Eutrophication, marine biotoxins, human health.

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Eutrophication, marine biotoxins, human health Romano Viviani Department of Biochemistry,

University of Bologna, Via Belmeloro 8/2, Italy

40126Bologna,

ABSTRACT Eutrophication phenomena in marine coastal waters can today be explained on the basis of natural or anthropogenic causes. Undesirable effects and also sanitary problems in both types of eutrophication are often produced, but they may differ greatly in frequency and significance. Some algal biotoxins can affect both marine animals and man, whilst others affect man alone. From data currently available it appears that the sanitary state of man can be affected through the digestive, respiratory and cutaneous apparatus. Four main dinoflagellate biointoxications are now recognized: paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), diarrhoetic shellfish poisoning (DSP), and venerupin poisoning. Other biointoxications are due to a diatom bloom responsible for amnesic shellfish poisoning (ASP) and to blue algae blooms which have effects on the skin and the respiratory tract. All these marine toxins are considered and particular attention is paid to: producing organisms, chemistry of the components, compromised sea foods, methods of analysis, occurrence worldwide, human intoxications, toxicology and mechanism of action on a molecular level, therapeutical notes, tolerance levels and remarks on safety. Attention is also paid to the relationship between the anthropogenic eutrophication and PSP and DSP since these are the most widespread biointoxications from toxic marine dinoflagellates in the world today and for which the European Economic Community (EEC) is proposing health legislation such as tolerance limits and methods for official analysis. In view of the harmful potential of coastal anthropogenic eutrophication, the main current committment of various countries concerns control. Finally, it is important to develop a suitable monitor research system using all the specific standards of allowed toxic substances, and also research on effective antiodotes against all biotoxins.

INTRODUCTION

Pollution by land-based and atmosphere-based sources, the emptying of waste directly into the sea and also natural phenomena such as upwelling, produces effects in the coastal marine environment. With respect to relationships with living organisms, the chemical composition of contaminants which flow into this environment are divided into

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conservative (metals, non-metals, synthetic organic compounds, petroleum hydrocarbons and combustion products) and non-conservative (nutrients containing N, P, Si) [1]. Both conservative and non-conservative contaminants produce effects, not only on marine organisms, but also on public health thus creating complicated problems for national and international programming of the protection of the environment and of public health by legislation for their control. In this overview, the role played by nutrients, on which the eutrophication phenomenon depends has been considered. Ecologically speaking, and in the broadest sense, eutrophication means a substantially increased trophic level beyond the prevailing conditions in a given ecosystem, due to an unusually rich supply of nutrients in the euphotic layer. Eutrophication phenomena in marine coastal waters can today be explained on the basis of natural causes or anthropogenic causes [2]. These natural and anthropogenic causes may also be simultaneously present in the same areas. The first and most important indices of natural and anthropogenic eutrophication phenomena are given by the visible characteristics of water: abnormal growth of macroalgae and/or increase in the phytoplankton biomass, which is indicated by the terms "coloured sea" and "red tides". The main characteristic which distinguishes the two types of eutrophication is the length of time before its appearance. Natural eutrophication is a relatively slow process (time scale 1 0 - 1 0 years). Anthropogenic eutrophication which occurs more frequently in coastal areas due to man's contribution of nutrients appears in a short space of time — 10 years or less on a time scale. Undesirable effects and also sanitary problems in both types of eutrophication are often produced, but they may differ greatly in frequency and significance. To clarify anthropogenic eutrophication further, an important role is played by the eutrophic phenomena that have appeared in recent decades which will be studied together with the determined lengths of time for the occurrence of eutrophication due to the creation of new urban settlements such as tourist, zootechnical and aquacultural ones. This overview defines methodologies in order both to distinguish the various forms of coastal eutrophication, especially anthropogenic eutrophication, and those caused by situations of sea conservative chemical pollution, and in order to discover means of preventing health hazards. From data currently available it appears that the sanitary state of man can be affected through the digestive, respiratory and cutaneous apparatus, therefore eutrophication may have an environmental impact affecting coastal inhabitants, fishing workers and bathing. As far as the biotoxins produced by micro- and macroalgae are concerned, some may result in the intoxication and death of molluscs, crustaceans and marine 3

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fish, some affect both marine animals and man, whilst others affect man alone. The ingestion by man of biotoxins present in aquatic plants or animals produces disorders that are termed biointoxications [31. The information currently available indicates that some biotoxins are synthesized in the phytoplankton, in the phytobenthos and in macroalgae and produce their effects on man as such or after being modified during metabolism in the food chain. Biointoxications are differentiated from pathological conditions caused by food poisoning bacteria, radioactive contaminants, poly cyclic aromatic hydrocarbons (PAH), toxic metals, persistent chlorinated hydrocarbons, parasites and allergies resulting from the consumption of fishing products. At present, toxins from blooms or red tide dinoflagellates are known to be responsible for four biointoxications: paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), diarrhoeic shellfish poisoning (DSP), venerupin poisoning. Another biointoxication is due to a diatom bloom: "amnesic shellfish poisoning" (ASP). Pathologic phenomena in the respiratory tract are present in association with NSP. Other biotoxins produced by blue algae blooms have effects on the skin. In this overview, attention is paid to PSP and DSP since these are the most widespread biointoxications from toxic marine dinoflagellates in the world today and for which the EEC is proposing health legislation such as tolerance limits and methods for official analysis. The present knowledge of NSP and ASP is also considered although, for now, limited to Florida and Canada. The ASP concerns biointoxication produced by a diatom (bearing in mind that in the Adriatic Sea where substantial winter-spring diatom blooms have occurred, a phenomenon known as "mucilage" which is believed to be caused by diatoms, appeared in the summer of 1988 and 1989). Also considered are the toxic cyanophyta for their effects on the skin and for the health problems linked to bathing and thus to tourism in coastal areas. Particular attention is paid to the chemical structure of the biotoxins and to their mechanism of action on a molecular level in order to better understand the biochemical lesion caused by these structures, the damage caused to cells and the consequent health risk and possible therapy. DINOFLAGELLATE TOXINS

Paralytic Shellfish Poisoning

(PSP)

In certain coastal areas oysters, mussels and clams become toxic sporadically or constantly in some months of the year and produce in man a neurotoxic syndrome known as "paralytic shellfish poisoning" (PSP) [4].

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Experimental proof of the algal origin of this food poisoning was given in 1937 by Sommer et al. [5] in California; they succeeded in making healthy molluscs toxic after they had been fed in the laboratory on G. catenella, following the isolation of the algal toxin directly from the cells of this dinoflagellate species. The final proof of the role of phytoplankton in causing PSP in man was obtained when the toxin of Mytilus californianus and the saxitoxin from the siphon of Saxidomus giganteus were isolated and characterized and shown to be chemically identical to the toxin obtained from axenic cultures of Gonyaulax catenella [6]. PSP-producing organism The water-soluble toxins of PSP type are produced in temperate water by members of the genus Gonyaulax, recently reclassified to Protogonyaulax or Alexandrium [7] including: G. tamarensis (G. excavata), G. catenella, G. acatenella and G. monilata. The dinoflagellate recognized to be source of PSP toxins in tropical waters is Pyrodinium bahamense var. compressa [7]. The dinoflagellates are propelled by two flagellae; some are bioluminescent. In addition to the motile form, such as G. tamarensis, they produce resting cysts (hypnozygotes), as a result of sexual reproduction. Thus there are two sources for contamination of shellfish with PSP: (1) motile cells of Gonyaulax species; (2) resting cysts of G. tamarensis in the sediment-water interface; the latter example is thus not associated with a bloom phenomena, and quantitative analysis for phytoplankton of seawater would be a totally inadequate measure to predict shellfish contamination from this source. Based on investigations carried out in North America, Dale and Yentsch [8] considered that cysts which had lain dormant for several months were at least 10 times more poisonous than vegetative cells. Toxin production in P. tamarensis, toxic and non-toxic, and in toxic P. catenella, was studied under varying culture conditions [9]. Cells growing under phosphorus limitation showed a dramatic increase in toxin content over t h e control cultures. Phosphorus limitation and also low temperatures increase the total toxin content per cell, while Fe and Ν limitation showed little effect in toxin production. Another species that is at present classified together with Gymnodinium and Ptychodiscus is Gymnodinium catenatum which it is pointed out, is found not only on the coast of north-west of Spain [10] but also on the west coasts of Mexico [11], and is also known in the gulf of California, the Mar del Plata and Japan. G. catenatum is an interesting species because, in general form and structure, it resembles Protogonyaulax but it lacks theca and produces a hydrosoluble toxin of PSP type.


