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2.0 AMNESIC SHELLFISH POISONING AND PSEUDO-NITZSCHIA BLOOMS

2.1 Nature of Amnesic Shellfish Poisoning ( ASP)

Amnesic Shellfish Poisoning ( ASP) was first recorded in 1987 when 107 persons became ill and three died after consuming toxic blue mussels ( Mytilus edulis) cultured in Cardigan Bay, Prince Edward Island, Canada, (Bates et al., 1998). The mussels contained the neurotoxin, domoic acid ( DA) (Figure 1). The clinical symptoms of domoic acid ( DA) intoxication leading to ASP are: nausea, vomiting, abdominal cramps, diarrhea, memory loss, decreased level of consiciousness, seizures, confusion and disorientation (Landsberg, 2002). Domoic acid is a water soluble tricarboxylic acid of low molecular weight (311), and a secondary metabolite produced by the condensation of a citric acid cycle derivative (probably glutamate) with geranyl (probably geranyl pyrophosphate) (see Maldonado et al., 2002). Domoic acid mimics the neurotransmitter L-glutamic acid, and in the presence of glutamate binds irreversibly to glutamate receptor sites. Binding causes massive depolarization of neurons increasing cellular Ca ⁺ which induces neuronal swelling and death. The affected nerve cells are located in the hippocampus and function in memory retention; hence, the memory loss characteristic of ASP (Bates et al., 1998).

An equally novel DA outbreak occurred in Monterey Bay, California, four years after the Canadian ASP event. Althought no human illness or fatalities were reported, this was the first Pacific incident and first aquatic animal mortality attributed to DA recorded. At least 43 brown pelicans ( Pelecanus occidentalis) and 95 Brandt's cormorants ( Phalacrocorax penicillatus) died from ingestion of northern anchovy ( Engraulis mordax) contaminated with DA (Work et al., 1993). Domoic acid has since been detected in the flesh and viscera of other phytophagous clupeoids, including bay anchovy ( Anchoa mitchilli) and sardines (Landsberg, 2002; Vale and Sampayo, 2001). In Mexican waters, brown pelicans died after feeding on chub mackerel, Scomber japonicus, which contained DA (Sierra Beltran et al., 1997).

Soon after the Monterey outbreak in 1991, DA presence in razor clam ( Siliqua patula) and Dungeness crab ( Cancer magister) populations along the U.S.A. Pacific Northwest coast led to closure of an important commercial and recreational fishery for these species (Horner et al., 1996). Four years later, in 1995, high concentrations of DA (1,100 µg g ^-1) were detected in sea scallops ( Placopecten magellanicus) from Georges Bank, one of the largest commercial shell fishery sites in the western North Atlantic (see Pan et al., 1996). In 1998, more than 400 California sea lions ( Zalophus californianus) died along the central California coast after ingesting DA during feeding on northern anchovy ( Engraulis mordax). Affected sea lions displayed the neurological symptoms associated with DA intoxication, including seizures, head waving, ataxia, depression, and abnormal scratching (Scholin et al., 2000). The latter symptom, accompanied by a loss of righting reflex, was also displayed by piscivorous brown pelicans ( Pelicanus occidentalis) poisoned after ingesting DA during feeding on toxin-bearing anchovy (Work et al., 1993) and chub mackerel (Sierra Beltran et al., 1997). Surviving pelicans remained weak and disorientated two months after their intoxication.

Discovery that DA is a phycotoxin that causes human ASP and mortality of marine fauna, ranging from invertebrates to marine mammals, provoked considerable alarm among public health officials and researchers. The lethal vectoring of DA from anchovies to piscivorous sea birds and marine mammals required resolution of whether human consumption of fish that ingest and store DA posed an ASP health threat. There is no evidence that this is the case. The early evidence suggested that DA "outbreaks" were primarily a North American problem, and demonstrated the need to document the global occurrence patterns of DA and the sites of potential ASP outbreaks. Routine monitoring for DA using shellfish as sentinel indicators has now established that this phycotoxin is found globally, its sources are species of the globally distributed diatom genus, Pseudo-nitzschia. [European occurrences of DA are discussed in Section 2.9.]

Multiple adverse affects accompany the presence and vectoring of DA among marine foodweb compartments: the threat of human illness and death ( ASP); seafood safety; financial loss resulting from closure of shellfish cultivation sites and fishing grounds; mortality of natural stocks; vectoring of DA through the foodweb leading to ecological disruption of key trophic components. Among these, the need to ensure seafood safety prompted the issuance of regulations in 1997 which defined the maximum concentration of DA permissible in shellfish marketed in Europe (see Gallacher et al., 2001). The monitoring protocols developed for shellfish safety applies a threshold level of 20 µg DA g ^-1 tissue (organ), above which harvesting and marketing restrictions are imposed, including closure of fishing areas (Gallacher et al., 2001; Vale and Sampayo, 2002).

2.2 Scottish coastal waters and ASP

In response to the 1997 directive, and after trial assays in 1996-1997 detected DA (Tett and Edwards, 2002), DA monitoring in Scottish waters initiated in 1998 detected its presence in several commercially important shellfish species and other invertebrates (Gallacher et al., 2001). Surveys in 1999 revealed DA levels increased dramatically: in the commercially important king scallop ( Pecten maximus) populations, DA levels considerably exceeded (maximum = 534 µg g ^-1) the threshold of 20 µg DA g ^-1 tissue (organ) (Figure 2). Domoic acid was also found in harvestable queen scallop ( Chlamys opercularis), but at much lower levels (maximum = 48 µg g ^-1), even on grounds where both scallop species co-occured. Edible mussel ( Mytilus edulis), in contrast, was relatively unaffected: DA, when detected, was usually at sub-threshold levels (maximum = 23 µg g ^-1). Trace levels of DA (=2.5 µg g ^-1) were found in oyster ( Ostrea edulis), cockle ( Cerastoderma edule), brown crab ( Cancer pagurus) and green shore crab ( Carcinus maenus), paralleling that reported for these benthic components in North American waters (Horner et al., 1996).

Exceedance of the DA threshold led to widespread closure of the Scottish scallop fishing areas, including the West Coast grounds covering an area of 8,500 square nautical miles (Figure 2; Gallacher et al., 2001). Scallop harvesting was prohibited in June 1999; the ban was still in effect in April, 2000. The regional scale and duration of DA toxicity greatly exceeded that observed in U.S.A. and Canadian waters, with three features of special interest: the high DA levels in king scallop relative to other shellfish; the prolonged presence of DA above the regulatory threshold; and the diverse habitats in which DA contaminated king scallop populations were found. Very high levels and prolonged periods of DA intoxication were found in both offshore and inshore waters, with particularly high intensity around the inner Hebrides, in the Sound of Jura, Firth of Lorne, Inner Sound and Minch (Figure 2; Tett and Edwards, 2002). The outbreak was less intense in the Moray Firth and to the east of Orkney.

Follow up surveys in late-1999 in Loch Sligahan, Tobermory Bay and along a transect in the Mull-Colonsay-Jura region assessed the variability of DA among individual and neighboring king scallop populations over spatial scales ranging from <5 m to >5 km (Campbell et al., 2001). The distribution of DA among scallop organs was also examined. Pronounced micro-habitat differences (patchiness) in DA levels were found, both between scallop groups and individuals only 25 m apart. The causes of this toxin patchiness are obscure, but mirror the pronounced macro-regional variations found (Figure 2). Domoic acid levels among individuals from within the same sub-population also varied greatly; some scallops had exceptionally large toxic burdens (<1,000-3,689 µg DA g ^-1). Variablity in bivalve toxicity within groups and between adjoining populations is not unique to DA toxicity, similar variation has been reported for paralytic shellfish toxins ( PST) (Bricelj and Shumway, 1998).

The causes of regional, interannual and micro-habitat variations in phycotoxin intoxications are obscure, but are not likely to result from either a fixed, or single factor regulation. Shellfish toxicity is governed by the interaction of multiple factors, including the magnitude, persistence, bloom period, patchiness in, and potency of the toxic bloom source(s); further modified by environmental effects on the metabolism of the shellfish species that have ingested the toxin (Bricelj and Shumway, 1998). Campbell et al. (2001) emphasized the complexity of managing the "roe-on" king scallop fishery during ASP events, given the high individual (whole organism) variation in toxicity and the occurrence of DA in the gonad at levels above the regulatory limit. They suggest that a king scallop fishery based on the adductor muscle, as in the U.S.A., would be much more manageable given that DA levels in this muscle rarely (if ever) exceed statutory limits, even when toxin levels in other body tissue are extremely high.

