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3.0 DINOPHYSIS BLOOMS AND DIARRHETIC SHELLFISH POISONING

3.1 Nature of Diarrhetic Shellfish Poisoning ( DSP)

Gastro-intestinal distress in humans, with symptoms of diarrhea, nausea and vomiting, following consumption of mussels was first reported from The Netherlands in the 1960s (Kat 1979, 1985). A gastro-enteritis outbreak, with similar clinical symptoms after consumption of mussels, also occurred in Norway that decade (Krogh et al., 1985), with evidence of a similar event in 1901, nearly a century earlier (see Dahl et al., 1996). Kat was the first to postulate that dinoflagellates, on which mussels feed, and specifically species of the genus Dinophysis, were the source of the toxic principle that caused gastro-enteritis. This linkage was confirmed in Japan where gastro-enteritis outbreaks correlated with mussel toxicity and seasonal blooms of the dinoflagellate Dinophysis fortii (Yasumoto et al., 1979, 1980). The syndrome was termed Diarrhetic Shellfish Poisoning ( DSP), and the responsible toxin(s) termed dinophysistoxin ( DTX-1) (Yasumoto et al., 1980). The chemical structure of the DSP toxins ( DST) isolated from toxic scallops corresponded to the polyether compound, okadaic acid ( OA), and was identical to the toxin isolated from Dinophysis (see Quilliam and Wright, 1995). The DSP toxin group is composed of four compounds, OA and its derivatives DTX-1, DTX-2, DTX-3, any or all of which may occur during a given DSP incident, with others still being discovered [Section 3.4]. The DSP toxins are accumulated mainly in the hepato-pancreas of bivalves which can transform the ingested DSP toxins, for example from DTX-1 to DTX-3 (Landsberg, 2002). The chemical and physical properties of the DSP toxin group are given in Quilliam and Wright (1995).

Upon ingestion, DSP toxins bind to protein phosphatase receptors, building up phosphorylated proteins that lead to the clinical symptoms of lethargy, general weakness, cramps, vomiting and diarrhea. Symptoms usually appear 30 minutes to several hours after consumption of contaminated shellfish, and can persist for several days (Larsen and Moestrup, 1992). Death from DSP has not been reported, but there is increasing awareness that OA and its derivatives may promote skin and stomach tumours (Landsberg, 2002). Mussels are primarily associated with DSP toxicity, but other bivalve species feeding on Dinophysis can become toxic . Bivalves differ in their rates of accumulation and depuration of DSTs, as found with other phycotoxins (Shumway, 1990). Blue mussels, Mytilus edulis, in Swedish waters remained toxic for up to five months (Shumway, 1990). When DSP toxin levels in shellfish exceed 200 ng g ^-1, closure of shellfishing grounds is recommended (Quilliam and Wright, 1995). Vectoring of DSTs through the food web and their effect on marine organisms are poorly understood (Landsberg, 2002).

3.2 Dinophysis species: DSP toxicity , taxonomic problems, autecology

Dinophysis is probably the most species-rich genus of dinoflagellates, with more than 200 species described (Larsen and Moestrup 1992),. However, a capacity for DST production has been confirmed in only eight dinophysoids to date, and suspected in two others (Table 2). Maximum reported concentrations of DSP toxins reported for Dinophysis species are given in Table 3.

Table 2.Dinophysis species reported to produce dinophysistoxins ( DST), or are suspected producers.

Table 3. Maximal concentrations of DSP toxins in Dinophysis species ( OA = okadaic acid; DTX-1 = dinophysistoxin-1).

** from Johansson et al., (1996); others extracted Andersen et al. (1996).

DST levels vary among dinophysoids; maximum and minimum OA levels differ about 2-fold, and DTX-1 levels 25-fold, excluding the trace amount reported for Dinophysis acuminata (Table 3). The major DTX-1 producers are Dinophysis fortii and Dinophysis rotundata; Dinophysis acuta has high levels of OA. Field studies indicate that cellular DST levels vary greatly during blooms of a given species, both locally and among regionally separated populations (see Blanco et al., 1996). Dinophysis rotundata, for example, is toxic in Spanish rías. but non-toxic in eastern Canadian and French coastal waters.