R1

H

R2

H

R3

Carbamate toxins

N-Sulfocarbamoyl toxins

DecarbamoyI toxins

H

STX ΝΕΟ GTX I GTXII GTX III GTX IV

B1 B2 C3 C1 C2 C4

de-STX de - ΝΕΟ de - G T X I d e - G T X II d c - G T X III d e - G T X IV

OH OH H H

H H H OSO3

H OSO 3 OSO" H

OH

OSO~3

H

R4

R4

R4

o-

oO

635

HO-

o 3s

Fig. 1. Structures of 18 naturally occurring PSP components. STX: saxitoxin; ΝΕΟ: neosaxitoxin; GTX: gonyautoxin.

From these morphological and biochemical similarities it has been suggested that it may be a theca-less mutant of Protogonyaulaux [7,11]. The dinoflagellate Gymnodinium catenatum Graham was collected for the first time in the Galician rias (Spain) in October 1976 during an outbreak of paralytic shellfish poisoning and also in 1981 [12]. In Southern Tasmania (Australia) shellfish toxicity has recently been documented in G. catenatum blooms [13]. Chemistry of the PSP components On the basis of the early investigations of Schantz [14], on the existence of different toxins in G. tamarensis as in G. catenella, other components have been isolated from algae and shellfish, and chemically characterized. The 18 PSP components make up three groups: carbamate, N-sulfocarbamoyl, and decarbamoyl components [15] (Fig. 1). Generally, they have chemical properties comparable with saxitoxin. The carbamate toxins are the dominant components in shellfish, whereas the N-sulfocarbamoyl are the dominant group in dinoflagellate cells. In terms of toxicity the components differ widely, as measured by mouse bioassay, with the carbamate toxins as the most toxic (as a group) and the N-sulfocarbamoyl toxins as the least toxic (5-100 times less); the decarbamoyl components have intermediate toxicity, as a group [4,15].

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The fact that such a large number of closely related compounds is responsible for PSP greatly complicates accurate detection and quantification of shellfish toxicity. PSP compromised seafoods PSP are a group of toxins produced by certain species of dinoflagellates present in phytoplankton. The toxins are taken up by predators feeding on plankton, such as bivalve molluscs, but also as fish plankton feed. Human exposure is principally brought about by consumption of PSPcontaining shellfish which accumulate the toxins. During the process of filter-feeding, a characteristic feature of bivalves, the dinoflagellate motile cells and resting cysts are transported from the gills in the mantle cavity to the oesophagus and stomach. The highest concentrations of PSP have been found in these digestive organs but PSP is also present in other soft tissues. Shellfish are generally not harmed by the presence of PSP. The most contaminated shellfish are Mytilus edulis and Saxidomus giganteus. Since the sulfamate toxin is far less potent than its corresponding carbamate group it is easy to convert sulfamate into carbamate: the sulfamate toxins, when present in bivalves constitute a reservoir of latent or cryptic toxicity [16]. Because of the importance of detoxification of toxic live shellfish, the effects of ozonation, thermal shock, cation exchange and chlorination have been studied on the biological process of detoxification [3]. Ozonation appears to be the most viable procedure to remove low levels of the toxins from soft-shell clams [17], but is ineffective when they have retained the toxins for long periods [18]. Several observations and studies in the past suggest that industrial processing (canning) may be a way of utilizing contaminated shellfish resulting in a pronounced decrease in PSP concentration [3]. In seafood a particular problem concerns finfish. Since finfish, unlike shellfish, are unable to accumulate the toxins in their flesh there would seem to be no problem in terms of the suitability offish plankton feed for human consumption, except possibly in instances where whole fish are consumed without processing [19]. A recently reported human death due to PSP from ingestion of fish in the tropics (Indonesia) was questioned. The response was made that whole fish are often eaten in the tropics. In this instance, the clupeid fish in question were caught by fishermen during a red tide, probably of Pyrodinium bahamense var. compressa [20]. The danger to the public health is through the consumption of whole fish (including viscera) and shellfish harvested from areas in which red tides occur.


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Methods of analysis for PSP The most commonly employed method is mouse bioassay, involving intraperitoneal injection of mice with an acidified extract of shellfish tissue and the determination of death time, which is then converted into PSP concentration. All PSP components are measured by this procedure [3,15]. Identification of numerous saxitoxin derivatives during the last two decades has led to bioassay for PSP detection being considered as a not entirely satisfactory assay for potentially contaminated food sources in various PSP toxins. The development of alternative assay procedures to the in vivo animal bioassay has gained increasing support [21]. An improved high pressure liquid chromatographic procedure (HPLC) for the PSP toxins has been developed by Sullivan and Wekell [22]. At present a radioimmunoassay (RIA) and indirect enzyme-linked immunoabsorbent assay (ELISA) were developed only for the detection of saxitoxin but not for all PSP toxins [23,24]. PSP occurrence worldwide The first descriptions of symptoms, disease and deaths caused by the consumption of poisonous mussels and clams were reported about 200 years ago by Captain Cook and Captain Vancouver during their expeditions to the coast of the Pacific Northwest, and were related to what was known by the Indians [25]. In Europe the first PSP-induced fatal cases in humans reported in the medical literature occurred in 1855 [26] in Germany. In this century most of the reported PSP episodes took place on the east and west coasts of North America. Most cases of PSP have been linked with the "red tides" caused by dinoflagellates [27-29]. A recent summary of PSP in Canada [19] indicates that since 1973 there have been more than 300 documented cases of paralytic shellfish poisoning, resulting in about 35 deaths. Causative dinoflagellates include Gonyaulax catenella, G. acatenella and G. excavata. In the Atlantic coast of Portugal, Spain, England, Norway and the Faroe Islands, cases of PSP have been described since 1960. Shellfish toxins associated with PSP have also been demonstrated annually since May 1968 when 78 people were affected after consuming mussels from the north-east coast of England [30]. In October and November 1976, an epidemic of PSP was recorded in Spain (63 cases), France (33 cases), Italy (38 cases), Switzerland (23 cases) and Germany (19 cases) [31-33]. These incidents in western Europe were caused by mussels (Mytilus edulis) originating from Vigo and Pontevedra (Atlantic coast of Spain) [31]. In the last decade, there has been an increased frequency of red tides in the coastal waters in Brunei Darussalam, Malaysia (Sabah), Philippines and Thailand. The toxic dinoflagellate causing red tides in Brunei

