Spatial and temporal evolution of PSP toxins along the Atlantic shore of Morocco

Spatial and temporal evolution of PSP toxins along the Atlantic shore of Morocco

Hamid Taleb

*, Paulo Vale

, Mohammed Blaghen

aInstitut National de Recherche Halieutique-2, Rue de Tiznit, Casablanca, Morocco

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

cUniversite´ Hassan II, Faculte´ des sciences Aı¨n chok, Casablanca, Morocco Received 17 July 2002; accepted 5 September 2002

Abstract

A monitoring program for bivalve molluscs contaminated by algal toxins was established in 1992 at different stations along the Atlantic Moroccan shore. The presence of toxicity in bivalve molluscs commercially exploited was tested fortnightly using the mouse bioassay method. Results obtained from this surveillance indicate paralytic shellfish poisoning is responsible for bivalve molluscs contamination along the Atlantic coastline of Morocco. Toxin profile was established by automated pre- column HPLC/FLD in selected contaminated tissues. The study of individual toxins in mussel during a bloom in the northern Atlantic coastline in 1994 showed a very complex profile, typical to that obtained with cultures of the toxigenic dinoflagellate Gymnodinium catenatum isolated from the Iberian region. However, toxin composition of mussels and marine beans from later blooms in the southern Atlantic coastline in 1999 showed a strong resemblance with that of Alexandrium minutum, due to dominance of gonyautoxins 1/4. A minor contamination by G. catenatum due to the presence of decarbamoyl-saxitoxin is hypothesized.

q

2002 Elsevier Science Ltd. All rights reserved.

Keywords:Paralytic shellfish poisoning; Morocco; Mouse bioassay; HPLC

1. Introduction

The last two decades have been marked by an expansion of harmful algal blooms throughout the world. The occurrences of toxic microalgae represent a significant threat to human health and fisheries resources throughout the world and intoxication after the ingestion of contami- nated bivalve molluscs is a worldwide problem (Hallegraeff, 1993).

With almost 3000 km of seacoast, Morocco is not spared from this phenomenon. The first case of intoxication following the ingestion of mussels was recorded in 1961.

The years 1971, 1975, 1981 and 1994 were marked by acute

episodes of human poisoning by phycotoxins (Bourhili, 1982; Tber, 1983).

Bivalve harvesting and mariculture have considerable importance for the economy of the coastal Moroccan population. However, the frequent occurrence of toxic algal blooms causes serious problems for the health of consumers and economic losses due to closure of shellfish harvesting grounds. We reported previously that blooms of harmful dinoflagellates affect the Mediterranean shore of Morocco quite frequently, mainly Gymnodinium catenatum, which causes seasonal outbreaks of paralytic shellfish poisoning (PSP) from January through April (Taleb, 1997;

Taleb et al., 1998). Long-lasting contamination of the cockle Acanthocardia tuberculata was also attributed to this microalge (Taleb et al., 2001).

To overcome this situation, an intense monitoring network was set up in major shellfish producing areas.

The main objective is to be able to protect the consumer and to defend fishermen against economic losses.

0041-0101/03/$ - see front matterq2002 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 4 1 - 0 1 0 1 ( 0 2 ) 0 0 2 7 7 - 5

www.elsevier.com/locate/toxicon

* Corresponding author. Tel.:þ212-62-029817; fax:þ212-22- 26-69-67.

E-mail address:[email protected] (H. Taleb), talebh@inrh.

org.ma (H. Taleb).

according to AOAC (1990). This test consists in mixing 100 g of homogenized tissues with 100 ml 0.1 M HCl. The mixture is boiled for 5 min and centrifuged for 15 min at

gradient used. For routine determination a single-point calibration was performed with a working solution of 2.6

GTX2

3/ml at the beginning of the analysis and after each

Fig. 1. Location of sampling sites.

six consecutive samples. For estimation of toxin compo- sition of dcGTX2

3, both peaks were added and taken as 100% of GTX2

3, for dcSTX both peaks were added and taken as 70% GTX2

3, for C1/2 and GTX5 as 200% of GTX2

3, and GTX1-4 as 135% of GTX2

3.

3. Results and discussion

3.1. Monitoring data

The results obtained from the monitoring along the Atlantic coastline showed the occurrence of a major PSP outbreak in November 1994. The PSP contamination affected bivalve molluscs in the northern Atlantic area, stretching from Larache to Essaouira (Fig. 1). Maximum

concentration of PSP toxins—6000

tissue—was detected in mussel samples near Casablanca (Fig. 2). Mussels from other sites were affected by levels of PSP toxins ranging between 1000 and 3000

100 g tissue (Fig. 2). The PSP levels recorded in other specimens of bivalve molluscs from the same area—oysters (C. gigas ) and clams (R. decussatus ) were lower and did not surpass 2608 and 738

respectively.