The prolonged and regionally extensive occurrence of DA in Scottish waters leading to closure of the commercially valuable scallop fishing grounds has triggered debate over the origin(s), and whether the synthesis and source(s) of this phycotoxin are being stimulated. It has been specifically suggested that salmonid fish farms may be changing water quality favoring blooms of the harmful algae capable of DA synthesis and/or its trophic vectoring. This hypothesis is the focus of the following evaluation, and addressed after first reviewing Pseudo-nitzschia autecology, the microalgal source of DA. Section 2.4 presents some caveats to this evaluation.

2.3 Domoic acid, the cause of ASP: its source and vectoring

The source of DA during the 1987 Canadian ASP event was the diatom Pseudo-nitzschia multiseries (= Pseudo-nitzschia pungens f. multiseries), on which the cultured mussels in Cardigan Bay were feeding (Subba Rao et al., 1988; Bates et al., 1998). This first report of toxin production by a diatom, was confirmed soon after. During the 1991 avicide in Monterey Bay, high DA levels and remnants of Pseudo-nitzschia australis cells were found both in the stomach contents of the dead birds and in the viscera of anchovies ( Engraulis mordax), on which the birds had been feeding. The anchovy, in turn, were feeding on Ps. australis ( = Pseudo-nitzschia multiseriata). Experiments confirmed that Ps. australis produces DA, and was the most probable source of the DA vectored to the piscivorous sea birds feeding on anchovy (Bates et al. 1998; Garrison et al., 1992; Work et al., 1993). Domoic acid is now known to be widely distributed among trophic compartments, indicating multiple, direct and indirect pathways occur by which DA is vectored along the food chain. There can be direct transfer from Pseudo-nitzschia to its grazers, as in the mussel and northern anchovy intoxications. Filter-feeding shellfish (mussels, scallops, clams, oysters) generally are major, direct recipients of DA transfer. Domoic acid can accumulate in invertebrates that do not graze phytoplankton, including crabs and spiny lobster ( Panulirus elephas); also in vertebrates, including planktivorous fish and seabirds, and in marine mammals (Bates et al., 1998; Landsberg, 2002; Scholin et al., 2000). Upper trophic levels therefore accumulate DA though food web vectoring, this transfer influenced by the degree of grazer tolerance to DA. Domoic acid is an avicide, for example, but the planktivorous fish on which sea birds prey tolerate higher DA body burdens without suffering mortality or neurotoxic effects, as also the case for shellfish (Landsberg, 2002). In zooplankton, DA ingestion is either inhibitory or without effect depending on the copepod species (Bates, 1998).

While copepods, shellfish and planktivorous fish vector DA to their predators, the details of this vectoring are poorly understood. For example, the sources of DA to surf-zone communities of razor clam ( Siliqua patula) in the Pacific Northwest are puzzling (Trainer et al., 1998). Pseudo-nitzschia spp. are not members of the surf-zone phytoplankton communities on which razor clams feed. This led Trainer and co-workers to suggest that dead or dying cells of Pseudo-nitzschia spp. containing DA were displaced by waves and tides into the surf-zone where they were filtered by razor clams. The copious secretion of DA by Pseudo-nitzschia spp. [see Section 2.6] available to the bacterial community and microbial loop, and their role in routing and vectoring DA have been ignored by researchers. Among the possible routings of DA through the food web, ASP contamination of the Scottish shellfish grounds (Figure 2) most likely results from a combination of direct transfer (ingestion) of DA from Pseudo-nitzschia cells to the shellfish, and indirectly via microbial loop processes.

The capacity of Pseudo-nitzschia species to form long, ribiform chains several millimeters in length and to entangle with other, chain-forming species (Figure 3) raises the issue of whether this enlargement deters grazing and restricts ingestion of DA. Size-based grazing deterrence does not appear to be a significant problem to benthic filter feeders or phytophagous fish. Grazing on Ps. multiseries, reported to have a maximal cell length of 140 µm (Hasle and Syvertsen, 1900), has been reported for the California mussel ( Mytilus californianus), blue mussel ( Mytilus edulis), sea scallop ( Placopecten magellanicus) and northern anchovy ( Engraulis mordax) (Bates, 1998). Chain formation of Pseudo-nitzschia spp. would not appear to be a factor affecting the direct vectoring of DA to shellfish.

The initial discovery of two toxic Pseudo-nitzschia species, Ps. multiseries and Ps. australis, in a genus that is globally distributed and whose species are capable of great abundance, raised great concern over whether a major, new harmful algal bloom type, stimulated by unknown habitat changes, was emerging. While nine Pseudo-nitzschia spp. are now known to produce DA (Table 1), and DA presence in shellfish has been recorded globally, incidences of human ASP affliction have not been reported since the 1987 Cardigan Bay event. [Roelke et al. (1993) report that two South Koreans became ill with ASP symptoms after eating smoked oysters. Examination of smoked oyster in samples from the same cannery lot number did not detect toxic Pseudo-nitzschia in gut contents, but Pseudo-nitzschia pungens f. pungens was present in tins of smoked oyster.] The rarity of ASP illness despite the global presence of detectable DA in shellfish stocks probably reflects the efficacy of DA monitoring as a means to ensure shellfish safety. The open question is whether toxic blooms of Pseudo-nitzschia species are being stimulated in coastal waters, and if so, by what factors.

2.4 What is Pseudo-nitzschia?

Taxonomists recognize two major diatom groups: centric diatoms, which are primarily planktonic, and pennate diatoms, which are primarily benthic. Pennate diatoms exhibit a group preference for growth in the benthic microflora, and are abundant on abiotic (sediments, ice, dock pilings, etc.) and biotic (macroalgae, epizoic) surfaces. The genus Pseudo-nitzschia is remarkable among pennate diatoms for its planktonic life-mode and pelagic abundance. It is one of the most common planktonic genera, occurring globally in polar, temperate, subtropical and tropical areas (Hasle, 1972, 1996, 2002). Only a few other pennate diatom genera have achieved such planktonic success. The genus Asterionellopsis, notably Asterionellopsis glacialis, blooms in coastal waters, but is species depauperate. Pelagic pennate diatoms are important in Antarctic blooms. Thalassionema and Thalassiothrix are ubiquitous genera, but depauperate in species and usually not abundant.

Pseudo-nitzschia is a relatively species-rich genus (19 taxa, 2 formae (Lundholm et al., 1994), nine species of which currently are known to produce DA in culture (Table 1). DA-producing Pseudo-nitzschia spp. are globally distributed in coastal waters and capable of achieving great abundance (>10 ⁷ cells L ^-1) (Hasle, 2002). Amphora coffeaeformis (Maranda et al., 1990) and Nitzschia novis-varingica, recently found at a Vietnamese shrimp aquacultural site, are the only other diatoms known to produce DA; both species are benthic (Bates, 2000; Kotaki et al., 2000; Lundholm and Moestrup, 2000). The unique capacity of the nine Pseudo-nitzschia species to produce DA is evident given that ca. 800 pennate and 1,000 centric species have been described (Sournia et al., 1991). Analyses of base pairs of nuclear-encoded large subunit ( LSU) rDNA from 42 species led Lundholm et al. (2002) to conclude that pennate diatoms evolved the ability to produce DA twice, with DA-producing Nitzschia novis-varingica distantly related to Pseudo-nitzschia. Lundholm and co-authors also concluded that Pseudo-nitzschia is polyphyletic, with emendation of the genus needed . [The older taxonomic literature placed the presently recognized Pseudo-nitzschia species in the genus Nitzschia.]

In summary, Pseudo-nitzschia species are unique among the phytoplankton for their DA production, unusual success as planktonic pennate species, in number of species, and ability to produce large blooms.

Table 1.Pseudo-nitzschia species shown to produce domoic acid ( DA), as pg cell ^-1, in culture and their cell size characteristics*

* Cellular volume and surface area were calculated using the maximum apical and transapical axis dimensions given by Hasle & Syvertsen (Tables 74 and 75 in 1990), excluding Ps. multistriata (from Sarno & Dahlmann, 2000), and assuming the geometric shape to be a double cone. Sources: (1) Trainer et al. (2001); (2) Bates et al. (2001); (3) Lundholm et al. (1994); (4) Sarno & Dahlmann (2000); (5) Bates (1998); (6) Rhodes et al. (1996a).

