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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.37 no.4 suppl.A Santiago  2004

http://dx.doi.org/10.4067/S0716-97602004000500002 

 

Biol Res 37: 721-731, 2004

ARTICLES

Simultaneous presence of Paralytic and Diarrheic Shellfish Poisoning toxins in Mytilus chilensis samples collected in the Chiloe Island, Austral Chilean Fjords

CARLOS GARCÍA1, PAMELA MARDONES1, ANDREA SFEIR2 and NÉSTOR LAGOS1*

1 Laboratorio Bioquímica de Membrana, Programa Fisiología y Biofísica, Facultad de Medicina, Universidad de Chile. Santiago, Chile
2 Centro Regional de Análisis de Recursos y Medio Ambiente. Campus Pelluco, Universidad Austral de Chile, Puerto Montt, Chile

Dirección para Correspondencia


ABSTRACT

The study shown here provides the first indisputable evidence that shellfish can be contaminated with Paralytic Shellfish Poisoning (PSP) and Diarrheic Shellfish Poisoning (DSP) toxins during the summer season in the Southern Chilean fjords. Quantitative analysis of the simultaneous presence of PSP and DSP toxins in Mytilus chilensis samples collected in the Chiloe Island are shown. The High Performance Liquid Chromatography (HPLC) analysis with pre-column derivatization method for DSP toxins and the post-column derivatization methods for PSP toxins, both with fluorescent on-line detections, showed that both type of toxins were concentrated by the filter bivalve Mytilus chilensis in amounts above the international safe limits. The phytoplankton analysis showed the presence of both Alexandrium catenella and Dinophysis acuta in the water column. The data shows stratification of the toxic dinoflagellates in the water column, since the lowest amount of both DSP and PSP toxins were measured in the superficial and deeper levels of the water column. Moreover, the highest toxicities of both types of toxins were shown by the shellfish samples collected at a depth of 6 meters with 190 nanograms of DTX-1 / gram of digestive gland and 709.8 mg of PSP toxins / 100 grams of mussel meat.

Key terms: DSP toxins, PSP toxins, Dinophysistoxin-1, Patagonia fjords, Chile.


INTRODUCTION

Toxic harmful algal bloom (HAB) arises due to the exponential growth of dinoflagellates or diatoms that produce toxins. Blooms are stimulated by multiple environmental factors, such as nutrient increase in the water column, the augmented utilization of coastal waters for aquaculture, eutrophication and unusual climatological changes (Hallegraeff 1993). The outbreaks of these species are constant threat to public health worldwide and have negative impacts in the ecology and severe economic losses to aquaculture, fisheries, and tourism (Lagos, 1998).

To date, five microalgal poisons in seawater have been described; four of these are associated with toxic dinoflagellates and one with diatoms. Of them, Paralytic Shellfish Poisoning (PSP) and Diarrheic Shellfish Poisoning (DSP) are the most frequent and widely distributed (Lagos, 1998; Lagos, 2003). Three morphologically distinct genera of dinoflagellates are recognized as primary sources of PSP toxins: Alexandrium sp., Pyrodinium sp., and Gymnodinium sp. (Hallegraeff, 1993). These dinoflagellates primarily produce the water-soluble PSP toxins such as saxitoxin (STX), neoSaxitoxin (neoSTX) and gonyautoxins (GTXs). Approximately 26 analogues of these naturally-occurring toxins have been described (Oshima, 1995; Compagnon et al., 1998; Lagos, 2003). The PSP toxins produce a clinical syndrome characterized by generalized malaise with facial paresthesia, asthenia, dystonia, ataxia, dyspnea, hypotension, tachycardia, vomiting, gastrointestinal disturbance, respiratory arrest and death depending of the amount of toxins intake (Andrinolo et al., 1999; Lagos and Andrinolo, 2000; Andrinolo et al., 2002).

