco Vernazza for the support provided to this EFSA scientific output. Suggested citation: EFSA Panel on Contaminants in the Food Chain (CONTAM); Scientific Opinion on marine biotoxins in shellfish Palytoxin grou.. [38 pp.]. doi: /j.efsa Available online: European Food Safety Authority,

2 SUMMARY Palytoxin (PlTX)-group toxins are marine biotoxins which have mainly been detected in marine zoanthids (soft corals) of the genus Palythoa and benthic dinoflagellates of the genus Ostreopsis. PlTX-group toxins were first reported in Hawaii and Japan but are currently distributed worldwide. Blooms of Ostreopsis spp. have recently been reported in four European countries: France, Greece, Italy, and Spain. Occurrence of Ostreopsis spp. may result in contamination of shellfish species intended for human consumption. PlTX-group toxins are complex polyhydroxylated compounds with both lipophilic and hydrophilic areas. At least 8 different PlTX analogues are known: PlTX, ostreocin-d, ovatoxin-a, homopalytoxin, bishomopalytoxin, neopalytoxin, deopalytoxin and 42-hydroxypalytoxin, but only for PlTX and ostreocin-d the chemical structure has been characterised. The occurrence data reported by European countries were limited and comprised only PlTX and ovatoxin-a in mussels and sea urchins. Currently there are no regulations on PlTX-group toxins in shellfish, either in the European Union (EU), or in other regions of the world. Signs and symptoms of PlTX-group toxins intoxication are not well-defined, but include myalgia and weakness, possibly accompanied by fever, nausea and vomiting. Fatalities appear to be rare although there are reports of severe cases, in which patients died after about 15 hours. The toxicological database is limited, comprising only acute toxicity studies via several routes of administration in various animal species. The toxicity of PlTX is strongly dependent on the route of administration; in experimental animals PlTX and ostreocin-d are much less toxic after oral than after parenteral administration. Acute toxicity and deaths have been reported from human outbreaks, but there are no reliable quantitative data on acute toxicity of PlTX-group toxins in humans. PlTX-group toxins are acutely toxic mainly by interference with Na + /K + -ATPase ion-pump. The absence of long term toxicity studies precludes the derivation of a tolerable daily intake (TDI), but in view of the acute toxicity of PlTX-group toxins the Panel on contaminants in the food chain (CONTAM Panel) decided to establish an acute reference dose (ARfD) using the lowest-observedadverse-effect-level (LOAEL) for oral toxicity (gavage) in mice of 200 µg/kg b.w. as the reference point. It was noted that sublingual administration of PlTX in the region of 200 µg/kg b.w. caused toxicity of internal organs. Since transmucosal transport of PlTX in the mouth could not be excluded and because mice seem to be less sensitive than other species, the CONTAM Panel decided to apply an extra uncertainty factor of 10 in addition to the default uncertainty factors of 10 for intra- and 10 for interspecies variation and derived an oral ARfD of 0.2 µg/kg b.w. This ARfD applies to the sum of PlTX and ostreocin-d. In order to ensure protection against the acute effects of PlTX-group toxins, it is important to use a large portion size rather than a long term average consumption in assessing the health risk of the consumers. In line with the previous scientific opinions on shellfish toxins the figure of 400 g shellfish meat was used in the acute exposure assessment as an appropriate estimate of a large portion size. In order for a 60 kg adult to avoid exceeding the ARfD of 0.2 µg/kg b.w., a 400 g portion of shellfish meat should not contain more than 12 µg of the sum of PlTX and ostreocin-d, corresponding to 30 µg/kg shellfish meat. The CONTAM Panel noted that the available occurrence data of PlTX-group toxins are from mussels and sea urchins sampled in contaminated regions and thus are not representative for PlTX-group toxins in shellfish currently reaching the market. These data however indicate that consumption of a large portion of 400 g of shellfish meat (i.e. mussels) harvested from contaminated regions as a worst case estimate could result in a dietary exposure of about 3 µg/kg b.w. of the sum of PlTX and ovatoxin-a for a 60 kg person. If ovatoxin-a had a similar potency to PlTX and ostreocin-d, this exposure would be well above the ARfD. 2

3 The mouse bioassay (MBA) has been used to detect PlTX-group toxins in fish and shellfish, but for reasons of animal welfare and poor specificity there is a growing concern with respect to its use. Alternative assays such as cell based assays have been developed. While cell based assays, taking advantage of certain PlTX functional properties, appear to have the lowest limit of detection (LOD) for PlTX-group toxins, some assays showed interference from other toxins and the positive results should be confirmed by chemical analysis. High performance liquid chromatography-fluorescence detection (HPLC-FLD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods can be valuable tools for the determination of PlTX-group toxins. The optimisation of these methods for application to shellfish extracts, their (inter-laboratory) validation and the development of standards and reference materials are necessary. 3

