Paralytic shellfish toxins in the marine gastropods Zidona dufresnei and Adelomelon beckii from Argentina: toxicity and toxin profiles.
KEY WORDS: gastropod, Zidona dufresnei, paralytic shellfish poisoning, liquid chromatography, mouse bioassay, toxin profiles
Paralytic shellfish poisoning (PSP) is a potentially fatal syndrome that occurs when shellfish consumers are exposed to harmful neurotoxins known as the saxitoxins (STXs) (Etheridge
2010). They are a family of more than 30 structurally related compounds that are present in certain species of dinoflagellates and that subsequently accumulate in bivalve shellfish tissues during filter feeding (Shumway 1990, FAO 2004). Dinoflagellates known to contain these harmful products include the genera of Alexandrium species, together with Gymnodinium and Pyrodinium (Shumway 1990, Taylor et al. 1995, Hallegraeff et al. 2012). Occurrences of PSP-producing harmful algal blooms have been recorded along the coast of Argentina for many years with the presence of Gymnodinium catenatum recorded in 1961/1962 around Mar del Plata (Balech 1964) and blooms of Alexandrium tamarense identified from 1980 onward in the Valdes Peninsula region of Patagonia (Andrinolo et al. 1999). Since the initial identification of A. tamarense, PSP outbreaks have occurred relating to high concentrations of paralytic shellfish toxins (PST) in shellfish tissues. During the 1980 event in Patagonia, the toxicity of mussel tissue was reported at 173,360 mouse units (MU)/100 g shellfish flesh (Elbusto et al. 1981). Similar events during 1985, 1988, and 1991 resulted in toxicities more than 20,000 M U/100 g in mussels (Santinelli et al. 1994). During 1991, high concentrations of A. tamarense were measured (16 x [10.sup.3] cells/L) (Santinelli et al. 1994). Such blooms have been recorded annually through the spring and summer seasons, with A. tamarense identified from South Argentina in Patagonia all the way up the Argentinean coast and into Uruguay (Gayoso 2001). Paralytic shellfish poisoning has also been associated with intense blooms of Alexandrium catenella within the Beagle Channel in South Argentina. One exceptional bloom during 1991 and 1992 was measured at a density of 8.21 x [10.sup.5] cells/L and resulted in a toxicity in mussels harvested near Ushuaia city in February 1992 that reached 1.20 x [10.sup.6] pg STX eq/kg (Goya & Maldonado 2014), most likely as a result of 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, toxic blooms of a range of different dinoflagellate species, typically with A. catenella occurring in the south (Beagle Channel), G. catenatum occurring in the north (shelf waters of Buenos Aires Province), and A. tamarense affecting much of the Atlantic coastline (Fig. 1). Each of these may produce human health risks associated with the consumption of toxic shellfish (Elbusto et al. 1981, Carreto et al. 1986, Esteves et al. 1992, Ciocco 1995, Carreto et al. 1998b), although the greatest risks are reported as being related to the increased intensity and geographic spread of A. tamarense and A. catenella (Carreto et al. 2002a, Guzman et al. 2002, Persich et al. 2006). Although PSP toxicity in spring is the result of A. tamarense (Carreto et al. 1998a), the origin of autumnal toxicity has been related to the advection of G. catenatum cells from the coastal area of Rio de la Plata (Akselman et al. 1998).
To comply with European regulations and to ensure consumer protection, monitoring of toxic phytoplankton in the water and PSP toxins in shellfish is a statutory requirement for EU member states and for other non-EU countries that wish to export shellfish products to countries within the EU (Anon 2004). The statutory limits of PST in flesh are described by EC Regulation 853/2004, with the maximum permitted limit (MPL) of 800 [micro]g STX eq/kg shellfish flesh (Anon 2005b). In Argentina, the Agri-Food Health and Quality National Service (Senasa) has been responsible, since 1980, for conducting the official control testing of bivalve shellfish and for ensuring that shellfish products containing PSP levels greater than the MPL are not placed onto the market. The EU reference method for detecting PSP toxins--the PSP mouse bioassay (MBA) (Anon 2005a, Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment 2006)--has been used to provide this control in Argentina. When PSP is detected at levels above the MPL, the area in which the toxicity is detected is closed to harvesting until the shellfish have been assessed as safe again.
The coastline of Argentina is extensive (4,989 km), and shellfish harvesting occurs over a wide and varied geographic area. Bivalves commonly harvested include a number of different mussels (Mytilus sp., Aulacomya ater), wedge clams (Donax hanleyanus), yellow clams (Mesodesma mactroides), scallops (Zygochlamys patagonica and Aequipecten tehuelche), and Pacific oysters (Crassostrea gigas).
