Lezioni, Tossicologia marina
Toxic algae (microalgae) are microscopic unicellular organisms that live in water bodies. The major part of
algae are producers on the food chain and without them all the rest of the food chain couldn't exist. There
are also food for molluscs like oysters, mussels and clams but also for crustacean and fish larvae.
Occasionally, these algae can grow very fast leading to blooms, so a copious proliferation that we usually
observe in summer on the surface of lake and little water bodies, but also into the sea. These blooms can
cause the phenomenon of eutrophication. Very famous blooms are the red tides, because of some algae
contain red pigments. The term “tide” is not really appropriate because it is not a real tide; also the color
can change, thus also the term “red” is inappropriate. Usually these big proliferations of planktonic algae
are useful for aquaculture, but in some cases they cause serious effects on environment, on human health
and on the commerce of aquaculture's products. There are now known about 5000 species of marine algae,
but just 300 of them can reach high concentration (>1 million cells/L) and only 80 can produce powerful
toxins. Some toxins are very powerful and 500-1000 ug can be lethal for a person; however, different toxins
have different power (and different LD50)
Red tides were very well known in the past. They were described in an opera of Exodus, that associated the
red tide to loss of blood. In 1793 Captain George Vancouver reported first algae poisoning during a trip in
British Columbia. Indigenous people didn't eat molluscs when water was lighting because of the presence of
bioluminescent algae. Both in Canada and USA there are endemic area for paralytic shellfish poisoning. You
can find some advertisement telling 'no collect shellfish' and so on.
Cycle of an algae. Cysts can germinate only during certain times of the year with warmer temperatures and
increased light stimulating germination. The cyst breaks open and a swimming cell emerge. The cell
reproduces by simple division within a few days (or hatching). If conditions remain optimal cells will
continue to divide, reproducing exponentially. A single cell can produce several hundreds of cells within a
couple of weeks. If other single cells reproduce similarly, then the toxicity in shellfish may occur. When
nutrients are gone, growth stops and gametes are formed. Two gametes join to form one cell, which
develops into a zygote and then into a cyst. The cyst fall to the ocean bottom and is capable of germination
There are 2 main types of algal proliferation: 1) that causes toxic effects in human beings, but they can
affect other organisms such as fishes; 2) that doesn't cause toxic effects in human beings. For example,
Noctiluca scintillans and Gonyaulax polygramma are not toxic for humans but they can cause death in
invertebrates and fishes subsequently to the oxygen depletion. Karenia mikimotoi (a dinoflagellate) and
Prymnesium parvum can cause serious damages in an aquaculture but these 2 species are not toxic for
humans. In the case of P. parvum, fishes are suffocated by the presence of a foam, produced by this
microalgae, that obstructs the gills; fish can not breath anymore. Also Karenia brevis can cause massive
fishes deaths: it is spread along the New Zealand and the Florida coast. If some people recognize a bloom of
this, have to inform immediately those responsible. Pfiesteria piscidica produces some toxins, in particular
two that affect seriously fishes. One is neurotoxic, the other one is dermotoxic and it can provoke dermatitis
with visible wounds. A massive death of pelicans and cormorants was detected along the west coast of
America and was attributed to Pseudonitzschia diatoms; they produced domoic acid, that passed through
the food chain until seabirds.
One dish of mussels can cause illness or even death if they contain the right toxin. Mussels filter water
continuously and all the compounds inside can be accumulated for this reason in shellfish you can have
huge amount of toxins. They can also transform toxins in compounds easily metabolized for them, but not
Algae and Shellfish Poisoning
usually, main vectors of this intoxication are molluscs. During the uptake of oxygen they open the valves
allowing the passage of big amount of water through the gills. The now filtered particulate passes to the
stomach, where the digestion begins. The substances useful for the mollusc are transferred into the
digestive gland. Filtering a lot of water, the mollusc will be exposed to anything is in the water: virus,
bacteria, toxic algae and so on. Since the principal food of mollusc is phytoplankton, if the filtered algae
produce toxins, the latter can be accumulated. As long the mollusc eat phytoplankton, the contamination
will be higher, moreover during an algae bloom. At the end of the bloom they start to excrete toxins until
they are not in the body anymore. In USA algae poisonings cover 7,4% of the overall marine intoxications.
Algae toxins represent a problem all over the world and there are more than 60000 cases of poisoning per
year. Although bacterial and viral intoxications in shellfish are more common, algae poisoning can be surely
more powerful, sometimes lethal, than them. When cooking mussels, some toxins are thermostable so they
are not eliminated. to recognize shellfish poisonings is quite difficult so proper data on the number of cases
are not available. We don't know actually the true percentage of sick people; for example, since ciguatera is
not lethal but it causes just acute diarrhea, a lot of cases are not registered.
Of the (about) 80 species that produce toxins, most of them belong to the taxon of Pyrrophyta, Dinofiaceae
class, commonly known as Peridineae or dinoflagellates. Toxins responsible of human intoxications are
produced mainly by two types of unicellular organisms: dinoflagellates and diatoms [and cyanobacteria].
Algae toxins are secondary metabolites that are produced only in certain taxonomic groups of microalgae.
The role of these metabolites is still not clear, maybe they can act as deterrent for predators. Production of
these metabolites can vary according to environmental factors. They are usually thermostable – so not well
destroyed with boiling – and passing through the food chain they can cause intoxications. If they find
optimal environmental conditions (nutrients, oxygen, temperature) in the sea they can bloom and be
filtered by shellfish. Biomonitoring systems allow for the block of harvest and commerce of contaminated
products (shellfish in aquaculture, for instance). Developing countries without monitoring system and
dependent from the sea for their feeding can have sanitary problems.
But, is there a real increase of algae toxins? Well, there are some reason because the answer can be NO.
Probably, a concomitance of factors can lead to a detected increase of algae poisonings:
Increase of scientific knowledge of toxic species;
• Increase on using coastal water for aquaculture;
• Increase of eutrophication;
• Unusual climate conditions;
• Transport through ballast waters of dinoflagellates and molluscs;
• Better monitoring system.
A continuous check of the quality of water is needed. When monitoring an aquaculture there can be found
several algae, more or less toxic, but the real thing to do is the analysis of cultivated molluscs because they
are the products that will be finish in the consumers' dish.
Ostreopsis ovata produces palytoxin, in particular ovatoxin. Environmental problem →
List of poisoning. Since 2000 maybe there is also palytoxin in the Adriatic sea, produced by bloom of
Ostreopsis (ovatoxin is an analog of palytoxin).
DSP – Diarrhetic Shellfish Poisoning
DSP is the main problem in Italy and is one of the most frequent poisoning. It was discovered in Japan in
1978 by Prof. Yasumoto. The last outbreak in Italy was in September 2010, in Turin and Aosta with more
than 300 cases of contamination caused by mussels coming from Trieste. In 99% of cases you have diarrhea
with a massive loss of water. Other symptoms are nausea, abdominal pain, vomit, weakness and chills.
