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2.3 Source organism(s) and habitat

2.3.1 Source organism(s)

The PSP toxins are present in some genera of dinoflagellates and one species of blue-green algae. Several species of the genus Alexandrium (formerly named Gonyaulax or Protogonyaulax) are identified as contaminators in shellfish. These are Alexandrium tamarensis, A. minutum (syn. A. excavata), A. catenella, A. fraterculus, A. fundyense and A. cohorticula. Other clearly distinct dinoflagellates have also been recognised as sources of the STXs. These are Pyrodinium bahamense and Gymnodinium catenatum (Mons et al., 1998). The toxicity of the dinoflagellates is due to a mixture of STX derivatives of which the composition differs per producing species and/or per region of occurrence.

In Marlborough, New Zealand, the toxin profile of A. minutum consisted predominantly of various proportions of GNTX1, GNTX2, GNTX4, neoSTX and STX (see Figure 2.1). These profiles were similar to those observed in other New Zealand isolates of A. minutum. They were, however, rather different from those observed in this species elsewhere in the world (MacKenzie and Berkett, 1997).

There is also an immobile form of some dinoflagellates, the resting cyst or the hypnozygote. The cysts sink to the bottom of the sea and accumulate at the borderline of water and sediment where they over-winter (Mons et al., 1998). When favourable growth conditions return, the cysts may germinate and reinoculate the water with swimming cells that can then bloom. In this way the survival of certain dinoflagellates from one season to the other season is assured. The cysts themselves are also toxic, however their exact toxicity is not clear. Some investigators claim a toxicity of the same order as the dinoflagellate itself but others mention a ten to thousand fold higher concentration of PSP toxins in the cysts than in the mobile cells (Mons et al., 1998). In Jakarta Bay, Indonesia, motile forms of Pyrodinium bahamense were recorded just after finding cysts of this species in surface sediments. Probably P. bahamense undergoes a complete life cycle in Jakarta Bay (Matsuoka et al., 1998). Cysts from three main groups of toxic or potentially toxic dinoflagellates were found along the coasts of Portugal: i) cysts of G. catenatum were present along the whole coast, dominated assemblages by up to 68 percent in the southwest coast; ii) cysts of P. bahamense were present on the eastern side of the Atlantic Ocean; and iii) cysts of the genus Alexandrium were present along the whole coast and accounted for 8 to 31 percent on the south coast (Amorim and Dale, 1998).

Apart from the protista, the freshwater cyanophyte Aphanazomenon flos-aquae has also been shown to contain STX and neosaxitoxin (neoSTX) (Mons et al., 1998). Other investigators indicated the presence of PSP components in shellfish and crabs without any sign of the appearance of toxic protista. These species were e.g. Spondylus butler and Zosimous acnus (Mons et al., 1998). It is not clear to what content the consumption of coral reef algae was responsible for this effect. During a recent investigation on dinoflagellate cyst production in the Gulf of Naples spherical smooth-walled cysts, which germinated into A. andersonii, were observed in the summer months. Although this species was reported in the past as non-toxic, Ciminiello et al. (2000c) found a clonal culture of this species positive for PSP in the mouse bioassay.

2.3.2 Predisposing conditions

It is not predictable when a bloom of dinoflagellates will develop; neither is the population density a predictable factor. A bloom begins as a small population of toxic dinoflagellate cells in the lag phase or in the form of resting cysts residing in the bottom sediment and the timing and location of a bloom depends on when the cysts germinate and where they were deposited. Climatic and environmental conditions such as changes in salinity, rising water temperature, and increased nutrients and sunlight trigger cyst germination to a vegetative stage that enables rapid reproduction. Once the dinoflagellate bloom begins, an exponential growth phase causes a tremendous increase in their population. In time, the depletion of nutrients and carbon dioxide in the water, and degraded environmental conditions caused by the bloom, decrease population growth. A stationary phase ensures levelling off the population. At this high level of the bloom, the water can have a fluorescent reddish colour referred to as red tide. Continued environmental degradation increases cell death and ultimately leads to a population crash. At this phase of the bloom, many dinoflagellate species form resting cysts that sink to the bottom, ready for the next bloom. Within this bloom cycle, the most toxic cells generally occur during the middle of the exponential growth phase (Mons et al., 1998).

Cyst beds of A. catenella are widespread in coastal and estuarine waters (13 to 25 °C) in New South Wales, Australia. Cysts from cultured isolates in Australia exhibited dormancy periods at 17 °C as short as 28 to 55 days. This contrasts with the usually longer dormancy requirements of temperate populations of A. catenella from Japan (97 days at 23 °C) and of A. tamarensis from Cape Cod (United States) or British Columbia (Canada). Sometimes a one hour temperature increase from 17 to 25 °C improved the germination process of some cultured Australian A. catenella cysts up to 100 percent (achieved after 98 days), but cold-dark storage did not produce the lengthened dormancy requirements reported overseas for over-wintering temperate cyst populations. This indicates that different geographic isolates of the same dinoflagellate taxon can have different cyst dormancy requirements (Hallegraeff et al., 1998).

