Marine Biotoxins

Vinod Kumar Verma, Prabjeet Singh, Dinesh Kumar and Shashank Singh.

College of Fisheries

G.B.P.U.A.T., Pantnagar, Uttrakhand (India)

Email: agnesv2008@gmail.com

A biotoxin is any toxin produced by a living organism (plant, animal, fungus, bacteria). The toxins produced by various marine organism can be summarized as:-

1. Dinoflagellates — Pfiesteria, Ciguatera,

2. Fungi — Stachy botyrs, Fusarium,

3. Bacteria — Pseudonomas fluorescens

4. Spirochetes — Borrelai (Lyme disease)

5. Blue-Green Algae — Microcystis

PARALYTIC SHELLFISH POISONS

Sources and Occurrence

It is caused by a group of toxins (saxitoxins and derivates) produced by dinoflagellates of the genera Alexandrium, Gymnodium and Pyrodinium. Dinoflagellates are among the major components of the marine phytoplankton. They are single-celled organisms, 40 - 50 μm in diameter.

The PSP toxins occur in, and are produced by, certain unicellular marine algae known as dinoflagellates, members of the phylum Dinophyta. Most of the PSP-producing dinoflagellates are found in the genus Gonyaulax.

Chemical Properties

The chemistry of paralytic shellfish poisons (PSP) has been reviewed by Shimizu (1978) and Schantz (1980). PSP are a group of toxins produced by dinoflagellates of the genus Gonyaulax. The first agent to be chemically characterized was saxitoxin, which, though it was initially discovered in shellfish in California, has since been found in greatest concentrations in the Alaskan butterclam, Saxidomus giganteus, from which the name is derived. Saxitoxin has been shown to be a derivative of tetrahydropurine. It is a white, very hygroscopic solid, soluble in water, slightly soluble in methanol and ethanol, but practically insoluble in most non- polar organic solvents. It is a very basic substance, with two titratable groups, pK_a 8.2 and 11.5, and a relative molecular mass of 299.

Symptoms of PSP

The signs and symptoms of PSP in man may range from a slight tingling and numbness about the lips to complete paralysis and death from respiratory failure. Typically, the tingling sensation around the lips, gums, and tongue develops within 5 - 30 min of consumption. In moderate and severe cases, this is regularly followed by a feeling of numbness in the finger tips and toes, and, within 4 - 6 h the same sensation may progress to the arms, legs, and neck, so that voluntary movements can be made only with great difficulty. In fatal cases, death is usually caused by respiratory paralysis within 2 - 12 h of consumption of the PSP-containing food. Sensitivity to PSP is so variable that estimates of the human dose resulting in death range from 500 μg to 1000 μg to 12 400 μg. There are no reports of late effects in survivors or of the effects of long-term, low-level exposure to PSP.

Prevalence in fish and fishery products

Blooms of toxic algae - and outbreaks of PSP - occur regularly throughout Europe, and the EU-monitoring programmes regularly detect high toxin levels. The dinoflagellates bloom as a function of water temperature, light, salinity, presence of nutrients and other environmental conditions. Blooms of toxic algae have recently become more prevalent, and many experts believe coastal pollution and shipping practices have contributed to this expansion. Water temperature must be (5-8°C for blooms to occur. If temperature decreases to below +4°C, the dinoflagellates will survive as cysts buried in the upper layer of the sediments. Shellfish that have fed on toxic dinoflagellates retain the toxin for varying periods of time depending on the shellfish. Some clear the toxin very quickly and are only toxic during the actual bloom. Others retain the toxin for a long time, even years.

Stability of toxin

The toxic compounds are water-soluble and heat stable. A 5-minute cook will reduce toxicity by only 30% and increasing this to 20 min will only effect a 40% denaturation.