Symptoms come out after 30 minutes up to some hours, rarely after 8-12 hours. Intensity of symptoms
depends on the assumed dose (at least 40 ug of OA or 36 of DTX1).
DSP is caused by okadaic acid and dinophysistoxins (DTX). There are three main types of DTXs identified
with numbers: DTX1, DTX2, DTX3. These 4 compounds are very similar and they differ only for the presence
of different substitutes in 3 positions. Once, DSP toxins were included with yessotoxins and pectenotoxins,
since they were extracted with OA and DTXs. Dinophysis fortii produces only OA and it was the responsible
for the first DSP outbreak in Italy. D. norvegica, D. acuta, D. acuminata and D. caudata produce mainly
OA; ??????? CHIEDO ALLA PROF PERCHE' NELL'ELENCO COMPAIONO PIU' VOLTE LE SPECIE. Prorocentrum
lima is sometimes (and not surely) responsible of DSP. Production of DTXs and OA depends on
environmental factors and on the geographic area.
The mechanism of action includes several compartment. The most quoted mechanism is the inhibition of
proteinphosphatase 1 and 2A (PP1 and PP2A). There is an increase on the phosphorylation of proteins that
control the secretion of Na from enterocytes; and of element of the cytoskeleton, that involved structural
modification of cells. The main result is the entrance of water in the gut, leading to diarrhea. In the case of
DTX1 there is also tumor promotion. Keep in mind that promoter needs an initiator, otherwise it cannot
work; moreover the promotion has to be continuous, thus with continuous exposures. The main target of
DTXs is the small intestine but in few cases can be involved even the liver. The thresholds for commerce are
160 ug of toxins (OA, DTXs and derivatives) per kg of mollusc meat.
The diagnosis of DSP is based on the anamnesis (the medical history) of patients. There are not available yet
specific diagnostic test. The minimum dose that provokes diarrhea in adult is 40 ug OA or 36 ug DTX1. The
diagnosis can be confused with microbial or azaspiracid intoxication that cause diarrhea too. For example,
the first case of DSP in Italy was confused with an azaspiracid poisoning. However, the treatment is the
same, i.e. the supply of minerals and water that the patient massively loses. The prognosis lasts 3-4 days,
without having pharmacological treatment. There is no a specific antidote, since the main symptom is
diarrhea. With regards to the promotion of tumor, OA and DTX1 are considered just weak promoters and is
still unknown their real role in this process. However, promoters need to be assumed several times
continuously and it is very rare, with the exception of countries that base their feeding on shellfish, that
someone eats several times a week mussels or clams. Microcystin is a stronger tumor promoter compared
OA better inhibits PP2A than PP1; the contrary is valid for DTX1.
The DSP is the main problem in Italy and in Spain along the coast (here also PSP, that can be lethal). DSP is
spread all over the world but the majority of cases are reported in Europe and in Japan, but there are some
new cases in USA. The first recognized case in Italy was in 1989, along the Emilia Romagna coast. It is likely
that beforehand there were some cases more, not recognized as DSP. Since 1989 there haven't been more
cases until 2010, when 300 people were intoxicated. From 1989 monitoring systems are doing their work;
there are spread along the Italian coast, with particular regard of Liguria, Adriatic sea and Sardinia.
People get, contract the intoxication eating molluscs like mussels (Mytilus galloprovincialis), clams (Tapes
decussatus, Venus gallina) and oysters (Ostrea edulis). Molluscs are not affected by the toxins and they
accumulate them in the digestive gland. There is no morphological change in a mollusc full of toxins.
Mytilus galloprovincialis is one of the most contaminated species. They are cultivated in ropes and they can
accumulate huge amount of toxins, mainly produced by Dynophysiaceae. This group moves as the
thermocline moves, in 10 cm. When a toxins or the putative presence of a toxin in a mollusc are recognized,
it starts a procedure that forbids the sale of the cultivated products and monitors the situation even after
the sale of products. These procedures are also ruled by the EU. Although these precautions, is not
uncommon to see people self-harvesting clams or other molluscs in the beach, and the intoxication can
derive also from these practices.
Toxins have to be individuated also at low concentration, even lower than 2 ppb. Some toxins have not a
chromophore, a part of the molecule by which it can be recognized; moreover there can be other
substances in the sample, covering the one we are trying to study. The optimal test should be rapid, able to
analyze a lot of sample in a brief time. It's important to have a zero point, a white sample, in order to
establish the quantity from which the toxin can be recognized. There are three main kinds of analyses:
Chemical analysis. HPLC is relatively slow and not used anymore, substituted by LC-MS (Liquid
• Chromatography – Mass Spectrometry), that is very rapid, although the cost, and it can recognize 1
ng of substance in 1 g of tissue. Moreover, there can be evaluate the presence of different toxins
together. Since 2011 LC-MS is operative in almost all European countries and it is preferred also to
some biological tests.
Biochemical analysis are classified into structural assays and functional assays. In the structural
• assays there is an interaction between a part of the toxin and a part of another molecule – for
instance, in the immune-enzymatic assay, the binding site of antibodies. The signal that comes out
is proportional to the amount of toxin in the sample. The most used structural assay is undoubtedly
ELISA. This test has a sensibility of 0,1 ug per g of tissue; they are rapid and easy to use. There are a
wide range of commercially available kit, but the disadvantage is that actually ELISA can recognize
just OA, DTX1 and DTX2. Another disadvantage of ELISA is the development of false positive,
because the part of toxin that bind the other molecule can be shared with other substances.
Functional assays rely on a biochemical action of the toxin and the amount of detected toxin is
usually proportional to its toxicity in vivo because this phosphatase binds with the same affinity. The
most used functional assay is that of inhibition of PP2A (more details below). The sensibility is 50
ng/g of tissue, and they are also available in commercial kits. Another, but not widely used,
biochemical test is the radio-immunological test, that involves the utilization of radionuclides.
Biological tests. The mouse bioassay has a sensibility of 200 ng/g tissue and it is cheap, rapid and
• easy; unfortunately it can produce false positive and recognized other 'lipophilic toxins', that can be
accumulated in the studied tissue. The mouse bioassay can be compared to an acute toxicity test.
In vivo test has the pro and con to show the overall effect of the substance, resulting from the
action of all the toxins, not only one. Advantages of mouse bioassay are: 1) it gives a measure of the
total toxicity, that correlate the response of the animal to the toxin; 2) it doesn't need particular
purification procedure of mussels neither particular machine tools; 3) it can reveal also other toxic
compound; 4) the toxicity detected is comparable to that on human. There are also many
disadvantage: 1) you need an animal house; 2) animals have not to be stressed and all in the same
condition (same weight, age, sex, etc.); 3) possible interference caused by substances extract with
the wanted toxins (false positive and false negative); 4) variability of the response between the
laboratories, and for this reason is needed an intercalibration among labs to assess the variability;
5) less precise than analytical method and less sensibility.
Otherwise one can carry out an in vitro test of cytotoxicity; most of time the cytotoxicity is too high
and you cannot recognize the effect of a toxin from the one of another toxin.