Outbreaks of PSP, including those in Argentina, have been traditionally associated with bivalve molluscs such as mussels and clams (e.g., Carreto et al. 2002b). There is evidence, however, for accumulation of PST in a number of nonfilter-feeding marine species such as the grazing and carnivorous gastropods, cephalopods, and crustaceans, and fish such as mackerel and anchovy (Shumway 1995, Lagos 2003, Deeds et al. 2008). Carnivorous gastropods in particular are able to accumulate toxins through predation on bivalve molluscs that themselves have bioaccumulated PST during periods of toxic phytoplankton blooms. Global surveillance has provided evidence for toxicity and the presence of specific PST in the carnivorous whelk Rapana venosa from Japan (Ming & Wong 1989), from a number of marine snail species from Japan (Kotaki et al. 1981), and from a variety of whelk and snail species from New England (Tufts 1979), East Canada (Worms et al. 1993), Washington state, Alaska, and Chile (Shumway 1995). Along the coast of Argentina, a number of species of carnivorous marine snails are found, and many are harvested regularly. Species include Adelomelon beckii, Adelomelon ancilla, Odontocymbiola magellanica from cold temperate waters, together with Zidona dufresnei and Adelomelon brasiliana from warm temperate waters. The dominant species in the landings of the trawl fleet is Z. dufresnei; it is found in the warm waters from Rio de Janeiro (Brazil) to San Matias Gulf of Argentina, living in sandy bottoms in water up to 100 m deep (Lasta et al. 1998). Snail fishing is incidental, with catches made by fisherman trawling for species of fin fish. Snails captured in trawler nets are separated from the main catch and boxed before sale back on land. Therefore, the exact locations of each snail harvest are difficult to identify. With hundreds of tons of snails landed in Mar del Plata and Necochea each year, the edible portion of the snails--the muscular foot--provides an important and nutritious seafood product. With these snails preying on toxic bivalves, outbreaks of PSP in these gastropod species have been recorded after MBA testing along the Mar del Plata coast (Carreto et al. 1996). In 1986, the first PSP assays were conducted in snails and showed strong evidence for the accumulation of PSP toxins in these species. Therefore, in 1987, official PSP controls in marine gastropods were initiated.
Although the MBA has provided a simple and useful monitoring tool for PSP in snails and bivalve molluscs in Argentina for many years, the full performance characteristics of the method remain unknown. In comparison with alternative nonanimal testing methods, the method is insensitive and prone to interference (Etheridge 2010). During the past 6 y, a number of countries worldwide have moved away from relying on the PSP MBA, and have moved toward implementing alternative testing methods involving either precolumn oxidation (PreCOX) liquid chromatography with fluorescence detection (LC-FLD) (Anon 2005b, Lawrence et al. 2005) or post-column oxidation (PCOX) LC-FLD (van de Riet et al. 2009). Both methods have been validated (Lawrence et al. 2005, van de Riet et al. 2011) and adopted by the AOAC as official, first-action methods (AOAC 2005.06 [Anon 2005a] and AOAC 2011.02 [Anon 2011a]). The PreCOX LC-FLD method was approved by the EU as an alternative to the MBA (Regulation EC 2074/2005, as amended) (Anon 2006), and the PCOX method was approved for use in the United States and Canada by the Interstate Shellfish Sanitation Conference. More recently, a third alternative method--the PSP receptor binding assay--has been validated through interlaboratory study and was also adopted by the AOAC as a first-action method (AOAC 2011.27 [Anon 2011b]). In the United Kingdom, the PreCOX method has been validated further to incorporate additional toxins, and extended to include shellfish species harvested in UK waters (Turner et al. 2009, Turner et al. 2010, Turner et al. 2011). This has generated method performance data for species of importance to the United Kingdom and has provided valuable early-warning data on the presence and variability of toxin profiles in shellfish of different species and in shellfish harvested from different geographic locations (Turner et al. 2014).
This article summarizes the work conducted on samples of 2 species of carnivorous gastropods harvested along the coast of Buenos Aires Province and from Patagonian waters between 1986 and 2012. Total PST content was determined by PreCOX LC-FLD and compared with the original MBA. Data derived from LC-FLD enabled the assessment of toxin profiles and any profile changes potentially relating to geographic or temporal sources. Profiles were also compared with those determined in mussel samples harvested from similar locations, and with the profiles reported previously in source phytoplankton (Carreto et al. 1996).
MATERIALS AND METHODS
Harvests of snails arrived in port at either Mar del Plata or Necochea, where samples from each landing batch were collected for official control testing. Analysis was performed in 2 stages--first, on the edible part of the snail before authorization for thermal processing and, second, on the final cooked product before export. Commercialization of the uneviscerated snail is not allowed. Between 1986 and 2011, more than 40,000 samples of raw muscular foot were tested for PSP by the MBA. For this comparative study, 41 snail samples were analyzed by both LC-FLD and MBA, including 36 Zidona dufresnei and 5 Adelomelon beckii samples (Table 1). Thirty-two of the samples consisted of the uncooked muscular foot (78%); the remainder consisted of the viscera (n = 5, 12%), mucus (n = 2, 5%), and with 1 cooked foot and 1 cooked viscera sample. Snails were harvested and collected between November 1986 and February 2012 throughout a range of latitudes (37[degrees]-43[degrees] S) along the coast (Fig. 1), with samples collected during all 4 seasons of the year. Twenty-four of the samples were collected in the winter/springtime, with 17 harvested in summer/autumn. Three of the 41 samples were obtained from end products associated with toxic outbreaks in 1996 and 2009 (Table 1). In addition, 24 samples of mussels (.Mytilus edulis) were also analyzed to enable a comparison of toxin profiles between the snails and mussels harvested in the same approximate geographic regions.
Snails and mussels taken for official control monitoring were transported to Senasa, where they were homogenized and extracted before MBA as described by AOAC 959.08 (Anon 2005a). After analysis, the remaining HC1 extracts were stored frozen (-20[degrees]C) until required for further analysis. During 2012, HC1 extracts were thawed and 10 mL subsamples of each sample were taken, refrozen, and shipped to Centre for Environment, Fisheries and Aquaculture Science (Cefas) in the United Kingdom. All samples were sent under temperature-controlled conditions and were received at the Cefas laboratory in May 2012 after 5 days of transport. On receipt at Cefas, the samples were checked before storing at -20[degrees]C until required for analysis.