4 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 4 Background as provided by the European Commission... 5 Terms of reference as provided by the European Commission... 9 Assessment Introduction Chemical characteristics Regulatory status Methods of analysis Supply of appropriate reference material Mammalian bioassay Mouse bioassay Biomolecular methods Cytotoxicity assays Haemolysis assay (erythrocytes) Assays with MCF-7 cells Assays with neuroblastoma cells Immunoassays Chemical methods HPLC-based methods HPLC-FLD HPLC-UV detection LC-MS/MS High performance capillary electrophoresis Proficiency tests Summary of methods Levels of PlTX-group toxins in shellfish from contaminated areas Data Collection Statistical description of PlTX-group toxins in shellfish Influence of processing Exposure assessment Toxicokinetics Toxicity data Mechanistic considerations In vitro toxicity Effects in laboratory animals Acute toxicity Toxicity following intravenous- (i.v.), intramuscular- (i.m.) and subcutaneous- (s.c.) administration Toxicity following intraperitoneal (i.p.) administration Toxicity following intra-tracheal administration Toxicity following sublingual administration Toxicity following oral administration Relative potency of analogues Observations in humans Hazard characterisation Risk characterisation Uncertainty Conclusions and recommendations References Abbreviations

5 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION Marine biotoxins, also commonly known as shellfish toxins, are mainly produced by algae or phytoplankton. Based on their chemical structure, the toxins have been classified into eight groups, namely, the azaspiracid (AZA), brevetoxin, cyclic imine, domoic acid (DA), okadaic acid (OA), pectenotoxin (PTX), saxitoxin (STX) and yessotoxin (YTX) groups, as agreed at the Joint FAO/IOC/WHO ad hoc Expert Consultation held in Two additional groups, palytoxins (PlTX) and ciguatoxins (CTX), may also be considered. STX and its derivatives cause Paralytic Shellfish Poisoning (PSP), and DA causes Amnesic Shellfish Poisoning (ASP). Diarrhetic Shellfish Poisoning (DSP) is caused by OAgroup toxins (OA and dinophysis toxins (DTX)), and AZA group toxins cause Azaspiracid Shellfish Poisoning (AZP). These toxins can all accumulate in the digestive gland (hepatopancreas) of filterfeeding molluscan shellfish, such as mussels, oysters, cockles, clams and scallops, and pose a health risk to humans if contaminated shellfish are consumed. Marine biotoxin-related illness can range from headaches, vomiting and diarrhoea to neurological problems, and in extreme cases can lead to death. To protect public health, monitoring programmes for marine biotoxins have been established in many countries, which often stipulate the use of animal models (for example, the mouse bioassay (MBA) and the rat bioassay (RBA)), for detecting the presence of marine biotoxins in shellfish tissues. In the European Union (EU), bioassays are currently prescribed as the reference methods. Various stakeholders (regulators, animal welfare organisations, scientific organisations) have expressed their concerns about the current legislation in Europe, not only with regard to the use of large numbers of animals, involving procedures which cause significant pain and suffering even though non-animal based methods are available, but also since the scientific community argues that the animal test may not be suitable for all classes of toxins and that the state-of-the-art scientific methodology for the detection and determination of marine biotoxins is not fully reflected in current practices. 1. Legal framework In 2004, the purported EU Hygiene Package of regulations, bringing together and replacing the existing hygiene regulations for the food sector previously contained in numerous individual vertical Directives was published. In Annex II Section VII Chapter V (2) to Regulation 853/2004/EC 5, are established maximum levels for ASP, PSP and DSP toxins. Annex III of Commission Regulation No 2074/2005/EC 6 of 5 December 2005 lays down the recognised testing methods for detecting marine biotoxins. Annex II Chapter II (14) to Regulation (EC) 854/2004 7, gives the monitoring authorities in the EU Member States the mandate to examine live molluscs for the presence of marine biotoxins. The EU Hygiene Package came into effect on 1 January The Council Directive 86/609/EEC Council Directive 86/609/EEC 8 makes provision for laws, regulations and administrative provisions for the protection of animals used for experimental and other scientific purposes. This includes the use of live vertebrate animals as part of testing strategies and programmes to detect identify and quantify marine biotoxins. Indeed, the scope of Article 3 of the Directive includes the use of animals for the safety testing of food, and the avoidance of illness and disease. Directive 86/609/EEC sets out the responsibilities that Member States must discharge. As a result of this use of prescriptive language, Member States have no discretion or flexibility, and most of the 4 ftp://ftp.fao.org/es/esn/food/biotoxin_report_en.pdf 5 OJ L 139, , pp OJ L 338, , pp OJ L 139, , pp OJ L 358, , pp