DTX3 is usually the product of transformation of DTX1, DTX2 and OA that mussels try to detoxify, so it is not
present in algae but just a product of mussels metabolism! In vitro, DTX3 cannot be more metabolized. In
USA there has been developed a quickly immuno-based test, usable directly after the harvest of mussels on
Acute toxicity tests must be performed under the same environmental condition:
T 22 +/- 1 °C;
Relative humidity 60-70%
Light-dark cycle of 12 hours. Differences in length of days and quantity of light can provoke
Controlled quality of administered water and food;
Rodents are kept in the animal house for one week before the test. They are divided for sex, possible not
more of 3 individuals per cage, to avoid fight between males. You can build up a self-made calibration curve
(death time and concentration of toxin on the axis), using logically the same animals (same sex, age,
standard and laboratory conditions); that curve will be utilized just for the actual test. In order to get results
as best as possible is useful to carry out several analysis in combo, as LC-MS and mouse bioassay
(quantitative and qualitative evaluation). The ideal way to monitoring is to combine method of rapid assay
for screening like biochemical assay (functional) and after use a LC-MS to confirm results.
Prophylaxis. What happens if the tests are positive to DSP? The first thing to do immediately is to stop the
harvest and commerce of mussels from that area. Molluscs are periodically checked from the sanitary
authority, according to regulatory programs, both European and national. The most used method is LC-MS,
this is validated from the EU and labs have to use this in a ring analysis to consolidate data. Mouse bioassay
was formally descarded in 2010, but it is still used for other correlated purposes. If 2 or 3 treated animals
died after the mouse bioassay, the assumption was that molluscs were contaminated with OA, DTXs or
pectenotoxins in amount over the one permitted. There is suggested by the protocol to perform further
extraction with different solvent, in order to identify the presence of other putative toxins.
The maximum amount of OA, DTXs, pectenotoxins and azaspiracids in molluscs is establish 160 ug/kg of
mussels meat. For yessotoxins the limit is 1 mg/kg. Other lipophilic toxins can be found using mouse
bioassay. The toxicological characterization is not yet completely clear: under investigation are
pectenotoxins and yessotoxins.
They don't induce diarrhea; they are produced by Gonyaulax grindlei (now Protoceratium reticulatum), G.
polyedra (now Lingolodinium polyedrum), G. spinifera and they are extremely toxic per parenteral way, but
practically no toxic if assumed by oral administration in mouse.
There are 9 different types of pectenotoxins. They are produced by D.fortii and D.caudata. The limit for
commerce is 160 ug/kg mussel meat, detected with LC-MS for DSP.
PP2A Inhibition assay – A functional method for diarrhetic shellfish toxins detection
DSP is caused by OA, DTX1 and DTX2, which are accumulated in bivalve molluscs. To avoid poisonings, EU
has established a threshold for the commercialization to 160 ug/kg of mussel meat. Methods to detect
these toxins, with an always increasing precision, are needed. One is the PP2A inhibition assay, a functional
assay based on the mechanism of action of DSP toxins. OA (okadaic acid) is the main DSP toxin and it is
an inhibitor of PP2A. These enzymes are involved
in the dephosphorylation of different protein,
which are involved in the regulation of many
cellular processes. When PP2A is inhibited there
is an increased phosphorylation degree of
proteins that control the secretion of electrolytes
through intestinal epithelium. This electrolytes
disequilibrium leads to big amount of water in
the intestinal lumen, that induces diarrhea. DTX3
cannot directly inhibit this enzyme (PP2A),
because this toxin is inactive if not hydrolyzed. However, hydrolysis can occur at gastrointestinal level and
DTX3 can induce diarrhea.
At pH 8-8.4 the PP2A can dephosphorylate an artificial substrate named p-NNP (p-nitrophenyl phosphate).
When this enzyme acts, p-NPP is transformed into p-nitrophenol (p-NP) and this reaction can be measured
by a spectrophotometer. OA can block this reaction avoid the synthesis of p-nitrophenol. Different
previously known concentrations of OA are used to build up a calibration curve plotting the absorbance
against the OA concentrations. Using this calibration you can measure the concentration of unknown
OA inhibits PP2a → PP2A cannot dephosphorylate n-NPP → no synthesis of n-NP
Here the pipeline to carry out a PP2A assay:
1. Collection and preparation of shellfish sample;
2. Shellfish meat extraction;
3. Dilution of shellfish extraction;
4. Preparation of OA reference solutions;
5. Preparation of enzyme and substrate solutions;
6. Performance of the assay;
7. Data analysis.
Collection and preparation of sample. Is needed a big amount of shellfishes, like 2 or 3 kg. Shellfish are
cleaned up with freshwater, then opened with a knife. The abductor muscle is cut and the meat is washed
again in order to remove sand and other undesirable material. There is now a passage on a sieve for 5
minutes, in order to drain the tissues (meat). After this, with a minipinner the meat is homogenized and the
sample is ready.
Shellfish meat extraction. 1 g of meat is extracted using 4 ml of 90% aqueous methanol. With an Ultra-
Turrax the content of the test tube is homogenized (1 min x 14000 rpm). Now it comes centrifugation at
3000 g for 10 minutes, and the supernatant is collected to carry out the assay. The extract need to be
hydrolyzed in order to liberate OA, DTX1 and DTX2 from their esters, which will inhibit PP2A.
Dilution of shellfish extract. Different solutions of the sample extracts have to be prepared, in order to have
a good probability that at least one of them will fall in the central part of the calibration curve (linear):
[A]: 50 ul extract + 950 ul of buffer (40 mM Tris/HCl pH 8,4)
• [B]: 500 ul [A] + 250 ul buffer
• [C]: 500 ul [B] + 1000 ul buffer
• [D]: 500 ul [C] + 1000 ul buffer
The buffer contains Mg, that is needed by the PP2A to function.
Preparation of OA reference solutions. Using a stock solution of OA (the standard reference compound) in
EtOH, 10 OA reference solutions are prepared by dilution adding the Tris/HCl buffer. Concentration range is
from 0 to 2,222 ng/ml. In this way, reference solutions that will be used in the assay are obtained.
Preparation of enzyme and substrate solutions. PP2A is isolated from the skeletal mass of rabbits, or
produced by recombinant bacteria. The concentration of PP2A will be 0,05 U/ml. The final concentration of
the substrate, p-NPP, will be of 141 mM.
Performance of the assay. In each well of the 96-well plate we have to add:
50 ul of the OA reference solution [for the calibration curve] or extract solution [for the analysis] (in
• the blank → 50 ul of Tris buffer);
50 ul of the enzyme solution (in the blank → 50 ul of Tris buffer);
• 100 ul of Tris buffer;
• 50 ul of substrate solution.
Now is time to shake the well plate for 1 minute with the microplate shaker. Then, incubate the plate for 1
hour at 36 °C, and finally read the absorbance with the spectrophotometer. Each determination is made in
duplicate. If the color in the well is an intense yellow, there is a lower concentration of toxin; if the color in
the well is a bland yellow, more transparent, there is a higher concentration of toxin.