The Diarrhetic Shellfish Poisoning is produced for dinoflagellates that belong to the Dinophysis sp and Prorocentrum sp genera; both produce lipid-soluble toxins such as Okadaic acid (OA), Dinophysistoxin-1 (DTX-1), Dinophysistoxin-2 (DTX-2) and Dinophysistoxin-3 (DTX-3) (Yasumoto and Murata, 1993). Clinical symptoms include diarrhea, nausea and abdominal cramps. No poisoning deaths have been reported, although these toxins are known tumor promoters (Suganuma et al., 1988; Rivas et al., 2000; Vale and Sampayo 2002, García et al., 2003).

In Chile the presence of PSP and DSP is endemic in the country's Austral Regions. In these fjords, Alexandrium catenella is the primary agent responsible for PSP toxin production and Dinophysis acuta (Zhao et al., 1993) and Dinophysis acuminata (Uribe et al., 2001) in the case of DSP. In both cases DTX-1 is the predominant DSP toxin detected (Uribe et al., 2001).

During the summer of 2002, a PSP bloom affected the geographic area of Chiloe Island, X Region, Chile. The PSP bloom produced contaminated shellfish, that produced 49 intoxications and one death after human consumption. Moreover, the total annual production of Mytilus chilensis was lost. The littoral around Chiloe Island was declared a catastrophic zone by the Chilean government due to the economic loss and the social problem produced by the harvest ban. The PSP bloom showed an unusually high toxicity in this area; the mouse bioassay revealed toxicities above 15,000 mg of PSP toxins /100 g of mussel meat (Mytilus chilensis) in February 15, 2002 in this island.

This study is the first to show a quantitative analysis of the simultaneous presence of PSP and DSP toxins in Mytilus chilensis samples collected at Chiloe Island. The HPLC analysis with pre-column derivatization method for DSP toxins and post-column derivatization methods for PSP toxins, both with fluorescent on-line detections showed that the filter bivalve Mytilus chilensis concentrated both types of toxins in amounts above the international safe limits. The phytoplankton analysis showed the presence of both Alexandrium catenella and Dinophysis acuta in water samples.

MATERIAL AND METHODS

Chemicals

1-heptenesulfonic acid sodium salt, periodic acid, potassium phosphate dibasic, tetrabutylammonium phosphate, okadaic acid (OA), dinophysistoxin-1 (DTX-1) standard toxins, deoxycholic acid (DOCA) were purchased from SIGMA (Sigma Chemical Co., St. Louis, MO, USA). 9-antryldiazomethane (ADAM) was purchased from Funakoshi Pharmacy (Tokyo, Japan). HPLC grade solvents (acetonitrile, acetone, methanol, chloroform, HCl, acetic acid) were purchased from Fisher Scientific (New Jersey, USA), phosphoric acid, ammonium hydroxide were purchased from Merck (MERCK, Darmstadt, Germany). The SEP-PAK® cartridges for solid phase extraction of silica and C-18 were purchased from Waters Corporation (Division of MILLIPORE, Milford, MA, USA). High grade pure water was obtained by elution through an ion exchange cartridge followed by boiling for 2 hours with nitrogen bubbling.

PSP toxins sample preparation

The mussel samples were collected in an aquaculture center in Cailin Island, X Region, in the south of Chile on April 24, 2002. The Mytilus chilensis samples were collected from the aquaculture shellfish lantern at different depths. Sample #1 at 1 meter from top of the water column, sample #2 at 3 meters, sample #3 at 4 meters, sample #4 at 6 meters, sample #5 at 7 meters, sample #6 at 8 meters and sample #7 at 10 meters deep. Tissue samples of 100 g each were homogenized in equal volumes by weight of HCl 0.1 N in a variable speed Tissue Tearor (Biospec Products. U.S.A.). The pH was adjusted at 4 and then extracted at 85 ºC during 10 minutes. The extract was centrifuged to 10,000 g for 5 minutes, the supernatant was concentrated in a speed vac plus SC210A (SAVANT) and filtered. Twenty microliters of the filtrate were applied to HPLC.