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Darussalam, Philippines and Malaysia w&sPyrodinium bahamense var. compressa [20]. A summary of PSP-related illness, number of hospitalizations and deaths are also reported [20]. During the last two decades PSP has been observed in temperate and tropical areas throughout the world with increasing frequency. Whether this is a true increase, or results from improvement in surveillance, detection and reporting systems, is not clear. The introduction of toxic dinoflagellates into all seas of the world can be made through their cysts in the ballast water of vessels collected during a red tide. Episodes of PSP caused by red tides attributed to hypertrophication of terrestrial origin and to marine aquaculture in the last ten years are reported. Thus, for example, the population of Shatin and Tai Po in Honk Kong's coast has increased from 70 000 in 1973 to 600 000 in 1988, and an ultimate population by 1990 of more than 1 million is anticipated [34,35]. The surface waters of the Tolo Harbour system, polluted by these cities and from agricultural waters have experienced a progressive increase in phytoplankton standing crop and in the incidence of red tides. The phytoplankton species composition in the inner harbour has shown changes since 1976, with diatoms gradually replaced by dinoflagellates. This structural change in the phytoplankton community can be attributed to hypertrophication and organic enrichment of Tolo Harbour. The occurrence of red tides as well as fish kills due to red tides, algal blooms and oxygen depletions in Tolo Harbour have shown a progressive increase since 1979 and have become regular phenomena in recent years [35]. Paralytic shellfish poisoning (PSP) from Protogonyaulax toxicity levels in shellfish collected from Tolo Harbour have, on average, tripled from 1984 to 1987 [35]. The intensification of aquaculture has also influenced the quality of the water leading to increase of toxic phytoplanktonic blooms and of the appearance of cases of PSP [36-38]. This has occurred both in the coastal waters of the Atlantic and in countries of the Far East. Water quality problems associated with cage farming include those due to wastes (faeces and uneaten food) and nutrient discharge, reduced dissolved oxygen and high BOD, especially underneath the cages. The reduction in water currents exacerbates water quality problems. Development of these conditions is considered to result in "self-pollution". This risk is also suggested for mollusc farms. It is known that sedimentation occurs in mollusc farms, resulting from excretory products and the trapping of suspended particles due to reduced water movement. In the Faroe Islands in 1984, the first proof was found that a relationship exists between aquaculture pollution and red tides caused by Gonyaulax excavata, which results in massive fish kills and cases of PSP in man after


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having eaten mussels from the same area [36]. Similar cases have occurred in the Far East [37,38]. Management measures to mitigate deteriorating coastal water quality and the adverse environmental impacts of aquaculture development are now required as a matter of urgency. Human intoxication: clinical toxicology PSP causes a widespread inhibition of impulse-generation in peripheral nerves and skeletal muscles by blocking the sodium channel, which may result in respiratory paralysis leading to death. Saxitoxin is one of the most lethal non-protein toxins known for man (fatal dose 1-2 mg) and approaches botulinum toxin in its lethal effects [3,4,15]. The symptoms of PSP usually appear in man within 30 minutes following the consumption of toxic bivalve molluscs: paraesthesia affecting the mouth, lips, tongue, and finger tips, profound muscular asthenia, inability to maintain an upright posture, ataxic gait, loss of balance. The gastro-intestinal symptoms in PSP due to Gonyaulax, such as nausea, vomiting, diarrhoea and abdominal pain, are less common or do not appear at all. In the most severe forms, the clinical setting is dominated by a progressive muscular paralysis beginning from the legs, and this paralysis prevents standing and results in death due to respiratory paralysis. Consciousness is rarely compromised. In lethal cases the evolution is very fast and the death occurs within 8 hours, on average, due to respiratory or cardiocirculatory deficiencies. The prognosis is favourable in cases of survival in the first 12-24 hours. The mortality index is equal to about 8-10% in the paralytic syndrome due to molluscs [15,39]. Toxicology. (a) Studies of acute toxicity in animals. Tests on various species of animals show that the primary site of action of saxitoxin is located in the periphery, with secondary sites located in the CNS. (b) Mechanism of action. Saxitoxin acts by inhibiting the temporary permeability to N a ions, and this has made a considerable contribution to the hypothesis that the N a and K ions move independently through the cell membrane by separate channels and not by a single common channel [41. At the molecular level all PSP toxins are water-soluble non depolarizing toxins [40]. The saxitoxin as guanidium toxin is regarded as a "blocking" agent that reduces the number of conducting Na channels by occupying some site near the outer opening [41]. Saxitoxin binds to specific receptors in the nerve membrane in a 1:1 stoichiometry [42] with high affinity (Kd = 2 nM) [42]). The potent inhibition of ion flux is not due to a plugging phenomenon but is rather the result of a lid on the sodium +

+

+

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channel, occupying a flat arrangement bound to the anionic surface of the membrane [43]. The recent experiments suggest that this action is not independent from the presence of other toxins [44]. Therefore, channels modified by lipophilic toxins have also to be altered in respect to STX. Therapeutical notes In cases where humans eat saxitoxin-contaminated shellfish, symptoms appear within minutes of ingestion, while death can occur anywhere from 1-12 h later [45,46]. This should give sufficient time to intervene with an injection of antiserum. Recent studies of Davio [47] examined antiserum neutralization of saxitoxin in greater detail. The effect of antiserum injected intravenously must essentially be immediate, since saxitoxin injected subcutaneously normally kills mice within 5-10 min. While the data demonstrate that antiserum A can counteract saxitoxin in vivo, this particular antiserum may not be effective against the many other "saxitoxin-like" paralytic shellfish poisons produced by Gonyaulax dinoflagellates and associated with toxic shellfish. Thus, a true antidote for the paralytic shellfish poisons must have a broader reactivity. Since, until now, effective antidotes against all biotoxins are not available, therapy remains essentially symptomatic [48]. Current therapy of saxitoxin poisoning includes gastric lavage with the help of activated charcoal and similar absorbents, to remove contaminated shellfish, and artificial respiration. Researches on antidotes for PSP are directed to natural active substances. In this respect during a red tide episode caused by Pyrodinium bahamense var. compressa in Western Samar, Philippines in 1983, those who were taken ill after ingesting the green mussel, Perna viridis, were reported to have taken coconut milk (gata, Pilipino) with brown sugar or unpurified sugar-lumps {tagapulot, Pilipino) as a temporary palliative pending medical attention. Many victims felt relief after this drink. It has been demonstrated in mice that substances active in detoxification of Pyrodinium toxins are present in coconut milk and in brown sugar [49]. Tolerance levels and remarks on safety The Task Group of the World Health Organization recognized serious difficulties in establishing the dose associated with the appearance of signs and symptoms and death [15] with reference to bioassay of contaminated food. The human dose resulting in death ranges from 500 |jg up to 12 400 μg. More than 30 years ago the USA and Canada adopted the tolerance


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level of 80 μg PSP/100 g (exercised on fresh shellfish at the production site). In Europe most countries have adapted the 80 μ&100 g tolerance; however, within the European Community (EC), three countries — the Federal Republic of Germany, Italy [50] and The Netherlands — have established a lower tolerance of 40 μg/100 g. During the PSP outbreaks in Italy in 1976 caused by imported mussels from the Atlantic coast of Spain, the lowest level causing symptoms was 566 μg/100 g [32,33]. The most recent PSP outbreak in Europe, published in the scientific literature, took place in Norway in 1981. Eight out of 10 people, who consumed mussels containing about 1600 pg, total PSP/100 g became affected. Two people developed no symptoms of intoxication at all, having ingested an estimated total dose of 320 |ig [51]. Since the tolerance employed in USA and Canada (80 |ag/100 g) is more than 10 times lower than the lowest level that has caused intoxications, as observed during the most recent PSP outbreak in Europe [32,33,51], in order to harmonize the PSP tolerance in the EC, it is recommended that the EC adapt a tolerance of 80 μg PSP/100 g for shellfish. In relation to the common methods to be used — in addition to the bioassay method — the fluorometric HPLC procedure [22] has been proposed, but the use of this procedure requires the availability of reference material for at least six PSP components [52], and that kind of reference material is not yet commercially available. In addition, studies should be undertaken to elucidate PSP distribution in shellfish under several environmental conditions such as blooms of PSP-producing dinoflagellates and absence of dinoflagellates but presence of resting cysts. Neurotoxic Shellfish Poisoning