DSP toxicity is not limited to Dinophysis species, although they are the primary sources of DST. Benthic (psammic) dinoflagellates in the genus Prorocentrum also produce dinophysistoxins, most notably Prorocentrum lima, a common and widely distributed species found also in Scottish coastal waters (Barbier et al., 1999; Maranda et al., 1999; Tett and Edwards, 2002). Prorocentrum lima can grow epiphytically on macroalgae, fouling aquacultural long-lines (Lawrence et al., 1998), and also swarms pelagically (Maranda et al., 1999). There is circumstantial evidence that P. lima epiphytic on the long-lines of culture-rafts was the source of the DSP toxicity of shellfish which ingested P. lima during its sporadic resuspension events (see Lawrence et al., 1998). There are no confirmed cases of shellfish DSP toxicity having resulted from the ingestion of other epibenthic prorocentroids. While the contribution of P. lima and related congeneric species to DSP toxicity appears to be relatively minor, the focus on Dinophysis may have overlooked the importance of P. lima. Unlike Dinophysis spp., P. lima is easy to culture; relevant information on its growth and toxicity is available (Barbier et al., 1999; Bauder et al., 1996; Marr et al., 1992; Tomas and Baden, 1993).

Dinophysis spp. form morphotypes that confuse their taxonomy and compromise taxonomic identification of DST producers collected in field samples, a problem aggravated by the differences in toxic potential found among species (Table 3). For example, cells having size and shape features intermediate between those of D. acuminata and D. sacculus, whose toxic potential differs, have complicated monitoring efforts and species identification in French, Spanish and Adriatic coastal waters (see Bravo et al., 1995). This has led to recognition of a " Dinophysis acuminata complex", which includes D. cf. acuminata, D. cf. sacculus and an unidentified Dinophysis sp., and to which these species are assigned in lieu of proper identification. Small cells of D. acuta resemble Dinophysis dens (Hansen, 1993). The morphological intergrades characterizing Dinophysis may be formed in response to environmental conditions, or represent different life cycle stages (Solum, 1962; Bravo et al., 1995; Hansen, 1993).

Investigators have been unsuccessful in establishing Dinophysis into experimental cultures despite repeated efforts. Basic ecophysiological information on the growth and nutritional requirements of Dinophysis species is lacking. It is known that Dinophysis species are slow growing, and that Dinophysis is a nutritionally versatile genus, with species having obligate photosynthetic, mixotrophic and phagotrophic capability, and various combinations of these metabolisms found (Granéli et al., 1995; Jacobson and Anderson, 1994; Maestrini, 1998; Subba Rao and Pan, 1993). Dinophysis ecophysiology is reviewed by Maestrini (1998).

3.3 Dinophysis blooms

Ecological insights into Dinophysis are based primarily on field observations. The evidence suggests that Dinophysis species are generally cosmopolitan and eurythermal, with a preference for open coastal and oceanic waters. Dinophysis spp. rarely achieve population levels sufficient to discolour seawater. Reference to Dinophysis blooms is usually to population densities of <10 ⁴ cells L ^-1, with abundance usually <10 ³ cells L ^-1. Population levels of 10 ⁴ to 10 ⁵ cells L ^-1 during DSP episodes are sometimes reported (Dahl and Yndestad, 1985; Kat, 1985), but these populations have often been physically aggregated and concentrated by current movements, rather than the result of local growth. An extreme example of this is the mass occurrence (23 million cells L ^-1) of D. acuminata, D. acuta and D. norvegica found in reddish bands 10 m in width and a few dm in thickness within a small, sheltered Norwegian bay concentrated there by onshore winds (Dahl et al., 1996). Thus, Dinophysis "blooms" can result from two different mechanisms: in situ growth at the observational site, or as the advected accumulation of cells produced elsewhere; e.g. active and passive local growth. Pazos et al. (1995) have described D. acuta blooms that developed in a Galician rîa corresponding to these two different bloom mechanisms. A mid-summer bloom was the result of an hydrography that supported local in situ growth; the late-autumn bloom that developed during a downwelling event was an "accumulation bloom" formed by cells advected into the rîa.

