Paralytic shellfish toxins and associated toxin profiles in bivalve mollusc shellfish from Argentina
Introduction
Paralytic Shellfish Poisoning (PSP) is a potentially fatal syndrome that occurs when shellfish consumers are exposed to harmful neurotoxins known as saxitoxins. These are a family of over 50 structurally-related compounds that are present in certain species of dinoflagellates and subsequently accumulate in bivalve mollusc shellfish tissues during filter feeding (Wiese et al., 2010). Dinoflagellates known to contain these harmful products include the genera of Alexandrium species (previously called Gonyaulax), together with Gymnodinium and Pyrodinium. (Llewellyn, 2006; Wright, 1995; Lagos, 2003; Etheridge, 2010). The impacts of PSP outbreaks include human intoxications and death from consumption of contaminated shellfish (Anderson et al., 2012).
Occurrences of PST-producing harmful algal blooms have been recognised along the coast of Argentina for many years (Akselman et al., 2006). Species identified include the autumnal Gymnodinium catenatum first recorded in the warm shelf waters around Mar del Plata, Buenos Aires Province in 1961–62 (Balech, 1964; Akselman et al., al.,1998; Montoya et al., 2006) and in the colder waters of the Chubut and Patagonian regions, blooms of Alexandrium (Elbusto et al., 1991; Santinelli et al., 1994, Andrinolo et al., 1999; Krock et al., 2015, B. 2018; N.G. Montoya et al., 2018) identified from 1980 onwards. Since these early reports, Alexandrium has been identified regularly at high cell densities during the Spring throughout nearly the entire 5000 km length of the Argentinean Atlantic coast, extending northwards into Uruguay (Carreto et al., 1981; Elbusto et al., 1991; Esteves et al., 1992; Carreto et al., 1998b; Gayoso, 2001; Akselman et al., 2006; Montoya et al., 2010; Fabro et al., 2018; Sastre et al., 2018).
Literature pertaining to this region reports two species of Alexandrium associated with toxicity, A. catenella and A. tamarense. As a result of reclassification (John et al., 2014), these two toxic species should be considered synonymous in this region and so will be reported as A. catenella within this paper.
Since the initial identification of A. catenella, links to high levels of paralytic shellfish toxins (PSTs) in shellfish tissues have been recorded, relating to PSP outbreaks (Carreto et al., 2002a; Guzmán et al., 2002; Perisch et al., 2006; Sunesen et al., 2014) and significant human health risks (Elbusto et al., 1981; Carreto et al., 1981, 1986, 1996, 1998, 2002; Esteves et al., 1992; Ciocco, 1995; Lagos, 2003; N.G. Montoya et al., 2018; Villalobos et al., 2019). The first record of PSP occurred in November 1980, with samples of mussels subsequently found to contain PSP at 173,360 Mouse Units (MU) per 100 g shellfish flesh (Elbusto et al., 1981), equivalent to 312,048 µg STX eq./ kg using a conversion factor of 0.18. Other outbreaks were reported in subsequent years, including human poisonings during 1985 in northern Patagonia (Vecchio et al., 1986; Santinelli et al., 2002) and during 1990 when another extraordinary outbreak occurred resulting in mussels close to 290,000 µg STX eq/kg (Elbusto et al., 1993). During 1985, 1988 and 1991, toxicity >20,000 MU/100 g was reported in mussels (Santinelli et al., 1994). During 1991, high concentrations of A.catenella (reported as A. tamarense) were measured (16 × 103 cells/L) (Santinelli et al., 1994). Such blooms have been recorded annually through the end of winter, spring and summer seasons, with A. catenella (reported as A. tamarense) identified from Patagonian and Buenos Aires coast, and into Uruguay (Gayoso, 2001). PSP has also been associated with intense blooms of A. catenella within the Beagle Channel in S. Argentina (Almandoz et al., 2011, 2019). One exceptional bloom during 1991–1992 was measured at a density of 8.21 × 105 cells/L and resulted in toxicity in mussels harvested from near to Ushuaia city during Feb 1992 reaching 1272,000 µg STX eq./kg (Goya and Maldonado, 2014), most likely due to the high toxicity of the A. catenella population (325 pg STX eq./cell) (Benavides et al., 1995). Consequently, the waters along Argentina are affected by periodic dense and toxic blooms of a range of different dinoflagellate species, with typically A. catenella occurring in the south (Beagle Channel), G. catenatum occurring in the north (shelf waters of Buenos Aires Province) and A. catenella (reported as A. tamarense) affecting much of the Atlantic coastline (Fig. 1).
The coastline of Argentina is extensive (approximately 6000 km). The Argentine Sea is a littoral and epicontinental sea, covering the South American continental shelf up to 200 m deep. It has an area of 990,000 km², from the Río de la Plata to the Malvinas Islands, making it one of the largest and richest fishing banks on the planet and shellfish harvesting occurs over a wide and varied geographical area. Exploited bivalve species are mussels (Mytilus edulis and Aulacomya ater), scallops (Aequipecten tehuelchus and Zygochlamys patagonica), and oysters (Crassostrea gigas). Other species fished less frequently include purple clams (Amiantis purpurata), hard shell clams (Ameghinomya antiqua), geoduck (Panopea abbreviata), razor clams (Solen tehuelchus and Ensis macha), and the oysters (Ostrea puelchana) (Lasta et al., 1998; Turner et al., 2014a). The yellow clams (Mesodesma mactroides = Amarilladesma mactroides) and wedge clams (Donax hanleyanus) are found in the intertidal zone within Buenos Aires Province, but commercial exploitation is unauthorized. Consumption does occur however as a result of illegal fishing and recreational harvesting. The mussel species Brachidontes rodriguezii is a smaller coastal bivalve that is often used as a sentinel species in regulatory monitoring programs.