In 1995, the phenomenon reappeared near Casablanca at the same period as the previous year but with much lower intensity, reaching only 200

mussels. The oyster (C. gigas ) and clam (R. decussatus ) populations from the same littoral area were partially contaminated at amounts just over the regulatory inter- national threshold of 80

Fig. 2. Maximum PSP toxin levels recorded in mussel from Casablanca region during the November 1994 algal bloom.

Fig. 3. Evolution of PSP toxins between mussels and marine beans from Plage Blanche in the Agadir region during the July – August 1998 bloom.

The southern Atlantic side of the coast stretching from Agadir until Tan Tan, which was not affected during the 1994 toxic outbreaks, was hit by contamination in the 1998 summer. Maximum PSP toxin levels recorded in marine beans from Plage Blanche were 720

meat, while mussels reached 216

(Fig. 3). Mussels in Mir Left attained 290

100 g meat (Fig. 4). September 1998 marked the complete detoxification of all bivalve molluscs.

However, in March 1999 the toxicity phenomenon reappeared again in the southern region causing toxification of shellfish. PSP levels ranged between 162 and 1142

STX equiv./100 g meat with the maximum recorded in

mussels from Taghazout (Fig. 5). Toxicity persisted until end of June, which marked the complete decontamination of shellfish resources.

3.2. Toxin profile

HPLC analysis conducted on a mussel sample from the 1994 outbreak at Casablanca, in the northern Atlantic coast, showed a very complex profile with numerous PSP analogues (Fig. 6(a)). All the 10 known oxidation products of PSP toxins (Lawrence et al., 1995, 1996) were found.

We described earlier the order of elution we determined for our HPLC conditions (Vale and Sampayo, 2001).

Fig. 4. Maximum PSP toxin levels recorded in mussel from Agadir region during the July – August 1998 algal bloom.

Fig. 5. Maximum PSP toxin levels recorded in mussel from Agadir region during the March – April 1999 algal bloom.

After periodate oxidation, compound

corresponds to N- sulfocarbamoyl 3

4 (C3

4); compounds

corre- spond to decarbamoyl-gonyautoxins 2

3 (dcGTX2

dcGTX3); compound

to N-sulfocarbamoyl 1

2 (C1

C2);

and

to decarbamoyl-saxitoxin (dcSTX);

to gonyautoxins 1 – 4 (GTX1/4);

to N-sulfocarbamoyl B1, and

to saxitoxin (STX) and/or neosaxitoxin (NeoSTX).

Peroxide oxidation confirmed the presence of dcGTX2

3 and dcSTX by inversion of peak height, respectively, as follows: 2 was higher than 3 and 6 was higher than 7

(Lawrence et al., 1996). Peroxide oxidation showed the absence of STX in peak 10, which must be attributed exclusively to NeoSTX. Presence of large amounts of NeoSTX was already suspected from periodate oxidation:

usually height of peak 7 is around four times that of peak 6, if only dcSTX is involved. As height of 7 was much higher than expected, presence of this N-

-hydroxylated toxin was hypothesized. Table 1 shows percentile distribution of toxins based on periodate oxidation, NeoSTX was estimated from height of peak 10.

Fig. 6. Chromatograms of PSP toxins from: (a) Casablanca mussels after periodate oxidation; (b) Taghazout mussels after periodate oxidation;

(c) Taghazout mussels after peroxide oxidation; (d) marine beans after periodate oxidation. Chromatogram (a) was obtained on a Macherey- NagelNucleosilcolumn; (b, c) and (d) were obtained on two distinct 125£3 mm²MerckLichrosphercolumns. Detector used for sample (a) was from a different series than the one used for the remaining.

Analyses conducted in samples from the southern coast showed a much simpler profile (Fig. 6(b) – (d)). A mussel tissue from Taghazout was essentially contaminated by gonyautoxins 1 – 4, and a smaller amount of STX (Fig. 6(b), Table 1). Peroxide oxidation confirmed the presence of STX, and a trace amount of dcSTX due to appearance of peak 6 which was not evident upon periodate oxidation (Fig.

6(c)). Absence of peak 3 in peroxide oxidation also confirmed presence of the N-

-hydroxylated analogues GTX1

4, besides the non-N-

-hydroxylated GTX2

3.

Marine beans were contaminated exclusively by GTX1/4 (Figs. 6(d)).

4. Discussion

The episodes of shellfish contamination by PSP toxins occur differently in various locations of the Moroccan Atlantic shore. In November 1994, the phenomenon affected the northern part, stretching from Larache to Essaouirra.

The identification in mussels of individual PSP toxins by HPLC revealed a complex toxin profile. This is similar to that of G. catenatum, which presents C1/4, GTX5

6, NeoSTX, and specially the presence of dcSTX and absence of STX (Franco and Ferna´ndez-Vila, 1993; Donker et al., 1997). Our data coincides well with those concerning the abundance of this species found by Tahri (1998). Moreover, the November 1994 bloom of G. catenatum occurred on a large scale and affected also the Atlantic coasts of Portugal (Moita et al., 1998).