The size and shape of Pseudo-nitzschia spp. are notable: the cells are long and needle-shaped, the tips of which attach to form step-shaped colonies (Figure 3a; Hasle and Syvertsen, 1990). Long, ribiform chains of 20 or more cells are formed that can reach several millimeters in length and entangle with other colonial species (Figure 3b). The number of cells per chain varies with growth conditions; it decreases with nutrient limitation when solitary cells predominate (Smayda and Boleyn, 1965). Cell length varies from 30 µm ( Ps. turgidula) to 160 µm ( Ps. seriata), with two morphometric groups recognized based on cell width (= transapical axis) which ranges from 1.0 µm ( Ps. delicatissima) to 8.0 µm ( Ps. australis) (Table 1; Hasle and Fryxell, 1995). The larger Pseudo-nitzschia spp. (i.e. those >3.0 µm in width) comprise the " Nitzschia seriata complex"; the smaller species the " Nitzschia delicatissima complex" (Hasle and Syvertsen, 1990). Cell size and the degree and pattern of silicification, on which Pseudo-nitzschia taxonomy is based, vary among species. This makes it difficult to identify properly the species present in field populations using standard microscopy. [When settling in counting chambers, the cells (chains) of larger species (" Nitzschia seriata complex") tend to orient visually in side (= girdle) view, whereas inspection of their cellular valve (top view) is required to distinguish among species.] The smaller Pseudo-nitzschia spp. (the " Nitzschia delicatissima complex") are also taxonomically difficult. Their silica cell walls (= frustules) often are so finely structured that inspection of micro-features essential for species identification can not be achieved when viewed in counting chambers or by routine microscopy. As a result, Pseudo-nitzschia spp. frequently are misidentified. Their proper identification requires use of transmission and scanning electron microscopy, techniques usually not applied in routine field studies.

Species misidentifications compromise evaluation of pre-1985 field observations on Pseudo-nitzschia spp. dynamics and DA occurrences. Domoic acid toxicity undoubtedly occurred previously, but went un-noticed; it is unlikely that the capacity of Pseudo-nitzschia spp. to synthesize this phycotoxin evolved recently. It took the large-scale, 1987 ASP outbreak in Cardigan Bay and the 1991 Monterey Bay bird dieoff to alert the scientific community to Pseudo-nitzschia toxicity and DA as a microalgal phycotoxin. These two outbreaks showed also the need for accurate species identifications is paramount. Prior to having been shown capable of producing DA, Ps. multiseries was distinguishable from morphologically similar, non-toxic species only by the number of poroids on its silica frustule, features visible only by electron microscopy! The limited availability of ecophysiological data is also a problem. Awareness that Pseudo-nitzschia is a somewhat species-rich genus with a capacity for DA synthesis is a recent discovery. Relatively few experimental studies have been carried out on Pseudo-nitzschia strains to provide the ecophysiological data needed to quantify DA synthesis, bloom behavior and vectoring in situ. This preclude reliable retrospective analysis of the population dynamics of DA-producing Pseudo-nitzschia species prior to the discovery of ASP. It is equally difficult to address the unanswered question of whether blooms of Pseudo-nitzschia species have been increasing in recent decades, an issue very relevant to the salmonid farming - ASP issue of interest here. Nonetheless, through application of first principles, together with available ecophysiological data, the proximate environmental causes of observed Pseudo-nitzschia bloom dynamics can be arrived at, and help put into perspective whether salmonid farming is contributory to Pseudo-nitzschia blooms and ASP occurrence in Scottish coastal waters.

2.5 Domoic acid ( DA) production by Pseudo-nitzschia species and strains

The capacity of Pseudo-nitzschia to synthesize DA varies among the nine known producer species (Table 1). Based on the amounts of DA per cell produced in batch culture, Cusack et al. (2002) have arrayed the capacity of Pseudo-nitzschia spp. to produce DA as follows: Ps. australis > Ps. seriata > Ps. multiseries > Nitzschia navis-varingica > Ps. pseudodelicatissima > Ps. multistriata > Ps. fraudulenta > Ps. pungens > Ps. delicatissima > Ps. turgidula.

The cellular DA levels given in Table 1 are maximal values reported in the literature; lower levels have also been reported. For Ps. australis, the range of 12-37 pg DA cell ^-1 (Garrison et al. (1992) is considerably lower than the reported maximum of 78 pg DA cell ^-1 (Table 1). Shellfish filter feeding on Ps. australis containing its maximal cellular DA content would have to filter only 15% of the population to ingest the same amount of DA when feeding on cells at the same population density, but whose DA content is at the lowest reported level. The maximal cellular levels given in Table 1, particularly for the purportedly minor producers, might actually be greater for several reasons. Domoic acid levels are a function of the degree of cellular stress; influenced by experimental conditions and reporting methodology, and there are clonal (strain) differences in DA production capacity (Bates, 1998). Pseudo-nitzschia cells also secrete copious amounts of DA into their growth medium (Bates, 1998). Most investigators calculate and report cellular DA levels based on actual cellular DA content; others base this on 'whole culture" levels, e.g. cellular + filtrate levels (see Cusack et al., 2002). These confounding factors are discussed in greater detail in Section 2.6

Differences in toxicity among strains of the same species are a common feature of harmful algal species, a trait well developed among Pseudo-nitzschia spp. Toxic and non-toxic strains have been reported for Ps. australis, Ps. delicatissima, Ps. multiseries, Ps. pseudodelicatissima and Ps. seriata (Bates et al., 1998; Lundholm et al., 1994; Martin et al., 1990; Trainer et al., 1998; Villac et al., 1993; Villareal et al., 1994). It is unknown why the same Pseudo-nitzschia species is toxic in one region of its distributional range and not in another (Bates et al., 1998). Metapopulation differences in DA toxicity of Ps. pseudodelicatissima appear to have a genetic basis (Skov et al., 1997), but the role of environmental induction of toxicity is unresolved. Despite numerous reports that Pseudo-nitzschia blooms are virtually monospecific, the DA synthesis capability of the bloom population can be polyclonal rather than monoclonal (Skov et al., 1997), i.e. there is unequal capacity among the cells of the bloom population to produce DA. The occurrence of strain differences in DA synthesis capacity is significant to monitoring strategies that are based on the occurrence of a given Pseudo-nitzschia species at a given abundance level. Used as an early warning indicator of potential ASP problems, these indices trigger initiation of DA monitoring that may require closure of shellfishery grounds. Strain differences in DA production capacity compromise the use of such indicator species and threshold abundances as the basis of this type of DA monitoring and public health strategies, particularly if applied indiscriminately throughout the distributional range of that species. An added complication is to use a fixed population level irrespective of the Pseudo-nitzschia species. Since Pseudo-nitzschia species vary in their capacity to produce DA (Table 1), the warning population levels used to initiate DA monitoring should not be kept constant; lower thresholds should be set for strong producers. Other complications of the variable capacity for DA synthesis among Pseudo-nitzschia species and strains are considered in Section 2.5.1.