Methods of Analysis
Reagents and Chemicals
Certified reference materials used were decarbamoylsaxitoxin (dcSTX), neosaxitoxin (NEO), saxitoxin dihydrochloride (STX di-HCl), gonyautoxins 1-5 (GTX1-5), N-sulfocarbamoylgonyautoxin-2 and -3 (C1&2), decarbamoylneosaxitoxin (dcNEO), and decarbamoylgonyautoxin-2 and -3 (dcGTX2&3). These reference standards were purchased by the Institute for Biotoxin Metrology, National Research Council Canada (NRCC; Halifax, Nova Scotia, Canada). Two well-characterized but noncertified standards of C3&4 and GTX6 were also obtained from NRCC. Certified Reference Materials were diluted in deionized water to produce concentrated stock standard solutions prior to dilution in 0.1 mM acetic acid for preparation of instrument calibration standards. Solvents and chemicals used for sample preparation and liquid chromatography (LC) analysis were either analytical or LC grade.
Shellfish Extraction and MBA
Muscular foot samples obtained during routine control testing were separated from the shells, rinsed with tap water, minced, and homogenized. Viscera and mucus were obtained only in some samples, for research purposes only. For mussel samples, animals were shucked and the whole flesh was homogenized. Snail and mussel tissue homogenates were extracted following AOAC Official Method 959.08 (Anon 2005a). A total of 100 g shellfish homogenate was mixed with 100 mL 0.1 M hydrochloric acid, and the pH was adjusted to less than 4.0. The mixture was boiled gently for 5 min before cooling, readjusting the pH to 3.0-4.0 if required. The mixtures were centrifuged, and the supernatants were used for the assays. The MBAs were performed using triplicate albino mice CF1 strain (weight range, 18-22 g). Testing was conducted on extracts of each shellfish sample following the guidance of the AOAC (Anon 2005a). Sample toxicities were calculated from the median death times of the mice and are expressed here in terms of micrograms STX equivalents per kilogram shellfish flesh. The conversion factor value applied was 0.19. The limit of detection (LOD) of the bioassay was found to range from 300-350 [micro]g STX eq/kg. The established federal guideline of 800 pg STX eq/kg was applied to determine whether harvesting areas were to be open or closed.
Sample Cleanup and Analysis
Acidic extracts were cleaned at Cefas using CIS-bonded solid phase extraction (SPE) cartridges (C18-T SPE; Phenomenex, Manchester, UK). Cartridge eluants were adjusted to pH 6.5 [+ or -] 0.5 before diluting to a final volume of 4.0 mL. All C18 SPE-cleaned samples were cleaned further using a refined ion exchange (COOH) SPE clean up (Turner et al. 2009), facilitating the separation of toxins into 3 fractions. Fraction F1, contained the C toxins (C1&2 and C3&4), fraction F2 contained the gonyautoxins GTX1-6 and dcGTX2&3, and fraction F3 contained STX, dcSTX, dcNEO, and NEO. all of which were subjected to further analysis.
C18-cleaned extracts of each sample were derivatized using periodate oxidation (Anon 2005a) prior to qualitative LC-FLD analysis to determine both the presence of PST in the samples and the potential presence of N-hydroxylated toxins. All samples were subsequently quantified against 5-6-level calibration standards after peroxide oxidation of C18-cleaned extracts. If the screen results showed the potential presence of N-hydroxylated PST, then periodate oxidation of fractions F1-F3 was conducted prior to additional LC analysis. Unoxidized C18-cleaned extracts were also analyzed, with the peak area responses of any naturally fluorescent chromatographic peaks with the same retention time as PST subtracted from the toxin peak areas of the oxidized sample. Toxicity equivalence factors were taken from those published by EFSA (EFSA 2009). The highest toxicity equivalence factor was used for each isomeric pair (GTX1&4, GTX2&3, C1&2, and dcGTX2&3). Concentrations of individual toxins were calculated in units of STX di-HCl equivalents per kilogram to enable the assessment of toxin profiles in terms of toxicity. Paralytic shellfish toxin concentrations were subsequently used to calculate estimated sample toxicities in terms of micrograms STX di-HCl equivalents per kilogram. Samples were analyzed blind with no prior knowledge of toxicity results obtained using the MBA.
For reverse-phase LC, mobile phase A--comprised of 0.1 M ammonium formate, adjusted to pH 6 [+ or -] 0.1 with 0.1 M acetic acid--and mobile phase B was prepared from 0.1 M ammonium formate with 5% acetonitrile, also adjusted to pH 6 [+ or -] 0.1 with 0.1 M acetic acid. The mobile phases were delivered by an Agilent (Manchester, UK) 1200 series LC at a flow rate of 2 mL/ min. Chromatographic separation was performed using a Gemini C18 reverse-phase column (150 X 4.6 mm, 5 pm; Phenomenex) with a Gemini Cl8 guard precolumn (both held at 35[degrees]C). The chromatographic gradient was as follows: 0%-5% mobile phase B in the first 5 min, 5%-70% phase B for the next 4 min, hold at 70% phase B for 1 min, and back to 100% phase A for the next 2 min. One hundred percent phase A was held for an additional 2 min to allow for column equilibration prior to subsequent sample injections. Agilent fluorescence detectors (1200 model FLD) were used to detect PSP toxin oxidation products. Fluorescence excitation was set to 340 nm, and emission to 395 nm.