Dinophysis spp. frequently become entrained and disperse within the streamlines accompanying small-scale, Langmuir circulation cells, and also within the larger-scale features of more dynamic current systems. As already pointed out, "blooms" of this slow growing HAB assemblage are often physical accumulation events, rather than the result of active growth (Lassus et al., 1993). Godhe et al. (2002) has provided a particularly vivid account of the influence of such advections on DSP toxin fluctuations in a Swedish mussel farm area. In addition to tolerating entrainment within currents, several Dinophysis species are capable of growth in fronts. In Irish coastal waters (McMahon et al., 1998), the sources of Dinophysis cells advected onshore are often frontal zone populations actively growing at those physically dynamic sites. The Irish Shelf Front, located 30 km offshore, was the source of D. acuminata and D. acuta populations advectively seeded into coastal shellfish farms leading to DSP toxicity of the cultured shellfish. Dinophysis spp. are also fast swimmers (Smayda, 2000), and exhibit strong diel vertical migrations (see Maestrini, 1998). Such behaviour facilitates the summer blooms of D. acuta that develop deep (15-20 m) within the stratified layer, and below the pycnocline in Spanish coastal waters at mussel culture sites (Reguera et al.,1995). Cells from this sub-surface bloom are redistributed and seeded into other areas by wind-induced upwelling or internal waves.

Smayda and Reynolds (2001) recognized nine distinct habitats in which dinoflagellates assemble and bloom, molded around an abiotic template of light energy, nutrient supply and physical mixing in permutative combinations. Dinophysis species have been classified as "drift" forms (Type VII) capable of achieving modest blooms during upwelling relaxations (see Reguera et al., 1995). However, they are better adapted to advection within the streamlines of small-scale, Langmuir convection cells than in more dynamic current systems in which shear/stress is more intense. Species that bloom in the latter habitats, termed "mixing-drift habitats" (Type IV-VI), are adapted to the increased velocities associated with frontal zones, the dampened, but still elevated vertical mixing during coastal upwelling relaxations, and when entrained within coastal currents. Such species are, respectively, components of the frontal zone life-form (Type IV = Karenia mikimotoi), the upwelling relaxation life-form (Type V = Gymnodinium catenatum), and coastal current entrained life-form (Type VI = Alexandrium tamarense). The Type IV-VI species listed here are common in the Scottish and European coastal waters , and are discussed in later sections.

Dinophysis spp. probably are insignificant in carbon cycling and marine trophodynamics, given their low standing crops and notwithstanding their species diversity and wide geographical distribution. Were they not the cause of diarrhetic shellfish poisoning hazardous to human health and a threat to mariculture, they probably would be primarily of taxonomic interest. The following Section evaluates the toxic Dinophysis communities found in European coastal waters, their impacts on aquaculture and the potential problems confronting development of shellfish cultivation in Scottish coastal waters.

3.4 Dinophysis and DSP in European coastal waters

DST contamination of cultured and wild shellfish stocks in European coastal waters has been reported from 10 countries: Denmark, France, Germany, Ireland. Italy, Norway, Portugal, Spain, Sweden and The Netherlands. Since Kat (1979) first reported DSP illness in The Netherlands 25 years ago, DSP outbreaks have become common in European waters. A major DSP event occurred in 1984 during a D. acuta bloom in Scandinavian coastal waters (Skagerrak) when several hundred persons became ill after eating mussels (Dahl and M. Yndestad, 1985; Krogh et al., 1985). In France, major DSP outbreaks were recorded in the early 1980s during blooms of D. acuminata (Lassus et al., 1985). In 1983, about 3,000 DSP intoxications were recorded in southern Brittany (Belin et al, 1993). In Spain, a 1981 DSP outbreak afflicted "several thousand" persons who had eaten cultured mussels (Fraga and Sanchez, 1985). Dinophysis blooms have become the main threat to the Galician mussel industry (Blanco et al., 1995). Market closures due to DSP usually occur during the late-summer and early autumn blooms of D. acuminata and D. acuta. During 1991-1993, these species and DST were present year-round. In Portugal, where D. acuminata and D. acuta also bloom, DSP toxicity was first confirmed in 1987, and (excluding 1993) has recurred annually in Aveiro estuary (Palma et al., 1998).