In cultures of A. fundyense toxin production was discontinuous, induced by light, and always occurred during a defined time frame (approximately 8 to 10 hours) within the early G1 phase of the cell cycle and dropped to zero for the remainder of the interphase and mitosis (Taroncher-Oldenburg et al., 1997).

Dinoflagellates develop at relative high temperatures and abundant sunlight. In Europe and South Africa cases of intoxications and mortality thus occurred mainly between May and November, whereas in North America the intoxications were reported between July and September (Mons et al., 1998).

The type of habitat in which PSP intoxications have been observed varies considerably. Hydrographic conditions probably play an important role; in particular, the presence of a thermocline is very important (an upper layer of seawater which does not mix with the underlying water). Indirectly windforce and turbulence in the water may influence the existence of this thermocline (Mons et al., 1998).

The growth characteristics of A. tamarensis were studied by artificial culture in the laboratory. The results demonstrated that the optimum situation is temperature 22 to 26 °C; salinity 28 to
31 ‰; light intensity 1500-2500 lux and light/dark period 16/8 hours. The average doubling time is 85 hours (Hao, 2001).

There was evidence for a coincidence between Pyrodinium blooms and El Niño-Southern Oscillation (ENSO) climatic events. El Niño is caused by an imbalance in atmospheric pressure and sea temperature between the eastern and western parts of the Pacific Ocean and results in a shoaling of the thermocline (Mons et al., 1998).

The amount of nutrients in the seawater has to be adequate to fulfil the needs of the organisms, especially the concentration of trace elements, chelators, vitamins and organic material in general. However, there are many uncertainties in the determination of the exact role of nutrients in the development of red tides. For example, the development of red tides is sometimes stimulated by low salt concentrations, whereas in other cases high concentrations of salt seem to induce the bloom (Mons et al., 1998).

Irradiance also has an effect on the growth of, for example, A. minutum. Growth of A. minutum cultured from a case outbreak in New Zealand (Bay of Plenty), was studied using 54 combinations of irradiance and different N sources (NO3-, NH4+, urea) and concentrations. Irradiance had more effect on growth in cultures enriched with NO3-, than with NH4+ or urea. Growth appeared to saturate at relatively low irradiance suggesting that A. minutum is able to sustain reasonably good growth rates, even in poorly illuminated depths within the water column (Chang and McLean, 1997). The optimal environmental conditions for cell growth and toxin production of A. minutum T1 isolated from southern Taiwan Province of China were temperature 25 °C, pH 7.5, light strength 120 mEm-2 s-1, and salinity 15 ‰. The optimal levels of nutrients supplemented in the 50 percent natural seawater medium were phosphate 0.02 percent, nitrate 0.01 percent, cupric ion 5.0 ng/g, ferric ion 270 ng/g and humic acid free. Both cell toxicity and total toxicity reached the maximum level at the post-stationary growth phase and decreased quickly (Hwang and Lu, 2000).

For A. catenella (in laboratory culture isolated from the waters of the Hong Kong Special Administrative Region, China) the highest amount of toxin/L of medium was recorded at 20 °C at the beginning of the stationary phase (four hours after the onset of darkness and lasting four to five hours), when cell density was highest and the amount of toxin/cell was still relatively high. At 10 °C the cell density was low while the amount of toxin/cell was high. At 30 °C, the population at full capacity was low and the amount of toxin per cell was also low (Siu et al., 1997).

The N:P ratio is expected to have a marked influence on the production of toxin during a bloom. Several studies are reported in the literature which describe the effect of N:P ratios on the growth of Alexandrium spp. and also the effect on their toxin content (Mons et al., 1998; Béchemin et al., 1999; John and Flynn, 2000). Nitrogen restriction reduced population growth and toxin production, while phosphorus restriction reduced only population growth but enhanced toxin production. When nutrients are non-limiting, the main limiting factors for A. catenella are temperature (20-25 °C), salinity (30-35 ‰) and pH (8.0-8.5) (Siu et al., 1997).