Analysis of data
Mean Abs (absorbance) values for the blank well is subtracted from the mean values of the other
The net Abs value for the well containing 0 ng/ml OA represent the 100% of enzyme activity
Calculation of the % residual enzyme activity; ratio between Abs of the test well and Abs of control;
• OA calibration curve: % residual enzyme activity of OA reference solutions is plotted against OA
• concentration of these solutions;
Calculation of OA concentration in samples: interpolation of the enzyme activity of the sample from
• the linear interval of the calibration curve obtained from the OA reference solutions, taking into
account the sample preparation and dilution;
Toxins concentration: expressed in mg of OA equivalent/ kg of meat;
• LOQ (limit of quantification): 64 ug OA equivalent/kg meat.
We will not consider all the point of the curve but just the middle part where we have a linear situation of
the calibration curve. There are also to consider all the dilutions made in the assay. OA equivalents means
that the assay cannot discriminate from all possible toxins (so, in these equivalents are comprised also DTX1
and DTX2). The LOQ – Limit of Quantification, namely the lowest toxic concentration is 64 ug of OA
Table on slide.
Algal toxins are secondary metabolites of algae, produced under some specific conditions; they are not toxic
for algae but for other organisms. These metabolites are accumulated in seafood and can induce some
poisonings in human. The vectors with which human can contract intoxication are mainly mussels, fishes
and crustaceans. Mussels accumulate toxins through filtration; fishes that eat mussels undergo the process
of bioaccumulation. Toxins are colorless, odorless and thermostable, and thus really difficult to detect.
Palytoxin is a complicated molecule from a chemical point of view. It is considered one of the most toxic
compound present in nature of non-protein origin. It was identified for the first time in 1971 in several soft
corals of Hawaii, known and Palythoa and Zooanthus species, that are used as decorative elements in
aquariums. This is one of the most problematic issue related to human intoxications. Palytoxin is not only
synthesized by soft corals but also from dinoflagellates of the genera Ostreopsis and Trichodesmium. These
are unicellular algae, originally diffused in the tropics but afterword identified also in more temperate areas.
Researchers were very curious about the presence of palytoxin in algae and corals and they found that this
toxin was present also in Trichodesmium spp., that is a cyanobacteria, and maybe the link between coral
and algae. There are different exposure routes, each one with specific symptomatology:
Oral exposure: GI problems, muscle cramps, myalgia, cardiac dysfunctions, respiratory stress,
• cyanosis, death. This kind of exposure is associated with the ingestion of contaminated seafood, the
oral exposure is the most dangerous;
Cutaneous exposure: edema, erythema, urticarial rush, itch, numbness and swelling, neurological
• problems as paresthesia and metallic taste. This exposure is associated to the handling of Palythoa
corals or to seawater contact during Ostreopsis blooms;
Aerosol: rhinorrhea, cough, respiratory distress, fever, conjunctivitis, dermatitis. It is associated with
• aerosolized water during Ostreopsis blooms and during cleaning of aquariums containing Palythoa
species. Ostreopsis produces palytoxin and through the action of waves people can inspire aerosols.
The most part of case of contamination was not investigated chemically but information were obtained just
on observation of symptoms and on the knowledge of vectors; palytoxin was chemically identified in only 2
cases. Only few cases were ascribed unequivocally to palytoxin. In Philippines there was a case in 1988:
after 15 hours from the ingestion of a crab a person died. Another case in Japan in 1987: a person
consumed a fish (Scarus ovifrons) and died after 4 days. The last one case of death in Madagascar, in 1999: a
woman died after have eaten a sardin-like fish. In Japan (2000) 11 people on 33 eating a fish soup were
intoxicated and hospitalized. In the Mediterranean sea no intoxication after ingestion of contaminated
seafood have been occurred. However, huge amounts of palytoxin and analogs have been found in mussels,
echinoderms ad crustaceans. Moreover, signs of toxicity were recovered.
Experimental studies have been done in order to study the toxicity of palytoxin. The first report on in vivo
toxicity of PLTX was achieved using a semi-purified toxin, because the procedure of extraction and
purification were not well developed yet; in 1981 the exact chemical formula was obtained. Lots of in vivo
studies were performed after parenteral and oral administration of the toxin in different animal models.
However, due to the limited availability of PLTX the majority of study was carried out in mice. In vitro studies
have been performed in order to identify the mechanism of action. The most useful data are those that
come from the oral exposure studies. The first LD50 per os was obtained in mice and it was equal to 510
ug/kg of body weight according to the OECD 425 pipeline. OECD pipelines are established from EU in order
to limit the use of treated animals in the study of toxic compound; the OECD 425 is the one to follow in case
of PLTX and it provides some statistical procedures that involve the use of only 2 animals. The LD50 is
calculated then by an algorithm. The lethality in mice starts from 600 ug/kg per os (oral exposure);
according to a new study on acute oral toxicity of PLTX the LD50 is of 767 ug/kg. Symptoms are mainly
spasm (86%), paralysis (80%) and respiratory problems (70%). The ultrastructural analysis has revealed a
fibrillar disorganization and aggregation of mitochondria in skeletal and cardiac muscle cells. Also NOAEL
and LOAEL are necessary to establish the limit. Seven days of PLTX administration caused lethality and toxic
effects at doses >30 mg/kg/die (LOAEL); the NOAEL was estimated to be equal to 3 mg/kg/die. At
histological level, macroscopic alterations were observed at GI level (gastric ulcers and intestinal fluid
accumulation); pulmonary level (severe inflammation, locally associated with necrosis); myocardial level
(hypereosinophilia and fiber separation). Lungs, heart, liver and GI trait are the main PLTX targets. However,
these studies are not so common since a very limited amount of the substance studied, palytoxin, is
The high toxicity of PLTX is related to the mechanism of action. PLTX transforms the K-Na ATPase in a
cationic channel and the gradient is not maintained anymore. The K-Na ATPase is a transmembrane pump
which is essential to maintain cellular homeostasis; it serves to transfer 3 Na ions out of the cell and 2 K ions
into the cell, in a cyclic process that exploits the hydrolysis of ATP. PLTX bond with a subunit of the ATPase
changes it from a transmembrane pump to a non-specific cationic channel; as a consequence, a consistent
ionic imbalance at cellular level is induced. The great amount of sodium ions into the cell provokes ROS
production, cell mass reduction, mitochondrial dysfunctions and an increase of calcium in the cell: this
latter effect leads to membrane and cytoskeleton damages.
In vitro effects are distinct according to excitable cells (neurons, muscle and cardiac cells) and non-excitable
cells. In excitable cells, the calcium increase is mediated by the reverse functioning of the Na-Ca exchanger
and by the activation of voltage-gated calcium channels. Calcium dependent toxic (and dramatic) effects
are: 1) disruption of the cardiac excitation-contraction coupling; 2) Uncontrolled muscle contraction; 3)
uncontrolled neurotransmitter release. In non-excitable cells, the big amount of intracellular sodium
induces cytoplasm acidification due to the reverse functioning of the Na-H proton pump, and the overload
of protons leads to mitochondrial dysfunction (the electrochemical gradient is not maintained anymore)
and ROS production. In this case, effects are more sodium-dependent instead of calcium-dependent (keep
in mind that in both situation all begin with an overload of sodium). The state of non-homeostasis induces a
necrotic cell death. Thus, palytoxin is able to block the cardiac beating.
