Comparative study on differential accumulation of PSP toxins between cockle (Acanthocardia tuberculatum) and sweet clam (Callista chione)

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Comparative study on differential accumulation of PSP toxins between cockle (Acanthocardia tuberculatum) and sweet clam

(Callista chione)

Reqia Sagou

^a,

*, Rachid Amanhir

^a

, Hamid Taleb

^a

, Paulo Vale

^b

, Mohamed Blaghen

^c

, Mohamed Loutfi

^c

aInstitut National de Recherche Halieutique, 2 rue Tiznit, Casablanca, Morocco

bInstituto de Investigac¸a˜o das Pescas e do Mar (IPIMAR), Av. Brasilia, 1449-006 Lisboa, Portugal

cUniversite´ Hassan II, Faculte´ des sciences Aı¨n Chock, Casablanca, Morocco

Received 13 January 2005; accepted 17 June 2005 Available online 15 September 2005

Abstract

At the western Mediterranean coast of Morocco, the cockle (Acanthocardia tuberculatum) contained persistent high levels of paralytic shellfish toxins for several years, while other bivalve molluscs such as sweet clam (Callista chione) from the same vicinity were contaminated seasonally to a much lesser extent. In order to understand the causes of this prolonged contamination, a comparative study on PSP decontamination between sweet clam and cockle was conducted from November 2001 untill June 2002. PSP toxicity was analysed by automated pre-column oxidation (Prechromatographic oxidation and LC-FD) in several organs of both species, namely digestive gland, foot, gill, mantle, muscle and siphon for sweet clams. The results showed that cockle sequester PSP toxins preferably in non-visceral organs (Foot, gill and mantle) contrary to sweet clam that sequester them in visceral tissues (digestive gland). The toxin profile of cockle organs indicated dominance of dcSTX, whereas sweet clam tissues contained especially C-toxins. Substantial differences in toxin profile between cockle and sweet clam, from the same area as well as from the composition of PSP toxin producer,Gymnodinium catenatum, confirm the bioconversion of PSP toxins in cockle.

Keywords: Acanthocardia tuberculatum;Callista chione; PSP toxins; Organs; Accumulation

1. Introduction

The cockle (Acanthocardia tuberculatum) is known to sequester PSP toxins for a long time in its tissues even when the potentially toxin producing microalgae are not present (Marquez, 1993; Tagmouti et al., 1996; Taleb et al., 1998, 2001; Vale and Sampayo, 2002). Due to permanent contamination of the later species with PSP toxins for

several years, the western Mediterranean shore of Morocco was closed for shellfish harvesting since 1992. This situation caused the economic loss for fishermen and a significant effect on the local economy due to decreased of fishery revenue.

Acanthocardia tuberculatumis mainly exploited in the canning industry in Morocco and Spain. A Spanish team has demonstrated that after a thermal treatment of cockles at 1168C for at least 51 min, toxicity drops to undetectable levels by mouse bioassay (Berenguer et al., 1993; Burdaspal et al., 1998). On the basis of the later processing, an exceptional European legislation allows harvesting in Spain www.elsevier.com/locate/toxicon

doi:10.1016/j.toxicon.2005.06.020

* Corresponding author. Fax:C212 22 26 69 67.

E-mail address:resagou@yahoo.fr (R. Sagou).

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of cockles with PSP toxins levels less than 300mg STXeq./

100 g Commission Decision 96/97/EC (OJEC, 1996).

Following the ulterior studies that attributed the high toxicity of cockles to the biotransformation of C-toxins (with low specific toxicity) into decarbamoylsaxitoxin (dcSTX) with relatively higher specific toxicity (Taleb et al., 2001), the present work focused on determining the causes of PSP toxins persistence in this species. In bivalve molluscs, the viscera are invariably found to contain the highest toxicity levels immediately following the exposure to toxic algal blooms. However, the uptake, elimination and the transfer of toxin in other organs follows a characteristic pattern in each bivalve species (Lassus et al, 1989; Bricelj and Shumway, 1998). Thus, a comparative study of PSP toxins distribution was conducted in different organs of cockle and a sweet clam (Callista chione) from the same area. The later species is contaminated occasionally during the occurrence of harmful algae bloom episodes.

2. Materials and methods

The cockle (A. tuberculatum) and the sweet clam (C. chione) were collected monthly at Martil station which is localized at the western Mediterranean coast of Morocco, between November 2001 and May 2002. Both shellfish species were washed and separated into digestive gland (hepatopancreas), foot, mantle, muscle and gills for cockle plus siphon for sweet clams.