Involvement of eubacteria other than cyanobacteria in the production of PSP toxins has proven to be a controversial subject. It is suggested that bacteria play a role in this area although the precise mechanisms are unclear. It is feasible that the production of PSP toxins is an inherent function of some marine bacteria required for their physiological processes and is incidental in relation to dinoflagellate and shellfish toxicity. Additionally, increasing evidence that bacteria are capable of metabolising PSP toxins may prove to be of practical importance in terms of both dinoflagellate and shellfish toxicity. It may be pertinent to conduct more detailed studies on bacterial and dinoflagellate interactions in marine environments (Gallacher and Smith, 1999).

2.3.3 Habitat

Dinoflagellates and their cysts have mainly occurred in the waters near North America, Europe and Japan but occurrences in Asia are increasingly reported (Mons et al., 1998). In northeastern Canada, PSP was reported more than 100 years ago. In the northeast of the USA, particularly in the New England region, where toxicity was restricted to the far eastern sections of Maine near the Canadian border, the first documented PSP case dates from 1958 (Anderson, 1997).

A. catenella has been observed particularly along the coast of North America, southern Japan and Venezuela, whereas A. tamarensis is found in North America, northern Japan, southern Europe, Turkey and Australia (Mons et al., 1998). Cyst beds of A. catenella are widespread in coastal and estuarine waters (13-25 °C) in New South Wales, Australia. Cysts from cultured isolates in Australia exhibited dormancy periods at 17 °C as short as 28 to 55 days. This contrasts with the usually longer dormancy requirements of temperate populations of A. catenella from Japan (97 days at 23 °C) and of A. tamarensis from Cape Cod or British Columbia (Hallegraeff et al., 1998).

A. fundyense occurs in the coastal waters of northeastern North America (Taroncher-Oldenburg et al., 1997) and blooms of Protogonyaulax tamarensis are a common, seasonal occurrence in the Gulf of Maine (Shumway et al., 1988).

A. excavata (syn. A. minutum) has been reported from the northeast coast of North America, Egypt, Australia, the North Sea (Denmark, Germany, the Netherlands, Norway and Great Britain), the Mediterranean coast (Mons et al., 1998) and New Zealand (Chang et al., 1997a). Since 1990, A. minutum has been reported from a lagoon in Sicily (Italy) where both an exploitation of natural settlements of clams (Ruditapes decussata and Cardium spp.) and small-scale farming of blue mussels (Mytilus galloprovincialis) are practised. No cases of human intoxication were reported. Cell densities are maximal in May (Giacobbe et al., 1996). Along the northwest of the Adriatic coast of Italy, A. minutum was found at the Emilia Romagna sampling stations in 1994, 1995 and 1996 from April to July (Poletti et al., 1998). In the Mediterranean Sea, only the potentially toxic A. minutum and A. tamarensis have been reported to be present until now. However, an investigation of Ciminiello et al. (2000c), performed on cultured material, namely cysts from the Gulf of Naples germinating to A. andersoni, showed positive effects for PSP in the mouse bioassay. The toxicity profile A. andersoni consisted mainly of toxins in the STX class, in particular STX and neoSTX.

Outbreaks of PSP in Japan, the northwest coast of North America, southern Ireland, Spain, Mexico, Argentina and Tasmania (Australia) have been caused by blooms of Gymnodinium catenatum. The present day distribution of G. catenatum includes the Gulf of California, Gulf of Mexico, Argentina, Venezuela, Japan, the Philippines, Palau, Tasmania, the Mediterranean, and the Atlantic coast of Spain and Portugal (Mons et al., 1998). G. catenatum is not endemic to Tasmania but was introduced some decades ago. The first bloom was seen in 1980 with major blooms in 1986, 1991 and 1993. Several lines of evidence suggest that ballast water discharge from cargo vessels originating from Japan and the Republic of Korea, or less likely Europe, was the most probable mechanism of introduction (McMinn et al., 1997).

The first harmful implications of Pyrodinium bloom became evident in 1972 in Papua New Guinea. Since then toxic Pyrodinium blooms have apparently spread to Brunei Darussalam, Sabah (Malaysia), and the central and northern Philippines. During a Pyrodinium bloom in 1987 in Champerico on the Pacific coast of Guatemala, 187 people had to be hospitalised and 26 people died (Rodrigue et al., 1990). In 1989 another bloom swept northward along the Pacific coast of Central America, again causing illness and death (Mons et al., 1998).

Direct measurement of the specific toxicity of cultured isolates of A. ostenfeldii suggested a low risk of PSP associated with this dinoflagellate species. A. ostenfeldii has been described from numerous locations on the west coast of Europe such as Iceland, the Faeroe Islands (Denmark), Norway and Spain, as well as Egypt, the west coast of the USA, the Gulf of St. Lawrence, Canada and East Asiatic region of the Russian Federation. Cysts of A. ostenfeldii were stated to be common in sediments around the New Zealand coast (Levasseur et al., 1998; Mackenzie et al., 1996).

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