Chromatographic conditions for HPLC analysis of PSP toxins

Toxins were determinate under the conditions described previously using ion par chromatography with post-column derivatization (Lagos, 1998). Briefly, 20 ml treated samples was injected (Rheodyne model 7725i with a 20-ml loop) to a silica-base reversed phase column (Supelcosil 5 mm, C-8, 4.6 x 150 mm, SUPELCO, Bellefonte, PA, USA). The following mobile phase of 2 mM 1-heptenesulfonic acid in 30 mM ammonium phosphate buffer pH 7.1. At a flow rate of 0.7 ml/min, acetonitrile (100:5) was used for detection and quantitation of STX group of toxins. For the GTXs group of toxins, the 2 mM 1-heptenesulfonic acid in 10 mM ammonium phosphate buffer pH 7.1 elution buffer was used. In both cases, the eluate from the column was mixed continuously with 7 mM periodic acid in 10 mM potassium phosphate buffer pH 9.0, at 0.4 ml/min, heated at 65 ºC by passing through a coil of Teflon tubing (0.5 mm i.d., 10 m. long), and then mixed with 500 mM acetic acid at 0.3 ml/min before entering the monitor. The fluorescent detector was set at an excitation wavelength of 330 nm and an emission wavelength of 390 nm. For HPLC chromatographic equipment, a Shimadzu LC_10AD liquid chromatograph apparatus with an on-line Shimadzu RF_551 spectrofluorometric detector was used. Both the oxidizing reagent and the acid were pumped by a dual-head pump (model SP-D-2502, Nihon Seimitsu Kagaku). Data acquisition and data processing were performed with Shimadzu CLASS-CR 10 software. Toxin concentrations were measured by comparing the peak areas for each toxin with those of the standard. As an external standard, pure toxin solutions calibrated by combustion analysis nitrogen measurements and HPLC-MS were used (Lagos, 1998).

In order to avoid false toxin peaks, the samples were reanalyzed with the same HPLC-FLD procedure but replacing the oxidizing reagent with distilled water. Under these conditions the oxidation does not occur.

Mussel extract for DSP toxins detection

Two grams of digestive glands were removed from Mytilus chilensis, (Blue mussel). They were then homogenized and extracted twice with 3 ml of chilled 80% methanol with mechanical stirring using a tissue tearor (BioHomogenizer M 133/2280, Biospec Products, Inc., Bartlesville, OK, USA). The methanolic phase was then centrifuged at 1,500 x g for 5 minutes. The 2.5 ml of the supernatant was diluted with water to a final 26.66% methanol. From this dilution, 5 ml were then transferred to a 250 mg C-18 SEP PAK® cartridge. The system was washed with 5 ml of 50% methanol to remove lipid components and then 5 ml of pure methanol was added to elute the DSP toxins. This eluted fraction was evaporated to dryness under reduced pressure in a Speed Vac Plus (Savant, SC 210A, Farmingdale, NY, USA). The clean and dry extracts were used for derivatization with ADAM.

Derivatization of DSP toxins with ADAM

The ADAM derivatives of standards and sample toxins were carried out according to a previously described method (García et al., 2003). Briefly, the solid mussel extract residues or standards were treated with a freshly prepared solution of 0.1 % ADAM (in 100 ml of acetone and 400 ml of methanol). After one hour in the dark at 25 °C, the sample was evaporated to dryness and the residue was diluted in 200 ml CH2Cl2 /hexane, 1:1 (v/v) and then transferred into a 500 mg Silica gel SEP PAK® cartridge. The system was washed successively with 5 ml of CH2Cl2 / hexane, 1:1 (v/v) and 5 ml CH2Cl2. Finally, the DSP toxins were eluted with 5 ml of CH2Cl2 / methanol, 1:1 (v/v). The last fraction was evaporated to dryness, dissolved in 1 ml methanol and then 10 ml was injected and analyzed by HPLC-FLD.