2.5.1 Influence of cell size

The major DA producers, Ps. australis, Ps. seriata and Ps. multiseries, are large cells (in length and cellular volume) and fall within the " Nitzschia seriata complex" (Hasle and Syvertsen, 1996). The three other species in this size-group ( Ps. multistriata, Ps. pungens, Ps. fraudulenta) are relatively minor DA producers (Table 1). [ Ps. multistriata is placed in this complex based on the width of its transapical axis applying Hasle and Syvertsen's (1996) criteria.] The three species in the narrower, lightly silicified " Nitzschia delicatissima complex", Ps. delicatissima, Ps. pseudodelicatissima, Ps. turgidula, are also minor DA producers. It has been argued that differences in cell size and not in intrinsic capacity (physiology) of Pseudo-nitzschia spp. to synthesize DA account for their species-specific differences in DA synthesis (Garrison et al., 1992; Martin et al. (1990). Garrison et al. believed that the low cellular DA levels (2-7 pg cell ^-1) reported for Ps. multiseries, and the higher levels (3-31 pg cell ^-1) in Ps. australis were comparable when adjusted for differences in their cell volumes Martin et al. (1960) concurred, having found that while the cell volume of Ps. multiseries and Ps. pseudodelicatissima differed by 18-fold, their DA content per unit cell volume differed by only 3.5-fold. I have reexamined this conclusion using newer data and the 3,900-fold difference found between the highest and lowest cellular DA levels among the nine producer species: 78 vs 0.02 pg DA cell ^-1 (Table 1). The DA content per unit cell volume for the three major producers ranged from ca. 13 to 32 fg µm ^-3 vs. only 0.12 to 5.6 fg µm ^-3 for the minor producers, Ps. turgidula and Ps. pseudodelicatissima. The difference in normalized DA production between the two end members ( Ps. australis and Ps. turgidula) was 275-fold, while their cell volumes varied only 10-fold (Table 1). This suggests, contrary to earlier findings, that the considerable differences in DA production capacity among Pseudo-nitzschia spp. reflect an unequal physiological capacity, rather than interspecific differences in cell size. This might help to explain the regional and micro-habitat differences in DA toxicity of king scallop observed in Scottish waters, and discussed earlier. The Pseudo-nitzschia species being filtered by benthic bivalves may influence their DA content.

Domoic acid production by Ps. multiseries in culture decreased over time (Bates et al., 1999), pointing to another aspect of DA synthesis. Diatom species generally exhibit significant decreases in cell size because of their cellular division mode, requiring an auxosporulation stage which increases the cell size. Cusack et al. (2002) reported the mean cell volume of Ps. australis (750 ± 140 µm ³) decreased over a one-year period by >50% in culture from that of cells in the field population from which it was isolated. Thus, a reduced capacity for DA production may be coupled to intracellular reductions in Pseudo-nitzschia cell size and volume. This would affect the threshold population densities used in monitoring strategies to regulate closure of shellfishing grounds. Initiation of domoic acid monitoring is sometimes based on the abundance of the Pseudo-nitzschia species selected for taxonomic monitoring which, upon reaching a threshold abundance level warning of potential ASP problems, triggers monitoring of DA. While the threshold level of 20 µg DA g ^-1 in shellfish tissue is applied for shellfish ground closure, irrespective of the source species, the potential of a given Pseudo-nitzschia species to supply DA to threshold levels is the product of its cellular DA level and population abundance. Ingestion of fewer cells of Ps. australis, for example, will be needed to reach threshold levels than ingestion of less toxic Ps. pseudodelicatissima. The use of a fixed population density irrespective of the Pseudo-nitzschia bloom-species as an early warning signal of potential ASP problems triggering initiation of DA measurement is risky. In the Norwegian monitoring program, a warning level of 10 ⁶ cells L ^-1 of Ps. pseudodelicatissima triggers analyses of the DA content of mussels (Aune et al., 1995). [ DA was not detected even though population levels reached several million cells L ^-1.] If the same population warning level was applied in monitoring Ps. australis and Ps. multiseries abundance in the coastal waters of Washington and Oregon, DA intoxication of shellfish, which occurred at population levels as low as 10 ⁴ cells L ^-1, would have been overlooked (Trainer et al., 2001). The occurrence of clonal (strain) differences in DA synthesis capacity must also be considered in developing monitoring strategies.

2.5.2 Influence of growth phase, cell division and bacteria

The synthesis of DA by Pseudo-nitzschia is not continuous; at the cellular level it is influenced by whether the cells are actively growing or not, and at the population level it is influenced by the stage of the population growth curve. Domoic acid production in stationary phase Ps. multiseries was 100-fold greater than during the exponential phase of its growth curve, similar to Ps. seriata. In Ps. australis, DA production began in the mid-exponential growth phase and continued into the stationary phase, when it increased (Bates, 1998; Bates et al., 2001). The relationship between DA production and the population growth curve of Ps. multiseries in batch culture is shown in Figure 4. The transition from undetectable DA production to copious production during the late exponential/early stationary phase of the growth curve is clearly evident. Cells in stationary growth are physiologically senescent; this suggests DA production is a stress-related response. The cell division rate of stationary cells also decreases, with a strong inverse relationship found between cellular DA content and the rate of cell division, as shown for Ps. multiseries in Figure 5. A reduction in cell division is usually in response to some limiting factor(s). Bates (1998) concluded that the primary trigger of DA production is the slowing and cessation of cell division, secondarily stimulated by factor(s) then limiting to growth, such as nutrient limitation. These conclusions, based on batch culture experiments, contrast with the results of chemostat experiments in which Ps. multiseries in continuous culture was perfused with nutrients (Si, P) at a constant flow (dosage) rate (Bates et al., 1996; Bates, 1998). Chemostat experiments are more representative of in situ growth conditions than batch culture experiments. In batch cultures, nutrients continuously decline because of cell growth. In chemostat experiments, nutrients are pulsed at a constant supply rate to establish a physiologically similar population in sustained growth at a population level optimal for that nutrient dosage. In chemostat experiments, nutrients are consumed and restored; in batch cultures, nutrients are consumed and reduced. In continuous culture, Ps. multiseries produced DA during active growth, unlike in nutrient-limited batch culture where growth cessation was required for DA synthesis (Bates et al., 1996). Bates and co-workers suggested their chemostat experiments mimicked conditions during the 1987 Cardigan Bay ASP event when field populations alternated between nutrient-limited and nutrient-sufficient conditions; yet, Pseudo-nitzschia increased both in abundance and cellular DA level. Extrapolating to the general condition, in situ populations are exposed to oscillations in nutrient influx and nutrient limitation, with DA synthesized during the latter condition and quenched or reduced during the nutrient enrichment period. This dynamic reconciles the apparent discrepancy between batch and continuous culture results.

Two mechanisms are projected to promote increased DA production and shellfish accumulation in situ based on these experimental results. Case I: Pseudo-nitzschia species is first stimulated to bloom; this is followed by another, different type of stimulation: the synthesis of DA. The population growth (ungrazed) and DA production curves would then be similar to those in Figure 4. While there are two, separate triggering events in Case I, the population increase results from growth stimulation, unlike the synthesis of DA which is a response to growth suppression. Case I is akin to a batch culture; the bloom that develops will be short-lived and intense before being dissipated because of nutrient limitiation and, in situ, by grazing and advection. In Case II, the population develops in chemostat fashion. The population level reached may not be very high, but growth is expected to continue for an extended period, unlike the "boom and bust" bloom behaviour of Case I populations. In Case II events, growth and grazing co-occur, nutrients are consumed and restored, and DA production is both stimulated (by low nutrient levels) and quenched (by high nutrient levels). The magnitude (carrying capacity) of the bloom population is determined by the nutrient concentration and its supply rate. Regional and local persistence of the population is influenced by physical oceanographic conditions, and would be favored at fronts and in eddies. In Case II dynamics, the population can be visualized as oscillating between the exponential and stationary growth phases (Figure 4), i.e. chemostat and batch culture modes, during which cell division is oscillatory, increasing and decreasing and accompanied, respectively, by decreased and increased DA synthesis.

The period of DA ingestion and accumulation by shellfish during Case I dynamics is expected to be abrupt and short-lived compared to Case II. Benthic communities are then exposed to, and filter the sinking, DA enriched senescent Pseudo-nitzschia cells, their DA content at high levels because of the nutrient limitation that caused the bloom to collapse. DA intoxication of shellfish is not limited to Case I blooms of Pseudo-nitzschia. Case II events leading to chronic exposure of shellfish to low toxin levels also allows accumulation of DA, as shown for razor clam in the Pacific northwest (Trainer et al., 1998). In this situation, there is a critical nutrient level which allows the population to continue low-grade growth sufficent to maintain the population, and also to synthesize DA. If the nutrient level increases, so does the population, but DA synthesis is then quenched. If nutrient (Si, P) levels decrease significantly, DA synthesis is stimulated, but the population collapses and the supply of DA to filtering bivalves is limited, both in amount and duration of supply. During Case II situations, while the degree of DA accumulation is species-specific and a function of the Pseudo-nitzschia population density, the depuration rate of DA is also important. High depuration rates are reported for mussel ( Mytilus edulis) and soft-shelled clam ( Mya arenaria), whereas DA is retained in the digestive gland of Atlantic deep-sea scallop for months, and razor clams maintained levels from 10 to 50 µg DA g ^-1 for three months (Trainer et al., 1998; Gilgan et al., 1990).