6 provisions of the Directive must be applied in all cases. It is clear that Member States have to ensure that: the number of animals used for experimental and other scientific purposes is reduced to the justifiable minimum; that such animals are adequately cared for; and that no unnecessary or avoidable pain, suffering, distress or lasting harm are caused in the course of such animal use. Member States may not (Article 7, 2) permit the use of live animals in procedures that may cause pain, suffering, distress or lasting harm: if another scientifically satisfactory method of obtaining the result sought and not entailing the use of live animals is reasonably and practicably available. When animal use can be justified, Directive 86/609/EEC specifies a range of safeguards that Member States must put in place to avoid or minimise any animal suffering that may be caused. All justifiable animal use should be designed and performed to avoid unnecessary pain, suffering, distress and lasting harm (Article 8). Member States must ensure (Article 19, 1) that user establishments undertake experiments as effectively as possible, with the objective of obtaining consistent results, whilst minimising the number of animals and any suffering caused. This latter requirement necessitates the use of minimum severity protocols, including appropriate observation schedules, and the use of the earliest humane endpoints that prevent further suffering, once it is clear that the scientific objective has been achieved, that the scientific objective cannot be achieved, or that the suffering is more than can be justified as part of the test procedure. The EC and Member States are also required (Article 23, 1) to encourage research into, and the development and validation of, alternative methods that do not require animals, use fewer animals, or further reduce the suffering that may be caused, whilst providing the same level of scientific information. 3. Recognised testing methods for marine biotoxins and maximum levels Commission Regulation (EC) No. 2074/ specifies a mouse bioassay (MBA) for the determination of paralytic shellfish poisoning toxins (PSP) and a MBA or the rat bioassay (RBA) for lipophilic marine biotoxins. Alternative test methods can be applied if they are validated following an internationally recognised protocol and provide an equivalent level of public health protection. Besides paralytic shellfish poisoning toxins, okadaic acid, dinophysistoxins, pectenotoxins, azaspiracids and yessotoxins, also cyclic imines, (gymnodimine, spirolides and others which are currently not regulated in the EU), all give a positive response in MBAs. The reference method for the domoic acid group (the causative agent of ASP) is based on highperformance liquid chromatography (HPLC). Chapter V (2) (c) and (e) of Section VII of Annex III to Regulation (EC) No 853/ establishes that food business operators must ensure that live bivalve molluscs placed on the market for human consumption must not contain marine biotoxins in total quantities (measured in the whole body or any part edible separately) that exceed the following limits: 800 micrograms per kilogram for paralytic shellfish poison (PSP), 20 milligrams of domoic acid per kilogram for amnesic shellfish poison (ASP), 160 micrograms of okadaic acid equivalents 9 per kilogram for okadaic acid, dinophysistoxins and pectenotoxins in combination, 1 milligram of yessotoxin equivalents per kilogram for yessotoxins, 160 micrograms of azaspiracid equivalents per kilogram for azaspiracids. 9 Equivalents: the amount of toxins expressed as the amount of okadaic acid that gives the same toxic response followed intraperitoneal administration to mice. This applies similarly for the group of yessotoxins and azapiracids, respectively. 6

7 4. Joint FAO/IOC/WHO ad hoc Expert Consultation on Biotoxins in Bivalve Molluscs (Oslo, September ) Based on the available information, the Joint FAO/IOC/WHO ad hoc Expert Consultation suggested provisional acute reference doses (ARfDs) 10 for the AZA, OA, STX, DA, and YTX-group toxins, respectively (summarised in the Table 1). The Expert Consultation considered that the database for the cyclic imines, brevetoxins and pectenotoxins was insufficient to establish provisional ARfDs for these three toxin groups. In addition, guidance levels were derived comparing results based on the consumption of 100 g, 250 g or 380 g shellfish meat by adults. However, the Expert Consultation noted that the standard portion of 100 g, which is occasionally used in risk assessment, is not adequate to assess an acute risk, whereas a portion of 250 g would cover 97.5 % of the consumers of most countries for which data were available. Available methods of analysis were reviewed for the 8 toxin groups and recommendations made for choice of a reference method, management of analytical results and development of standards and reference materials. The Joint FAO/IOC/WHO ad hoc Expert Consultation, however, did not have sufficient time to fully evaluate epidemiological data and to assess the effects of cooking or processing for deriving the provisional guidance levels/maximum levels for several toxin groups (especially the AZA and STX groups). The Consultation encouraged Member States to generate additional toxicological data in order to perform more accurate risk assessments and to facilitate validation of toxin detection methods in shellfish. 10 The acute reference dose is the estimate of the amount of substance in food, normally expressed on a body-weight basis (mg/kg or µg/kg of body weight), that can be ingested in a period of 24 hours or less without appreciable health risk to the consumer on the basis of all known facts at the time of evaluation (JMPR, 2002). 7