There are many palytoxin-like compound; omopalytoxin,
neopalytoxin, 42-hydroxypalytoxin, etc. according to different
substitutes in several positions. The molecule of the leader
compound – palytoxin – has 64 chiral centers, so the number
of stereoisomers is really exponential. Each of these analogs
could have different toxicity. 42-hydroxypalytoxin is completely
equal to palytoxin but a hydroxyl group on position 42. It was
initially identified in Palythoa toxica. In Palythoa tuberculosa, a sister of Palythoa toxica, produces a 42-
hydroxypalytoxin with a conformational change in C50. The 42-OH-PLTX has a lethality in mice starting from
300 ug/kg per os. The LD50 is 651 ug/kg, overlapping the LD50 of palytoxin; also the symptoms and the in
vivo cytotoxicity are very similar to those induced by PLTX. However, the conformational inversion on C50
reduce the cytotoxicity of the Palythoa tuberculosa 42-OH-PLTX of almost 2 orders of magnitude.
Ostreocin-D is produced by Ostreopsis ovata. Generally, this toxin is less toxic than PLTX. In vitro studies
revealed that Ost-D is less toxic than PLTX by 3 orders of magnitude.
For ovatoxin-a no complete toxicological evaluations have been carried out so far; however, ovatoxin-a
should be less toxic of 2 orders of magnitude compared to PLTX.
EC50 is a parameter used to compare the cytotoxicity of compounds and indicates the concentration that
causes the same effect in 50% of the treated cells.
These test are important to establish the limit of this toxins in the seafood. PLTX is still not regulated but
there is a suggested limit from EFSA and FDA of 30 ug of PLTX equivalents (PLTX + Ost-D) / kg of mussel
meat. However, new data regarding the repeated oral toxicity of PLTX should be considered to revise this
limit. Moreover the toxic potential of the new PLTX analogs should be evaluated to deeply characterize the
real risk on human health. The method employed to detect palytoxin in seafood should be rapid, sensitive,
specific and user friendly. There are chemical, biological and antibody-based methods available to detect
PLTX. The two official methods employed are LC-MS and HR (high resolution) LC-MS (chemical) and the
mouse bioassay. Mouse bioassay and LC-MS are thus the most commonly used. LC-MS is difficult to
interpret because we could have a lot of interference. For mouse bioassay is the same: the extraction
procedure can extract other lipophilic toxins, so the toxicity can be due also by other toxins.
The ELISA (Enzyme Linked ImmunoSorbent Assay) assay is very sensitive and specific and uses antibodies;
it is very easy to use and requires a basic lab equipment. It is a colorimetric assay, so the color is
proportional related to the concentration of analyte: the more intense the color the higher the
concentration of analyte (do not confuse with PP2A inhibition assay!). Antibodies are linked to the plastic of
the wells of the plate. Antibodies are able to capture (so called capturing antibodies) analyte molecules.
There are 2 types of ELISA. In the competitive ELISA, only one antibody is employed; there is a competition
between the sample antigen and the enzyme-linked antigen, that produces the colorimetric reaction. In the
sandwich ELISA, the antigen is detected by 2 antibodies. In this case we have 2 kind of detection:
Direct immunodetection: the antibody is conjugated with an enzyme system;
• Indirect immunodetection: a secondary antibody recognizes the detecting antibody (that bind the
• antigen) and is conjugated with the enzyme system. In this case we use 3 antibodies: the first is
attached to the well surface, the second recognizes the antigen bound to the first antibody and the
third has the purpose to amplify the signal. The latter (an unspecific antibody) is conjugated with an
enzyme which leads to the colorimetric reaction.
Commonly used enzyme are the HRP – Horse Radish Peroxidase and AKP – Alkaline Phosphatase. The
substrate that gives the colorimetric reaction is TMB, a chromogen that become blue when reacting with
the enzyme. Antibodies can be monoclonal or polyclonal. The monoclonal antibodies recognize all the same
epitope of the antigen because they are produced from clones of a single plasma cell. Polyclonal antibodies
recognize different parts (epitopes) of the antigen.
A newly developed sandwich ELISA assay for PLTX is now developed. Main features:
Very sensitive (LOD = 1,1 ng/ml and LOQ = 2,2 ng/ml)
• Accurate (bias = 2,1%)
• Specific for PLTX and analogs (ovatoxin-a, 42-OH-PLTX, PLTX-biot)
Monoclonal antibodies obtained in mice are used as capturing agents; the recognition of palytoxin and
analogs is get from polyclonal antibodies obtained in rabbit. The third antibody will be thus an anti-rabbit
antibody conjugated with the HRP enzyme. Procedure:
1. Preparation of the sample (mussels, algae or seawater);
2. Extraction of the toxins (performed using methanol or butanol);
3. ELISA assay (performed in parallel with the calibration curve);
4. Interpretation of data (extrapolation of PLTX content in the sample based on the calibration curve).
The hemolytic assay is another method used to quantify palytoxin, exploiting its ability to hemolyze
erythrocytes of different species. The hemolytic activity of palytoxin was discovered casually by Habermann,
that after incubation of red blood cell with the toxin forgot the experiment; on his return he found
erythrocytes hemolyzed. PLTX is a potent but slow hemolysin, in pigs, rats, mice, rabbits, guinea pigs and
human erythrocytes. The hemolytic assay is based on the skill
of PLTX to convert the Na-K ATPase into
an unspecific cationic channel, leading to
a late lysis of red blood cells. The release
of hemoglobin can be measured with a
spectrophotometer. The release of
hemoglobin was found to be time,
temperature and PLTX concentration
dependent. If the T increases from 37 °C
to 42 °C, the hemolysis occurs faster.
Under 15 °C there is no hemolysis even
after 8 hours of incubation at high
concentration. PLTX induces hemolysis
after 4 hours of incubation of the samples with the toxin and no differences were observed from samples
incubated for 6 hours or more. The presence of borates (boric acid oe sodium tetraborate 5,0x10-6 M) and
calium ions (>2,0x10-5 M) in the buffer solution is reported to increase hemolysis induced by PLTX, due to
its ability to promote interaction between the toxin and the Na-K ATPase. This assay shows pros and cons.
No sterile conditions;
• Easy to perform;
Variability depending on the species of origin of the erythrocytes;
• Possible interindividual variability depending on sex, age and other factors;
• Marked matrix effects.
Mice and rats are in fact more sensible to PLTX activity.