In Danish coastal waters and fjords, D. acuminata and D. norvegica can be abundant, while D. acuta and D. rotundata also commonly occur, but are less abundant (Andersen et al., 1996). Dinophysis toxins are detected in mussels only when D. acuminata is abundant. In cases where D. norvegica and DSP toxicity co-occur, toxicity is found only when D. acuminata is present.

In Ireland, DSP toxicity is a growing threat to shellfish cultivation, with new DSP toxins being discovered - features that distinguish Dinophysis and DSP behaviour in Irish coastal waters from other European waters. The annual yield of rope-cultured mussel ( Mytilus edulis) in the bays of southwest Ireland is about 5,000 tonnes (McMahon et al., 1998). But further development of the shellfish industry " has been hindered by regular closure due to accumulation of DSP " associated with the presence of D. acuta and D, acuminata (p. 128 in McMahon et al., 1998). Since DSP monitoring began in 1984, harvesting closures at shellfish cultivation sites, some lasting up to 10 months, have been common, required because of elevated DST levels in mussels and oysters ( Crassostrea gigas) (McMahon et al., 1995, 1998). The "toxic season" usually occurs during summer (June to September), and growers and processors adapt their marketing strategies to this "toxic season" effect. In 1994, toxicity ( DTX-2) persisted until February even though toxic Dinophysis species were not recorded after September. This led McMahon et al. (1995) to state that surveillance of Dinophysis spp. presence alone should not be relied upon to prevent marketing of toxic shellfish; bioassay and chemical confirmation of toxin presence are needed.

The DSP toxins being reported from Irish coastal waters are notable. DTX-2, isolated from Bantry Bay in 1991 (Hu et al., 1992; Carmody et al., 1995), appears to be the predominant OA derivative in Irish coastal waters, and not DTX-1 and DTX-3 that are typically found (see Draisci et al.,1998). A new okadaic ( OA) class toxin, DTX-2B, has also been isolated from Irish mussels (Draisci et al., 1998). Killary toxin-3 ( KT-3) was isolated from mussels collected from Killary Bay after at least eight persons in The Netherlands became ill after consuming mussels cultured in this west Ireland embayment (Satake et al., 1998). Satake et al. (1998) suggested that winter-toxicity of mussels in Killary Harbour, both chemically and etiologically, is distinct from DSP, and that KT-3 may belong to a new class of toxins. KT-3 also differed from other known nitrogen-containing dinoflagellate toxins - procentrolide, pinnatoxin, gymnodimine and spirolide. [Okadaic acid and its derivatives do not contain nitrogen (Quilliam and Wright, 1995).] Recently, James et al. (2001) recognized KT-3 as a new class of shellfish toxins, and renamed azaspiracid ( AZP). AZP has caused human intoxications in The Netherlands (1995), Arranmore Island (1997), France (1998) and Italy (1998) following consumption of mussels and oysters cultivated in four different regions along the Irish west coast. This toxin has also been reported from England and Norway (James et al., 2001). Azaspiracid is slowly depurated by shellfish, with toxicity having persisted up to eight months in mussels cultivated in Killary Harbour. While the human symptoms of illness induced by AZP are similar to DSP, azaspiracids differ toxicologically and chemically from other shellfish toxins. This led James et al. to declare a new toxic syndrome named Azaspiracid Poisoning. The phytoplanktonic source of azaspiracid poisoning has not been identified; field samples suggest that it may be D. acuta.