8 Table 1: Summary data used in the derivation of the ARfD and current guidance levels. Group toxin LOAEL(1) NOAEL(2) µg/kg body weight AZA 0.4 (1) 10 (H) BTX Cyclic Imines DA 1,000 (1) 10 (H) OA 1 (1) 3 (H) PTX STX 2 (1) 3 (H) YTX 5,000 (2) 100 (A) Safety Factor (Human data (H) Animal data (A)) Provisional Acute RfD µg/kg 2.4 µg/adult (a) N/A N/A Derived Guidance Level/ Max Level based on consumption of 100 g (1), 250 g (2) and 380 g (3) mg/kg SM (1) mg/kg SM (2) mg/kg SM (3) 60 mg/kg SM (1) 100 µg/kg 6 mg/adult (a) 24 mg/kg SM (2) 16 mg/kg SM (3) 0.33 µg/kg 0.2 mg/kg SM (1) mg/kg SM (2) µg/adult (a) 0.05 mg/kg SM (3) N/A 0.7 µg/kg 0.42 mg/kg SM (1) mg/kg SM (2) µg/adult (a) 0.11 mg/kg SM (3) 30 mg/kg SM (1) 50 µg/kg 3 mg/adult (a) 12 mg/kg SM (2) 8 mg/kg SM (3) Limit Value currently implemented in EU legislation 0.16 mg/kg SM 20 mg/kg SM 0.16 mg/kg SM 0.16 mg OA equivalents/kg SM 0.8 mg/kg SM 1 mg/kg SM SM = shellfish meat, LOAEL = lowest-observed-adverse-effect level, NOAEL = no-observed-adverse-effect level, N/A = not available, EU = European Union (a): Person with 60 kg body weight (b.w.) The Joint FAO/IOC/WHO ad hoc Expert Consultation also indicated that there were discrepancies between different risk assessments, especially for determining methods of analysis for certain marine biotoxins and in relation to established maximum limits. Test methods for the eight toxin groups were reviewed and recommendations for Codex purposes made. Mouse bioassays are widely used for shellfish testing but for technical and ethical reasons it is highly desirable to move to new technologies which can meet Codex requirements more adequately. Most currently available methods do not meet fully the strict criteria for Codex type II 11 or III 12 methods and have therefore not been widely used in routine shellfish monitoring. However, the recommendations made by the Expert Consultation represent the best currently available methods. Liquid chromatography-mass spectrometry (LC-MS) has much potential for multi-toxin analysis and has been recommended for consideration and recommendation by Codex. The Joint FAO/IOC/WHO ad hoc Expert Consultation is of the opinion that the complexity and chemical diversity of some toxin groups is such that validated quantitative methods to measure all toxins within a group will be extremely difficult. Thus the implementation of a marker compound concept and the use of functional assays should be explored. 11 A Type II method is the one designated Reference Method where Type I methods do not apply. It should be selected from Type III methods (as defined below). It should be recommended for use in cases of dispute and for calibration purposes. 12 A Type III Method is one which meets the criteria required by the Codex Committee on Methods of Analysis and Sampling for methods that may be used for control, inspection or regulatory purposes. 8

9 5. Working Group Meeting to Assess the Advice from the Joint FAO/IOC/WHO ad hoc Expert Consultation on Biotoxins in Bivalve Molluscs, Ottawa, Canada, April 10-12, 2006 The working group (WG) discussed available reference methods in particular and concluded that they should be highly specific, highly reproducible, and not prone to false positives or false negatives. The methods are expected to be definitive and may well result in significant rejections of products and must therefore withstand the most robust legal and scientific scrutiny. In considering their weaknesses and merits, the meeting noted that the various mouse bioassays should be discussed individually since the level of performance and success differs markedly between the official method for PSP by mouse bioassay, the American Public Health Association (APHA) method for brevetoxins and the multiple mouse bioassay DSP procedures employed for the other lipophilic toxins such as okadaic acid, azaspiracids and others. Recognizing that the majority of the currently available methods do not meet all Codex criteria for reference methods (Type II), the WG concluded that Codex Committee for Fish and Fishery Products (CCFFP) should consider a variety of biotoxin analytical methods. Wherever possible, reference methods should not be based on animal bioassays. Functional methods, biochemical/immunological and chemical-analytical methods currently in use, and considered to be validated according to Codex standards, should be recommended by CCFFP to the Codex Committee on Methods of Analysis and Sampling (CCMAS) for review and designation as Type II or Type III methods. Because the Expert Consultation has offered 3 different guidance limits associated with three levels of consumption (100 g, 250 g and 380 g) for most toxin groups, it is important to determine which consumption level is appropriate for the protection of consumers. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION In accordance with Art. 29 (1) (a) of Regulation (EC) No 178/2002, the Commission asks EFSA to assess the current EU limits with regard to human health and methods of analysis for various marine biotoxins as established in the EU legislation, including new emerging toxins, in particular in the light of - the report of the Joint FAO/IOC/WHO ad hoc Expert Consultation on Biotoxins in Bivalve Molluscs (Oslo, September ), including the ARfDs and guidance levels proposed by the Expert Consultation, - the conclusions of the CCFFP working group held in Ottawa in April 2006, - the publication of the report and recommendations of the joint European Centre for the Validation of Alternative Methods (ECVAM)/DG SANCO Workshop, January 2005, - the report from CRL Working group on Toxicology in Cesenatico October 2005, - any other scientific information of relevance for the assessment of the risk of marine biotoxins in shellfish for human health. 9