These two scenarios suggest that the seasonal (or longer) persistence of DA in Scottish coastal waters (Figure 2) is not dependent upon major or minor blooms of Pseudo-nitzschia. A combination of chronic exposure to low Pseudo-nitzschia population densities developing as Case II events and low depuration rates and/or the biofiltration of advected cells which contain DA can be the mechanism of accumulation and prolonged retention of DA by shellfish. The prolonged occurrence of DA in the king scallop population in Scottish coastal waters (Figure 2) probably reflects Case II dynamics. This assumes that shellfish directly ingest Pseudo-nitzschia cells containing DA. Section 2.6 deals with the secretion of DA by Pseudo-nitzschia cells and its vectoring to shellfish.

There is evidence that bacteria are involved in the cellular production of DA. Domoic acid production In bacterized cultures of Ps. multiseries was 20-fold greater than in bacteria-free cultures (Bates, 1998). Synthesis of DA does not appear to depend upon a specific metabolic type, since it was inducible by contaminating bacteria-free Pseudo-nitzschia cultures with a variety of bacteria from different sources. The mechanism of bacterial enhancement of DA production is obscure. The observation that Ps. multiseries lost its viability and capacity to produce DA after one or more years in laboratory culture may be related to this effect of bacteria.

2.5.3 Influence of irradiance, phosphorus, silica, nitrogen

Domoic acid synthesis requires irradiance (Bates, 1998). Exposure to the photosynthesis inhibitor DCMU (=3-(3,4-dichlorophyenyl)-1,1 dimethyl urea) quenches DA production; this indicates that photosynthesis is essential for the production of DA (Pan et al., 1991, 1996a). The rate of DA production in senescent cells decreases at lower irradiance levels (Figure 4) and ceases during the dark phase of an L:D photoperiod, or when stationary cultures are placed in darkness and even though DA production has begun (Bates, 1998). The relationship between DA production and irradiance in experimental cultures, if applicable to natural populations, suggests that the DA content of Pseudo-nitzschia cells sinking out of the euphotic zone into darker layers, where they become increasingly available for shellfish consumption, is partly a function of the average irradiance experienced by the Pseudo-nitzschia cells during DA production.

The photosynthetic energy requirement for DA production might suggest that its synthesis is under classical Redfield Ratio control. The Redfield Ratio stoichiometrically links the photosynthetic fixation and respiration of carbon to the assimilation of nitrogen and phosphorus: 106C:16N:1P (by atoms). For every 106 atoms of C fixed during photosynthesis (or respired), 16 atoms of N and 1 atom of P are assimilated (or released) in Redfield Ratio kinetics, with an increase in N and P uptake accompanied by increased C production. While this relationship is a general trait of the marine microalgae, and can be applied to the synthesis of carbon by Pseudo-nitzschia, it does not apply to the production of DA. The converse occurs: DA production begins when the macronutrients silica and phosphorus become limiting to photosynthesis and cell division - that is, there is an indirect relationship between DA production and P and Si concentrations (Bates, 1998; Bates et al., 1996; Pan et al., 1996a,b). Low irradiance does not appear to be as stressful as Si and P limitation (Bates, 1998).

The demonstration that Si and P limitation stimulates DA production is unexpected. Silica is required by diatoms for frustule (= cell wall) formation, for DNA replication, and influences phasing of the cell division cycle (Pan et al., 1996a). Otherwise, Si is considerably less involved (relatively inert) in biochemical processes than N and P. The experimental demonstration that Si limitation can lead to DA production is very relevant to in situ populations. Increasing evidence suggests that diatom growth in coastal waters, usually considered to be N-limited, is more often Si-limited than acknowledged. For example, at the peak of the 1987 bloom of Ps. multiseries in Cardigan Bay, Si concentrations were depleted, a limitation which Pan et al. (1996a) suggested enhanced the observed DA production. Pan and co-workers proposed that the 3-month persistence of this toxic bloom was the result of a repetitive sequence of land run-off or tidal mixing delivery of Si, followed by intervening periods of Si limitation. In the case of P limitation, DA production occurred even during cellular synthesis of alkaline phosphatase, with a positive correlation found (Bates, 1998). Alkaline phosphate synthesis by cells triggered by inorganic P limitation allows the assimilation of dissolved organic P compounds. While this appears to satisfy the need for P, it does not quench production of DA. It is uncertain whether the experimental demonstration that P limitation triggers DA production is realized in situ. Marine coastal waters are traditionally viewed as N-limited systems, and freshwater systems as P-limited systems. Nonetheless, triggering of DA production by P limitation is similar to that reported for saxitoxin production (the cause of paralytic shellfish poisoning) by some dinoflagellates (see Cembella, 1998). Unless the production of these toxins in natural populations is triggered by other factors, their toxicity would suggest that P limitation can occur in situ.

Pan et al. (1996, 1996b) sought to explain the metabolic basis for the apparent requirement DA synthesis has for irradiance and limitation by Si or P. They argued that a competition for free energy ( ATP) occurs between primary cellular metabolism and DA production, a secondary metabolite. During active growth, i.e. during the exponential growth phase, photosynthesis rates and the rates of N, P and Si uptake are high. The high demand for metabolic energy during this growth phase reduces the amount of ATP available for secondary metabolism and the synthesis of secondary metabolites such as DA (see Bates, 198). Domoic acid synthesis requires ATP. This free (photosynthetic) energy source is diverted to DA production when cell division (growth) slows and then ceases. Addition of Si or P to stationary phase cultures temporarily reduces their limitation and the production of DA until these nutrients become limiting again. These experimental results and the theorized biosynthetic pathways of DA suggest that two events must take place leading to DA accumulations in shellfish such as observed in Scottish waters (Figure 2). Either a bloom must be triggered, or there is persistence of toxic Pseudo-nitzschia at low population levels, followed by stimulation of DA synthesis, as discussed in Section 2.5.2. Fehling et al. (2004b) recently reported their experimental results of the influence of silicate and phosphate on DA synthesis in a strain of Ps. seriata f. seriata isolated into culture from Scottish waters. They demonstrated that both P and Si limitation enhanced DA synthesis, but comparison of the magnitude of DA production indicated a greater threat of Ps. seriata -generated amnesic shellfish poisoning events under Si rather than P nutrient limitation.

Nitrogen is essential for the synthesis of DA, an amino acid. Domoic acid contains only 4.5% N on a molecular weight basis compared to 33% for saxitoxin in Alexandrium tamarense and, unlike saxitoxin, is probably not a significant N storage compound (Bates et al., 1991). Although only 1.5%, on a molar basis, of the N taken up in stationary phase cultures of Ps. multiseries was allocated to DA production, this limited uptake was fundamental to its biosynthesis. There is a significant difference between NO ₃ and NH ₄ in their regulation of DA production and potential influence on the bloom ecology of Pseudo-nitzschia. NO ₃ is essential for DA production. If NO ₃ is absent, stressed cells can not synthesize DA. NH ₄ can not replace this NO ₃ requirement even though NH ₄ and urea are important N sources (Bates et al., 1991), with glutamine uptake also reported for Ps. multiseries (Hillebrand and Sommer, 1996). DA production is quenched in the absence of NO ₃, and inhibited during its limitation (Hillebrand and Sommer, 1996). That is, there is an inverse relationship between DA production and the degree of N limitation. This contrasts with the direct relationships found between DA production and the degree of Si and P limitation. Pseudo-nitzschia spp. appear to be sensitive to NH ₄ concentrations, which interfere with NO ₃ metabolism. The photosynthetic assimilation number (g C g chl ^-1 hr ^-1) and growth rate of several clones of Ps. multiseries and Ps. pungens relative to those grown on NO ₃ were inhibited at NH ₄ concentrations >110 µM (Bates et al., 1993). Hillebrand and Sommer (1996) found Ps. multiseries " was barely able to grow" with NH ₄ as the sole N source at high concentrations, but growth increased when the NH ₄:NO ₃ ratio decreased to low levels. Growth rates were inversely related to NH ₄ concentrations, but when provided NO ₃ growth followed a hyperbolic relationship akin to Michaelis-Menten kinetics (see Hillebrand and Sommer, 1996).