Apart these disadvantages, the hemolytic assay is one of the most promising method to recognize and
quantify PLTX; for this reason, it would be useful a characterization in order to detect unequivocally PLTX all
over the world with a standardized pipeline. A general and uniform protocol is still lacking for this assay and
it has never been characterized as screening tool to quantify PLTX in shellfish. Practically, erythrocytes are
Samples of erythrocytes are diluted in a red blood cell preservation solution;
• Samples are centrifuged ffor 10 minutes at 4°C and 2400 rpm;
• After the centrifuge, the surnatants are removed;
• Red blood cells pellet is washed 3 times with centrifuge;
• The final pellet is resuspended with 1:10 dilution in the red blood cell preservation solution;
• Samples are now stored at 4°C in red blood cell preservation solution (PBS + 1 mM EDTA + 5 mM of
After purification the assay can be carried out. Samples are put in the wells of the plate (125 ul) with 125 ul
of 0,1% v/v of tween 20. and a certain concentration of the toxin. Tween is a buffer that forms a complex
with the membrane cholesterol of erythrocytes, leading to the lysis. The negative control is composed of
125 ul of red blood cell suspension and 125 ul of K-free D-PBS, without the toxin. The percentage of
hemolysis is calculated by a formula:
[(Abs of treated samples – Abs of negative control)/ (Abs positive control – Abs negative control)] x 100.
A calibration curve has to be constructed in order to extrapolate the amount of toxin in unknown samples.
The resulting parameters are:
EC50 = 6,2 x 10-9 M;
• Working range = 3,91 x 10-10 to 2,5 x 10-8 M
• LOD (Limit of Detection) = 2,36 x 10-10 M (6,3 ug/kg);
• LOQ = 6,09 x 10-10 M (16,3 ug/kg)
This method is repeatable, accurate and specific. The low intense color is index of a lower concentration of
PLTX in the sample. The hemolytic assay cannot be used so far in quantification of PLTX in mussels at level
below the maximum limit suggested by EFSA of 30 ug of PLTX/ kg of mussel meat.
AZP – Azaspiracid Shellfish Poisoning
AZP is quite new: it was identified in 1995 after an intoxication characterized by symptoms similar to those
of DSP occurred in the Netherlands. This poisoning was caused by mussels coming from Killary Harbor in
Ireland. Chemical analysis were performed and there was a positive response for DSP with low
concentration of okadaic acid and analogs, but too low to explain this intoxication; in fact a new toxin was
found and named killarytoxin. Three years later more complete chemical analyses were carried out and the
name of the new toxin recovered was azaspiracid, due to its particular structure. Azaspiracid is a lipophilic
toxin produced by Azadinium spinosum, a very little dinoflagellate. This toxin can be accumulated in edible
seafood like mussels, oysters, clams, scallops, snails, crabs and sponges. So far, human AZP were exclusively
due to contaminated mussels coming from Ireland. Symptoms are very similar to those of DSP and of a
generic bacterial intoxication: nausea, vomit, diarrhea. Almost surely, the incidence of this intoxication is
underestimated because it can be confused easily with DSP. DSP and AZP differ in that AZP symptoms
appear after few hours and remain for 2-5 days. At now, AZA (azaspiracid) has been detected along the
western coastline of Europe, especially along the Norwegian coast, and also in Chile. However, cases of AZP
8 cases, Netherlands 1995;
• 24 cases, Ireland 1997;
• 10 cases, Italy 1998;
• 16 cases , UK 2000;
• 30 cases, France 1998; 219 cases, France 2008 (last intoxication).
All these cases are due to ingestion of mussels coming only from Ireland. In 2008 began a monitoring
program and since that moment outbreaks have not been recognized anymore. In 1995 anything was
known about azaspiracid, neither the producers, and the chemical structure was identified recently. AZA is
present in plankton. Initially, the heterotrophic dinoflagellate Protoperidinium crassipes was believed to be
the main producer; another dinoflagellate was discovered to produce this toxin in 2007, Azadinium
spinosum, prey of Protoperidinium crassipes. Thus, the producer is now believed to be A.spinosum.
Afterward, some other species of Azadinium genus were discovered in Italy, Chile, Korea, Denmark, Ireland,
Scotland, Argentina, France and Mexico. Not all the species are known to produce azaspiracid. Recently,
another Azadinium species (Azadinium dexteroporum) able to produce AZA (mainly AZA3 and AZA7, analogs
of the lead compound azaspiracid) was found in the gulf of Naples.
The name azaspiracid was given to the toxin because of the presence of a cyclic amino group called AZA
group, of a 3-rings spiranic group and of a carboxyl residue (that provokes acidity). Up to now more than 30
analogs of azaspiracid have been characterized. Different substitutions on side chains determine different
azaspiracid molecules. From a toxicological point of view they are classified with a number in order of
discovery. AZA1, AZA2 and AZA3 are actually the most studied; AZA4 and AZA5 are now under investigation.
EU imposed an admissible threshold in mussels of 160 ug of AZA1 equivalent (AZA1, 2, 3) / kg of mussel
meat. Most of 30 analogs of AZA metabolites of AZA1 and AZA2 biotransformation in mussels and/or
artifacts due to mussels conservation. Recent studies demonstrate that already after 24 hours it is possible
to find AZA1 and AZA2 metabolites in mussels (AZA 3, 5, 8, 11, 12, 17, 19). Other metabolites are detected
after some days: AZA 4, 10 and 21 after 2 days, AZA23 after 3 days, AZA9 after 6 days.
Notably, in tissue samples at T > 100°C AZA are prone to degradation. Cooking at high T could perhaps
degrade AZA and analogs. However, no information are available regarding the resulting toxicity of most of
these compounds. Among these analogs, the main products due to mussels bioconversion are AZA17 and
AZA19, which take origin respectively from AZA3 and AZA6. These analogs are known for their toxicity
arising problems for public health, since they are not regulated by EU.
According to data obtained by in vivo experiments and on epidemiology, GI apparatus seems to be the
target organ of these toxins. Although experiments have been performed from 20 years ( in vitro and in
vivo), the mechanism of action of AZA is still unknown, as the intracellular pathways and the molecular
targets: we know just symptoms!
In in vitro studies, multiple effects have been observed on various cell lines (monocytes, lung cells,
enterocytes, lymphocytes), including cell death both by necrosis and apoptosis, alteration of cytoskeleton
and decrease of cells volume. In vivo studies in mice by intraperitoneal and oral administration have shown
multi-organ damages (liver, pancreas, intestine, lungs, stomach, spleen and thymus). Very unspecific
damages have been observed in different cell models:
AZA reduces cell viability even at very low concentration, by the activation of apoptotic processes;
• AZA reduces ATP levels, cell adhesion, cell volume and activates some kinases.;
• AZA is able to alter intracellular levels of calcium, cAMP and pH: these effects might modulate
• apoptosis induction;
AZA can modulate ionic fluxes through plasma membrane, especially in neurons (modulating
• bioelectric activity) and cardiomyocytes (modulating K fluxes on hERG channels, responsible for the
auto-generation of the membrane potential in these cells).
However, in vivo studies show no damages at cardiac level.