(NSP)

A milder neurotoxic form (NSP), occurring in Florida after ingestion of bivalve molluscs and showing similarities in some aspects to ciguatera, has been shown to be linked to another red tide-forming dinoflagellate [4]. All red tides reported in Florida are associated with mass mortality in marine animals. This phenomenon is due to a bloom of Gymnodinium breve which was identified in 1947 as the aetiological agent and is considered to be the sole agent responsible for all the outbreaks described since 1844. These phenomena were observed 24 times from 1844 to 1971 [53] and the fact that they occurred before the development of agriculture, towns, industries and tourism indicates their natural origin. Besides the environmental and economical damage, health problems caused by consumption of toxin-infested shellfish and by inhalation of the wind-sprayed cells were noticed.

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Organisms producing NSP toxins The red tide-forming dinoflagellate, Ptychodiscus brevis (= Gymnodinium breve), is one of the most notorious species for its mass fish kills and destruction of other marine life along the coast of Florida. Resting cysts of P. brevis are not present in the sediment-water interface. The motile form of P. brevis produces several neurotoxins, collectively called brevetoxins (or P. brevis toxins). These accumulate in filterfeeding shellfish (oysters, clams) causing neurotoxic shellfish poisoning (NSP) when consumed. An atoxic strain of G breve was found in the Inland Sea, Japan [54]. P. brevis appears to be not only restricted to the gulf of Mexico, the east coast of Florida and North Carolina coast [55], but in reports of some blooms from northern Spain, the eastern Mediterranean coast and Japan [53]. Gymnodinium catenatum, collected for the first time in Galician rias (Spain) in October 1976 and also in 1981, produces PSP toxins but not brevetoxins [12]. Chemistry of NSP components The first structure elucidation of the most abundant P. brevis neurotoxin was by Lin et al. [56] and consisted of an eleven-number heterocyclic oxygen-containing fused ring system culminating in an unsaturated lactone at one end and an unsaturated aldehyde at the other, designated brevetoxin-B (BTX-B). Other brevetoxins were characterized [57-59] (Fig. 2, Table. 1). OH

Me

D

> ^ O H

2

BvTX-X E

ο ^ O ^ C H . CH2

Fig. 2. Structures of the brevetoxins (BvTX). The backbone of BvTX-A differs from that öf brevetoxins B, C, D, E, which vary only in the substituents (R) on carbon 41 [581.

C H


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TABLE 1 Brevetoxins nomenclature [44] New nomenclature

Old nomenclature

BvTX-A

BTX-A

BvTX-B

BTX-B, GB-2, T2, T34, T47

BvTX-C BvTX-D BvTX-E

BTX-C dihydro-BTX-B, GB-3, T17 GB-3-acetate

Brevetoxin-compromised seafoods The major seafoods containing brevetoxins are shellfish [60]. From the preliminary stages of studies on brevetoxins isolated from P. brevis and from neurotoxic shellfish it appears that the toxins are not always identical in amounts or type. The approximate ratio of T17 (GB-3) to T 3 4 (BTX-B, GB-2) is 1:3.1 in P. brevis obtained either from culture or from field population. There is little qualitative data on rates of accumulation and depuration of brevetoxins in bivalves. Oysters accumulate the toxins in less than 4 h in the presence of 5000 cells/ml, and depurate (60%) of the accumulated toxin in 36 h [61]. Potency of depuration is species-specific and highly variable, even under controlled laboratory conditions [62]. Commercial bivalves, therefore, are generally safe to eat 1-2 months after the termination of any single bloom episode. Canning cannot be a way to decrease NSP concentration in bivalves. However, while no cases of death have ever been confirmed in man following ingestion of mollusc after Florida red tides, the fishing industry also suffers due to adverse publicity concerning dead fish washed ashore. The fish usually start dying when P. brevis counts reach the 250 000 cellsAitre range. Methods of analysis for NSP The potency of P. brevis blooms is generally determined by an ichthyotoxicity assay of either the contaminated seawater or crude and purified toxin extracts [3]. Toxicity of contaminated shellfish is determined by mouse bioassay, which evaluates the cumulative effects rather than determining concentrations of individual toxins. The bioassay is based on the dose that provokes a fixed death time in mice injected intraperitoneally with a crude toxic residue extracted from bivalves with ethyl ether [63].

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Recently, methods using high performance liquid chromatography (HPLC) have been developed for the qualitative and quantitative analysis of the P. brevis toxins [64,65]. Human intoxication Early records (pre-1900) report illnesses following consumption of shellfish taken from Florida red tide areas [66], but only since 1952 has the relationship between "red tide" caused by G. breve [67] and neurotoxic shellfish disease (NSP) become known. This disease occurs when people are poisoned by eating bivalve molluscs containing G. breve toxins. Two intoxicative phenomena that occur in man during Florida red tides are NSP and respiratory irritation. Neurotoxic shellfish poisoning is a rather mild form of shellfish poisoning. According to McFarren et al. [67] the G. breve toxins produce, in man, sensations of paraesthesia in the mouth and digits, ataxia, slow pulse, sensations of hot and cold, dilatation of the pupil (mydriasis), and mild diarrhoea, followed by recovery in two days. The degree of the reaction depends on the quantity of toxin consumed, but all victims recover in a few days. The neurotoxic form occurring in Florida after ingestion of bivalve molluscs is similar in some respects to ciguatera, a syndrome caused in the Caribbean and in tropical coral reef areas in the Pacific Ocean by the benthic dinoflagellates Gambierdiscus toxicus and Prorocentrum concavum. A differential diagnosis for comparing ciguatera with DSP is needed. Ecological data (phytoplankton, red tide) can provide elements for a differential diagnosis (among them fish mortality), but they need to be confirmed by ichthyotoxicity in laboratory. P. brevis cell fragments, upon becoming airborne in sea spray, elicit nonproductive sneezing and coughing when inspired [68]. All toxins isolated from P. brevis possess this activity and during purification, if they become airborne on silica gel particles, cause the same effect. Toxicology (a) Studies of acute toxicity in animals (in vivo). Two direct intoxicative phenomena unique to Florida red tide are extensive fish kills and human respiratory irritation. Fish kills are the most prominent visible effect of P. brevis red tides. NSP is, in contrast, the secondary or indirect acute phenomenon which is due to consumption by man of bivalve molluscs contaminated by toxins. Toxins Τ π and T34, isolated from Florida's red tide dinoflagellate Ptychodiscus brevis, cause a dose-dependent response in several in vivo biological systems. Potent fractions responsible for in situ fish lethalities,