The heightened sensitivity of Pseudo-nitzschia spp. to NH ₄ is unexpected. Diatoms generally tolerate high NH ₄ levels (see Hillebrand and Sommer, 1996). The common and abundant coastal diatoms Skeletonema costatum and Asterionellopsis glacialis (both found in Scottish waters), for example, were unaffected by 200 µM NH ₄, whereas there was significant suppression of photosynthesis, growth and cell yield of Ps. multiseries at >110 µM NH ₄ (Bates et al., 1993). In contrast, Ps. multiseries was not inhibited at >440 µM NO ₃. It is well known that diatoms preferentially assimilate NH ₄ over NO ₃, with NO ₃ assimilation usually inhibited at >2 µM NH ₄. Although the requisite N assimilation experiments have not been carried out, it would appear that in this feature also, Pseudo-nitzschia spp. may differ from other diatoms in their N physiology. The uptake of NO ₃ requires the enzyme nitrate reductase, whose synthesis is light-dependent, a coupling consistent with the dependency of DA production on both irradiance and NO ₃. The role of NO ₃ and NH ₄ excretion at fish farm sites and toxic Pseudo-nitzschia blooms is considered in Section 2.9.

2.5.4 Iron metabolism and DA synthesis

Domoic acid is not needed for cellular growth. It is a secondary metabolite produced in response to stress, raising the question whether its production alleviates stress and, if so, how. Phytoplankton cells experience two major stresses: nutrient limitation and grazers. The evidence that DA is allelopathic against grazers is mixed, particularly copepods and shellfish, the two primary grazers (Bates, 1998). Domoic acid does not appear to play a role in macronutrient assimilation [see Section 2.5.3], but there is mixed evidence, and debate, that it is active in iron metabolism and resupply (Bates et al., 2001; Maldonado et al., 2002; Rue and Bruland, 2001).

Domoic acid, an analogue of kainic acid, contains the three carboxyl groups in its chemical structure (Figure 1) which suggest that it should chelate, or bind trace metals (Bates et al., 2001). Domoic acid produced in Ps. multiseries cultures chelated Fe and Cu (Rue and Bruland, 2001). At elevated Cu concentrations the amount of Cu-binding chelator produced was 20-fold greater than in populations reared at low concentrations. Rue and Bruland attributed two benefits to this putative chelation, the chelator presumed to be DA: it detoxifies the habitat of trace metals, such as high Cu concentrations, and by solubilizing particulate Fe it increases the availability of Fe to the cells. They mention that in large scale, in situ Fe enrichment experiments to test Martin's Fe limitation hypothesis, blooms of Pseudo-nitzschia spp. were preferentially selected for.

Maldonado et al. (2002) have vigorously advanced the notion that Pseudo-nitzschia spp. secrete Fe-binding organic ligands during Fe limitation, functioning to mute this limitation. Experiments with Ps. multiseries confirmed DA has a high affinity for Fe and Cu; that DA production increases with Fe stress, and when added to culture media DA increased Fe uptake. They concluded that Pseudo-nitzschia spp. have lower Fe uptake rates than diatoms generally, but are more sensitive to Cu than other coastal diatoms. They considered DA synthesis to be an adaptive strategy to alleviate Fe limitation given experimental observations that showed Fe-deficient Ps. multiseries cells secreted DA in amounts 6 to 25-fold greater than when Si- and P-limited and at equivalent growth rates; that DA production began in the exponential growth phase (note: in Si- and P-limited cultures secretion is primarily a stationary phase response); and the cellular residence time of DA molecules was only 40 minutes in Fe-deficient cells versus about 13 hrs in Fe-sufficient cells. This high DA turnover time accompanying extracellular secretion, and inception of DA biosynthesis during the exponential growth phase were considered consistent with their theory. Maldonado et al. (2002) suggested that secreted DA probably does not function as a siderophore in binding Fe because of its low stability constant for Fe in sea water compared to siderophores. Organisms that secrete siderophores for Fe uptake, such as Prorocentrum minimum (Trick et al., 1981), typically exhibit higher (by 20-30-fold) Fe uptake rates during Fe limitation than when Fe-sufficient, and when siderophores are not active.

Bates et al. (2001), also working with Ps. multiseries, came to the opposite conclusion - that DA secretion does not alleviate Fe deficiency since it is not an Fe sequestering compound. They report Fe deficiency inhibits DA synthesis in Ps. multiseries, and that Fe-replete cells had a 10-fold higher DA production rate. They concluded the decrease in DA production during Fe stress argues against a function in Fe uptake, pointing out that DA synthesis decreased during the stationary phase, when cells are physiologically stressed. This also makes it unlikely that DA functions in Fe uptake to meet metabolic requirements. Further, despite the ability of DA to bind Fe, key physiological processes remain depressed during stationary phase.

The debate over the role of DA in Fe nutrition reflects the complex, interactive effects of N, Si, P and Fe in regulating its synthesis. As pointed out, NO ₃ is essential for DA production. The assimilation of NO ₃ is mediated by the enzyme nitrate reductase, whose synthesis requires Fe. Iron-limited cells lose their ability to assimilate NO ₃ (Maldonado et al., 2002). The interdependence of Fe and N assimilation and DA production might explain the contradictory results, should NO ₃ have been co-limiting in the Bates et al. (2001) experiments, but not in those carried out by Rue and Bruland (2001) and Maldonado et al. (2002). Methodological and clonal differences might also have been contributory. Bates et al. (2001) did not exclude that DA plays a role in Fe metabolism; they suggested that it may serve as an Fe storage molecule similar to ferritin. Production of DA during stationary phase would help to solubilize cellular Fe during cell lysis, and aide its recycling into new biomass (cells). Whatever the role of DA in Fe metabolism of Pseudo-nitzschia spp., its synthesis, stimulated by Si or P limitation, followed by secretion will not alleviate limitation by the latter macronutrients. The role of DA in the autecology of Pseudo-nitzschia remains obscure; its production, leading to potential ASP illness, can best be described as a stress response during growth limitation.

2.5.5 Summary

The synthesis of DA by Pseudo-nitzschia spp. is the most complex of the phycotoxins produced by harmful algae. Domoic acid synthesis is under multifactorial control, with at least three conditions required for its production: irradiance, a supply of NO ₃, and nutrient stress resulting from either Si or P limitation. Micro-nutrient (Fe and Cu) stress also enhances DA production. The presence of bacteria appears to be necessary. DA synthesis is also under tight cellular control: photosynthesis is essential for the production of DA, but most of the DA is produced during the stationary growth stage when photosynthesis becomes nutrient limited. The influence of Si, P and NOY on DA production is indirect; the effect of these nutrients, when limiting, on the rate of cell division is more important since there is an inverse correlation between the rate of cell division (µ) and DA production. As the cell division rate slows down, the population growth rate does likewise and abundance ultimately reaches its environmental carrying capacity. During this growth stage copious amounts of DA are secreted [see Section 2.6]. The cellular mechanisms involved in triggering DA production and its secretion as cellular growth decreases are unknown. The time (day) of inception of DA production upon entry into the senescent phase varies with the growth rate and irradiance level; that is, it remains under environmental control. Restoration of nutrients (Si or P) will reduce DA production until limiting once again. There may be an indirect role of irradiance through its effect on NO ₃, which requires NO ₃-reductase, synthesis of which is light dependent. However, the synthesis of NO ₃-reductase is sensitive to NH ₄, and the question remains open as to whether NH ₄ can also provide the N needed for DA production. The problem of NH ₄ toxicity is considered in Section 2.11.

Extrapolation of these laboratory results to field conditions suggests that whatever the stress that triggers DA production through a reduction in growth rate, the level of production and the potential ASP impact will vary with habitat conditions, including nutrient and irradiance levels, thin layer dynamics (which influence irradiance exposure), nutrient pulsing accompanying physical conditions, population abundance levels, the Pseudo-nitzschia species in bloom, the ratio of the euphotic zone depth to benthic depth, and benthic shellfish community. An important finding relevant to the relationship between Pseudo-nitzschia bloom events and DA accumulation in shellfish is that the production and supply of DA to filter feeding bivaves are maximal during the collapse of the bloom, and not during the exponential phase of the bloom.