A recent study has reported the spatiotemporal variability of PST levels in shellfish in the Chubut region of Argentina, between 2000 and 2011 as determined using routine MBA toxicity testing (Villalobos et al., 2019). Whilst the MBA has for many years provided a reliable method for regulatory testing of PST in bivalve molluscs in Argentina (Turner et al., 2014a), other methods are now available for regulatory testing. Chemical detection methods such as those utilising chromatographic separation with fluorescence detection (Lawrence et al., 2005; Anon, 2005a; van de Riet et al., 2011; Anon, 2011) are able to quantify both total PST toxicity and individual saxitoxin analogue concentrations. Methods such as these provide a useful tool to aid the risk assessment and risk management process, specifically providing information that can enable the relationship between toxicity and toxin profiles and bivalve species to be assessed, together with insights into geographical and temporal variability throughout any given area (Turner et al., 2014b). Furthermore, the pre-column oxidation LC-FLD method has recently become the reference method for PSP determination in the EU (Anon, 2017; Turner et al., 2019).
This study aimed to conduct LC-FLD analysis on 151 shellfish samples comprising eight species of bivalve molluscs harvested throughout the waters of Argentina over more than 31 years between 1980 and 2012, with the aim of understanding the types of PST analogues typically seen in Argentinean molluscs. Several of these samples are related to toxic outbreaks that occurred during those years, and of which until now the toxin profiles were unknown. Samples were analysed using the pre-column oxidation LC-FLD AOAC 2005.06 method to determine individual PST concentrations, calculate toxin profiles and total PST content. LC-FLD data enabled the assessment of toxin profile dependency, specifically examining the potential relationships to species type, geographical and temporal source and the effects of bioaccumulation of toxins from different algal species throughout the coastal waters of Argentina.
Section snippets
Samples
Live bivalve molluscs were sampled from different regions of the Argentine Sea (Fig. 1) between 36°21′ S (San Clemente del Tuyú, Buenos Aires Province) and 54°54′ S (Bahía Aguirre, Tierra del Fuego Province), between 1980 and 2012 as part of the official control monitoring programme. 151 mollusc samples, all in whole animal form, incorporating eight species were included in the study samples: 3 hard shell clams (Ameghinomya antiqua), 13 wedge clams (Donax hanleyanus), 27 yellow clams (Mesodesma
Total pst
A total of 151 bivalve mollusc samples were analysed by LC-FLD, with quantified PST concentrations summed to estimate total PST toxicity. Across the entire sample set, toxin concentrations varied considerably, with high toxicity confirmed throughout the entire coastline (Fig. 2). All toxin analogues available at the time as CRMs were detected in at least one of the samples. No chromatographic peaks indicative of either C3&4 or GTX6 were detected in any of the samples. In terms of sample
Toxicity
Application of the pre-column oxidation LC-FLD method further confirms the high level of risk from PSP intoxication present at times in bivalve molluscs harvested from Argentinean waters as previously reported using MBA (Elbusto et al., 1981; Carreto et al., 1981, 1986, 1996, 1998, 2002a; Guzmán et al., 2002; Perisch et al., 2006; N.G. Montoya et al., 2018; Villalobos et al., 2019). The maximum toxicity quantified here, from a mussel sample harvested from Banco Constanza (42°31′S/62°42′W) a
Conclusions
This study has quantified the huge variability in PST toxicity encountered throughout the entire coastline of Argentina using the pre-column oxidation LC-FLD, now the EU reference method for PST determination in bivalve molluscs, confirming the significant risk to shellfish consumers. The toxin analogue profiles determined show a remarkable variability, with some regional, seasonal and species-related differences. Dominant analogues were confirmed as being GTX1&4, GTX2&3 and STX, showing some
Declaration of Competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We recognize and thank Ana Ballabene, Horacio Sancho, Alicia Fernández and Carlos Elbusto from the SENASA Mar del Plata Laboratory for their valuable contribution provided by assays and records between 1980 and 1997, and to Débora Bellonio and Ana Luz Candelo for their valuable assistance with classification of samples. We wish to thank Professor Michael Rychlik from the Technische Universität München for funding the time of Sophie Tarnovius during her Masters studies at Cefas. We would also
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2022, Marine Pollution BulletinCitation Excerpt :However, aquaculture is frequently impacted by harmful algal blooms (HABs), with a negative influence on the ecological and economical aspects of aquatic systems because of the production of several marine toxins, such as paralytic shellfish toxins (PSTs), which can accumulate in molluscs and cause paralytic shellfish poisoning (PSP) syndrome in humans. There are 57 analogues of PSTs (Wiese et al., 2010; Anderson et al., 2012; Goya et al., 2020), and three sub-groups can be determined: the carbamoyl (gonyautoxins: GTX1–4 and saxitoxins: STX, NeoSTX), the decarbamoyl (dc-GTX1-dc-GTX4, dc-STX, and dc-NEO) and the N-sulfocarbamoyl (C-toxins: C1-C4, GTX5 and GTX6) subgroups, in decreasing order of toxicity (Oshima, 1995). These toxins are water-soluble, tetrahydropurine, and heat-stable.