10 ASSESSMENT 1. Introduction Palytoxin (PlTX)-group toxins have mainly been detected in marine zoanthids (soft corals) of the genus Palythoa (e.g. Palythoa toxica, P. tuberculosa, P.vestitus, P. mammilosa, P.carobaeorum, P. aff. Margaritae) and benthic dinoflagellates of the genus Ostreopsis (e.g Ostreopsis siamensis, O. mascarenensis, O. ovata). They were first reported in Hawaii and Japan but are currently distributed worldwide (Moore and Bartolini, 1981; Deeds and Schwartz, 2009). Blooms of Ostreopsis spp. have also been reported in European countries (Ciminiello et al., 2009), such as in France, Greece, Italy and Spain. Occurrence of Ostreopsis spp. may result in contamination of shellfish intended for human consumption, e.g. bivalve molluscs. O. ovata mainly produces ovatoxin-a and traces of PlTX. Signs and symptoms of PlTX-group toxins intoxication are not well-defined, but include myalgia and weakness, possibly accompanied by fever, nausea and vomiting. Fatalities appear to be rare although there are reports of severe cases, in which patients died after about 15 hours (Deeds and Schwartz, 2009). Currently there are no regulations on PlTX-group toxins in shellfish, either in the European Union (EU), or in other regions of the world. 2. Chemical characteristics PlTX-group toxins are complex polyhydroxylated compounds with both lipophilic and hydrophilic areas. The basic molecule consists of a long, partially unsaturated aliphatic backbone containing cyclic ethers, 64 chiral centers, hydroxyl and 2 amide groups (Figure 1). The primary amino-group at the C-115 end of the molecule accounts for the basicity of PlTX-group toxins (Katikou, 2007). The molecular formula and molecular weight of PlTX analogues differ depending on the Palythoa species from which they are obtained (Moore and Bartolini, 1981). The chemical formula of PlTX from P. toxica is C 129 H 233 N 3 O 54, 115 of the 129 carbons being in continuous chain. Its molecular weight is 2,677 Da and it was found to exist as a dimer in aqueous solution (Uemura, 2006). Due to their two chromophores, PlTX-group toxins exhibit an ultraviolet (UV) absorption spectrum with a λ max at 233 and 263 nm. PlTX-group toxins are insoluble in nonpolar solvents, sparingly soluble in methanol and ethanol and soluble in pyridine, dimethyl sulfoxide and water. They are heat resistant. In addition to PlTX, other toxins of the same group are produced by several Ostreopsis species. For example O. siamensis produces the major toxic constituent ostreocin-d which is a chemical analogue of PlTX, also referred to as 42-hydroxy-3,26-dimethyl-19,44-dideoxypalytoxin. For these two compounds preparative isolation and characterisation by nuclear magnetic resonance (NMR) has allowed elucidation of the planar structure. Five further analogues have been found to co-occur with PlTX in P. tuberculosa, e.g homopalytoxin, bishomopalytoxin, neopalytoxin, deopalytoxin and 42-hydroxypalytoxin, but have not been reported to be produced by Ostreopsis spp. (Uemura et al., 1985; Ciminiello et al. 2009). O.ovata produces ovatoxin-a, for which the molecular mass (2647,5 Da) and structural evidence from mass spectrometry show that it is an analogue of PlTX. Ovatoxin A has two oxygen atoms less than PlTX but it is not known from which part of the molecule the oxygen atoms are missing (Ciminiello et al. 2006, 2008; Guerrini et al., 2009). 10

11 3. Regulatory status Currently there are no regulations on PlTX-group toxins in shellfish, either in the EU, or in other regions of the world. During the first meeting of the working group on Toxicology of the national reference laboratories (NRLs) for Marine Biotoxins (Cesenatico, Italy, October 2005), a provisional limit of 250 µg/kg shellfish was proposed (CRLMB, 2005). 4. Methods of analysis Several published methods exist for the determination of PlTX-group toxins. The mouse bioassay (MBA) is considered a simple way to detect PlTX-group toxins, but for ethical and scientific reasons there are growing concerns with respect to its use. Some functional assays and chemical methods have also been developed. Most of these methods were developed for monitoring PlTX in seawater and phytoplankton (Arzul et al., 1994; Habermann et al., 1981; Igarashi et al., 1999; Neely and Campbell, 2006; Oda et al., 2001; Paul et al., 1995; Rangel et al., 1997) and they have found application for the analysis of shellfish. However, standards are not readily available for PlTX-group toxins, which hampers the method development. None of the methods to determine PlTX-group toxins have been formally validated in interlaboratory validation studies Supply of appropriate reference material There is a lack of certified standards and certified reference materials for PlTX-group toxins Mammalian bioassay Mouse bioassay The MBA has been used to detect PlTX-group toxins in fish and shellfish tissues, e.g mussels, oysters, scallops and clams. Two different extraction protocols have been used for the MBA to detect PlTXgroup toxins. The first protocol, developed by Yasumoto et al. (1978) is an investigative tool for the determination of the causative agents responsible for a food poisoning outbreak associated with the consumption of molluscs in Japan, and uses acetone extraction of whole flesh or hepatopancreas of shellfish followed by evaporation and resuspension of the residue in a 1 % solution of Tween 60 surfactant. Taniyama et al. (2002) proposed a modified protocol using 75 % aqueous ethanol for extraction of shellfish whole flesh and defattening with diethylether, after removal of the ethanol. This second protocol was found to be more selective for detection of PlTX-group toxins since it allows for discrimination between samples containing okadaic acid (OA) and its analogues, and those containing PlTX-group toxins (Aligizaki et al., 2008). Due to the partitioning behaviour of PlTX-group toxins, as outlined in Aligizaki et al. (2008), the EU harmonised protocol of the MBA (Yasumoto et al., 1985) does not efficiently extract PlTX-group toxins. In the MBA typical signs for PlTX include jerking and stretching of hind limbs and lower back, weakening of fore limbs, creeping paralysis, ataxia, cyanosis, decreased locomotion, convulsion, gasping for breath and death within 15 minutes. The reported values for the intraperitoneal (i.p.) lethal dose ( LD 50 ) of PlTX in mice range from 150 to 720 ng/kg body weight (b.w.) (Onuma et al, 1999; Rhodes et al., 2002; Riobó et al., 2008). The main advantages of the MBA are: the provision of a measure of total toxicity based on the biological response of the animal to the toxin(s); it does not require complex analytical equipment. 11

