Download EMERGING MARINE BIOTOXINS – report from the seminar 2012

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 2–585 ng/g (2) to
11–7950 ng/g (3) in shellfish from France and Italy, respectively, whereas reported values for Spanish mussels
were in the range of 13–20 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
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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.
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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.
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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
[email protected]
References
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