HPLC Chromatographic conditions for analysis of DSP toxins

The DSP toxins analyses were performed on a Shimadzu Liquid Chromatograph System equipped with a pump Shimadzu LC-6A, a rheodyne injector (7725i Rheodyne. Cotati, CA, USA) and a fluorescence detector Shimadzu RF-535. Ten microliters of toxin-ADAM derivatives were injected on a reversed phase column Supelcosil LC-18 (5 mm; 25 cm x 4.6 mm, Supelco, Bellefonte, PA, USA). An isocratically mobile phase of CH3CN / CH3OH / H2O 8:1:1 (v/v) with a flow rate of 1 ml/min were run at room temperature. The excitation and emission wavelengths were set at 365 and 415 nm respectively. Peaks in the resulting chromatograms were identified by comparison with the retention times of DSP phycotoxin analytical standards derivatized with ADAM. This method corresponds to a High Performance Liquid Chromatography with fluorescent on-line detection and with a pre-column derivatization procedure.

Collection and Analysis of phytoplankton

Phytoplankton samples were collected using a Rutner net with a 10.4 cm mouth opening and a mesh size of 20 mm. Vertical hauls were carried out from a depth of 15 meters. The phytoplankton sample (water sample) was examined directly in the living state and in samples preserved with Lugol's iodine solution (Tangen, 1978; Franks, 1995).

Toxic species were first identified using a Zeiss inverted microscope and then counts according to the standard Utermöhl technique (Utermöhl 1958). The result of the count was expressed in liters, thus establishing the numerical density of toxic species and total phytoplankton. The taxonomic analyses were also performed by observing directly in a contrast phase microscope Unilux-12.

RESULTS

Figure 1 shows a map of Chiloe Island in which the PSP contaminated areas are identified by red dots. These contaminated places were found during the monitoring program of Chiloe Island done by the marine toxins laboratory of Castro City Hospital. This monitoring program was developed due to the massive intoxication occurred in this area in February 10, 2002. The monitoring program is detects the contaminated shellfish using the mouse bioassay (Japanese Ministry, 1981 for DSP toxins and for PSP toxins; Williams, 1984). According to this monitoring program, the bloom started in the south and moved north; Cailín Island- 43º09'55''S; 73º31'30''W (red circle) was one of the first locations affected. This place is one of the major areas of Mytilus chilensis aquaculture in the Chiloe Island. The red dots in the north correspond to places were the PSP blooms were detected during March and April. The localities were: Dalcahue 42º 15´/ 42º 30´S; 73º 30´/ 73º 45´W, Lemuy Island 42º 30´/ 42º 45´S; 73º 30´/ 73º 45´W, Quenchi 42º 00´/ 42º 15´S; 73º 15´/ 73º 30´W, Queilen 42º 45´/ 43º 00´S; 73º 15´/ 73º 30´W, Achao 42º 15´/ 42º 30´S; 73º 15´/ 73º 30´W, Ancud 41º 45´/ 42º 00´S; 73º 45´/ 74º 00´W, all within an area of 100 kilometers. The monitoring program done by the Castro City marine toxins laboratory only focused on PSP toxin detection, primarily due to the human intoxications. Beginning in May 2002, the area surrounding Cailin Island showed shellfish with PSP toxins under the regulatory level (80 mg / 100 gr. of mussel meat). Nevertheless, the phytoplankton analysis showed the presence of D. acuta and A. catenella in the water sample in April 24, 2002.

 

Figure 1. Map of Chiloe Island, X Region, Chile. Cailin Island is located inside the red circle. The red dots on the map show the other places along the littoral where PSP-contaminated shellfish were detected by the monitoring program of Castro City Hospital marine toxins laboratory.