The M BA toxicity assessment was conducted by Senasa and was used to determine the levels of toxicity in both the raw and cooked snail tissues. For the purpose of this study, MBA toxicity results are reported for 40 of the 41 samples provided for analysis by LC-FLD. The remaining sample was found to be PSP positive by MBA, but no quantitative toxicity results were available. The LC-FLD results from the 41 samples analyzed at Cefas were used to estimate total sample toxicities for each of the snail samples received. These results were compared with the toxicity results determined previously by MBA at Senasa, to assess the degree of correlation between the 2 sets of data. Individual toxin concentrations were also calculated for each sample and were assessed to determine any clear patterns in toxin profiles. Profiles were examined in relation to the potential effects of the harvesting season, spatial variability, and the part of the shellfish sampled. Mean profiles were also assessed in comparison with the profiles determined in mussels sampled from the same region. A combination of visual assessment and a K-means cluster analysis was used to assess the presence of any specific profile patterns not identified through any of the previously mentioned analyses.
RESULTS AND DISCUSSION
Eighty-five percent of the more than 40.000 samples of muscular foot tested since 1986 were found to contain PSP content either below the MPL or lower than the MBA LOD (data not shown). Therefore, more than 6,000 foot samples analyzed to date by MBA as part of routine control testing have shown PSP at levels greater than the MPL of 800 pg STX eq/kg.
Of the 41 samples examined in this study, 38 were found to contain PSP toxins at concentrations greater than the LOD by PSP MBA, with the 3 negative results all relating to tests on samples for which clinical signs in mice were reported (Table 1). One of the remaining 38 samples was found to be PSP positive, but no quantifiable value was reported. Paralytic shellfish poisoning was found to range from just more than the detection limit to a maximum value of 22,468 [micro]g STX eq/kg in 1 viscera sample. The maximum value of PSP in the edible foot tissue was found to be 3,365 [micro]g STX eq/kg in 1 sample harvested during winter 1991, but many of the foot samples were found to contain PSP at levels many times greater than the MPL.
The limited PSP MBA data available from the analyses conducted on different parts of the same animals showed strong evidence for the high level of toxin accumulation in the viscera of the snails (Table 2). Paralytic shellfish poisoning toxicity was found at levels 20-120 times higher in the viscera compared with the edible foot, and 6-14 times higher in comparison with the samples of mucus secretion. Therefore, the greatest risk to consumers was found to relate to the consumption of whole snails or via the viscera during the cooking process. The evidence, however, still highlights the dangers related to the consumption of the edible foot muscle, when levels of PSP toxicity are high enough in the viscera. These risks are highlighted in Table 3, which summarizes the PSP results measured in uncooked and/ or cooked whole snail tissues from samples implicated in PSP outbreaks between 1989 and 2009. A range of mild and severe symptoms were described in these outbreaks, with symptom descriptions and onset times appropriate for toxic PSP. With death occurring in 1 case in 1992, after consumption of viscera from whole snails in which the foot was found subsequently to contain from 855-1,500 [micro]g STX eq/kg, there is clear evidence of the risks associated with PSP in these food products. For official control monitoring of snails, the detection of PSP in the foot muscle at levels greater than 800 pg STX eq/kg triggers the recall of the entire batch of snails obtained from the same harvesting zone as the test sample. Therefore, the greatest risk is to consumers of whole snails that have been harvested illegally and thus are not subjected to proper official controls.
Total PSP by LC-FLD
Table 1 summarizes the PSP data calculated in terms of total STX equivalents after quantitative LC-FLD assessment using AO AC 2005.06. The qualitative identification of PSP toxins by LC-FLD agreed well with the detection of PSP using the MBA. The 3 samples found to be less than the LOD by MBA, but which exhibited clinical signs during the assay, were shown to contain only low concentrations (52-115 [micro]g STX eq/kg) of PSP toxins--well below the limit of sensitivity of the biological assay. The 1 additional MBA result designated as positive, but with no quantitative result assigned, showed high concentrations of PSP toxins, with total PSP estimated by LC-FLD at 2,049 pg STX eq/kg. For the remaining 37 samples associated with quantitative PSP results from both methods, the comparison of the 2 approaches appeared to be reasonable, with all samples positive by MBA showing PSP concentrations greater than the limit of quantitation of the LC-FLD method. Of the 7 samples analyzed by MBA containing PSP less than the MPL, all except 1 sample were quantified as containing a total STX equivalent less than the MPL.
Figure 2 illustrates a good correlation between the 2 sets of data, with a Pearson correlation coefficient of 0.88 and a mean LC:MBA ratio of 1.13. This, therefore, indicates that the AO AC 2005.06 LC-FLD agreed well, on average, with the results obtained by MBA. The scatter around the mean LC:MBA ratio was found to be relatively large, resulting in part from a low number of outliers. Two samples in particular were found to have LC:MBA ratios of 0.15 that, if removed from the data set, resulted in a visual improvement to the overall method comparison. A 2-tailed paired t-test calculated from the 2 data sets (n = 37; 95% confidence) gave a t value of less than 0.5 that, when compared with the t critical value of 2.03, indicates there was no statistically significant difference in the results returned by the 2 methodologies. The degree of scatter in the correlation appeared similar to that observed in previous comparative studies at our laboratory (e.g., Turner et al. 2009, Turner et al. 2010, Turner et al. 2011).
