Sample assignment of biochemistry coursework.


Repeated closures of Irish mussel shellfisheries have been necessary since the mid-late 1990s.  These closures were due to toxic events and reports of mussel consumers falling ill with symptoms unlike any others previously reported for shellfish poisons.  Additionally there were no reports of known shellfish toxins (eg. PSP, DSP) present in the mussels. Subsequent tests on mussels and toxic extracts determined the presence of a novel compound, Azaspiracid, as the cause of the toxic effect.

The most well documented of these cases is the 1997 Arranmore incident where locals were affected with severe nausea, vomiting, cramps, headaches, and diarrhoea after consuming mussels containing azaspiracid.  Poisoning cases have since been reported in the Netherlands, France, England and Spain, partly as a result of importing Irish mussels.

You are asked to determine the properties of this toxin, the causative organism, the risk shellfish toxicity poses to humans and the environment and to outline a method of approaching such a shellfish toxicity event.

In your assessment you should do the following:

1. Describe the substance: e.g. what is it.

2. Describe its physico-chemical properties.

3. Describe its pathway from causative organism to human (route of contamination).

4. Describe its toxicology.

When assessing the toxicity of a substance you will have to consider the following: properties of the substance, acute/chronic toxicity, lethal/sublethal toxicity, potential to bioconcentrate, potential to be passed on food chains, media in which the substance will be dispersed (e.g. water, air, soil), quantity, potential hazard to human health.

5. State if you think this is a serious human/environmental problem. Include considerations of immediate – acute and lethal – toxicity, as well as potential chronic and sublethal effects.

6. Describe if there should be any immediate measures imposed.  Include pros and cons and evaluate using an evidence-based approach.

7. Describe if there should be any longterm measures imposed, and if current regulations are enough to address this toxicity issue.


Shellfish is regarded a “super food” as it is a highly nutritive has a high level of poly unsaturated fatty acid (PUFA) especially omega 3. Azaspiracids (AZA) are polyether marine toxins that are found in various shellfish species and can cause severe gastrointestinal human intoxications. The first outbreak of human illness was in Netherlands because of the consumption of contaminated shellfish originating from Killary Harbour, Ireland. People who consumed were reported by severe nausea, vomiting, cramps, headaches, and diarrhoea. Oysters were the only shellfish that accumulated azaspiracids at levels that were comparable with mussels.


High content of toxins are found in these species due to their grazing mechanism. Shellfish feed them by filtering suspended algae and food particles present in the water through their body and gills, where food particles are absorbed by the organism and they have a potential to concentrate both bacterial pathogens and phychotoxins at the dangerous toxins levels. However when this shellfish are consumed by humans it shows several toxic syndrome related to shellfish including severe nausea, vomiting, cramps, headaches, it targets liver, lungs etc. The highest levels of total azaspiracids (microg/g, µg/g) recorded to-date were in mussels (4.2), oysters (2.45), scallops (0.40), cockles (0.20), and clams (0.61).

Azaspiracids (AZA):  Author K. J. James; M. J. Fidalgo S ez; A. Furey; M. Lehane- Any of several organic compounds with a complex polycyclic structure that is responsible for the toxicity of some mussels. Azaspiracid poisoning (AZP) is a recently discovered toxic syndrome that was identified following severe gastrointestinal illness from the consumption of contaminated mussels (Mytilus edulis). The toxins, azaspiracids, are polyethers with unprecedented structural features. Studies toward total toxin synthesis revealed that the initial published structures were incorrect and they have now been revised. These toxins accumulate in bivalve molluscs that feed on toxic microalgae of the genus Protoperidinium. The first identified AZA in shellfish from Ireland, azaspiracid contamination of several types of bivalve shellfish species has now been confirmed throughout the western coastline of Europe.

Physical chemical properties of AZA:

AZA1 was initially reported as colourless and ououdrless amourphous solid , having the chemical formula C47H71NO12 and a molecular weight of 841.5 g/mol .

Later it was reported as colourless oil .Other studies reported the toxin to be a.

No UV absorption maxima were found above 210 nm wavelength and the refractive index of AZA1 was determined to be [α]20-21 (c 0.10, MeOH)  .AZA is assumed to be a stable compound.

At physiological pH, AZA1 exists as a zwitterion (i.e., contains both a positive and negative charge but is electrically neutral),

This overall neutral but potentially.Ionic character may result in enhanced possibilities for interaction of AZA with its biological target.

Little information is available about the stability of AZAs. During the production of a tissue

reference material, certain techniques were tested to stabilise the tissue material for long-term storage.

During a heat treatment study the toxins were observed to degrade when heated over 90 °C

However, the use of gamma irradiation, which is often used to stabilise tissue reference materials, had little effect on AZA analog stability when contained in mussel matrix. The toxins were observed to undergo rapid degradation when irradiated as a pure compound in solution.