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respiratory irritation, and neurotoxic shellfish poisoning have been identified [69]. Both toxins are potent ichthyotoxins. Most investigators use, in fact, a fish bioassay to identify potent fractions during purification. (b) Mechanism of action. While the water-soluble toxins STX and GTX responsible for PSP act as non-depolarizing agents in the membrane of the excitable cells, lipid-soluble neurotoxins — brevetoxins, responsible for NSP — act as depolarizing substances (Fig. 2). One of the toxic fractions (T47) acts to open membrane channels permeable to N a , leading to a N a influx. K analysis precludes the effect of T47 acting on the K channels [70]. Studies with T17 and T34 permitted the evaluation of different potencies and the extension of knowledge about the mechanism of action at the level of the molecular structure of membrane and results indicated that T17 exerts its depolarizing action by activating sodium channels and that the Τ17 binding site is separate from the tetrodotoxin site and perhaps lies in proximity to the procaine binding site [71]. According to more recent research, the lipophilic toxins profoundly affect Na channels, modifying virtually every aspect of their physiology and also the interaction of the channel with nearly every other known class of active drug, including polypeptide toxins, local anaesthetics, and the guanidinium toxins [44]. It is worth mentioning that at 10-15 g/ml of GBTX , C a uptake in skeletal sarcoplasmic reticulum (SR) vesicles is inhibited but the C a dependent ATPase activity and Ca efflux are enhanced [72]. Concerning health problems caused by inhalation of the wind-sprayed cells of P. brevis, this condition arises from the opening of sodium channels by the toxin(s), releasing acetylcholine and causing smooth tracheal muscle contraction. The effects are only temporary [73]. +

+

42

+

2+

a

2+

Tolerance levels and safety considerations The Florida Department of Natural Resources (DNR) has run a general control program since the mid 1970s. Only in 1984 were Ptychodiscus blooms specifically noted in control regulations. Closures are made when the dinoflagellates exceed 5000 cellsAitre near harvesting areas. Closures have lasted between a few weeks and six months. Two weeks after Ptychodiscus concentrations drop below 5000 cellsAitre, the first mouse bioassays of shellfish are carried out. When levels are below 20 MU (mouse unit)/100 g the grounds are reopened [74]. The bioassay system is slow; results take nearly one week. A field assay kit is in development. Also in Italy provision of law is based on this bioassay but established "no detectable amount" [50].

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Venerupin

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poisoning

Venerupin poisoning is a non-paralytic human biointoxication different from the DSP. Organisms producing toxins Prorocentrum minimum var. mariae lebouriae and var. triangulatum, which co-occur in blooms [75] have been incriminated in Japan in venerupin poisoning. According to Tangen [76], Prorocentrum minimum Schiller, probably responsible for the shellfish poisoning in Norwegian coasts, is a phytoplanktonic species so common that, if it is the source of the highly toxic "venerupin", the toxin must be only in rare strains. Prorocentrum minimum Schiller red waters have often been observed in Obidos Lagoon (Portugal) and have caused toxicity of bivalves there. Particular attention is given to two of those blooms, separated by about 10 years, in May-June 1973 and in January-February 1983 [77]. A comparative study of environmental conditions during the two red water of P. minimum indicates that the instances of P. minimum red water in 1973 and 1982-83 were both preceded by long periods of heavy rain. Phosphate in the lagoon waters increased during the observed phytoplankton blooms, with the two maximum peaks found during the P. minimum bloom. Also nitrate and ammonium proved to be important for the start of P. minimum growth in 1982-83. Research on the components of venerupin poisoning The toxic principles were found in the digestive glands (hepatopancreas liver or dark gland) of the bivalves by Japanese investigators [78]. Toxicity of 75% methanol extracts of cultured Prorocentrum minimum var. mariae-lebouriae, which is supposed to produce venerupin poisoning [75] was determined using mice as test animals. The chemical nature of the toxins is not established. Venerupin-compromised seafoods Venerupin poisoning was first reported in Nagai, Japan, in 1889, following the ingestion of the oyster Crassostreagigas. Of the 81 persons poisoned, 51 died [27]. A second outbreak occurred in 1941, when of 6 patients, 5 died, and from 1942 to 1950 there were 455 additional cases involving the eating of oysters and the shortnecked clam Tapes japonica [79]. Several hundred cases have been reported in the area of Lake Hamana with more than 100 deaths [80]. Also in Norway symptoms of venerupin poisoning have been described in 70 persons after consumption of mussels collected close to the centre of the massive bloom of P. minimum in the autumn 1979 [76].


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Methods of analysis for venerupin Demonstration of a Prorocentrum minimum bloom and determinations of toxins in mouse. The toxins are determined by assay in mice using procedures described by Okaiki and Imetomi [75] and also used by Tangen [76]. Human intoxication Venerupin shellfish poisoning is a public health problem in the Shizouka and Kanagawa prefectures of Japan, where these shellfish are not eaten from January through April. The poisoning is characterized by a long incubation period (24-48 h and sometimes longer [27,75,76]), anorexia, halitosis, nausea, vomiting, gastric pain, constipation, headache and malaise. These symptoms may be followed by increased nervousness, haematemesis, and bleeding from the mucous membranes of the nose, mouth and gums. In serious cases, jaundice may be present, and petechial haemorrhages and ecchymosis may appear over the chest, neck, and arms. Leucocytosis, anaemia, and a prolonged blood-clotting time are sometimes observed. The liver is usually enlarged. In fatal poisoning, acute yellow atrophy of the liver, extreme excitation, delirium and coma occur. Diarrhoetic Shellfish Poisoning (DSP) Diarrhoetic shellfish poisoning (DSP) is a term proposed by Yasumoto et al. [81,82] for a shellfish poisoning distinctly different from PSP and NSP in both symptomatology and etiology. The clinical symptomatology is of the gastrointestinal type, consisting of nausea, vomiting, and diarrhoea. Organisms producing DSP toxins In Japan Dinophysis fortii has been incriminated as the organism producing DSP toxins [55]. On European Atlantic coasts other dinoflagellate species are involved in DSP intoxications: Dinophysis acuminata in Spain [84]; D. acuminata, D. sacculus, Prorocentrum lima in France [85]; D. acuminata, Prorocentrum redfieldii, P. micans in The Netherlands [86]; D. acuminata, D. norvegica, P. micans in Scandinavia [87]; Dinophysis sp., D. sacculus, D. fortii in Adriatic sea [88]. Also various members of Dinophysis and Prorocentrum should be regarded as shellfish contaminants that may have caused diarrhetic poisoning. Chemistry of the components of DSP toxins Polyether toxins were isolated from infested shellfish. They are classified into acidic and neutral toxins. The acidic group comprises

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okadaic acid

(OA) :

= H . R2 =H

dinophysistoxin-1

( D T X 1 ) : R1 = H , R 2 r C H

dinophysistoxin-3

( D T X 3 ) : R 1 =acyl ( C

41

pectenotoxm-1

14

-C

3 2 2

), R2 = C H

3

O

( P T X 1 )

:

R

= C H 2O H

pectenotoxin

- 2 ( P T X 2 )

: R

= C H 3

pectenotoxin

- 3 ( P T X 3 )

: R

= C H O

Fig. 3. Structures of two groups of DSP toxins.

okadaic acid, dinophysiotoxin-1 and -3 (DTX-1 and DTX-3). The neutral polyether lactones were named pectonotoxins (PTX) after the family of the scallops (JPecten sp.) from which the toxins were first isolated [83] (Fig. 3). Another polyether toxin, yessotoxin, has been identified in Patinopecten yessoensis [89]. Only the acidic components (okadaic acid, DTX-1, DTX-3) have been shown to cause diarrhoea in experimental animal studies. The remaining four components have not been reported to have a diarrheagenic effect. Intraperitoneally administered in mice PTX-1 causes liver damage. DSP compromised seafoods Causative shellfish in Japan were the mussels Mytilus edulis and M. coruscum, the scallops Patinopecten yessoensis and Chlamys nipponesis akazara, and the short-necked clams Tapes japonica and Gomphina melaegis, while in European Atlantic coasts M. edulis in particular but also Ostrea sp. In Japan and in the Atlantic coast of Spain and France the infestation period ranges from April to September and the highest toxicity of shellfish is observed from May to August, though it may vary locally [81-85]. In Scandinavia, in contrast, oysters in February and mussels in October have caused DSP [90]. In the Adriatic Sea the data of 1989 (first of DSP episode) indicate that the infestation period in some coastal areas ranges from May to November [88].