12 Figure 1: Chemical structures of the palytoxin (PlTX)-group toxins, PlTX and ostreocin-d. 12

13 The main disadvantages of the MBA are: a MBA protocol, different from the Yasumoto et al. (1985) protocol is needed to efficiently extract PlTX-group toxins; if the Yasumoto et al. (1978) protocol is used, multiple interferences may occur from domoic acid (DA), saxitoxin (STX)-group toxins, yessotoxin (YTX)-group toxins and cyclic imines (CI); if the Taniyama et al. (2002) protocol is used, interferences can be reduced, but still include YTX-group toxins and the water-soluble toxins; it cannot be automated; it requires specialised animal facilities and expertise; the inherent variability in results between laboratories due to e.g. specific animal characteristics (strain, sex, age, weight, general state of health, diet, stress); the injection volume of one ml exceeds good practice guidelines (<0.5 ml) intended to minimise stress to mice; in many countries the use of the MBA is considered undesirable for ethical reasons Biomolecular methods Biomolecular methods for PlTX-group toxins are based on two different approaches using cell death or antibodies Cytotoxicity assays Haemolysis assay (erythrocytes) Haemolysis assays for the detection of PlTX-group toxins in algal samples and in shellfish are based on the capacity of the toxins to interact with the Na + /K + -ATPase converting it into a non specific cation channel. This leads to ion imbalance in the red blood cell resulting in delayed haemolysis. The characteristic delayed lysis and the suppression of haemolytic activity of samples after pretreatment with ouabain (an ATPase blocker), are indicative of haemolytic compounds in the sample. Several studies (Bignami, 1993; Taniyama et al., 2002, 2003; Riobó et al., 2006, 2008; Lenoir et al., 2004; Aligizaki et al., 2008) used a haemolysis neutralisation assay (HNA) including ouabain to estimate the PlTX-group toxin content of algal, fish or shellfish extracts. The assay developed by Aligizaki et al. (2008) can detect PlTX-group toxins at concentrations equivalent to 1.6 ng PlTX/kg shellfish tissue. The main advantages of haemolysis assays are: they have good detection capabilities; they are capable of detecting all biologically active PlTX-group toxins that convert the Na + /K + -ATPase into a non specific cation channel; the use of microplates enables multiple samples to be analysed in a single run. The main disadvantages of haemolysis assays are: they do not provide any information on the toxin profile; they may show interference from other haemolytic compounds possibly occurring in the marine environment. 13

14 Assays with MCF-7 cells Bellocci et al. (2008a) developed a cytotoxicity assay using the MCF-7 breast cancer cell line, where the extent of cell lysis is measured by the release of lactate dehydrogenase (LDH) in the culture supernatant. In this assay cells are exposed to materials to be analysed for the presence of toxins for a short time (1 hour), the culture medium is then removed and cell lysis is induced by the addition of a phosphate buffered saline solution. The removal of culture medium before induction of cell lysis renders the assay more selective for PlTX, allowing discrimination between components acting through the Na+/K+-ATPase, and those that cause cell lysis and/or display cytotoxic activity through a different mechanism of action (Bellocci et al., 2008a). In addition, the removal of culture medium from the cell monolayer at the end of the first incubation is accompanied by the disposal of dead cells and other cellular contents that could have been released into the culture medium, and therefore could be responsible for false positives, due to subsequent detection of LDH activity in culture supernatants. The assay developed by Bellocci et al. (2008a,b) can detect PlTX-group toxins at concentrations equivalent to about 10 ng PlTX/kg shellfish tissue. The main advantages of cytotoxicity assays using MCF-7cells are: they have good detection capabilities; they are capable of detecting all biologically active PlTX-group toxins that convert the Na+/K+-ATPase into a non specific cation channel; the use of microplates enables multiple samples to be analysed in a single run; they do not show interference from other haemolytic and or cytotoxic compounds possibly occurring in the marine environment. The main disadvantages of the cytotoxicity assays using MCF-7cells are: facilities are needed for maintenance and handling of cell cultures; they do not provide any information on the toxin profile Assays with neuroblastoma cells Cytotoxicity assays involving the use of neuroblastoma cells and including ouabain pretreatment were developed by several groups (Cañete and Diogène, 2008; Ledreux et al. (2009) and Espiña et al. (2009). Cañete and Diogène (2008) and Ledreux et al. (2009) used Neuro-2a cell-based bioassays in which the effect of PlTX-group toxins was estimated by using the 1-(4,5-dimethylthiazol-2-yl)- 3.5-diphenylformazan (MTT) assay for mitochondrial oxido-reductase activity, as an indicator for cell number. A similar assay has been developed by Espiña et al. (2009), using BE(2)-M17 human neuroblastoma cells and adding alamar blue to measure the mitochondrial oxido-reductase activity. The assays developed by Ledreux et al. (2009) and Espiña et al. (2009) can detect PlTX-group toxins at concentrations equivalent to about 50 μg PlTX/kg shellfish tissue. The main advantages of the cytotoxicity assays using neuroblastoma cells are: they have appreciable detection capabilities; the use of microplate format enables multiple samples to be analysed in a single run. The main disadvantages of cytotoxicity assays using neuroblastoma cells are: facilities are needed for maintenance and handling of cell cultures; they do not provide any information on the toxin profile; they are prone to interferences from other toxins (e.g. OA, azaspiracid (AZA)-, YTX-group toxins etc). 14