Diarrheic shellfish poisoning (DSP)

An intoxication characterized by gastrointestinal disturbances, often occurring as outbreaks associated with the consumption of shellfish, and consequently named diarrhoeic shellfish poisoning (DSP), has been reported from several parts of the world, including the Europe, Japan, South East Asia, North- and South-America. The causative dinoflagellates, which produce the toxins, are within the genera Dinophysis and Prorocentrum. These dinoflagellates are widespread, which means that this illness could also occur in any other parts of the world. A great number of toxins has been identified including okadaic acid (OA) and associated toxins (DTX 1-4)

Sources and Occurrence

Dinophysis fortii, an armoured marine dinoflagellate, has been identified as a producer of DSP in Japan, whereas D. acuminata is suspected of being the toxin producer in recent outbreaks in the Netherlands, based on epidemiological evidence. DSP has not been detected in cells of D. acuminata because attempts to cultivate the organism isolated from Dutch waters have been unsuccessful. Cases in Chile were associated with the occurrence of D. acuta, though detailed information is not available. Occurrence of one of the DSP toxins, okadaic acid, has been confirmed in a benthic dinoflagellate, Prorocentrum lima, though involvement of this species in DSP has never been known.

Species of Dinophysis are distributed widely but seldom form red tides. It has been noted that in the presence of D. fortii at a low cell density of 200 cells/litre, mussels and scallops become toxic enough to affect man. The infestation period in Japan ranges from April to September.

Chemical Properties

The presence in shellfish of 9 toxic components has been recognized and the chemical structures of 5 components have been established. These toxins are classified into two groups: okadaic acid and its derivatives named dinophysistoxins, and the novel polyether lactones named pectenotoxins. The chemical structure for dinophysistoxin-2 is not yet known because of its limited availability, while pectenotoxin-3, -4 and -5 are closely related to pectenotoxin-1, in chemical structure.

Symptoms of DSP

Frequency of signs and symptoms of DSP are diarrhoea (92%), nausea (80%), vomiting (79%), abdominal pain (53%), and chill (10%). The time from consumption of shellfish to the onset of illness ranged from 30 min to several hours, but seldom exceeded 12 h. About 70% of patients developed symptoms within 4 h. Suffering may last for 3 days but leaves few after-effects.

Neurotoxic shellfish poisoning (NSP)

A disease in human beings associated with red tides involving the dinoflagellate Gymnodinium breve has been encountered around the coasts of Florida, USA, named neurotoxic shellfish poisoning (NSP). According to symptoms and mode of exposure, two syndromes can be identified: (a) NSP associated with the consumption of shellfish containing cells or metabolites of toxic G. breve. The symptoms are predominantly neurotoxic in nature and resemble PSP, except that paralysis does not occur; (b) NSP characterized by respiratory symptoms and associated with exposure to aerosols of G. breve cells. There is however much less data available for this disease, compared with the other diseases caused by dinoflagellate toxins and tetrodotoxin.

Sources and Occurrence

The NSP toxic compounds have been isolated exclusively from G. breve, a non-thecate (naked) dinoflagellate, encountered around the coasts of Florida, USA, particularly during red tides, which are initiated in offshore waters primarily in the late summer and autumn months. The iron content of the water might be used as a predictive guide, as a maximum of iron has been observed immediately preceding red tides. Taxonomically, the organism has recently been transferred to Ptychodiscus brevis.

Chemical Properties

These components share the same skeleton made up of a single carbon chain locked into a rigid ladder-like novel structure consisting of 11 continous transfused ether rings. The compounds are soluble in organic solvents but are unstable in chloroform; this has caused difficulties in isolating the toxins in earlier investigations. The toxins are not fluorescent and do not have properties that make detection and quantification easy. Hence, no chemical method for analysis exists.

Symptoms of NSP

In human beings, consuming shellfish contaminated with G. breve cells, causes paraesthesia, alternating sensations of hot and cold, nausea, vomiting, diarrhoea, and ataxia occur within 3 h. Paralysis has not been observed, and the disease (NSP) appears to be milder than PSP. An upper respiratory syndrome of NSP has been reported, associated with aerosols of G. breve cells and/or toxins, in coastal areas of Florida, USA. The rapidly reversible syndrome is characterized by conjunctival irritation, copius rhinorrhoea, and nonproductive cough. There have been no recorded human deaths from NSP, but the toxin is fatal to fish and can cause massive fish kill.

Amnesic shellfish poisoning (ASP)

ASP is the only shellfish poison produced by a diatom. Disease was first identified in Canada in 1987, where more than 100 people became ill after consuming contaminated shellfish. Outbreaks have so far been confined to Canada and the USA, although the responsible algae have been found in many other areas.

