EMERGING MARINE BIOTOXINS report from the seminar 2012

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1 EMERGING MARINE BIOTOXINS report from the seminar 2012 Mirjana Andjelkovic Marine biotoxins (also called phycotoxins) are produced by certain phytoplankton species (diatoms and dinoflagellates) and can accumulate in various marine species such as fish, crabs or filter feeding bivalves (shellfish) such as mussels, oysters, scallops and clams. In shellfish, toxins mainly accumulate in the digestive glands without causing adverse effects on the shellfish itself. However, when substantial amounts of contaminated shellfish are consumed by humans this may cause severe intoxication. Approximately 60,000 human intoxications yearly with overall mortality of approximately 1.5% are related to toxins produced by algae (including freshwater cyano toxins) (1). This is both public health and economic problem. The necessary regular monitoring of specifically susceptible sites has a serious economic impact, moreover if the site gets closed during harvest, collection of that year as well as that of the following one can be hindered. In the case that toxic products make it to market resulting in fatalities or illness, consumers need to be compensated. Currently five groups of marine toxins are regulated in Europe: amnesic shellfish poisoning toxins, paralytic shellfish poisoning toxins, okadaic acid and pectenotoxin group, azaspiracid group, and yessotoxin group (EC/853/2004). Another group, neurotic shellfish poisoning toxins (e.g. brevetoxin) is regulated in US, New Zealand and Australia. These toxins are regularly produced in the Gulf of Mexico, particularly in coastal regions of southwest Florida. Regulated toxins have been for years detected by bioassays. Recently effort is made to accept HPLC and more specifically LCMS based detection methods as reference methods due to their increased international use and sensitivity. Their implementation in both, research and routine monitoring, led to detection of new marine toxins and toxin congeners. These are seen as emerging marine toxins. They are not on the radar of the European legislation or in other regions of the world. Scientists and legislative authorities are aware of these toxins but do not have sufficient amount of information to: 1. elucidate the activity of all unknown toxins; 2. estimate the toxicity to humans; 3. evaluate risk to humans. The emerging group of marine biotoxins have been principally monitored in shellfish. Countries as France, Italy, Spain and Norway report mostly palytoxins (PlTX, ostreocin-d, ovatoxin-a, homopalytoxin, bishomopalytoxin, neopalytoxin, deopalytoxin and 42-hydroxypalytoxin) and cyclic imines [spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs) and pteriatoxins (PtTXs)]. Toxin levels of some cyclic imines vary from ng/g (2) to ng/g (3) in shellfish from France and Italy, respectively, whereas reported values for Spanish mussels were in the range of ng/g (4). Putative palytoxin was for the first time detected in Italian waters at 1,350 ng for plankton pellet and 1,950 ng for butanol extract. It was therefore suggested that this toxin was the causative agent responsible for the Genoa 2005 outbreak of respiratory illness in people exposed to marine aerosols. Therefore some of these toxins are not only a concern of consumers but also may be seen as a public health problem related to environment. The cyclic imines group of toxins have been identified as nicotinic receptor blockers and are recognised as fast acting toxins. PlTX is one of the most toxic non-peptide substances known, showing remarkable biological activity even at very low concentration. This toxin and its analogs have become a global 30