15 Immunoassays Several antibody-based methods have been used for the detection of PlTX-group toxins, but there have been no recent developments. Levine et al. (1988) developed a radio-immunoassay for the detection of PlTX-group toxins. Bignami et al. (1992) developed five PlTX-specific enzyme-linked immunosorbentassays (ELISA) for the determination of PlTX-group toxins in crude extracts of P. tuberculosa. The authors reported that the ELISA methods, although having low detection capabilities, might be subject to matrix interferences. Whether or not matrix interferences could be reduced by dilution is unknown. There is no information about the detection capabilities in shellfish, although the reported limits of detection (LODs) of ELISA methods using PlTX standard were about 1 ng/ml in the assay (Bignami et al., 1992). The main advantages of the antibody-based methods are: they are fast, easy to use, and can be applied to screen many samples for possible further confirmatory analysis. The main disadvantages of the antibody-based methods are: antibodies are not readily available; detection capability for PlTX-group toxins in shellfish tissues needs to be clarified; they do not provide any information on the toxin profile; the accuracy is questionable as the cross-reactivity does not necessarily reflect toxic activity Chemical methods Several chemical methods are available for the determination of PlTX-group toxins: high performance liquid chromatographic (HPLC) methods with fluorescence detection (FLD) or UV detection, liquid chromatography-mass spectrometric (LC-MS) methods and capillary electrophoresis. Extraction methods for several marine biotoxins in shellfish are available but there is no information on their efficiency for PlTX-group toxins HPLC-based methods HPLC-FLD Riobó et al. (2006) developed a method based on a pre-column derivatisation and solid phase extraction (SPE) clean-up followed by HPLC coupled to FLD and applied it for the determination and quantification of PlTX-group toxins in benthic dinoflagellates of the genus Ostreopsis. The results correlated well with those obtained with a haemolysis assay. The instrumental LOD for derivatised PlTX was 0.75 ng standard injected. This instrumental LOD cannot be translated into a LOD for the whole analytical procedure as the method has not been applied to shellfish tissue yet. The main advantage of the HPLC-FLD method is: it can be automated. The main disadvantages of the HPLC-FLD method are: it has not been applied to shellfish tissues yet; LOD and limit of quantification (LOQ) information in shellfish tissues is lacking HPLC-UV detection PlTX exhibits two UV-absorption peaks (at 233 and 263 nm). HPLC-UV methods have been widely used for the determination of PlTX-group toxins, applying different combinations of columns and mobile phases (Lenoir et al., 2004; Mereish et al., 1991; Oku et al., 2004; Penna et al., 2005; Riobó et al., 2006; Yasumoto et al., 1986). HPLC-UV methods have mainly been used for analysis of 15

16 Ostreopsis extracts and, besides the method reported by Yasumoto et al. (1986) concerning determination of crab toxins, no HPLC-UV methods have been published to date for the determination of PlTX-group toxins in shellfish tissues. Reported LODs range from 0.1 to 2 µg injected and are much higher than reported LODs for other chemical methods. The main advantages of the HPLC-UV methods are: they can be automated; they are relatively inexpensive and easy to use. The main disadvantage of HPLC-UV methods is: they are about 1000-fold less sensitive than the liquid chromatography-fluorescence detection (LC-FLD) methods, which makes them inappropriate for the detection of PlTX-group toxins in shellfish tissues LC-MS/MS Different LC-MS/MS methods have been used for identification and quantification of PlTX-group toxins in seawater and phytoplankton (Lenoir et al., 2004; Penna et al., 2005; Riobó et al., 2006; Ciminello et al., 2006, 2008). Riobó et al. (2006) used FLD and MS/MS detection for analysis of PlTX-group toxins in the benthic dinoflagellate of the genus Ostreopsis. The authors showed that the LODs and LOQs of the liquid chromatography-mass spectrometry/mas spectrometry (LC-MS/MS) method were significantly higher than those reported for the LC-FLD method. This may be due to instrumental limitations when using ion trap systems in MS-MS mode over a broad mass range. The detection capability of the LC-MS/MS methods was significantly improved using a triple quadrupole (Ciminiello et al., 2006). This method was applied for the determination of PlTX-group toxins in seawater, plankton and macroalgal extracts (Ciminiello et al., 2006, 2008). Although LC-MS/MS methods are being used by official control laboratories for the quantitative determination of PlTXgroup toxins in shellfish, no validation studies on LC-MS/MS methods have been published to date and detailed performance characteristics of these methods are thus unknown. The LODs of LC-MS/MS methods for PlTX in shellfish were reported to be 2 and 36 µg/kg in France and Italy, respectively (Table 2, with differences probably due to differences in instrumentation used and the French protocol being based on analysis of hepatopancreas in mussels and roe in sea urchins). The LOQs were reported as 9 and 120 µg/kg in France and Italy, respectively. The main advantages of the LC-MS/MS methods are: they are rapid; they are sensitive, if triple quadrupole instruments are used; they can screen and measure PlTX-group toxins individually; they give information on the profile of PlTX-group toxins; they can be automated. The main disadvantages of the LC-MS/MS methods are: they require costly equipment and highly trained personnel; no validation studies have been published and detailed performance characteristics are not known High performance capillary electrophoresis As an alternative to poorly sensitive HPLC-UV methods for the determination of PlTX, high performance capillary electrophoresis (HPCE) methods with UV detection have been developed (Mereish et al., 1991; Lau et al., 1993). The LODs ranged from 0.25 to 0.5 pg/injection of PlTX solution, depending on the wavelength used (Mereish et al., 1991), but the methodology is not suitable for the analysis of shellfish, due to strong matrix effects. 16