Spatial and Temporal Variability of Snail Toxicity
Given the wide geographic area associated with the snail harvests and the large number of years during which snails have been collected for food consumption, there is the potential for patterns to be present that relate toxicity levels to each of these factors. Table 4 summarizes the mean toxicity data determined by both MBA and LC-FLD in relation to the date and location of the sample harvesting points as well as the season of collection. On initial inspection, the mean toxicity results indicated some potential for differences in toxicity in snails harvested at different times of the year and in different locations. The variability associated with these mean values, however, was very high, consequently making any inferences regarding differing toxin accumulation patterns impossible. Similarly, the mean toxicities appear substantially greater during the 1980s and 1990s, but, again, the variability of the values is very high, making any comparison among years meaningless. It is noted that the data from the 41 snail samples examined in this study represent only a small subset of the total number of snail samples analyzed between 1986 and 2012. Therefore, any investigations into variability of toxin uptake over time and in different geographic areas would need to be conducted on a larger data set.
The quantitative LC-FLD data obtained from each of the snail samples enabled the calculation of relative proportions of each toxin or toxin epimeric pair in terms of contribution to total STX equivalents. Figure 3A illustrates the mean toxin profiles determined for both Adelomelon beckii and Zidona dufresnei separately and together. In addition, the mean profiles are included for the 24 samples of Mytilus edulis sampled from a similar geographic area for comparative purposes. The chart shows the dominance of the parent compound STX in both of the snail species, with the toxin representing an average of 85% of the sample toxicity. Other toxins present at quantifiable levels included GTX2&3, dcSTX, and NEO, with only occasional trace levels of GTX1&4, C1&2, and dcGTX2&3. Gonyautoxin 5 was not observed, and dcNEO was detected only in 1 of the snail samples. Toxin profiles quantified in the 2 different snail species were similar, with no obvious differences noted. Therefore, there was no evidence for any significant differences in toxin accumulation or transformation between the 2 snail species assessed during this study.
The mean profile calculated from the 24 mussel samples was notably different from the profiles determined in the snails, as exemplified by the chromatograms shown in Figure 4. The mussels were dominated by the GTX toxins (GTX1&4 most notably, as well as GTX2&3), with only low relative concentrations of STX. Neosaxitoxin was detected at low levels in some mussels, with only trace concentrations of C1&2 picked up in approximately 25% of the samples. Therefore, there was evidence for markedly different profiles between the mussels and snails harvested from the same approximate locations. Given the wide range of years incorporated in both the mussel and snail sample set, and the relatively low variabilities associated with the mean profile proportions, there was good evidence for toxin transformation of the GTX toxins present in the mussels to STX in the snails, presumably as a consequence of the carnivorous snails feeding on the toxic mussels.
Carreto et al. (1996) used a postcolumn oxidation LC-FLD method (Oshima 1995) to assess the PSP toxin profile in an Alexandrium tamarense isolate obtained from the coast near Mar del Plata. They reported the dominance of C1&2 toxins in terms of absolute concentration, together with GTX1&4 and NEO at lower relative concentrations. Saxitoxin was the lowest of the 10 detected toxins. From those results, GTX1&4 toxins would have been the dominant toxins in terms of contribution to total STX equivalents. Carreto et al. (1996) also reported the toxin profiles in 1 sample of mussels (Mytilus eclulis) and 5 snail samples comprising Zidona dufresnei, Adelomelon brasiliana, and Adelomelon beckii. In terms of toxic equivalence, their results also showed the dominance of GTX toxins in the mussel sample, with the conversion to the dominant STX in all snail samples.
Consequently, the work presented here provides additional evidence for the conversion of GTX toxins into STX within the tissues of a larger number of carnivorous snail samples, obtained over a period of more than 25 y throughout a wide geographic extent and during each of the 4 seasons of the year. Therefore, there is further evidence here to support the hypothesis that the source toxins present in phytoplankton and accumulating in bivalve molluscs are transformed to the more potent parent STX during the feeding of snails on the mussels.
Spatial and Temporal Profile Variability
Figure 3B illustrates the mean toxin profiles and associated SDs in snail samples captured during each of the 4 decades of study to date. The data were summarized in this manner to assess whether any significant changes in toxin profiles may have occurred from the 1980s to 2012. Overall, the graph clearly shows that the dominance of STX in the snail samples has been a consistent feature of the contaminated gastropods, with no indications of any changes throughout time.
Figure 3C summarizes the mean profile results obtained after analysis of snails captured along different parts of the Argentinean coastline. With no exact location data available, the results were separated into 4 different areas of latitude (37[degrees], 38[degrees], 39[degrees], and 40[degrees] 43[degrees] S). For the 3 highest latitudes, profiles appeared almost identical, with the dominance of STX observed throughout these regions. The 1 visual difference was for snail samples harvested between 40[degrees]-43[degrees] S, where greater relative proportions of GTX2&3 and dcGTX2&3 were determined, with lower relative proportions of STX being quantified as a result. It was noted, however, that a large SD was associated with the mean profile for these samples, indicating some significant differences in profiles present in samples harvested from this area of the coast. On examination of the raw data, approximately half the samples obtained between 40[degrees]S and 43[degrees]S also exhibited the STX-dominant profile. The other half showed much lower levels of STX, with proportions of GTX2&3 exceeding 50% and with the decarbamoyl toxins dcSTX and dcGTX2&3 present around approximately 10% of the total STX equivalents (raw data not shown). These samples were harvested between November and January, whereas the STXdominant snails from the same area were harvested earlier in the year--during September to October. There may, therefore, be some indications of profile differences within this area of latitude, but without more study samples, it is impossible to reach conclusions regarding the causes for these changes.