Toxin profiles were performed by automated pre-column oxidation method with periodic acid, based on the method of Lawrence et al. (1995)with minor modifications described inVale and Sampayo (2001).

2.1. Sample preparation for HPLC

Shellfish samples were extracted according to AOAC method (1990): 100 g homogenized tissues were mixed with 100 ml 0.1 N chlorydric acid and boiled for 5 min, centrifuged for 5 min at 3000 rpm and pH adjusted to 2–3.

1000mL of supernatant was transferred to eppenddorf tubes (1 mL), those tubes were centrifuged at 12,000!g for 10 min on an eppendorff centrifuge and filtered into screw-cap autosampler vials (1.5 mL) using a 0.45mm nylon (:11 mm) disposable syringe filter. The equivalent of 1.25 mg extract (2.5mL) was oxidized and injected on the column without any further clean up.

2.2. Pre-column oxidation

The oxidizing mixture was prepared daily by mixing 500ml each of 0.03 M periodic acid, 0.3 M Na2HPO4 and 0.3 M ammonium formate, plus 60ml of 1 M NaOH in an autosampler vial. The neutralizing solution consisted of a 1:1 mixture of water and glacial acetic acid; 500ml were dispensed daily into another autosampler vial. Reactions

were controlled through the ‘Injector Program’ (part of the HPLC ‘Chemstation 6.0’ software). For peroxide oxidation vial 1 was replaced by 1000ml of 1 M NaOHC100ml of 10% H2O2. Reaction’s sequence was similar to that described for periodate inVale and Sampayo, 2001 2.3. LC separation and detection

Liquid chromatography was performed on a Hewlett- Packard (HP) machine equipped with Model 1050 quaternary pump and autosampler, Model 1046-A fluorescence detector and Model 1100 in-line degasser. The HP Chemstation software performed data acquisition and peak integration. Toxins were separated on a MerckLichrospher 100 RP-18 (5mm, 125!3 mm) column, protected by a guard-column (4!4 mm) also packed withLichrospher100 RP-18 (5mm). Column temperature was kept at 308C.

Detection wavelengths were set at 330 nm for excitation and 390 nm for emission, and gain set at 2¹⁴.

The mobile phases were as follows: A: acetonitrile;BZ 20 mM ammonium formate. Phase B was prepared as following: to 990 mL of Milli-Q water was added 10 mL of 2 M ammonium formate, and pH lowered with 1 mL 1 M acetic acid. The column was equilibrated with 100% phase B. During each analysis a gradient from 0 to 8% phase A was run over 10 min, and then re-equilibration at the starting conditions for 5 min prior to the next oxidation cycle. The time for derivatizing and the 5 min re-equilibration time was added to a total of 12 min. for re-equilibration between consecutive injections. Flow rate was set at 0.5 mL/min.

2.4. Toxin determination

The set of four ampoules, PSP-1B, and recently the CRM-dcSTX, were purchased from National Research Council of Canada (NRC), 2002. All standard solutions were diluted with distilled water as required. Calculations were based on peak height due to the incomplete separation of all toxins by the short gradient used. For routine determination a single-point calibration was run at the beginning and after each six consecutive samples with a working solution prepared by mixing equal volumes of dcSTX, GTX2C3, STX and toxin-free oyster extract, to reach a final concentration of: 0.46, 0.67 and 0.63mg/mL, respectively.

3. Results

3.1. Toxin identification

HPLC analysis conducted either in cockles or sweet clams showed a complex profile with numerous PSP analogues (Fig. 1a–d). In cockles we found after periodate oxidation dcGTX2C3, C1C2, dcSTX, GTX1K4, B1, STX (Fig. 1a).

Peroxide oxidation confirmed the presence of dcGTX2C3

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and dcSTX by inversion of peak height, respectively as follows: 2 were higher than 3 and 6 was higher than 7 (Fig. 1b) (Lawrence et al., 1996). Detection of peak 8 after peroxide oxidation indicated that at least GTX2 or GTX3 were present (Fig. 1b). Increase in height of peaks 5 and 9 after peroxide oxidation pointed to their identification as C1/2 and B1, respectively (Lawrence et al., 1995).