This extraordinarily complicated combination of habitat-cellular-population events and conditions regulating the synthesis of DA by Pseudo-nitzschia spp., and exceedance of ASP threshold levels in bivalve grazers, tempers ad hoc explanations that fish farm activities are responsible for the ASP "epidemic" that has characterized commercial scallop grounds in Scotland [Section 2.2]. This is further considered in Section 2.11.

2.6 Secretion of domoic acid: importance and trophic vectoring

Cellular DA levels are used as an index of the capacity of Pseudo-nitzschia spp. to produce DA (Table 1). Ecologists have generally ignored that DA production is primarily a stress response; that its production accelerates in aging populations, and that the stressed cells secrete, rather than retain, the bulk of the DA produced. The debate over the role of DA production in Fe metabolism [section 2.5.4] has focused on a major, neglected feature of DA synthesis: its copious secretion (Bates et al., 1991, 1998; Maldonado et al., 2002). Once DA synthesis begins, cellular levels increase initially, then decrease from progressive secretion. The intracellular residence time of DA molecules varies with nutritional status; it is only 40 minutes in Fe-deficient cells versus about 13 hrs in Fe-sufficient cells (Maldonado et al., 2002). The extracellular secretion of DA requires active transport across the cell membrane because DA is a small molecule and very hydrophilic (Maldonado et al., 2002). Figure 4 shows the gradual increase in extracellular levels and decrease in cellular DA content that occur during stationary phase populations of Pseudo-nitzschia. Cultured Ps. multiseries attained maximum cellular DA levels seven days after the stationary phase began, then declined. Extracellular concentrations progressively increased and exceeded cellular DA levels by 5-fold; eventually, DA was detected only extracellularly. During metal stress, 95% of the DA produced was secreted (Maldonado et al., 2002).

The high turnover and secretion rates of DA clearly impact its vectoring, but there is no information on this, or of the fate and potency of secreted DA. Two supply "waves" and types of DA availability to shellfish can be visualized: intracellular and extracellular. During the initial stages of DA production, and when DA is retained intracellularly by Pseudo-nitzschia, shellfish can directly filter feed on the toxic cells and ingest and accumulate the DA. This cellular packaging of DA is transient, and gradually becomes secondary as extracellular DA levels increase as a result of cellular excretion. The fate of extracellular DA is unknown, including whether it can be vectored and, if so, the vectoring pathway(s), and whether extracellular DA accumulated by shellfish retains its potency to pose an ASP threat. The excreted DA is in soluble form, and parenteral feeding on DA by shellfish is not expected. For shellfish to accumulate DA from its dissolved state, the required ingestion pathway is as follows: secreted DA must be adsorbed onto particles, or taken up by microorganisms within the microbial loop, and these particles and microbial elements are then filtered by the shellfish. Alternatively, the shellfish ingest microzooplankton that had been feeding on microbial loop organisms. It is recommended that should ASP continue to be a problem in Scottish coastal waters, then the relative importance of intracellular vs. extracellular DA as sources to king scallop be evaluated in experimental systems in which scallops are fed toxic Pseudo-nitzschia species, and when exposed only to DA conditioned medium. This may help to establish whether the primary source and pathway of DA leading to ASP is via cellular or extracellular vectoring.

2.7 Pseudo-nitzschia blooms and ASP in Scottish coastal waters

Routine monitoring for ASP in wild and cultivated shellfish in Scotland began in 1998, after DA was detected in 1996 (Gallacher et al., 2001; Macdonald and Davidson, 1998). Phytoplankton monitoring during 1996-1998 revealed Pseudo-nitzschia species were widespread around the west coast and the northern isles. Maximal population levels (up to 2 x 10 ⁵ cells L ^-1) were similar to those reported in Loch Creran during 1979-1980 (Tett and Edwards, 2002). A sample collected off southwest Shetland in June 1996 contained 3.5 x 10 ⁶ cells L ^-1. In 1999, a major ASP outbreak occurred during a diverse, widespread and abundant Pseudo-nitzschia bloom (Gallacher et al., 2001). Seven of the nine known DA producing Pseudo-nitzschia species (Table 1) were found, excluding Ps. multistriata and Ps. pseudodelicatissima. Fehling et al. (2004a,b) recently demonstrated that Ps. seriata f. seriata isolated from Scottish waters produces domoic acid. They concluded that minimally, of the nine species present locally, it and Ps. australis can be responsible for the the ASP toxicity in Scottish waters. Peak abundance at most of the 32 inshore monitoring sites (upper 10 m sampled) was ca. 10 ⁵ cells L ^-1, with a maximum of 2.3 x 10 ⁶ cells L ^-1 recorded. The Pseudo-nitzschia species were present year-round, with maximal abundance from April to August depending on the site. In June 1999, after exceedance of the regulatory limit of 20 µg DA g ^-1, most of the Scottish scallop fishing areas were closed (the ban was still in effect in April, 2000), with the most severe effect on the West Coast grounds (ca. 8,500 nautical miles ²) (Gallacher et al., 2001). Figure 2 shows the magnitude and geographic extent of the DA epidemic: the highest levels and most persistent toxicity occurred around the inner Hebrides, in the Sound of Jura, Firth of Lorne, Inner Sound and Minch, with weaker loci found offshore in the Moray Firth and to the east of Orkney (see Tett and Edwards, 2002).

The nine Pseudo-nitzschia species that occur in Scottish coastal waters are indigenous, cosmopolitan, and commonly bloom in global coastal waters (Figure 6). The variety of habitats occupied by Pseudo-nitzschia spp. within this distribution also accords with that reported for Pseudo-nitzschia generally. Prior to evaluating the relationship between local hydrography and potential bloom stimulation by salmon fish farm operations, a summary of Pseudo-nitzschia bloom behavior in European coastal waters is provided. Consideration of those observations, combined with the experimental findings discussed in earlier sections, is needed given the paucity of direct observations on Pseudo-nitzschia dynamics in Scottish coastal waters.

2.8 Distribution of Pseudo- nitzschia and DA in European waters

Pseudo-nitzschia species are common in Irish coastal waters, where their populations can exceed 10 ⁶ cells L ^-1 (Roden et al., 1981). DA above the regulatory threshold level of 20 µg DA g ^-1 wet weight was first detected in 1999, in king scallop ( Pecten maximus) (McMahon and Silke, 2000). Cusack et al. (2002) have linked this toxicity to Ps. australis based on the DA produced by a strain of this species isolated into culture from the southern coast of Ireland in 1997. Cusack et al. report that Ps. australis is commonly found during autumn off this coast, along with five other potentially toxigenic Pseudo-nitzschia species: Ps. delicatissima, Ps. fraudulenta, Ps. multiseries, Ps. pseudodelicatissima, Ps. pungens. This Pseudo-nitzschia assemblage is virtually identical to that reported by Gallacher et al. (2001) for Scottish coastal waters. Also similar to that reported by Gallacher et al., high DA levels were found in Pecten maximus along with sub-regulatory thresholds of DA intoxication of shellfish: mussel ( Mytilus edulis), oyster ( Crassostrea gigas, Ostrea edulis) and razor clam ( Ensis siliqua) (Cusack et al. 2002).

In Norway, blooms of potentially toxic Pseudo-nitzschia species were observed along a sampling grid of 27 monitoring station extending from 58°N to 70°N (Dahl et al., 2001). DA has not been documented in bivalves along this coast during a 10 year monitoring period. However, for precautionary reasons, warnings against collection of wild shellfish are issued when a Pseudo-nitzschia population threshold of 10 ⁶ cells L ^-1 is reached. In Denmark, DA has been found in Ps. seriata blooms (Lundholm et al., 1994, 1994a).

Elsewhere in European coastal waters, Ps. australis has been linked to DA toxicity of the mussel Mytilus galloprovincialis in Spanish rias (= fjords) at rope culture sites (Míguez et al., 1996). This October 1994 event was the first reported occurrence of Ps. australis and DA toxicity in Europe; the bloom population reached about 5 x 10 ⁵ cells L ^-1. Hasle (2002) has since reported Ps. australis occurs in Portuguese coastal waters. This distribution, extending from Scotland to Portugal, suggests Ps. australis widely occurs in European coastal waters, but this species remains enigmatic. As Hasle (2002) has reported, Ps. australis seems to have been ignored or misidentified until its novel 1991 DA event in Monterey Bay (Work et al., 1993). While subsequent reports, particularly from Pacific waters, suggest that Ps. australis is cosmopolitan (Figure 6) , its historical occurrence in European coastal waters is unknown. Given that it was not reported prior to its 1994 bloom in Spanish coastal waters, Hasle (p. 142 in 2002) has suggested that it "may be considered a newcomer in this area until examination of any archived material proves the opposite". Although the reported European occurrences of Ps. australis are confined to the coastal waters of three regions (Scotland, Ireland, Spain) where fish farm or shellfish culture activities are prominent, a direct association between aquaculture and bioinvasion of Ps. australis, if it indeed has occurred, as Hasle suggests, can not be demonstrated. The presence of DA in shellfish has been reported also from France (Amzil et al., 2001) and Portugal (Vale and Sampayo, 2001), and in Pacific waters from Japan and New Zealand (see Landsberg, 2002).