The potential effect of sampling season was also examined through the calculation of mean profiles for snails sampled at different points throughout each year. Clustering the data into season-dependent groups showed some differences in mean levels of STX; but, with large SDs associated with these means for samples harvested outside of winter, there were no significant differences noted (Fig. 3D).
Profile Variability Throughout the Animal
With samples analyzed either as the foot, viscera, or mucus secretion, profile differences in each matrix type could be assessed. Figure 3E illustrates the mean profiles in each of the 3 matrices, showing clear similarities between the profiles in both the foot and the viscera samples. Although the mean proportion of STX was greater in the foot, the associated SDs around the mean values indicated no significant difference between the 2 profiles. Consequently, there appeared to be no strong evidence for additional toxin transformation occurring during the metabolic processes governing the incorporation of PSP toxins into the muscular foot muscle, as suggested by previous evidence of Carreto et al. (1996). The mean profile for toxins in the mucus samples appeared slightly different, with a greater relative proportion of GTX2&3 toxins and a lower proportion of STX. These results were, therefore, different from those reported by Carreto et al. (1996), who described high proportions of STX in the mucus secretions in comparison with the levels in the viscera or the foot. It would be difficult, however, to determine the exact nature of any differences from these results, given that only 2 samples of mucus were included in the current study and 2 samples were described in the study of Carreto et al. (1996). In addition, the 2 samples in the current study showed notably different profiles, 1 with a dominant (96%) STX profile and the other containing more than 50% GTX2&3, resulting in very high SDs associated with the mean profiles. Although these differences are interesting, more analysis would be required on a greater number of mucus samples to determine whether the profile differences in this matrix would be expected. Nevertheless, there may be other factors that affect the toxin profiles that have not been captured by the parameters explored in this collection of results from 41 snail samples.
The K-means cluster analysis and visual interpretation of profile results showed that the toxin profiles were separated into 2 main groups. The first group were those samples (n = 34) containing STX as the dominant toxin (>90% total STX equivalents), with only minor proportions of the other congeners quantified. The second group consisted of the remaining 7 samples that were found to contain lower proportions of STX. Of this second group, 3 subgroups were identified, containing high proportions of dcNEO, C1&2, dcSTX, and GTX2&3 (profile 2); dcSTX only (profile 3); and GTX1&4, dcSTX, and GTX2&3 (profile 4). Although the variabilities associated with the mean profiles in some of these groupings were still found to be relatively high, there was still good evidence for a number of snail samples containing much lower proportions of STX and greater relative proportions of the GTX and decarbamoyl toxins (Fig. 3F). Of the 7 samples containing lower proportions of STX, all were of the species Zidona dufresnei, but were harvested over a wide range of geographic locations (37[degrees]-43[degrees] S), in spring, summer, and autumn in each of the decades in the study samples (1987 to 2012). The cause of these differences remains unknown. There is some potential for the source phytoplankton to affect the profiles, given the accumulation of different PSP congeners in any mussels feeding upon Gymnodinium catenatum in comparison with the more commonly occurring Alexandrium tamarense. Mussels feeding on the former will contain greater relative proportions of decarbamoyl toxins as well as GTXs and C toxins (Montoya et al. 2006), which in turn will provide a different toxin source to the carnivorous snails. On further examination, however, only 3 of the 7 samples coincided with the time and location of the appearance of G. catenatum in the marine waters around the province of Buenos Aires (Montoya et al. 2006). The other 4 samples were harvested either from locations where G. catenatum has not been reported and/or from times of the year when only A. tamarense would be expected to bloom. Overall, further studies on a larger data set of samples that also include specific information on phytoplankton species at each snail sampling point would be required to examine these potential relationships more thoroughly.
Forty-one samples of carnivorous snails were analyzed for PSP by the reference MBA method. Maximum levels of toxicity were found within the viscera of the animals, with significantly lower levels present in the mucus secretion and foot muscle. Even so, PSP was still found to be present in samples of the foot at levels many times greater than the MPL of 800 [micro]g STX eq/kg. A precolumn oxidation LC-FLD method based on AOAC 2005.06 was used to confirm the levels of total PSP toxins in each of these samples. Results indicated a good correlation between the 2 methodologies, with no statistical difference between the 2 sets of data. The LC-FLD method also confirmed the presence of low concentrations of toxins in 3 samples previously found to be less than the LOD by the MBA, yet exhibited clinical signs. The levels of toxicity determined by the 2 methods were examined in relation to sampling location as well as season and year of sampling. No significant differences were observed in relation to any of these variables. The LC-FLD analyses also allowed the determination of toxin profiles in each of the studied samples. Results showed the majority of snail samples of both species to contain a dominance of STX, usually representing more than 90% of the total STX equivalents. This profile contrasted notably with the profiles observed in 24 mussel samples harvested from the same approximate locations and with the profiles described by previous studies in both mussels and the source Alexandrium tamarense. This study therefore confirmed the apparent conversion of GTXs and other PSP congeners present in the algae and mussels to the parent STX after feeding by the carnivorous snails. A comparison of toxin profiles against the year and season of harvest showed no significant differences in results. Snails harvested within the areas contained by 37[degrees]-39[degrees] S also showed very similar toxin profiles, whereas samples from farther south (40[degrees]M3[degrees] S) showed lower average proportions of STX and greater relative concentrations of GTXs and decarbamoyl toxins. There appeared to be no significant differences among the profiles determined in the foot, mucus, and viscera matrices, given the large SDs associated with the mean profile results in each of these matrices. Seven of the Zidona dufresnei samples, however, were found to have different PSP toxin profiles in comparison with the STX-dominant majority. There appeared to be 4 different toxin profile clusters within the data set, but no indication regarding the parameters that may have affected this small subset of snail samples. For Adelomelon Beckii, these variations were not observed, with a consistent STX-dominant profile measured in each of the samples received.