Firsts in vivo studies aimed to evaluate the differences between AZA and okadaic acid. AZA and OA have a
completely different chemical structure. After few hours, mice develops a progressive paralysis of legs and
difficulties on breathing because of the block of muscular lung system. With low doses of injected AZA, mice
doesn't develop diarrhea; furthermore, instead of OA, AZA does not inhibit proteinphosphatase. According
to these findings, the molecular mechanism of AZA could be really different to that of OA. After
intraperitoneal administration, the minimum lethal doses were identified:
AZA1: 200 ug/kg of body weight, TEF = 1
• AZA2: 110 ug/kg of body weight, TEF = 1,8
• AZA3: 140 ug/kg of body weight, TEF = 1,4
The limited toxicological information does not allow the setting of robust toxic equivalence factor (TEF) for
AZA analogs. TEF are number that give us an idea on the potency of an analogs in respect to the reference
compound. AZA4 and AZA5, which are products of a metabolic bioconversion, have been found to be
significantly less toxic than the main toxins AZA1, 2, 3. TEF of AZA4 and AZA5 are 0,4 and 0,2. These TEF
values are not so reliable because the studies were done on a little number of mice.
After per os exposure (the most likely exposure route for human), AZA1 was demonstrated to cause
widespread organ damages, mainly to intestine (villi erosion, necrosis of the lamina propria), liver
(hepatocytes vacuolisation, liver hypertrophy, accumulation of fatty acid and mild inflammation) and lung
(interstitial pneumonia). Based on these effects, it can be hypothesized that liver and lungs can be the
target organs of this toxin after oral administration.
In 2001, the FSAI – Food Safety Authority of Ireland performed the first AZA risk assessment in shellfish
using LC-MS. Mussels were collected from the accident site. They established a putative concentration that
had led to symptoms to 5,7-10,7 ug AZA/g hepatopancreas, but they didn't take into account the cooking
stability of AZA and the analogs presence in hepatopancreas. In America, FDA sets the action level at 0,16
ppm of AZA equivalents. In Europe a different threshold is suggested according to a revision of the FSAI limit
including the now-available data on toxicity. The FSAI revision led to an estimate of 113,4 ug AZA per a 60 kg
person. Based on this limit, EU imposed in 2001 the regulatory level to 160 ug AZA equivalents/kg of
shellfish meat. Considering the new insights on this issue, in 2008 EFSA revised the limit to 30 ug of AZA1
equivalents/kg of shellfish meat. According to EFSA, the acute reference dose (ArfD) id of 0,2 ug of AZA1
equivalents/kg body weight, corresponding to 12 ug of AZA1 equivalents for a 60 kg person.
Since there are no information on the mechanism of action, no functional assay is available. EFSA suggested
the LC-MS/MS as reference method to detect AZA. Mouse bioassay was however frequently used, but at
the end of 2014 this method should be completely substituted by LC-MS/MS.
Brevitoxins are neurotoxic polyethers produced by dinoflagellates belonging to the Karenia genera, in
particular by Karenia brevis. Karenia brevis blooms have occurred in the gulf of Mexico and along the
southern east coast of the USA, mainly in Florida. These blooms (also called Florida red tides) consist on red
tides, where the water is brown/red colored. There are 2 different structural backbones of these molecules:
A backbones show 10 trans-fused cyclic rings, while B backbones 11. Brevitoxins can also present an
additional structure where the lacton ring is opened. Depending on the backbone and the substitutions,
there are 15 different brevitoxins: they all derived from brevitoxins 1 (A backbone) and 2 (B backbone).
Karenia brevis can produce other brevitoxins-related compounds as hemibrevitoxins and brevenals.
Hemibrevitoxins are incomplete products of brevitoxins. Brevenals are instead antagonist of brevitoxins.
These metabolites are synthesized by shellfish and have been identified and structurally characterized; it is
now know that they are involved in marine mammals toxicity. Filter-feeding shellfish can accumulate these
toxins and act as vectors, leading to human and animals intoxications. For aquatic species there is a high
mortality: fishes, aquatic birds, sea turtle, crabs, lion's mane jellyfish, dolphins and manatees are affected.
Massive mortality of fish in Japan, China and Australia was caused by blooms of Chantonella marina, which
is a brevitoxins producer.
Human can be exposed through different routes. Ingestion of contaminated foodstuffs is a risk factor of NSP
– Neurotoxic Shellfish Poisoning, that presents GI and neurological symptoms: nausea, diarrhea, vomit,
headache, tingling in the face, difficulty in speaking, paresthesia, loss of coordination and bradycardia.
Brevitoxins can be also aerosolized from the water by the action of wind and waves; inspiring this aerosol
can cause skin irritation, conjunctivitis, rhinorrhea, bronchoconstriction, cough. People can get intoxication
also through cutaneous exposure. However, like other toxin poisonings, the oral route is the most
Until now, poisonings have occurred mainly in Florida and Texas, with 4 major outbreaks. Fortunately no
lethality has been reported; cases of NSP were not so common. The intoxication caused by brevitoxins
shows symptoms that appear after 30 minutes and last 2-3 days.
A lot of studies were focused on ADME of brevitoxins, finding that liver is the main target. After intravenous
administration, 70% of the toxin was concentrated in skeletal muscle, 18% in liver and 8% in intestinal tract;
lower concentrations were recovered in heart, kidneys, brain, spleen, testes and lungs. After intratracheal
instillation, the toxin was accumulated in liver, skeletal muscle and GI tract. Skeletal muscle might be a
storage compartment for the toxin. After intraperitoneal injection in mice, 39% of the toxin was bound to
components in mouse plasma; the 7% of the toxin in the blood was associated with serum albumin. Large
amount of toxin was associated with plasma high-density lipoproteins (HDLs). An in vitro study on possible
bonds of brevitoxin to human serum albumin have found covalent and non-covalent interactions between
them with the formation of an adduct made up from one or two toxin molecules and one HSA molecule.
The elimination happens mainly through urine and feces; there is no more toxin in the body after 3-4 days
after exposure. Summarizing, brevitoxins are accumulated mainly in the skeletal muscle tissue and undergo
to hepatic metabolism and biliary excretion. The tissue distribution and elimination patterns of brevitoxin
metabolites however depend on the structural features resulting from different reaction occurring in
The molecular targets of brevitoxin are the voltage-gated sodium channels in excitable cells. VGSCs are
transmembrane proteins formed by an α-subunit and one or more β-subunits. The α-subunit is composed
of 4 homologous domains, consisting of 6 transmembrane α-helices connected by internal and external
polypeptide loops. S5 and S6 helices form the ion-conducting pore and the S4 helix works as a voltage
sensor. These channel exist in 3 states: activated, deactivated and inactivated.