The Dinophysis species found in Irish coastal waters commonly occur in European coastal waters, and include D. acuminata, D. acuta, D. norvegica and D. rotundata, species that are confirmed DST producers (Tables 2, 3), with the former two species most frequently implicated in European DSP reports. In Japan, where DSP is a continuous problem, D. fortii and D. acuminata are the primary DST sources, with D. fortii having a higher toxin content (Sato et al., 1996; Yasumoto et al., 1980). Given the common pool of Dinophysis spp. in European coastal waters, the unique occurrence of novel DSP toxins in Irish coastal populations is puzzling. Neither the toxic profiles, nor bloom dynamics would appear to reflect special growth conditions at the aquacultural sites. McMahon et al. (1998) have demonstrated, for example, that offshore populations of D. acuminata found at thermocline depths of ca. 30 m in the Irish Shelf Front bloom during upwelling stimulations. Exceptionally large populations (124,000 cells L ^-1) of D. acuminata develop which are then advected onshore into shellfish culture areas, and become the source of the DSP toxins accumulated by the shellfish. That is, DSP toxicity in those instances is not the result of local growth and blooming of seeded Dinophysis populations, behavior that might lead to the unique toxic profiles being reported for Irish coastal waters. Dinophysis blooms at Irish aquacultural sites appear to be primarily "accumulation blooms", i.e. the toxic profiles and abundance of the advected cells reflect growth conditions elsewhere. It is well-known that toxicity of harmful algae is under genetic and environmental regulation, and that strain (clonal) differences occur. For example, the OA toxicity of French strains of P. lima was weaker than in Spanish strains (Barbier et al., 1999). It is unknown to what extent Dinophysis populations, their dynamics and regulation in Irish coastal waters diverge from those in continental European waters, contributing to the more or less distinctive DSP features compromising shellfish cultivation in Ireland.

Evidence for the view that the increasing reports of DSP events and the discovery of new OA class toxins indicate that a global increase in DSP is in progress is not convincing, partly because of failure to qualify what is meant by an "increase". If it is meant that blooms of toxigenic Dinophysis spp. are increasing, and/or their distributional ranges are expanding, confirmatory evidence for such ecophysiological change is lacking. If it refers to reports of DSP toxicity as a statistic, then this interpretation is justified. However, the more likely explanation, then, is that the global increase in cultivation and consumption of shellfish, particularly of edible mussel, is revealing a shellfish-borne toxicity that is normally present and latent. A statistical increase in DSP episodes is quite different from an increase that requires an altered ecology favorable to Dinophysis blooms.

3.5 Dinophysis blooms and DSP in Scottish coastal waters

Routine monitoring has established that diarrhetic shellfish poisoning is an occasional problem in Scottish coastal waters (Tett and Edwards, 2002). Dinophysis spp. occur commonly throughout Scottish coastal waters, are indigenous to sea lochs and offshore coastal waters, and usually occur in low abundance (<2,000 cells L ^-1), even when DSP is detected. Dinophysis populations have been reported from Lochs Creran, Etive, Long, Striven, Firth of Clyde, Edrachillis Bay and The Minch (see Tett and Edwards, 2002). This widespread distribution is consistent with Tett and Edwards' observation that DSP affected sites can occur anywhere on the Scottish coast, without any particular region being more affected than others, or without a clear regional pattern evident. The potential for significant Dinophysis bloom events and DSP toxicity in these waters is demonstrated by the exceptional bloom of D. acuminata that reached 942,000 cells L ^-1 in upper Loch Long in July, 1992, where it caused a brown-red discolouration of the surface layers, and mussels tested positively for DSP (Macdonald, 1994). Macdonald reports that " this was the first reported incident of a Dinophysis bloom on the west coast of Scotland". Dinophysis acuta and D. norvegica were also present. In 1997, okadaic acid was found in mussels harvested from Loch Seaforth and Loch Roag in the outer Hebrides.