We thank Horacio Sancho and Alicia Fernandez from the Senasa Mar del Plata Laboratory for their valuable contribution provided by assays and records between 1986 to 1997. Thanks are also extended Prof. Michael Rychlik from Technische Universitat Munchen for funding the time of Sophie Tarnovius during her Master's studies at Cefas and to Myriam Algoet for reviewing this manuscript. The work described in this article has been conducted in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for animal experiments.
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ANDREW D. TURNER, (1) * SOPHIE TARNOVIUS (1,2) AND ALEJANDRA B. GOYA (3)
(1) Centre for Environment, Fisheries and Aquaculture Science (Cefas), Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, UK; (2) Technische Universitat Munchen, Walther-Meifiner-Strafie 3,85748 Garching, Germany; (3) Marine Biotoxin Department, Mar del Plata Regional Laboratory, Agri-food Health and Quality National Service (Senasa), Argentina
* Corresponding author. E-mail: Andrew.email@example.com
TABLE 1. Summary of 41 snail samples analyzed in this study, showing species, part of snail analyzed, harvesting area, sample collection date, and PSP toxicides (micrograms saxitoxin equivalents per kg of shellfish) by MBA and LC-FLD. Zone or area Sample (n) Species Part of snail (harvest/catch) 28 * Zd Viscera 38[degrees]00' 29 Zd Viscera 38[degrees]15' 30 Zd Viscera 38[degrees]15' 32 Zd Mucus 38[degrees]15' 33 Ah Cooked foot 37[degrees]00' 40 Zd Foot 37[degrees]00' 41 Zd Foot 43[degrees]00' 43 Zd Foot 38[degrees]10' 46 Zd Foot 42[degrees]00' 47 Zd Foot 41[degrees]00' 48 Zd Foot 41[degrees]00' 50 Zd Foot 39[degrees]40' 52 Zd Foot 38[degrees]10' 57 Zd Foot 40[degrees]00' 60 Zd Foot 38[degrees]30' 61 Zd Foot 37[degrees]00' 62 Zd Foot 39[degrees]00' 64 Zd Mucus 38[degrees]00' 63 Zd Foot 39[degrees]00' 92 Zd Foot 39[degrees]00' 77 Zd Foot 38[degrees]00' 81 Zd Foot 37[degrees]53' S/57[degrees]04' W 80 Zd Viscera 37[degrees]50' 86 Zd Foot 38[degrees]00' 87 Zd Foot 37[degrees]50' 88 Zd Foot 38[degrees]00' 78 Zd F'oot 39[degrees]00' ([dagger]) 79 Zd Viscera 39[degrees]00' ([dagger]) 91 Ah Foot 37[degrees]50' 116 Ah Foot 38[degrees]00' 119 Ah Foot 38[degrees]00' 5,669 Ab Foot 38[degrees]00' 122 Zd Cooked viscera un ([dagger]) 20,209 Zd Foot 37[degrees]00' 22,809 Zd Foot 39[degrees]17' 23,000 Zd Foot 38[degrees]00' 23,002 Zd Foot 39[degrees]17' 23,424 Zd Foot 37[degrees]00' 130 Zd Foot 38[degrees]00' 131 Zd Foot 38[degrees]00' 132 Zd Foot 38[degrees]00' Sample (n) Date Season MBA LC-FLD 28 * 11/7/1986 Spring 22,468 9,250 29 3/23/1987 Autumn 20,727 18,275 30 3/24/1987 Autumn 31,314 28,532 32 4/28/1987 Autumn 7,317 5,226 33 4/28/1987 Autumn 1,159 2,031 40 12/19/1987 Spring 1,986 1,791 41 1/29/1988 Summer 2,206 2,098 43 5/18/1988 Autumn 3,164 3,793 46 9/15/1989 Winter 1,759 1,840 47 10/17/1989 Spring 1,911 3,197 48 11/8/1989 Spring 2,010 275 50 8/13/1990 Winter 1,820 3,076 52 8/28/1990 Winter 1,615 2,322 57 11/30/1990 Spring 2,662 3,608 60 3/6/1991 Summer 2,367 1,713 61 7/18/1991 Winter 2,242 1,221 62 7/22/1991 Winter 2,660 5,002 64 7/23/1991 Winter 2,557 4,835 63 7/29/1991 Winter 3,365 2,348 92 9/03/1993 Winter 2,101 3,354 77 5/11/1994 Autumn 3,830 3,418 81 11/08/1994 Spring 1,520 2,168 80 11/24/1994 Spring 1,7102 21,636 86 2/10/1995 Summer 1,934 3,129 87 3/13/1995 Summer 1,554 3,106 88 4/25/1996 Autumn 1,677 1,768 78 10/7/1996 Spring 540 946 79 10/10/1996 Spring 16,810 10,744 91 5/31/1997 Autumn Pos 2,049 116 6/6/2005 Autumn 960 1,325 119 3/8/2006 Summer 1,070 1,666 5,669 7/3/2006 Winter 990 701 122 11/1/2009 Spring 400 60 20,209 9/8/2010 Winter 580 373 22,809 7/21/2011 Winter 360 442 23,000 8/19/2011 Winter 410 490 23,002 8/19/2011 Winter 420 475 23,424 10/7/2011 