649

Comparative analysis for DSP in various shellfish collected from the same area was conducted in Japan and the highest toxicity was found in the blue mussels, with less toxicity in scallops, and very little in oysters. The differences were noted between mussels cultivated at different depths, with concentrations differing by factors of two to three [81,82]. Also the first results obtained in the Adriatic Sea [88] show that not all species of bivalve molluscs, living in the same habitat infested by the microalgae, manifest an analogous functional attitude towards the absorption and concentration of the enterotoxin in their tissues. In particular, Mytilus galloprovincialis, Chamelea gallina, Tapes decussata and Venus verrucosa have been monitored for toxin mouse bioassay and DSP was detected only in mussels, although they were drawn out of the same habitat. This uneven distribution of DSP will have an impact on the developments of sampling plans for shellfish, as part of monitoring schemes for control purposes. The method of cooking did not alter toxicity of the causative shellfish but intoxication could be avoided if the digestive glands were eliminated beforehand [81,82]. Methods of analysis for DSP The bioassay of all DSP components is based on the dose that provokes a fixed death time in mice injected intraperitoneally with a toxic residue extracted from shellfish with acetone [83]. The procedure is the official method in Japan and in several other countries. In France toxicity is expressed differently from the official Japanese biological test. Other bioassay methods are: suckling mouse bioassay [91], rat bioassay [92], Tetrahymena test [93]. More recently fluorometric determination of okadaic acid and DTX-1 has been developed using HPLC [94]. The bioassay and the HPLC method have not been studied collaboratively, and no attempts have been made to study the scientific parameters, such as precision, sensitivity and specificity. The intercalibration procedure is not applicable since at present no reference material for any of the DSP components is commercially available. DSP occurrence worldwide The most obvious phenomenon indicating a rapid production of algal toxins is the appearance of red tides along the coast. On the other hand not all DSP outbreaks are accompanied by macroscopic blooms of Dinophysis sp. or Prorocentrum sp. The first studies on DSP were carried out in Japan and continued in Europe, in which diagnostic investigations were made into different gastrointestinal disorders caused by some food-borne bacteria and viruses from DSP. In Japan more than 1300

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cases of DSP intoxication were reported in the period 1976-1982 [83]. In Spain approximately 5000 cases of gastrointestinal disorder were encountered in September 1981 [95]. Bacteria were ruled out as a cause, but no attempts were made to detect DSP components in the shellfish, as no methodology was available at that time. In France outbreaks of DSP intoxication amounted to about 2000 cases in both 1984 and 1986, with only 10 cases in 1985 [96,97]. Similar disease descriptions have been reported from outbreaks of DSP-intoxication in The Netherlands [92]. In Scandinavia 300-400 cases were encountered in the DSP outbreak in the fall of 1984 [98]. In June 1989 the presence of Dinophysis fortii cells in the hepatopancreas of mussels and lipid-soluble toxin of DSP type in mussel tissues collected in the coastal water of the Emilia-Romagna region [88] proved that the cause of certain cases of diarrhoea in consumers of molluscs was not due to bacteria or viruses but to biointoxication by DSP. This phenomenon, brought to light by the Research Center of Marine Biological Resources of Cesenatico (University of Bologna, Italy), has subsequently extended over the coastal areas of Abruzzo, Veneto and Friuli-Venezia Giulia. The existence of the enterotoxin in seafood was initially revealed by the McFarren method (biological test for the research into fat-soluble algal biotoxins) according to the provisions of Italian Law [50]. In the second stage the Yasumoto method [83] was used [88]. Human intoxication: clinical toxicology Frequency of signs and symptoms of DSP in patients were as follows: diarrhoea (92%), nausea (80%), vomiting (79%), abdominal pain (53%), and chill (10%). The incubation period ranged from 30 min to several hours but seldom exceeded 12 h. Around 70% of the patients developed symptoms within 4 h. Suffering may last for 3 days in severe cases but leaves no after-effects [81,97]. Thus no fatal case has ever been recorded. The minimum amount of DSP required to induce disease in adults has been estimated from analysis of left-over food to be 12 MU [83]. In Scandinavia mussels associated with the outbreak contained approximately 17 MU per 100 g [98]. In an inventory of phytoplankton perturbation along the French coast in 1986 the highest DSP levels in shellfish were 10.6 MU/100 g [96]. Mechanism of action Okadaic acid, being hydrophobic [99], can enter cells and operate on particulate as well as cytosolic fraction of various mouse tissues [100]. It is a very potent inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), two of the four major protein phosphatases in

651


the cytosol of mammalian cells that dephosphorylate serine and threonine residues [101,102]. Of the other two major protein phosphatases, the Ca 7calmodulin-dependent protein phosphatase 2B (PP2B) is far less sensitive, while the Mg-dependent protein phosphatase 2C (PP2C) is unaffected [103]. Other protein phosphatases [103-105] and numerous protein kinases are insensible to the toxin [104,106]. These and other studies have established that okadaic acid is extremely useful for studying regulatory phenomena in eukaryotic cells that are controlled through the reversible phosphorylation of proteins and in identifying ion channels that are controlled by phosphorylation. Knowledge about these biological processes which are included in the second messenger field can help us to understand the biochemical lesion of okadaic acid and other constituents of DSP as pectenotoxins. Okadaic acid probably causes diarrhoea by stimulating the phosphorylation that controls sodium secretion by intestinal cells as in the disease cholera caused by a toxin secreted by Vibrio cholerae, but with another mechanism. One of the subunits of cholera toxin can permanently activate the Gs protein, leading to continuous adenylate cyclase activity [107]. The resulting increase in cAMP activates cAMP-dependent protein kinase, which then phosphorylates one or more proteins that control sodium secretion by intestinal cells. Since cAMP or Ca /calmodulin dependent protein kinases or protein kinase C [104-106] are unaffected by okadaic acid, the inhibition of PP1 and PP2 is probably responsible for phosphorylation control of ion channels. Okadaic acid acts also through the variations of the cellular concentration of the C a second messenger. Okadaic acid strongly increases the L-tyjje inward C a current in isolated guinea pig cardiac myocytes [108] and increases the open state probability of C a dependent K channels in tracheal myocytes [109]. Are the PP1 and PP2 the okadaic acid-sensitive enzymes which dephosphorylate and inactivate the Ca channel? Recent data indicate that okadaic acid may function, not only as a tumor promoter, but is also capable of reversing cell transformation by some oncogenes. Carcinogenesis involves at least two stages, namely initiation and promotion. The tumor initiation stage is caused by agents that produce damage in DNA. The most well-known tumor promoter is 12-o-tetradecanoylphorbol-13-acetate (TPA), a deterpenoid ester from Croton oil. Unlike carcinogens which act directly on the cellular DNA, tumor promoters exert their effects by binding to receptors. These receptors somehow control cell growth and differentiation, for some cells can be induced to proliferate while others are induced to differentiate on treatment with very small quantities of tumor promoter. So, for example, low levels of a typical carcinogen insufficient to produce tumor alone 2