Dinophysis acuminata occurs most frequently in offshore waters, from which it is often advected onshore where it can become concentrated and/or grow as a "cove" bloomer, e.g. in semi-enclosed, coastal indentations. This behavior is probably responsible for the Loch Long bloom, which is similar to the sites of unusual D. acuminata bloom events reported for Danish (Anderson et al., 1996) and Norwegian waters (Dahl and Aune, 1996). Although mussels in Loch Long tested positively for DSP during the D. acuminata bloom, there were no reports of human illness. Macdonald points out that shellfish in this area are not regularly harvested commercially. Two aspects of the Loch Long bloom are of special interest relative to Dinophysis blooms elsewhere in European coastal waters. The advection of Dinophysis populations into lochs where fish farms are located raises the question of the potential role salmonid waste excretion might have on local Dinophysis blooms increasing the possibility of a DSP-toxic shellfish outbreak at fish farm sites. Shellfish cultivation in France, Ireland and Spain has been compromised by Dinophysis blooms, and the new derivatives of DSP toxins being discovered. Despite the low incidence of DSP related problems in Scottish coastal waters, the widespread distribution of the causative Dinophysis spp. in those waters (Table 2) and the aquacultural / fish farming issues raised by the Loch Long event indicate the potential for heightened, future DSP problems in Scottish coastal waters.

From the perspective of the potential stimulation of blooms of DSP-toxic Dinophysis species by fish farm operations - fish farming in Scottish waters would appear to be relatively non-threatening. Dinophysis species primarily occur in offshore coastal waters of relatively low nutrient content where they are adapted to survive entrainment within coastal currents and to grow at weak frontal systems. Dinophysis spp., even though generally capable of mixotrophy, are slow growing, with a very curtailed bloom capacity. They are classified as a Type VII dinoflagellate life-form types (Smayda and Reynolds, 2001). Type VII dinoflagellates prefer habitats that are intermediate between those of the mixing-drift adapted species (Types IV-VI) and those species adapted to highly stratified, oligotrophic watermasses (Types VIII-IX). The relatively low grade, broadly dispersed and infrequent episodes of DSP toxicity reported in Scottish coastal waters, despite intensive fish farming (Tett and Edwards, 2002), is consistent with the conclusion that Scottish fish farm activities most likely have minimal influence on Dinophysis blooms.

The situation is less clear with regard to the potential effect of shellfish aquaculture on Dinophysis toxicity in Scottish waters. The Irish experience of the need for frequent closures of shellfish culture sites because of advective transport of toxic Dinophysis spp. from offshore (Type VII) population growth sites into the shellfish growing areas is potentially of greater concern than fish farm waste nutrient stimulation of Dinophysis blooms. The mechanisms of advection and onshore concentration and the toxic Dinophysis spp. pool that occur in Irish coastal waters also occur off Scotland [see Section 11]. Should shellfish culture in Scotland increase both in yield and in the number of growth sites, then periodic DSP outbreaks are expected to impact this industry adversely. Given the experience in Ireland and Spain, shellfish aquaculture increases toxin transfer from the naturally occurring Dinophysis cells to the filter feeding, cultured shellfish (because of their higher population densities) over that of natural shellfish beds.

The related issue of whether cultured shellfish secrete nutrients, or alter phytoplankton communities during their filter-feeding which would favor Dinophysis blooms and DSP events is considered in Section 10 . Given the similar habitat conditions in Irish and Scottish coastal waters, it is important to establish whether the novel AZP and OA class DSP toxins reported from Irish shellfish cultivation sites, and unique to European coastal waters, are regionally coherent and extend into Scottish coastal waters. The appropriate DSP monitoring model to be applied to Scottish coastal waters may be that used in Ireland, rather than at shellfish cultivation sites in continental Europe. Expansion of shellfish culture in Scotland [see Section 10.5] must be accompanied by an upgraded program of DSP monitoring, and analyses of the toxin profiles of contaminated shellfish.

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