Symptoms of ASP

The disease was named after one of the more curious symptoms, which was loss of short-term memory. Other symptoms include nausea, vomiting, diarrhoea, headache and neurological effects including dizziness, disorientation and confusion. In severe cases seizures followed by coma and death may occur. The short-term memory loss seems to be permanent in surviving victims.

Ciguatera fish poisoning (CFP)

A variety of fish inhabiting tropical and subtropical seas may become toxic, and, by ingestion, cause an intoxication in human beings named "ciguatera", which is characterized by neurological and gastrointestinal symptoms. The term ciguatera is of Spanish origin, derived from cigua, which is a Carribean trivial name for a marine snail, Turbo pica that, when eaten, is said to cause indigestion. CFP is one of the most common food-borne illnesses related to finfish consumption. Its true incidence is not known, but it has been estimated that 10,000-50,000 people a year suffer from this disease. It is caused by consumption of fish that have become toxic by feeding on toxic dinoflagellates or toxic herbivore fish.

Sources and Occurrence

A dinoflagellate, Gambierdiscus toxicus, has been identified as the source of ciguatoxin which is found primarily in the tropics where it lives in association with macro algae, usually attached to dead corals. More than 400 species of fish are known to be vectors of ciguatoxins. Toxins can be detected in the gut, liver and muscle tissue by means of mouse assay. Some fish may be able to clear the toxins from their systems. In general, ciguatoxic species are limited to fish that feed on algae and the detritus of coral reefs, particularly the surgeon-fish (Ctenochaetus striatus), parrot-fish (Scarus gibbus), and the larger reef carnivores that prey on these herbivores. Thus, the larger carnivores such as moray eels, snappers, groupers, carrangs, Spanish mackerels, and emperors, certain in-shore tunas, and barracuda are most toxic.

Chemical Properties

The chemical properties of ciguatoxin have been reviewed by Scheuer (1982). The chemical structure of ciguatoxin is still largely unknown. The toxin has been extracted from the the moray eel liver and, after elaborate purification, has been obtained in a pure crystalline form as a white solid (Scheuer, 1982). Ciguatoxin is a highly-oxygenated lipid, soluble in polar organic solvents but insoluble in water. The relative molecular mass is estimated to be 1111.7 ± 0.3, and possible molecular formulae are C₅₃H₇₇NO₂₄ or C₅₄H₇₈O₂₄, but other combinations cannot be excluded.

Symptoms of CFP

Clinical symptoms vary widely but are characterized by gastrointestinal, neurological and cardiovascular disturbances often within 10 min but also up to 24 h after ingestion of toxic fish. The initial gastrointestinal symptoms are similar to any other food poisoning (abdominal pain, nausea, vomiting, and diarrhea). The neurological symptoms most often encountered are tingling and numbness in the mouth, hand and feet, muscle cramping and weakness, temperature reversal, superficial hyperesthasia with a sensation of burning. Headache, vertigo, stiffness, convulsions, hallucinations, transient blindness, salivation, perspiration are symptoms that may occur. A slow, irregular pulse and low arterial pressure may follow. Cardiovascular disorders usually disappear within 48-72 h while neurological effects may persist for weeks, even years in severe cases. Death from CFP is rare (<1% worldwide).

Puffer fish (Tetrodotoxic) poisoning (PFP)

Tetrodotoxin (TTX) is one of the most potent non-proteinacous toxins known and responsible for numerous fish poisonings. The toxin is named after the order Tetraodontidae (common names: puffer fish, balloon fish, globe fish, fugu, toad fish, blow fish), since many of these fish often carry the toxin. Apart from Tetraodontidae toxin has been found in goby, blue-ringed octopus, various gastropods and horseshoe crab. PFP has frequently occurred in Japan, where these fish are a traditional food item.