This regional comparison shows that the Pseudo-nitzschia spp., their blooms, and DA accumulation in shellfish found in Scottish coastal waters are not unique occurrences. What is unique is the prolonged and extensive DA occurrence in Scottish shellfish grounds relative to other European waters.

2.9 Vulnerability of scallops to DA intoxication relative to other shellfish

There is frequent reference that scallops (king, queen, sea, New Zealand) accumulate high levels of DA irrespective of the species, and in contrast to the lower degree of contamination reported for other shellfish species (mussels, oysters, clams). Why this apparent heightened vulnerability of scallop populations to DA accumulation? The colonies formed by Pseudo-nitzschia species can exceed 1 cm in attenuated length (Figure 3a), a large size that may be a barrier to filtration by the smaller shellfish. The scallop Pecten novazelandiae was shown to be capable of filtering a wide range of phytoplankton size classes, from the colony-enhanced large size of Ps. australis (Table 1) to the diminutive coccolithophore Gephyrocapsa oceanica (ca. 10 µm) (Rhodes et al., 1996b). It is unlikely, however, that the reduced DA loadings characterizing other shellfish groups can be explained by reduced filtration rates because of Pseudo-nitzschia cell (colony) size. Pseudo-nitzschia production of DA, as pointed out previously, is a stress response to nutrient limitation. During cellular senescence, pseudo-nitzschioids increasingly lose their capacity for colony formation: their chains break up into smaller units, with the predominance of single cells usually the terminal result. This "size reduction" behavior during the life cycle stage at a time when DA production is maximal should remove any size barrier to filtration by smaller shellfish, if indeed there is such a barrier.

Slower depuration rates by scallops may explain their characteristically high levels, prolonged retention and persistence of DA far after termination of causative Pseudo-nitzschia blooms. Toxin contamination of shellfish is species-specific, and may depend on the amount of algal cells available for ingestion (Shumway, 1990). Bivalve species can be broadly classified as rapid (e.g. Mytilus edulis) or slow detoxifiers (e.g. Placopecten magellanicus) (Bricelj and Shumway, 1998). In the case of saxitoxin, Mytilus edulis takes weeks to detoxify (depurate) to regulatory levels (up to 15% toxin loss day ^-1), while sea scallop, Placopecten magellanicus, takes months to years to detoxify (<3% toxin loss day ^-1). In the case of DA, >50% of the gut lumen content in Mytilus edulis was eliminated the first 24 hours, whereas DA levels in sea scallops remained high up to 15 days after exposure, and razor clams, Ensis siliqua, retained DA for up to six months (Landsberg, 2002).

It is recommended that DA retention and depuration rates in king scallop, Pecten maximus, be investigated, given ASP problem in Scottish waters, to help clarify the relationship between DA intoxication and Pseudo-nitzschia blooms. Field investigation of Pseudo-nitzschia population dynamics is also recommended.

2.10 Pseudo-nitzschia, ASP and fish farms in Scotland

Section 2.5.5 summarized the experimental data that show DA synthesis is under multifactorial environmental control. This type of regulation, the fish farm locations, and the production and dispersal of their nitrogenous wastes argue against fish farm activities having a significant impact stimulatory to Pseudo-nitzschia blooms. Therefore, I conclude that DA contamination of the commercial scallop beds in Scottish waters was not influenced by fish farm activities. The mode of stimulation of Pseudo-nitzschia blooms by fish farm activities is restricted to one pathway: bloom stimulation by excreted nutrients. Grazing on the natural food web components is not a factor. Fish are ammonotelic; there is copious excretion of NH ₄, urea and other organic nitrogenous compounds at fish farms [see Section 10.0]. Information on the nitrogen nutrition of Pseudo-nitzschia spp. is available for Pseudo-nitzschia multiseries and Pseudo-nitzschia pungens (Bates et al., 1993; Hillebrand and Sommer, 1996). While these species assimilate NO ₃, NH ₄, urea and glutamine, Ps. multiseries is NH ₄ -sensitive, inhibited by high experimental concentrations, including waste concentrations (>100 µM) expected at fish farm sites. High NH ₄ concentrations, unlike NO ₃, prolonged its lag growth phase, decreased its rate of photosynthesis and final population yield, and enhanced DA production 2- to 4-fold (Bates et al., 1993). In the presence of NO ₃, growth inhibition by NH ₄ was enhanced, and its assimilation influenced by the NH ₄:NO ₃ ratio (Hillebrand and Sommer, 1996). The sensitivity of Pseudo-nitzschia to NH ₄ contrasts with other diatoms. The important bloom species Skeletonema costatum and Asterionellopsis glacialis, both of which occur in Scottish waters, are much more tolerant of high NH ₄ concentrations and thrive in eutrophicated habitats (Thomas et al. 1980).

Although NH ₄ may inhibit Pseudo-nitzschia growth at fish farm sites, the effect is localized because dilution and dispersion will decrease its concentration, but blooms will not be stimulated much beyond the zone of initial dilution. The considerable spatial distances between fish farm sites and the widespread offshore distribution of DA (Figure 2) do not favor offshore seedings of Pseudo-nitzschia populations stimulated by fish farm nutrients, nor the transport of fish farm nutrient wastes to offshore sites. Dilution of nutrients and seed stocks minimizes this contributIon. It is important to recognize that the factors regulating growth of Pseudo-nitzschia differ from those regulating DA synthesis, as discussed previously. The seasonal persistence of DA in offshore scallops may be due to the continuous presence of Pseudo-nitzschia spp.; to regional advections of local, offshore blooms, to low depuration rates, or reflect sustained biofiltration of Pseudo-nitzschia cells present in low abundance and leading to gradual accumulation of DA in shellfish. The role and source of NO3 required for DA synthesis to occur are also relevant. In Nova Scotian waters, blooms of Ps. multiseries are closely associated with NO ₃ pulsing. Pseudo-nitzschia pseudodelicatissima is found in nutrient (NO ₃) enriched oceanic surface waters of the North Atlantic and equatorial Pacific (Buck and Chang, 1994). Pseudo-nitzschia fraudulenta forms thin layers that vary in thickness from a few cm to several m, and extend over distances measureable in km. NO ₃ provisions these blooms, as well. NO3 is usually referred to as "new N" because it is pumped from the deep water, aphotic zone reservoir during vertical mixing, upwellings, and at frontal zones. This physical oceanographic delivery of NO ₃ contrasts with the biological recycling and excretion that characterize NH ₄. Pseudo-nitzschia blooms do not depend on a supply of NH ₄; NO ₃, alone, supports growth, blooms and DA synthesis.

It must be reemphasized that DA synthesis is a stress response induced by Si and/or P limitation. Whether the Pseudo-nitzschia population is advected, or results from local growth, and whatever regulates its bloom dynamics, DA synthesis and bivalve accumulation are the result of repressed Pseudo-nitzschia growth, rather than stimulated growth. Growth of Pseudo-nitzschia stimulated by NH ₄ excreted at fish farms, at whatever scale, is not evidence that the observed DA levels are a consequence of fish farm activities. Rather, the requisite physical oceanographic sources of NO ₃, the coastal character of Pseudo-nitzschia spp. (Fryxell et al., 1997; Parsons et al., 1998), and the distributional pattern of DA in Scottish offshore waters (Figure 2), together with the evidence presented in Section 2.5.5, suggest that DA contamination of the commercial shellfish stocks is not coupled to a fish farm stimulus. All known Pseudo-nitzschia cellular and population growth parameters, and the conditions required for DA biosynthesis, occur at those offshore sites.

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