2+

2+

2+

2+

+

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when painted on mouse skin previous to application of the tumor promoter (TPA) yielded many tumors. It was found, employing the twostage carcinogenesis model, that okadaic acid [110] and DTX-1 [111] acted as promoters of skin tumors in the mouse, using dimethylbenz(a)anthracene (DMBA) as tumor initiator. There was no promoting effect using okadaic acid or DTX-1 alone. DTX-1 also induces ornithine decarboxylase activity in mouse skin comparable to the activity of okadaic acid. In addition NIH3T3 cells transformed by either the raf or ret-U oncogenes partially revert to the normal phenotype after incubation for two days with 10 nM okadaic acid [112]. Since PP1 and PP2A are likely to be the chief enzymes that reverse the actions of protein kinase C, they may function as either tumour promoters or suppressors, depending on the transforming agent or the cell type. Tolerance levels and remarks on safety All human episodes of DSP intoxication are characterized by signs and symptoms of gastro-intestinal disorder, suggesting that a tolerance should encompass diarrhoeagenic components only. No evaluation of DSP tolerances has yet been performed by international organizations, like the World Health Organization. Whilst awaiting these definitions a surveillance plan for DSP has been introduced in several European countries, comprising of frequent sampling of seawater for phytoplankton analysis and shellfish for detection of dinoflagellates in the digestive tract as well as for toxin analysis. A number of countries have established tolerances for DSP, applicable for domestic shellfish production sites as well as by importation of shellfish.^ The tolerances established vary greatly from country to country. In Denmark, The Netherlands and Spain the mouse bioassay method established "no detectable amount". In France the mouse bioassay established a tolerance of 0.044 MU/g digestive glands [113], while in Japan with another principle for MU calculation the level is 5 MU/100 g soft tissue as it is in Norway [83,114]. Sweden is the only country in Europe which monitors shellfish for DSP by the HPLC procedure [94] and maintains a tolerance level of 60 μg/100 g soft tissue (as okadaic acid and DTX-1). As no case of DSP contamination is known without the presence of at least one of the acidic components, a EEC tolerance level for DSP is suggested to include the acidic components, such as okadaic acid and DTX-1, which can be monitored by chemical procedures such as HPLC [115]. It should be explored whether the tolerance used in Sweden can be considered a safe level as improved analytical performance will increase the safety of shellfish consumers. IUPAC is presently organizing a collaborative study of the HPLC procedure by Lee et al. [94]. However, it


653

is essential that the EEC makes reference material of DSP components, at least the acidic components (okadaic acid, DTX-1), available to member countries. Documentation available until now from the EEC show that there is a tendency to standardize the analytical methodology and limits of tolerance with reference to the Swedish regulations, the use of a chemical method in HPLC of the tolerance limits of 60 μg of okadaic acid and DTX-1 per 100 g of soft parts of molluscs, was proposed [115]. Okadaic acid and DTX-1 are two of the chemically defined toxins, found in European shellfish.

DIATOM TOXINS

Amnesic Shellfish Poison (ASP) As regards eutrophication phenomena, diatoms were not considered to be as problematic as dinoflagellates until the end of November 1987 when 153 cases of acute intoxication related to ingestion of toxic mussels were documented in Canada. Symptoms included vomiting and diarrhoea which, in some cases, were followed by confusion, loss of memory, disorientation and even coma. The term Amnesic Shellfish Poison (ASP) has been proposed for this new shellfish toxin [116]. Chemistry of ASP component A task force was organized jointly by the Canadian Department of Fisheries and Oceans (DFO) and Health and Welfare Canada (HWC) to establish the extent of the contamination, the chemical nature of the toxin and its origins. Chemical analysis soon established that the toxicity was not due to any of the marine toxins that are usually monitored, nor to any of the known anthropogenic xenobiotics (e.g., heavy metals, pesticides, PCBs, and PAHs). Within 5 days it was established that the mollusc toxin was domoic acid, a relatively rare neurotoxic amino acid [116] (Fig. 4). ASP producing organisms Collaborative work in Canada led to identification of the pennate diatom, Niteschia pungens Grunow forma multiseries Hasle, as a source of domoic acid. Samples of the diatom were taken from the water through the ice in the vicinity of the Cardigan River estuary and were found to be associated with domoic acid levels as high as 1% dry wt following the toxicity outbreak [117].

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.COO HOOC

•COO

Quisqualate

Kainate

N - methyl - D-aspartate (NMDA)

Fig. 4. Structural formulas for domoic acid and other selective exogenous excitants.

Methods of analysis for ASP The biotoxicological method of analysis on mice for ASP is the same as that used for PSP. Mice injected intraperitoneally with a dilute hydrochloric acid extract of mussel tissue containing domoic acid showed that the relative potency of domoic acid is lower than that of PSP. When the observation time was extended to 3 h, to allow simultaneous testing for PSP and domoic acid, it was found that extracts of oysters from certain locations showed occasional low-level toxicity [118]. It was found that the mouse deaths in this case were due to a zinc level of over 900 μg g" wet wt [119]. Oysters are known to naturally accumulate zinc [119]. Chemical methods have also been defined for the demonstration and quantification of domoic acid in molluscs [120]. In order that both domoic acid toxicity and PSP may be identified in shellfish in Atlantic Canada, half the dilute hydrochloric acid extract, from shellfish being tested, is used for mouse bioassay and the other half for HPLC tests [120]. 1

ASP occurrence worldwide ASP now occurs only in Canada. One wonders why mussels in the Cardigan Bay area of Canada were particularly affected by domoic acid containing diatoms. Concentrations of the AT. pungens in Cardigan Bay were 10 million cells per litre in 1987. Some suggest that the proper mix of nutrients, sunlight and stratification due to fresh-water runoff contributed to the diatom blooms. It has also been suggested that fertilizers from intensive tobacco farming in the drainage basin of the Cardigan River may have provided the necessary nutrients for anthropogenic eutrophication [121]. Human intoxication: clinical toxicology Domoic acid is a mild neurological poison compared to PSP. When mussels contaminated by domoic acid were eaten in eastern Canada they produced 153 cases of gastro-intestinal distress with nausea, vomiting

655


and diarrhoea within 24 h; but added to that disorder, they also caused neurological illness within 48 h in older victims (ca. over 60 years old). Three elderly patients died. In the most severely affected cases neurological symptoms still persisted [116,121]. Toxicology The mechanism of action of domoic acid is actually known on excitatory amino acid receptors and on synaptic transmission. Excitatory amino acids, most notably L-glutamate and L-aspartate, have long been considered to be the most likely neurotransmitters [122]. These amino acids are known to act on several receptor types, the best characterized of which are named after the selective exogenous excitants iV-methyl-Daspartate (NMDA), kainate and quisqualate (Fig. 4). Glutamate and also NMDA subclass act to open membrane channels permeable to N a , leading to a N a influx and membrane depolarization [123]. Only the channel opened by NMDA receptor accessible to kainate, quisqualate and to domoic acid is, in addition, highly permeable to C a and induced lethal cellular C a entry. Actions at NMDA receptors can be selectively antagonized by micromolar concentrations of magnesium ions, organic antagonists such as D-2-amino-5-phosphonovalerate (APV) and dissociative anaesthetics, such as phencyclidine (PCP) [123]. +