Figure 2 shows a plankton sample collected by horizontal dragging in the surrounding areas of the Cailín Island on April 24, 2002. Figure 2A clearly shows the presence of both Dinophysis acuta and Alexandrium catenella, along with the presence of Thalassiosira sp. and Pseudonizschia sp. Figures 2B and 2C show isolated Dinophysis acuta and Alexandrium catenella respectively; both correspond to the dinoflagellates associated with the presence of DSP and PSP respectively in Chile (Uribe et al 2001; Lagos 1998; García et al., 2003; Lagos 2003). The cell densities for D. acuta reached 120 ± 11 cel/Lt and 235 ± 35 cel/Lt for A. catenella (April 24, 2002).

Figure 3 shows the HPLC-FLD chromatograms of the Mytilus chilensis mussel sample extract, along with those of PSP toxin standards. The PSP toxin standard mixture (Fig. 3A) shows three peaks: gonyautoxins (GTXs), neosaxitoxin (neoSTX) and saxitoxin (STX) with retention times of 7:11, 11:22, and 19:04 minutes respectively. The mussel extract also shows the same three peaks with identical retention times (Fig. 3B). In these chromatographic runs, the mobile phase used was 2 mM 1-heptanesulfonic acid in 30 mM ammonium phosphate buffer, pH 7.1, containing 3% of acetonitrile v/v. This elution buffer resolves the PSP toxins that belong to the group of saxitoxins (STX, neoSTX and dcSTX). With this mobil phase, gonyautoxins (GTXs) elute as a single fraction that run close to the front run with an Rt = 7:07 minutes. From the saxitoxins group, the presence of neoSTX (Rt = 11:20 minutes) and STX (Rt = 19:03 minutes) are clearly seen. Figure 3C and 3D show the chromatograms of the HPLC-FLD run associated with the gonyautoxins detection. The mussel sample (Fig. 3D) shows the same gonyautoxins that appear in the toxin standards run (Fig. 3C). Now the elution mobile phase used was 2 mM 1-heptanesulfonic acid in 10 mM ammonium phosphate buffer, pH 7.1. Under this condition it is possible to resolve only the PSP toxins that belong to the group of gonyautoxins (GTX4/GTX1 epimers, GTX5 and GTX3/GTX2 epimers). The five gonyautoxins are resolved in a 20-minute run as is shown in Fig. 3C. Here the chromatogram shows the peaks corresponding to GTX4, GTX1, GTX5, GTX3 and GTX2 with retention times of 9:55, 11:18, 14:36, 16:62 and 19:35 minutes respectively (Fig. 3C). The mussel sample chromatogram (Fig. 3D) shows the five peaks corresponding to GTX4/GTX1 (Rt=9:50 and Rt=11:25 minutes) GTX5 (Rt= 14:15 minutes) and the GTX3/GTX2 epimers (Rt=16:59 and Rt=19:45 minutes). In this chromatographic run the gonyautoxins eluted far from the front run, where extra peaks can be seen, these correspond to pigments present in the samples and have no relation to PSP toxins.

Seven mussel samples collected in Cailin Island were analyzed for PSP toxins according to the procedure described above. Table I shows the amounts of STX, neoSTX and the total of Gonyautoxins expressed as PSP toxins mg /100 g of mussel meat. Six of them showed amounts of PSP toxins above the international safe limited of 80 mg of PSP toxins/ 100 grams of mussel meat.

Figure 2. A. Typical phytoplankton sample (water sample) collected at Cailin Island in April 24, 2002. B. Isolated Dinophysis acuta and C. Isolated Alexandrium catenella, both were found in the water samples collected on April 24, 2002.

Figure 3. Chromatograms of PSP toxin standards and Mytilus chilensis samples.
A. Analytical standard mixture for Saxitoxins group: GTXs (Rt = 7:11 minutes); neoSTX (Rt= 11:20 minutes); STX (Rt = 19:04 minutes). B. Mytilus chilensis extract, showing GTXs (the major peak at Rt = 7:07 minutes, saturated signal), then neoSTX (Rt = 11:20 minutes) and STX (Rt = 19:03 minutes). C. Analytical standard mixture for Gonyautoxins group: GTX4 (Rt = 9:05 minutes), GTX1 (Rt = 11:18 minutes), GTX5 (Rt = 14:36 minutes), GTX3 (Rt = 16:62 minutes) and GTX2 (Rt = 19:35 minutes). D. Mytilus chilensis extract, showing GTXs group of toxins with identical elution Rt times of the five GTX standards.  