2 concern due to the poison s effects on animals and especially on humans. On the other hand, there are more frequent reports on ciguatera fish poisoning including frequent fatalities. Ciguatera fish poisoning is caused by the consumption of coral reef fish contaminated by ciguatoxin and related toxins from dinoflagellates (microalgae) and cyanobacteria. This poisoning, which is a prevalent tropical and subtropical disease, has been long known but now increasing number of incidences makes it more worldwide problem. Likewise the increasing analytical possibilities make it possible to respond the need to know and control it. At the recent Seminar on emerging marine toxins in Vigo, Spain during two days the experts were discussing these groups of toxins. The European reference laboratory of marine toxins hosted the meeting at the new location which was also an official opening of the labs. The lecturer session was opened with the talk of prof dr T. Yasumoto a leading figure in marine toxin research. His group in Tokio is currently collecting data and making a database of fish species related to ciguatera fish poisoning in Pacific waters. They are extracting the toxins and working on the production of the reference material which would enable detection of the toxins. The material will be available even this year for a limited number of laboratories. The conclusions of prof Yasumoto are that ciguatoxins are region- and fish species specific but the intraspecies variations were small. While some congeners like CTX1B type toxins are dominant in Okinawa (south of Japan), CTX3C type is more present Miyazaki (south-west part), and in fish from Marcus Island toxins of both types. Moreover the tendency of finding this toxins in northern part of Japan is rising. The lab is performing the chemical analysis (LC-MS/MS) of all the material with low sensitivity. Seriousness of the ciguatera toxins in Canary Islands was further presented during round table sessions. Madeira Island (Portugal) was also pointed as a problematic spot for these toxins. Nevertheless, other countries also face the problem due to import or shipping of fish caught during sport fishing in Caribbean. Since 2004 there are some episodes of ciguatera toxins implicating big amberjack fish (Seriola sp) and affecting around 70 people. Several causative possibilities were listed. Pollution, climate change have been already brought in correlation with toxic algal blooms. Similarly, the Canary Islands authorities and scientists are relating the occurrence to climate change, inflow of balast water of incoming boats and ships, possibility of fish migration from Caribbean, or appearance of an algae foreign to those waters (Gambierdiscus excentricus). Official control program of the region covers since 2010 these toxins and has introduced some new control strategies in fish. The samples are immediately checked with immunoassay kit (Ciga-Check) and then referred to the lab for: mouse bioassay, cytotoxicity test and additionally confirmed by LC-MS/MS in the EURL-MB. The big disadvantage in the efforts is the detection method which is not sensitive enough. To improve its performance the standard should be developed and an interlaboratory validation study should be organised. Studies of these toxins were pointed out as being of common interest and topic for the future work in many European countries. Following group of toxins were palytoxins on which prof. dr Aurelia Tubaro from University of Trieste gave some insights. She performs an extensive work on toxicity of palytoxins. Research with mice as well as cell cultures delivered the knowledge on the dose of these highly toxic compounds. It is noticed that already at 30 µg/kg dose functional impairment of skeletal and myocardial muscles can be seen. However prof Tubaro stressed that the knowledge on pharmacokinetics, repeated oral toxicity (basis for NOAEL) as well as assessment of cardiotoxicity of palytoxins but also ovatoxins is scarce. It is noteworthy to mention here the symptoms of the intoxication with those compounds. Experimental animals exerted the behaviour as: scratching, jumping, respiratory distress and paralysis; if the animal would recover it would usually lose weight. 31