17 The main advantage of the HPCE methods is: they can be automated. The main disadvantages of the HPCE methods are: they have not been developed for the analysis of PlTX in bivalve molluscs (and this may prove difficult due to matrix effects); they require highly trained personnel Proficiency tests PlTX is not a regulated toxin and there is no proficiency testing ongoing for this group of toxins Summary of methods The MBA has been used to detect PlTX-group toxins in fish and shellfish tissues, e.g mussels, oysters, scallops and clams, but for reasons of animal welfare and poor specificity there is a growing concern with respect to its use. Alternative assays taking advantage of certain PlTX functional properties have been developed. While cell based assays appear to have the lowest LOD for PlTX-group toxins, some of them showed interference from other toxins and positive results obtained with biological methods would require further confirmation by chemical methods. HPLC-FLD and LC-MS/MS methods could be valuable tools for the determination of PlTX-group toxins. The optimisation of these methods for application to shellfish extracts, their (inter-laboratory) validation and the development of standards and reference materials are necessary. 5. Levels of PlTX-group toxins in shellfish from contaminated areas 5.1. Data Collection EFSA issued in 2008 a call for data on the occurrence of PlTX-group toxins in European countries. Only Greece answered the call, providing analytical results for 11 targeted samples (including 10 mussels and 1 clam) analysed by a HNA and showing estimated values well above the LOD. Presently, PlTX-group toxins in shellfish do not have regulatory limits, and monitoring data are not available regarding this group of toxins. Nevertheless, Ostreopsis spp. have been in recent years increasingly found in the European Mediterranean countries as well as in other temperate areas worldwide (Shears and Ross, 2009). Monitoring plans and research projects on algae blooming have been initiated and some research projects have also been targeted to evaluate the possible presence of PlTX-group toxins in shellfish. Data obtained from some of these projects were included in the data collection. France 13 provided results for 19 sea urchin and 16 mussel samples, and Italy 14 for 21 sea urchin and 35 mussel samples. Overall, 102 samples from Greece 15, Italy 14 and France 13 were considered. The Greek samples were analysed by HNA while the samples from Italy and France were analysed by LC-MS/MS. All samples from Greece and Italy and sea urchins samples from France represented wild shellfish collected in contaminated regions during Ostreopsis spp. bloom. Mussel samples from France originated from experiments where uncontaminated mussels were intentionally placed in areas contaminated by 13 The data were obtained as part of the French national surveillance network for phytoplankton and phycotoxins (REPHY), as implemented by IFREMER, partly financed by the French Ministry for Food, Agriculture and Fisheries, Directorate General for Food, programme Italian monitoring project co-ordinated by Agenzia Regionale per la Protezione Ambientale della Campania (ARPAC). 15 National Reference Laboratory 17

18 Ostreopsis spp. in order to study the PlTX-group toxin uptake of the mussels. Table 2 gives an overview of the occurrence data for samples collected between 2004 and Table 2: Occurrence data reported on levels of PlTX-group toxins in contaminated areas of the Mediterranean sea (collected between 2004 and 2009). Country Year(s) of harvesting Number of samples Purpose of testing (a) Method of testing LOD (µg/kg) LOQ (µg/kg) Greece France research, pre-mc research, pre-mc HNA LC-MS/MS 2 9 Italy research, pre-mc LC-MS/MS Total 102 Pre-MC: pre-market control; HNA: haemolysis neutralisation assay; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LOD: limit of detection; LOQ: limit of quantification; (a): PreMC are samples collected at the place of origin, before or during harvesting. In the case of the French samples the digestive gland (hepatopancreas) and remaining tissue were analysed in mussels, and digestive tube and roe in sea urchins. The results were converted to whole shellfish by the reporting laboratory. This procedure generally allows the sensitivity of the method to be improved. In the samples from Italy the whole shellfish was analysed. This fact, together with differences in instrumentation, may explain the different LODs and LOQs shown by the two LC-MS/MS datasets Statistical description of PlTX-group toxins in shellfish Two different and not directly comparable analytical methods were used: HNA and LC-MS/MS. HNA determines the haemolytic activity of PlTX-group toxins present in the sample, whereas LC-MS/MS can detect separately the different PlTX-group compounds. Italy and France reported PlTX and ovatoxin-a for the samples analysed by LC-MS/MS. The LC-MS/MS method can also detect ostreocin-d but so far only PlTX and ovatoxin-a have been found. PlTX-group toxin concentrations were reported as a sum, assuming identical instrument response factors. The bounding approach was applied for values reported below LOD or below LOQ in order to identify the possible range of the data. The lower bound (LB) is obtained by assigning a value of zero (minimum possible value) to all the samples reported as <LOD or <LOQ. The upper bound (UB) is obtained by assigning the value of LOD to values reported as <LOD and LOQ to values reported as <LOQ (maximum possible value). Table 3 shows the statistical descriptors for the available data grouped by quantified PlTX-group toxins and analytical techniques. 18