In case of membrane depolarization,
the VGSC opens its gate allowing an
intracellular flux of sodium ions
(activated state). When the
transmembrane potential rises, the
channel inactivates by closing the gate
and sodium ions flux is blocked and
membrane repolarization occurs until
the normal resting potential is
restablished. At this point, the channel
returns into its deactivated state with
closed gate. Brevitoxin acts on S5 with a
''head-down'' orientation, with the A
ring (see structure) extending towards
the intracellular opening of the gate and the J or K ring near the extracellular surface of the pore. Brevitoxin
bond to the VGSC lead to:
1. A shift in the voltage dependence of activation to a lower membrane potential. In presence of toxin,
the channel opens under conditions in which it is normally closed;
2. An inhibition of channel inactivation;
3. An increase in mean open times, allowing big amount of sodium ions to enter in the cells.
These effects lead to the alteration of membrane properties of the excitable cells, and this represents the
basis of the toxic effects of this group of toxins. Several studies were also performed in vivo to establish the
acute toxicity. After a single administration, LD50 of brevitoxin 2 were: 200 ug/kg intraperitoneal; 200 ug/kg
intravenous; 6600 ug/kg oral. Even after a single administration, LD50 of brevitoxin 3 were: 170 ug/kg
intraperitoneal; 94 ug/kg intravenous; 520 ug/kg oral. Most of studies have been performed with brevitoxin
2 and 3 because they are more available.
After single intraperitoneal administration, mice treated with brevitoxin 3 presented a syndrome
characterized by hypersalivation, lachrymation, excessive urination and defecation. These symptoms
appeared 30 minutes after administration. Mice treated orally with brevitoxin 3 presented tremors,
muscular contraction, breathless and death, that are completely different symptoms compared to those of
the intraperitoneal route. Furthermore, these symptoms appeared 5 hours after administration. Rats, after
inhalation of 500 ug of brevitoxin 3/m3/2h/day for 5 consecutive days shown reduction of body weight, no
macroscopic or microscopic lesions, no inflammation nor immune system dysfunctions.
A NOEL (No Observable Effect Level) for brevitoxin in human has not been established yet, although toxicity
occurs in nanomolar concentration range. FDA suggested an admissible level for brevitoxin in shellfish of 0,8
ppm of brevitoxin 2 equivalents. It is clear that the consumption of even a few contaminated shellfish may
result in poisoning and that the severity of the disease may be different upon many factors including dose,
body weight, underlying medical condition, age.
Since the '80s, monitoring systems in Florida have been employed. There is no specific treatment for NSP.
This poisoning shares several symptoms with other poisonings and its diagnosis can be a challenge. To verify
the diagnosis, analysis on meal remnants or samples taken from the same harvesting area should be
performed. The analysis of urine sample can be used to confirm the presence of brevitoxin. Analgesics, fluid
replacement and respiratory support are putative primary solutions. GI decontamination can be performed
with activated charcoal. A potential employment of brevenals as therapeutic agents might be considered in
the treatment of NSP.
PSP – Paralytic Shellfish Poisoning
PSP Is one of the most dangerous intoxication that can be recovered and it is widespread all over the world.
It was the first intoxication by shellfish discovered. In Italy there were several intoxications, but no one felt
sick. Poisoned people developed just some irritation on lips and on the mouth. The amount of toxin was
very low, considering the fact that if the toxin is concentrated in only one mussel it can kill a person. This
intoxication has a very high rate of mortality, until 22% (1 person on 5). The mean around the world is
however 6%. Having different analogs with different toxicities, this family of compound can cause a variety
of effects. The oral exposure at concentration from 2 to 1500 ug/kg can provoke a paralytic intoxication. The
lethal dose in an adult man is of 1-2 mg. This poisoning is characterized by neurological symptoms, which
come out after 30 minutes from the consumption of contaminated shellfishes. In a less severe intoxication
the main symptoms are: perioral paresthesia (around the mouth), with inflammations of lips and tongue,
and some gastrointestinal symptoms (they are less common). In an intermediate poisoning the paresthesia
spreads on the face, in the hairs, in the harms and in the legs, where it shown off also muscular weakness.
Subsequently, the patient undergoes to an intorbidimento generale. Severe intoxications provoke
neurological effects, like double visions, ataxia, vertigo, confusion; the patient feels a sensation of slightness
and he has difficulty in the assessment of weight. Subsequently comes the paralysis of limbs, and it can
spread to the respiratory muscles leading to a respiratory block and finally death. During the poisoning, the
patient remains conscious. Unspecific GI symptoms are nausea, vomit, diarrhea, abdominal cramps.
Diagnosis. If the patient goes in dyspnoea he needs a respiratory support immediately. Up to now, specific
tests are not available. Apart hypotension, symptoms are tantamount to these of the tetrodotoxin in puffer
fish; saxitoxin and tetrodotoxin have a quite similar structure and the same mechanism of action.
Prophylaxis. The monitoring relies on AOAC LC and other LC-MS methods (chemical assays) and on the
AOAC Mouse bioassay. The latter consists in an injection of an acidic extract of molluscs on mice. The death,
characterized by neurological symptoms, comes after 5-7 minutes (if there is saxitoxin). The sensibility of
this test is equal to 400 ug of STX per kg of mussel meat. There are some promising receptor assays, useful
for dosing toxins that act on the sodium channels, based on the tenet that the affinity of a toxin for its
receptor is proportional to its toxicity in vivo. These tests are still in phase of assessment.
Saxitoxin is an alkaloid positively charged with more than 30 analogs, among which
neo-saxitoxin and gonyautoxins 1, 2, 3 and 4.
Saxitoxins (STX), more than 6 compounds
• Decarbamoyl-STX, more than 6 compounds
• Sulphodecarbamoyl-STX, more than 6 compounds
• Deoxydecarbamoyl-STX, more than 6 compounds
These compounds are produced by several microalgae, like Alexandrium tamarense, Alexandrium minutum,
Gymnodinium catenatum, Pyrodinium bahamense. These microalgae are endemic of North America, but
they are spreading all over the oceans, with a sporadic presence in the Mediterranean sea. There have been
some cases in Italy, for example in Emilia Romagna; from this case it began a monitoring program to avoid
the risk of intoxications, with good results. The presence of this algae is ascertained even in the gulf of
Trieste, where they cannot bloom because of the environmental conditions.
Saxitoxin acts blocking the voltage-dependent sodium channel, and thus stopping the transmission of
nervous impulse. The bond between STX and the sodium channel does not allow the membrane potential
to spread, with effect on the transmission of the nervous stimuli and the muscular contraction. For its
toxicity, STX is included in the convention of Paris of 1993 on armi di distruzione di massa. It cannot be sold
without special permit; even the laboratory use is extremely controlled.
Main vectors are edible molluscs, that accumulate toxins without having effects. Only one shellfish can
accumulate an amount enough to kill a person. Different species have different ability on accumulating and
excreting these toxins. Even other organisms, that eat shellfishes, can work as vectors, like crabs, puffer
fishes, codfishes, herrings and salmons. Some other important species die before they reach high
The limit established by the law is 800 ug of STX equivalent per kg of mollusc meat.
+1 anno fa
I contenuti di questa pagina costituiscono rielaborazioni personali del Publisher stefano.scilipoti di informazioni apprese con la frequenza delle lezioni di Tossicologia marina e studio autonomo di eventuali libri di riferimento in preparazione dell'esame finale o della tesi. Non devono intendersi come materiale ufficiale dell'università Trieste - Units o del prof Tubaro Aurelia.
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