Spring 370 206 130 2/15/2012 Summer <LOD 74 131 2/17/2012 Summer <LOD 115 132 2/17/2012 Summer <LOD 62 Ab, Adelomelon beckii; LC/FLD, liquid chromatography with fluorescence detection; LOD, limit of detection (300/350 mg saxitoxin equivalents/ kg); MBA, mouse bioassay; Pos, paralytic shellfish poisoning positive (>LOD), but toxicity not quantified; un, unknown location; Zd, Zidona dufresnei. * Sample not part of official control testing program, ([dagger]) Products associated with paralytic shellfish poisoning outbreak. TABLE 2. PSP toxicity by MBA (micrograms saxitoxin equivalents per kg of shellfish) in uncooked Zidona duf esnei showing results and associated ratios between the foot, viscera, and mucus. Date Zone (LS) Viscera Foot 07/11/1986 38[degrees]00 22,468 710 20/03/1987 37[degrees]15' 4,270 350 23/03/1987 38[degrees]15' 20,720 <LOD 24/03/1987 38[degrees]15' 31,314 350 25/03/1987 37[degrees]00' 5,010 490 28/04/1987 38[degrees]15' 32,800 520 23/07/1991 38[degrees]00' 56,000 460 25/07/1991 39[degrees]00' 5,830 420 24/11/1994 37[degrees]50' 17,102 770 Date Ratio of Mucus Ratio of Mucus: Viscera:Foot Viscera:Foot 07/11/1986 31 na -- 20/03/1987 12 na -- 23/03/1987 59 na -- 24/03/1987 89 na -- 25/03/1987 10 na -- 28/04/1987 63 7,310 14:63:1 23/07/1991 122 2,557 6:122:1 25/07/1991 12 na -- 24/11/1994 22 na -- LOD, limit of detection (300/350 [micro]g saxitoxin equivalents/kg); LS, Latitude South; MBA, mouse bioassay; na, not analyzed; PSP, paralytic shellfish poisoning. TABLE 3. PSP results (micrograms saxitoxin equivalents per kg of shellfish) from Z. dufresnei samples implicated in PSP outbreaks, and associated symptoms. Date n Predominant symptoms January 1989 1 Nausea, vomiting, paresthesia, feeling of weightlessness, paresis; recovery on day 3 August 1992 5 (3 mild. Nausea, vomiting, 2 severe) headache, paresthesia; respiratory arrest and death in 1 case October 1994 1 Paresthesias in the mouth, tongue, fingers, legs; respiratory distress October 1996 3 (2 mild, Gastrointestinal and 1 severe) neurological disturbances November 2009 2 Vomiting, perioral paresthesias, aphagia, weakness, descending ataxia, immobility, respiratory distress PSP toxicity by MBA Time of onset Date to symptoms Uncooked snail Cooked snail January 1989 30-60 min na na August 1992 30 min-2 h Foot: 855-1,500 na October 1994 30-60 min na Foot and viscera: 1,900 October 1996 30-60 min Foot: 540-830, Foot: 1,330, viscera: 16,810 viscera: 1,440 November 2009 30 min-5 h na Foot: <LOD, viscera: 400 LOD, limit of detection; MBA, mouse bioassay; na, not analyzed; PSP, paralytic shellfish poisoning. TABLE 4. Summary of mean toxicity data (micrograms saxitoxin equivalents per kg of shellfish; [+ or -] 1 SD) grouped according to date, season, and location of harvest of snails. MBA LC-FLD Date 1986-1989 8,729 [+ or -] 10,776 6,937 [+ or -] 8,783 1990-1999 3,146 [+ or -] 4,073 4,296 [+ or -] 4,998 2000-2009 855 [+ or -] 307 938 [+ or -] 709 2010-2012 428 [+ or -] 89 280 [+ or -] 185 Season Winter 1,606 [+ or -] 985 2,037 [+ or -] 1,639 Spring 6,162 [+ or -] 8.269 4,898 [+ or -] 6.613 Summer 5,531 [+ or -] 8.509 3,517 [+ or -] 6,096 Autumn 8,565 [+ or -] 11,318 7,233 [+ or -] 9,595 Location 37 LS 3,314 [+ or -] 5,608 3,842 [+ or -] 6,734 38 LS 7,041 [+ or -] 10,035 4,899 [+ or -] 7,572 39 LS 3,510 [+ or -] 5,487 3,299 [+ or -] 3,401 40-43 LS 1,825 [+ or -] 764 1,846 [+ or -] 1,459 LC-FLD. liquid chromatography with fluorescence detection; LS. Latitude South; MBA, mouse bioassay.
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|Author:||Turner, Andrew D.; Tarnovius, Sophie; Goya, Alejandra B.|
|Publication:||Journal of Shellfish Research|
|Date:||Aug 1, 2014|
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