+

2+

2+

Tolerance levels and remarks on safety An effect on certain consumers of domoic acid contaminated shellfish was inferred at an estimated concentration of 200 \xg g" wet wt. Therefore, an application factor of 0.1 was applied for safety and a concentration of 20 [ig g wet wt was set as the level of domoic acid above which a shellfish operation should be closed [121]. This compares with 0.8 Lig g for saxitoxin in shellfish, above which an area is closed for shellfish harvesting owing to PSP. With regard to health safety, a concentration of 20 \xg/g of domoic acid in a fresh weight of molluscs is considered tolerable [121]. With regard to health, particular attention must today be paid to the diatoms, in the high and middle Adriatic sea, in relation above all to the appearance of the "mucilage", which seems to originate from the diatoms among which there is a species of Nitzschia. This phenomenon has in fact created considerable ecological problems and has given rise to worries over health in Italy and Yugoslavia. The evaluation of any biotoxins in the "mucilage" was carried out both in 1988 and 1989 at the Research Center of Marine Biological Resources of Cesenatico (Italy) using the PSP method which, according to current Canadian legislation, is valid for ASP monitoring. The analyses always produced negative results and up to now the presence of domoic acid has always been excluded [124]. 1

- 1

_1

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CYANOPHYTA TOXINS

Cyanophita in freshwater are the main organisms responsible for eutrophication effects and for producing toxins [3]. Esotoxins are also produced by planktonic bloom forming genera of marine cyanobacteria belonging to the Oscillatoriaceae family, which poses potential public health problems for respiratory (Trichodesmium erytraeum) [125] or cutaneous (Lyngbya majuscula) symptoms of poisoning [126]. As far as Trichodesmium erytraeum respiratory symptoms are concerned, these are related to the presence of sea water aerosol containing fragments of this cyanophyta during blooms in coastal waters of Brazil [125] and in the Thailand Gulf [127]. This occurs almost every year, usually in February or March in Tamandarè bay or on the north-west coast of Brazil, and is locally indicated by the terms "Tingui" or "Tamandarè fever" [125]. It is characterized by respiratory symptoms accompanied by asthma, temperature increase, articular and periorbital pains, and burning on thorax and arms. On average, the symptomatology lasts three days. Eutrophication of coastal waters has only become apparent as a problem in the Gulf of Thailand and the species found to bloom most frequently is the cyanophyta Trichodesmium erytraeum [127]. The filamentous cyanophyta L. majuscula which grows abundantly in many areas of the sub-tropical and tropical Pacific basin and also in Caribbean, is the causative agent of a severe contact dermatitis that affects swimmers and bathers at the beaches [126,128]. Greater knowledge of the chemical nature and mechanism action of the marine cyanophyta toxins concern L. majuscula. Toxins of Lyngbya majuscula causative agents of contact

dermatitis

Chemical structures The active principle of the blue-green alga L. majuscula have been isolated and identified as two phenolic bis-lactones, aplysiatoxin and debromoaplysiatoxin [129], and an indole alkaloid, lyngbyatoxin A [130]. All of these three substances have been shown to be potent irritants, producing erythema, blisters and necrosis when applied to the skin [131]. Human intoxication The most recent major outbreak of this severe contact dermatitis that affects swimmers and bathers at beaches on the windward side of Oahu occurred in August 1980, at Kailua, Kalama, and Pilapu beaches. A total of 86 cases were reported to the Hawaii State Department of Health. The

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severe contact dermatitis was described as similar to a burn and generally involved the genital and perianal areas. The initial symptoms, which appeared after a few hours, were erythema and a burning sensation, followed by blister formation and deep desquamation which lasted for several days [128]. Toxicology The mechanism of action at cutaneous and respiratory organ level can be explained on the basis of knowledge about tumor-promoting properties, in that lyngbyatoxin A, debromoaplysiatoxin, and aplysiatoxin induce irritation in mouse skin to the same degree as TPA [132]. The most well known tumor promoter is 12-o-tetradecanoylphorbol-13-acetate (TPA), a deterpenoid ester from Croton oil. Recent studies suggest that the phorbol ester, teleocidin, and aplysiatoxin tumor promoters (Fig. 5) operate by activating a phospholipid and calcium ion dependent phosphorylating enzyme, protein kinase C [133]. The activity of protein kinase C is also stimulated by unsaturated diacylglycerol. It has been suggested that the reputed endogenous analog of these tumor promoters might actually be a diacylglycerol and that

debromoaplysiatoxin, R = H

TPA

Fig. 5. Structures of toxins of Lyngbya majuscula which behaved like the phorbol esters (TPA) and teleocidin Β typical tumor promoters.

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protein kinase C may be a receptor for the tumor promoters or at least a component of the receptor complex [134], In a search for new antineoplastic agents from blue-green algae, a cytotoxic substance active against P-388 lymphocytic mouse leukemia from a deep-water variety of L. majuscula showed that it was identical to debromoaplysiatoxin [135]. In the same species of cyanophyta there is a molecule which, depending on whether it contains Br or not, shows either tumor-promoting or antineoplastic properties. CONCLUSIONS

In view of the harmful potential of coastal eutrophication, there is an evident need for a reliable control and prevention of red tides. As far as the environmental conditions responsible for eutrophication are concerned, the measures to be taken for its prediction, prevention and control are being considered. As far as health, economic, aesthetic and environmental consequences are concerned, the main current commitment of various countries concerns control. Once the health risks have been defined and quantified and once the safety limits for seafood and for the effects on the skin and respiratory apparatus have been outlined, a control can be put into operation thus covering the health responsibility. A control can be made by decontamination of the shellfish and, once a red tide has begun, by finding the means, possibly biological, of accelerating its disappearance. A control by purification of the shellfish can be obtained for hydrosoluble biotoxins notwithstanding its limitations. This method is economically impracticable for the lipid soluble biotoxins due to the lengthy procedures required. A biological control solution would be preferable to a chemical control, but for red tides this still remains within the realms of science fiction. The closest to such a control is the possibility of using a parasitic dinoflagellate to attack the red tide organism. Amoebophrya ceratii is known to parasitize a variety of different dinoflagellates responsible for PSP, but greatly prefers Protogonyaulax catenella [136]. Also evaluated was the use of "grazing" by protozoa Zooplankton for PSP [137], whilst the nude dinoflagellate Ptychodiscus brevis, responsible for NSP, has been found to be particularly sensitive to "aponin", a surface-active natural compound produced by cyanobacterium Gomphosphaeria aponina [138]. Always in correlation to control, it would be opportune to develop a suitable monitor research system using all the specific standards of allowed toxic substances, and also research on effective antidotes against all biotoxins.


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As regards prevention, man is unable to control the natural conditions, but he is able to moderate the anthropogenic coastal nutrient load thus reducing both eutrophication and toxic red tides in the marine environment. In some cases, however, comparison of natural versus anthropogenic impacts is used by those responsible for pollution or degradation of the aquatic ecosystem to minimize the significance of anthropogenic effects on this planet. A living resource that is vulnerable to occasional natural catastrophe is rendered doubly vulnerable by unfavourable anthropogenic effects [139]. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

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A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids.

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Eutrophication of Dutch coastal waters.

Confidence rating for eutrophication assessments.

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Potential threats posed by new or emerging marine biotoxins in UK waters and examination of detection methodology used in their control: brevetoxins.

Thresholds in eutrophication of natural waters.

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The effects of marine carbohydrates and glycosylated compounds on human health.

Marine harmful algal blooms, human health and wellbeing: challenges and opportunities in the 21st century.

β-Lactam antibiotic-degrading enzymes from non-pathogenic marine organisms: a potential threat to human health.

Potential Threats Posed by New or Emerging Marine Biotoxins in UK Waters and Examination of Detection Methodologies Used for Their Control: Cyclic Imines.

Solid-phase extraction-based ultra-sensitive detection of four lipophilic marine biotoxins in bivalves by high-performance liquid chromatography-tandem mass spectrometry.

Has eutrophication promoted forage fish production in the Baltic Sea?

Coastal eutrophication in Europe caused by production of energy crops.

Mercury and Arsenic Bioaccumulation and Eutrophication in Baiyangdian Lake, China.