Sweet clams presented after periodate oxidation dcGTX2C3, C1C2, GTX1K4, B1, STX (Fig. 1c). Per- oxide oxidation indicated the presence of dcGTX2C3 and dcSTX by inversion of peak height respectively: Peak 2 was higher than 3 and 6 was higher than 7 (Fig. 1d). Sweet clam extracts presented an interference (peak X) that did not allow determination of dcSTX by periodate oxidation.

Time (min)

0 2 4 6 8 10

Time (min)

0 2 4 6 8 10

Time (min)

0 2 4 6 8 10

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2 4 6 8 10 12 14 16 18 20

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X

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Fig. 1. Chromatograms of PSP toxins from whole edible parts of: (a) cockles harvested November 2001, after periodate oxidation; (b) same sample after peroxide oxidation; (c) clams harvested November 2001 after periodate oxidation; (d) same sample after peroxide oxidation. After oxidation compounds2C3correspond mainly to decarbamoyl-gonyautoxins 2C3 (dcGTX2CdcGTX3); compound5to N-sulfocarbamoyl 1C2 (C1CC2);6and7to decarbamoyl-saxitoxin (dcSTX);8to gonyautoxins 1–4 (GTX1/4);9toN-sulfocarbamoyl B1, and10to saxitoxin (STX) and/or neosaxitoxin (NeoSTX).

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All dcSTX levels reported were obtained by peroxide oxidation. Similar concentrations of STX were found after periodate and peroxide oxidation. This showed that NeoSTX was not present in a concentration that allowed an unambiguous confirmation. In this case too, the presence of peak 8 after peroxide oxidation indicates at least to the presence of GTX2 or GTX3 (Fig. 1d) and equally the increase in height of peaks 5 and 9 confirmed the presence of C1/2 and B1, respectively.

3.2. Toxin tissue distribution and depuration

Fig. 2a shows total toxin concentrations determined by HPLC in selected cockle tissues harvested from November 2001 to May 2002. The most toxic organ was often the foot followed by the mantle and digestive gland. The gill and muscle presented continuously similar toxin concentration and always lower than any of the other tissues. Toxin concentration in the foot attained as high as three times the toxin content in the least toxic organ, the muscle. All tissues seem to eliminate PSP toxins slowly being difficult to find a non-detectable level in any of them throughout all the months sampled.

In sweet clams, PSP toxins were detectable in all organs only from November to January (Fig. 2b). The PSP toxin content in the digestive gland is higher than other organs

until May. Taking into account that many toxins were detected near the quantification limit not much conclusions could be drawn from the following samples. PSP toxins concentrations drop to levels undetectable by HPLC much faster than cockles.

Toxin profiles for cockle organs showed that dcSTX was the predominant toxin, constituting around 50–60% of total toxin burden in the organs of the most contaminated sample from November (Fig. 3a). Remaining toxin groups accounted no more than 5–15% each in any of the organs.

Two months later—in January—all organs had lost PSP toxins and toxin profile had changed slightly (Fig. 3b).

DcSTX now accounted for 60–70% of the toxic burden.

STX and its N-sulfocarbamoyl precursor B1 amounted to around 10% each. Gonyautoxins and their N-sulfocarbamoyl precursors C1/2, plus dcGTX2/3, accounted for less than 5% each. All organs presented a toxin profile more or less similar to that in the November sample.

Toxin profile in the most contaminated sweet clam sample studied (harvested November 2001) showed predominance of C1/2 and dcSTX (20–30% each) in most tissues (Fig. 4). Remaining toxin groups were between 5–20% each. No such predominance of a single toxin as encountered in cockle was found in sweet clam. All organs seemed to behave more or less similar amounts. In December, the hepatopancreas was the most toxic organ.

0 50 100 150 200 250 300 350 400 (a)

(b)

Nov 01 Dec 01 Jan 02 Feb 02 May 02

µg STXeq/100g

Digestive gland Foot Gills Mantle Muscle

0 10 20 30 40 50 60 70 80

µg STXeq/100g

Digestive gland Foot Gills Mantle Muscle Siphon

Fig. 2. Seasonal variation of PSP toxins in dissected organs in (a) cockle and (b) clams.

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In the May samples, it was the only tissue where any toxins were detectable. The last toxin to be detected in edible parts was dcSTX, and additionally gonyautoxins 1–4 in the hepatopancreas.