 

TABLE I

PSP toxin content in the Mytilus chilensis samples


Samples Nº

GTXs (mg/100 g)
neoSTX (mg/100 g)
STX (mg/100 g)

1 (1 m)

78.3
n.d
n.d
2 (3 m)
98.7
n.d
n.d

3 (4 m)

110.3
10.7
3.6

4 (6 m)

679.6
25.5
4.7

5 (7 m)

567.8
34.2
4.1

6 (8 m)

116.7
13.5
2.9
7 (10 m)
87.9
n.d
n.d

Nº = Number
GTXs = Gonyautoxins
neoSTX= neoSaxitoxin
STX= Saxitoxin
µg/ 100 g = micrograms/ 100 grams
n.d.= none detected
m = meters

The same seven mussel samples were extracted now for DSP toxin analysis. These analyses were performed according to the derivatization conditions described by García et al., 2003. Figure 4A shows the chromatographic run of mussel extract estherifies with ADAM, a fluorescent chromophorus. The estherification reaction with ADAM and the HPLC chromatographic run used in this article were the same described by García et al., (2003). A single signal of DSP toxin was observed in the chromatogram, showing a retention time of 11:47 minutes that appears when eluting in the same retention time of DTX-1 standard (Fig. 4B). Both chromatograms correspond to the fluorescent on-line detection of a pre-column derivatization procedure used as routine analysis for detection of DSP phycotoxins.

All seven mussel samples were analyzed and quantified using the same pre-column derivatization procedure. Each of the samples produced only one DTX-1 as DSP toxin. The amount of DTX-1 detected in the samples analyzed ranged from 33.5 to 190 nanograms of DTX-1 / grams of digestives gland (Table II). These amounts are below the international safe limited of 200 nanograms of DSP toxins/gram of digestives gland.

TABLE II

DSP toxin content in the Mytilus chilensis samples


Samples Nº

OA (ng/g)
DTX-1 (ng/g)

1 (1 m)

n.d.
33.5

2 (3 m)

n.d.
36.7

3 (4 m)

n.d.
92.5
4 (6 m)

n.d.

190

5 (7 m)

n.d.
90
6 (8 m)

n.d.

78.9

7 (10 m)
n.d.
67.2

Nº = Number
OA = Okadaic Acid
DTX-1= Dinophysistoxin-1
ng/g = nanogram/gram
n.d.= none detected
m = meters

 

Figure 4. Chromatograms of DSP toxin standards and Mytilus chilensis samples.

A. Mytilus chilensis extract, showing DTX-1 as the only DSP toxin present in the sample. The peaks in the front run correspond to the excess of ADAM chromophorus. B. DSP toxins analytical standard mixture, including DTX-1 (Rt = 11:47 minutes) and DOCA (internal standard with Rt = 18:02 minutes).

DISCUSSION

The presence of PSP and DSP toxins has been endemic in the southern Chilean fjords during the spring-summer period since 1991 (Lagos, 1998). Nevertheless, until now it has been not possible to show quantitative evidence that demonstrated the presence of both toxic dinoflagellates in water samples and at the same time the PSP and DSP toxins accumulated by shellfish harvested in the same place that the toxic phytoplankton was found.

The results shown in this article provide the first indisputable evidence that shellfish can be contaminated with both poison toxins during the summer season in this southern area. This data also shows the importance of quantitative analyses of both types of toxins in the shellfish samples, as the mouse bioassay for PSP toxins can cover the presence of DSP toxins as well. In places such as Chile's southern fjords, where the presence of the PSP and DSP poisons are endemic, the monitoring programs and the regulatory reference laboratories should be aware of the need to perform toxin quantitative analyses rather than mouse bioassays alone. When only the latter is used, the most toxic poison will mask all others. In this case, PSP toxins mask any other poison present, producing a potential risk for the population.