Source and Occurrence

It is mainly found in the ovaries, liver, and intestines of various species of pufferfish, lesser amounts being found in the skin; the body muscle is usually free of the toxin, with the exception of Lagocephalus lunaris lunaris, which often contains fatal amounts of tetrodotoxin in the muscle tissue (Tabeta & Kumagai, 1980). The most toxic pufferfish are members of the family Tetraodontidae, but not all the species in this family contain the toxin. The most toxic ones are caught along the coasts of China and Japan, and the meat of these species is considered a delicacy. The amount of toxin in the roe is related to the reproductive cycle, and is greatest just before spawning (early summer).

Chemical Properties

The compound has been obtained from an extract of pufferfish viscera in the form of colourless crystal prisms that are slightly soluble in water. It is an aminoperhydroquinazoline compound, with a relative molecular mass of 319. It has a guanidinium group with a pKa of 11.6, and a unique intramolecular hemilactal bond. The toxin is unstable at pH levels above 8.5 and below 3.

Symptoms of PFP

Symptoms of PFP occur within minutes and rarely more than 6 h after ingestion of toxic fish. Nausea and vomiting may or may not occur, but the most common symptoms are tingling or pricking sensation and dizziness. Disease may progress to muscle and respiratory paralysis. Where death occurs it is usually within 6 h and sometimes as rapidly as 20 min following toxin ingestion. Persons who have not died within 24 h generally recover completely. The distribution of the toxin in the fish is mainly in the ovaries (eggs), liver and skin. The muscle tissue is normally free of toxin.

Control and prevention of natural toxins

The molluscs have to undergo the following operations sequentially:

1. Preliminary cleaning in fresh water for a minimum of two minutes at a temperature of 20°C, plus or minus 2°C.

2. Pre-cooking in fresh water for a minimum of three minutes at a temperature of 95°C, plus or minus 5°C.

3. The separation of flesh and shells.

4. Second cleaning in running fresh water for a minimum of 30 seconds at a temperature of 20°C, plus or minus 2°C.

5. Cooking in fresh water for a minimum of nine minutes at a temperature of 98°C, plus or minus 3°C.

6. Cooling in running cold fresh water for approximately 90 seconds.

7. The separation of the edible parts (foot) from the non-edible parts (gills, viscera and mantle) mechanically with water pressure.

8. Conditioning in containers closed hermetically in a non-acidified liquid medium.

9. Sterilization in autoclave at a minimum temperature of 116°C for a time calculated according to the dimension of the containers used but which cannot be lower than 15 .

Conclusion

This document deals with outbreaks of certain human diseases associated with human exposure to compounds produced by algae. Predators feeding on the algae become contaminated by these compounds which, in this way, enter the human food chain.

Diseases such as paralytic shellfish poisoning (PSP), ciguatera, and the more recently identified syndromes, neurotoxic shellfish poisoning (NSP) and diarrhoeic shellfish poisoning (DSP), are discussed in the document as well as the evidence of their association with dinoflagellate toxins present in human food. Tetrodotoxin intoxication (pufferfish poisoning) is discussed because the compound, which is produced by certain fish in various areas of the world, has a similar action to that of saxitoxin, one of the main components causing PSP. Direct dermal contact with toxins causes a particular type of acute dermatitis, observed in certain areas of the world.

References

Abbott, B.C., Siger, A., & Spiegelstein, M. 1975.Toxins from the blooms of Gymnodinium breve. In: LoCicero, V.R., ed. Proceedings of the First International Conference on Toxic Dinoflagellate Blooms, Wakefield, Massachusetts, Massachusetts Science and Technology Foundation, pp. 355-365.

Acres, J & Gray, J. 1978. Paralytic shellfish poisoning. Can. Med. Assoc. J., 119: 1195-1197.

Adachi, R & Fukuyo, Y. 1979. The thecal structure of a marine toxic dinoflagellate Gambierdiscus toxicus gen. et sp. nov. collected in a ciguatera-endemic area. Bull. Soc. Jpn. Sci. Fish., 45: 67-71.

Baden, G.G., Mende, T.J., Lichter, W., & Wellham, l. 1981. Crystallization and toxicology of T34: A major toxin from Florida's red tide organism (Ptychodiscus brevis). Toxicon, 19: 455-462.

Kao, C.Y. 1966. Tetrodotoxin, saxitoxin and their significance in the study of excitation phenomena. Pharmacol. Rev., 18: 997-1049.