AZAs .stored in methanol were shown to slowly form methyl esters of the toxin

These esters were only observed in methanol extracts stored at room temperature or higher for prolonged periods (i.e., several months).

Toxilogy of AZA:

Toxicological studies have indicated that azaspiracids can induce widespread organ damage in mice and that they are probably more dangerous than previously known classes of shellfish toxins. It is a unique toxin group that targets the liver, lung, pancreas, thymus, spine and digestive tract.

They pose a treat to humans, when it is eaten, the Biotoxins created during algal blooms are taken up by shellfish (mussels, oysters), leading to these human foods acquiring the toxicity and poisoning humans. Examples include paralytic, neurotoxic, and diarrhoetic shellfish poisoning

Many reaserch have been done to study the toxic effect of AZA. All information regarding AZA toxicology has been obtained from controlled in vitro and in vivo experiments.

In vitro toxicology:

Mouse bioassay and intraperitoneal injection:

Mouse or rat bioassays were the first methods used to determine the toxic effects of AZA. Mice exposed to AZA by intraperitoneal injection react in a different manner than those exposed to other shellfish toxins. After dosing of AZA to male mice, he became sluggish, sat still in the corners and showed progressive paralysis and laboured breathing. No diarrhoea was observed. At low doses the animals died two to three days after dosing. The minimal lethal dose for mice was reported to be 150 µg/kg.I P injection of a lethal dose (>150 µg/kg) caused swelling of the stomach and liver, parallels with reduction in size/weight of the thymus and spleen.

The lethal dose cause excessive damage to the spine, thymus, pancreas, extensive tissue injury in the stomach. Studies also conducted in the mice with sub lethal dose mice died after 4 hrs, there were damage to the epithelial cells on the surface of the finger like villi.

Toxicological studies showed that repeated administration of AZAs caused prolonged damage in the intestine and induced lung tumors in mice (Ito et al. 2002)

An interesting study done by McCaron et al, that they found the level of AZA3 increases significantly when heating in absence of water for a short duration. They also found that the AZA3 increase in the level of concentration even in a very low temperature, However the concentration of AZA1 and AZA2 remain unchanged.This work was informative for shellish processing and analysis of shellfish products.

Cytotoxicity :

The original observation that AZAs can cause cytotoxicity was performed by Flanagan et al.

Using HepG2 hepatoblastoma cells and human bladder carcinoma cells (ECV-304) exposed to crude mussel extracts. Additional studies have since confirmed these findings, in a time- and concentration dependent manner, for a variety of other cell types from various mammalian sources.

Intracellular signaling molecules:

Many AZA analogs have been shown to cause a variety of effects on intracellular signalling

molecules. In mammalian cells, cytosolic calcium is an important secondary messenger for a variety of pathways, including cell death. Human lymphocytes exposed to AZA1 (200 nM) were shown to elevate cytosolic calcium levels by  ca. 50% above basal. This response was shown to be sensitive to extracellular calcium, PKC (protein kinase C) activation, PP inhibition, and cAMP (cyclic adenosine monophosphate) elevation. Differences in the effects of the

various AZA analogs may be a function of solubility and/or purity as it would be highly unusual for a class of structurally related compounds (with only single methyl and/or hydroxyl substitutions) to elicit completely different mechanisms of action rather than, more  commonly,various degrees of affinity and efficacy. Although these studies were unable to identify a specific mechanism of action, the modulation of cytosolic calcium and cAMP may be influenced by modulation of a membrane protein.

The ARfD panel recommends the minimum permissible level of AZA1 in shellfish is 30 µg AZA1 eq/kg shellfish meet this means that the dose should not exceed 12 µg eq/60 kg person , the panel also recommends that the use of validated LC-MS/MS methods as it is more structured way of reporting the toxins in the shellfish.

Much progress has been made in the areas of AZA toxicology and associated human health issues. Much progress has been made in the field of AZA 1 research and its application to protect the health of sea food consumers. Various shellfish monitoring programs , implementation of safe regulatory limits of consumption of shellfish have been imposed for the safety of humans . The strict regulatory control of azaspiracids in shellfish now requires frequent testing of shellfish using highly specific and sensitive methods involving liquid chromatography-mass spectrometry i.e. LCMS.

The measures imposed should be:

1. Reducing eutrophication should be the key concern while considering the future policies, because it does not only pose a problem to ecosystem but a treat to humans also.

2. Sampling of shellfish should be done regularly.

3. Analysis of sample like measurement of the toxicity in the shellfish should be done is regular basis.

4. Evalution of the result of the sampling should be dome in a proper way.

5. Some pamphlets or booklets should be distributed to the people to spread awareness about the harmful effect cause by this toxic shellfish.

6.  Consumers should purchase only inspected shellfish from reliable licensed retailers.

7. Retailers are required to maintain the certification records for their shellfish stock.