The mouse bioassay for PSP toxins is a very impressive assay for its speed _ its effects are fulminate and take place in minutes - and for its severity _ the symptoms of intoxication are very characteristic. This bioassay is therefore easy to perform and useful for testing PSP toxins.

The performance of the mouse bioassay for DSP toxins is completely different. This long assay can take 24 hours, and the symptoms are not easy to detect. In most cases, after the little mice are injected with the poison, they just move to a corner of the cage and remain there for hours. This assay is also a non-quantitative assay. Moreover, this bioassay has major problems associated with poor reproducibility and false positives; the latter appears to be due primarily to the presence of high levels of fatty acids in shellfish in certain periods of the year and in samples of oil-packed canned shellfish (Takagi et al., 1984; Kogawa et al., 1988; Lawrence et al., 1994).

The Mytilus chilensis samples collected in a Cailin Island aquaculture center proved to be contaminated with both type of toxins. The differing amounts of each type of toxins detected in the seven samples are associated with the location of the sample in the water column, since each sample was collected at different depths of the shellfish aquaculture lantern, meaning that each sample was located at a different depth in the water column and therefore exposed to different densities of toxic dinoflagellates. Mollusks that were at lowest depth (10 meters) and in the most superficial positions (1-3 meters from the surface) showed the lowest amount of DSP toxins, 67.2 and 33.5 _ 36.7 nanograms of DTX-1 / gram of digestive gland respectively (Table II). Similar results were obtained with the PSP toxin analysis. At a depth of 10 meters the shellfish PSP toxin content was 87.9 mg / 100 g of mussel meat and at 1-3 meters it was 78.3 and 98.7 mg of PSP toxins/100 g of mussel meat respectively (Table I).

These data show a clear stratification of the two toxins (PSP and DSP toxins) in the shellfish samples in the water column. This stratification measured in the mussel extracts obtained from shellfish collected at different water column depths can only be explained by the stratification of the toxic dinoflagellates in the water column, since the Mytilus chilensis mussels were attatched to the rope of aquaculture shellfish lantern at different depths. Moreover, the lowest amount of both DSP and PSP toxins are in the upper (surface) and lower levels of the water column. The highest toxicities of both toxin types are shown by the shellfish samples collected at a depth of 6 meters (sample # 4) with 190 nanograms of DTX-1/gram of digestive gland and 709.8 mg of PSP toxins/100 g of mussel meat.

The overlapping of DSP and PSP toxins in the same shellfish sample and the simultaneous presence of the dinoflagellates A. catenella and D. acuta in the water column as shown in this article reinforce the need to perform other tests in addition to the mouse bioassay in order to detect and quantify other types of toxins that may be masked by the effect of the more toxic one. This recommendation should be required in all places where the presence of two or more toxic phytoplankton species are endemic or break out in close proximity, as is the case of the fjords in southern Chile, where PSP and DSP blooms have occurred every spring-summer period for the last 30 years.

ACKNOWLEDGEMENTS

This study was supported by FONDECYT 1020090 and DID, Universidad de Chile, CSMAR 02/5-2, and Programa de Cooperación Científica Internacional GRICES / CONICYT.

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Corresponding Author: Néstor Lagos, Ph.D., Lab. Bioquímica de Membrana, Dept. de Fisiología y Biofísica, Facultad de Medicina, Universidad de Chile, Casilla 70005, Correo 7, Santiago, Chile, Phone: (56-2) 678-6309, Fax: (56-2) 777-6916, E-mail: nlagos@med.uchile.cl

Received: March 29, 2004. In revised form: August 11, 2004. Accepted: September 9, 2004

 

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