Temporal evolution of toxin profile in cockle showed an increase in dcSTX and STX proportions with a slow

decrease in remaining PSP group constituents over the time of toxin depuration (Fig. 5a). DcSTX and STX are the toxins that account for most of the toxicity in cockle. In sweet clams, taking into account that many toxins were detected near the quantification limit not much conclusions can be drawn for as to particular tendencies, except for the increase (a)

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Muscle Gills Mantle Digestive gland Foot

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dcGT X2/3 dcSAX C1/2 GTX1-4 GTX5 SAX/NEO

Fig. 3. Organ distribution of PSP toxins in (a) November and (b) January cockle samples.

Muscle Gills Mantle Digestive gland Foot Siphon 0%

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Fig. 4. Organ distribution of PSP toxins in the November clam sample.

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in proportion of GTX1K4, which as mentioned above, was the last toxin to be detectable in the following months (Fig. 5b).

4. Discussion

At the term of the present study, the obtained results show a large variation of accumulated PSP toxins in different organs of both studied species from the same area.

Thus, the maximal PSP toxins contents are recorded in cockle and sweet clam tissues at November. This period coincides with the beginning of Gymnodinium catenatum bloom in this region as reported by Tahri (pers. Com.).

Since December, PSP toxin levels are evacuated gradually from all sweet clam tissues. However, the high amounts are conserved in the digestive gland, where they are eliminated gradually until their complete disappearance at May. In the clams PSP toxins are accumulated mainly in the digestive gland.

Therefore, all cockle organs are contaminated at November. Then, from December the PSP toxin level starts decreasing in digestive gland. This lost is accompanied by an increase in PSP toxins content in other organs mainly the foot. Thus, the pattern of PSP toxins distribution showed the following trend: FootOOmantleOdigestive glandOgillZ muscle. Similar study carried out in Spain (Berenguer et al., 1993) and Portugal (Vale and Sampayo, 2002) on the same species, revealed the conservation of PSP toxins mainly in the foot.

Thus, it was reported that in all bivalves, the digestive gland contains the majority of total toxin during the toxic

bloom period (Bricelj and Shumway, 1998). Only, in slow detoxifying bivalves, the relative toxin proportion increases gradually in non- visceral tissue and can even surpass that of the digestive gland, this particularity concords with those of cockle (A. tuberculatum) which accumulate PSP toxins in non-visceral tissues particularly in foot. In contrast, the rapidly detoxifying bivalves retain PSP toxins preferentially in digestive gland that, as demonstrated by several study, detoxify faster than other tissues. This is in agreement with results for sweet clams (C. chione) which conserve PSP toxins in digestive gland.

Due the lack of G. catenatum toxin profile from Moroccan coast, the profile in bivalve tissue here was compared to that of the strain from Spain and Portugal.

G. catenatumhas been identified on the Atlantic coasts of Morocco, and also in the Alboran Sea (Western end of the Mediterranean Sea), the body of water shared by Morocco and Southeast Spain (Tahri-Joutei, 1998). Toxic outbreaks associated withG. catenatum, with a northern Atlantic limit in Cape Finisterre (Galicia, Spain), have been found as far south as Essaouira, in Morocco. Strains of this dinoflagellate from Iberian Peninsula present a complex profile, with C1–4, GTX5/6, neoSTX, and specially the presence of dcSTX and absence of STX (Donker et al., 1997; Anderson et al., 1989; Franca et al., 1996; Rodriguez-Vazquez et al., 1989; Oshima et al., 1990, 1993; Vale and Sampayo, 2001;

Taleb et al., 2001, 2003).

The PSP toxin composition of sweet clam tissues reflect well the toxin profile of G. catenatum,predominance of N-Sulfocarbamoyl and the presence of other PSP toxins compounds in low proportions. Thus, the dominance of C-toxins in sweet clam tissues in November indicates

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dcGTX2/3 dcSAX C1/2 GTX1-4 B1 SAX/NEO TOTAL

Fig. 5. Evolution of toxin profile and total toxicity in whole flesh of (a) cockle and (b) clam samples. Legend is the same for both figures.

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an eventual exposure to a toxic bloom. This date coincides as well with the proliferation ofG. catenatumin this region as confirmed by Tahri (pers. Com.)

Nevertheless, the high level of dcSTX in cockle tissues, the decrease in amount of C-toxin and the appearance of toxin in cockle that were not detected in toxigenic algae nor in sweet clam tissues may possibly be explained by both chemical and enzymatic conversion as suggested byTaleb et al. (2001).

In summary, the permanent contamination of cockle can be explained not only by the specific retention of dcSTX (Taleb et al., 2001) but also by differential accumulation of PSP toxin in non visceral organs principally in the foot.

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