3 Palytoxins were mentioned several times during the seminar both due to their high toxicity, and allergic-like reaction that they may trigger in persons handling them in the lab or tourist and inhabitants around the area with algal bloom. There are hypotheses that some other components, like some volatile compounds released by microalgae (Ostreopsis) can be blamed for these reactions. Northern European countries are also facing some toxins that were previously mainly recognised to appear in warmer waters. Norway is reporting since 2009 presence of spirolides produced by Alexandrium ostendfeldi and pinnatoxins produced by Vulcanodinium rugosum. The latter group of toxins is proven to have more congeners with two formed (PnTx F and G) in algae and the other metabolising out of this two via hydrolysis in shellfish. Pinnatoxins are found all over the coast of Norway in low levels but with only one hot spot where concentrations of the toxins may be higher. Dr Chris Miles (Norwegian Veterinary Institute) presented the actions and objectives that Norway has to overcome this problem. According to him a monitoring program for these toxins is in place already for some years. The shellfish and non-shellfish species are monitored (blue mussel, European oysters, scallops, cockles, horse mussel, sea snail, crabs, and sea worms) by SPATT discs. The authority is very interested in decoding the pattern of hot spot locations, to answer the question why are these toxins present all year around, and characterising the causative organism or whether a mixture of organisms could be responsible for this subtropic occurrence in northern waters. Likewise, dr P. Hess (IFREMER) reported presence of pinnatoxins all year around in low concentrations (PnTx G around 100 mg/kg) in some specific areas of France. These are warm lagoons. The situation is monitored via Official Control Program and a scientific project in collaboration with an institute in New Zealand. Although there are no reports of human illness due to SPXs, GYMs, PnTXs or PtTXs episodes of toxicity, some cases involving nonspecific symptoms such as gastric distress and tachycardia were recorded in individuals in Nova Scotia, Canada consuming shellfish during times when SPXs were known to be present. However these could not be definitively ascribed to SPXs since they were not consistent with the signs of toxicity shown in mice. These toxins are seen as very toxic with LD50 of 10 µg/g. The cyclic imines may be analysed by bio-analytical and physicochemical methods to describe the active mechanism as explained by dr R.Araoz (National Research Centre, France). They act on nicotininc acetylcholine receptors which can be present in the brain. Decline, disruption or alterations of nicotininc cholinergic mechanisms contribute to dysfunctions such as epilepsy, schizophrenia, Parkinson s disease, autism, dementia, Alzheimer s disease and addiction. The neurotoxic doses of PnTx and SPX is comparable to those of paralytic shellfish poisoning. Since these toxins are not regulated and less known all information is necessary and functional methods would provide many answers. However to overcome lack of sensitivity of e.g. receptor binding assay these test should be coupled to MS. The management of the emerging toxins may be explained as contemporary approach (dr J.Diogene, IRTA). The classical approach on toxins is based on the toxic episodes and experience that scientist got while the contemporary approach lies on the understanding the source of toxins, vectors of intoxications, structure and risk assessment of new occurrences. Therefore we make difference between known and classical toxins that are regulated and those which are non-regulated; between local and foreign and those with identified source and unknown source. 32

4 It can be concluded that there is no list of emerging toxins partially due to interpretation of emergent in the local context. However a lot was said about: palytoxins, ovatoxins, brevetoxins, cyclic imines (gymnodimins, pinnatoxins, pteriatoxins and spirolides), azaspiracids and ciguatera toxins. What is necessary is the systematic identification of toxin vectors and the toxins. The evolution from the classical approach to the contemporary is enabled by technical advances and awareness when conducting monitoring program. Finally, at the end of symposium and a meeting of a special working group formed on emerging toxins, participants came to a conclusion that ciguatera toxins may be a topic of common efforts for validating a detection method in an international validation study. This work would include work on reference materials and their easier availability. Mirjana Andjelkovic, WIV-ISP Brussels Mirjana.Andjelkovic@wiv-isp.be References 1. Lina Kantiani & Marta Llorca & Josep Sanchís & Marinella Farré & Damià Barceló. Emerging food contaminants: a review; Anal Bioanal Chem (2010). 398: ) 2. Amzil Z, Sibat M, Royer F, Masson N, Abadie E. Report on the First Detection of Pectenotoxin-2, Spirolide-A and Their Derivatives in French Shellfish (2007) Marine Drugs 5: Ciminiello P, Dell Aversano C, Fattorusso E, Forino M, Tartaglione L, Boschetti L, Rubini S, Cangini M, Pigozzi S, Poletti R. Complex toxin profile of Mytilus galloprovincialis from the Adriatic sea revealed by LC MS (2010) Toxicon 55: González AV, Rodríguez-Velasco ML, Ben-Gigirey B, Botana LM. First evidence of spirolides in Spanish shellfish (2006) Toxicon 48:

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Testing for Marine Toxins and DSP

Canadian DSP Symposium Vancouver, BC November 27, 2012 Testing for Marine Toxins and DSP Wade Rourke Canadian Food Inspection Agency Dartmouth Laboratory Dartmouth, NS 2007 Her Majesty the Queen in right