In contrast, the South Atlantic coast was not affected by the November 1994 phenomenon. PSP outbreaks occurred later at different periods in summer 1998 (July and August) and in spring 1999 (from February to May).

Toxin composition found in mussels and marine beans

the cockle Achanthocardia tuberculatum showed dcSTX and STX were the toxins retained longer (Taleb et al., 2001; Vale and Sampayo, 2002).

Periodicity of blooms in the Atlantic coast seems to be much wider in time, opposite to the restriction of periodicity found so far in the Mediterranean coast, which is usually from January until April (Taleb et al., 2001). According to data obtained so far, G. catenatum and A. minutum are both capable to contaminate bivalves in the Atlantic coast with dangerous PSP levels. By contrast, in the close Portuguese Atlantic coast A. minutum has been detected but has not been held responsible for any contamination of shellfish above the regulatory level for the past years (since establishment of phytoplankton monitoring in 1986) (Sampayo et al., 1997). With such a dispersion of months when toxicity above the regulatory level was detected, and given the human fatalities that already took place, the importance for a strict periodic control of shellfish resources is reinforced.

Acknowledgements

We knowledge M. Abdellatif Berraho for supporting this study and all the staff of the RSSL (Re´seau de Surveillance de la Salubrite´ du Littoral) for bivalve molluscs sampling.

References

AOAC, 1990. Paralytic shellfish poison, biological method, final action. In: AOAC, (Ed.), Official Methods of Analysis, 15th ed., Arlington, VA, method n8959.08.

Bourhili, E.H., 1982. Intoxication alimentaire par les moules (51 cas), Agadir, Octobre et Novembre 1982. The`se n833, Fac.

Med. et Pharm., Rabat. 84 p.

Donker, S., Reyero, M.I., Reguera, B., Franco, J.M., 1997. Perfil de toxinas PSP de seis cepas de Gymnodinium catenatum de Galicia. In: Vyeites, J.M., Leira, F. (Eds.), V Reunio´n Iberica sobre Fitoplancton To´xico y Biotoxinas, Anfaco-Cecopesca, Vigo, pp. 69 – 76.

STX ND 2% ND

ND, not detected;þ, presence hypothesized.

a MBA, mouse bioassay, inmg equiv.STX/100 g.

Forteza, V., Quetglas, G., Delgado, M., Reyero, M.I., Fraga, S., Franco, J.M., 1998. Toxic Alexandrium minutum bloom in Palma de Mallorca harbour, (Balaearic Islands, western Mediterranean). In: Reguera, B., Blanco, J., Ferna´ndez, M.L., Wyatt, T. (Eds.), Harmful Algae, Xunta de Galicia and IOC of UNESCO, Spain, pp. 58 – 59.

Franco, J.M., Ferna´ndez-Vila, P., 1993. Separation of paralytic shellfish toxins by reversed phase high performance liquid chromatography, with post-column reaction and fluorimetric detection. Chromatographia 35 (9 – 12), 613 – 620.

Hallegraeff, G.M., 1993. A review of harmful algal blooms and their apparent global increase. Phycologia 32, 79 – 99.

Lawrence, J.F., Me´nard, C., Cleroux, C., 1995. Evaluation of prechromatographic oxidation for liquid chromatographic determination of paralytic shellfish poisons in shellfish.

J. AOAC Int. 78 (2), 514 – 520.

Lawrence, J.F., Wong, B., Me´nard, C., 1996. Determination of decarbamoyl saxitoxin and its analogues in shellfish by prechromatographic oxidation and liquid chromatography with fluorescence detection. J. AOAC Int. 79 (5), 1111 – 1115.

Moita, M.T., Vilarinho, M.G., Palma, A.S., 1998. On the variability of Gymnodinium catenatum Graham blooms in Portuguese waters. In: Reguera, B., Blanco, J., Ferna´ndez, M.L., Wyatt, T.

(Eds.), Harmful Algae, Xunta de Galicia and IOC of UNESCO, Spain, pp. 182 – 183.

Sampayo, M.A.M., Franca, S., Sousa, I., Alvito, P., Vale, P., Botelho, M.J., Rodrigues, S., Vieira, A., 1997. Dez anos de

monitorizac¸a˜o de biotoxinas marinhas em Portugal (1986 – 1996). Arq. Inst. Nacional Sau´de 23, 187 – 194.

Tahri, L.J., 1998. Gymnodinium catenatum Graham blooms on Moroccan waters. In: Reguera, B., Blanco, J., Ferna´ndez, M.L., Wyatt, T. (Eds.), Harmful Algae, Xunta de Galicia and IOC of UNESCO, Spain, pp. 66 – 67.

Tahri, L.J., Berraho, A., Orbi, A., 2000. Harmful algae blooms and the upwelling regime in atlantic coastal waters of Morocco. IX International Conference on Harmful Algal Blooms, Hobart, Australia, 7 – 11 February 2000. Abstract.

Taleb, H., 1997. Re´sultats de la surveillance des phycotoxines le long des coˆtes marocaines. Travaux et Documents de l’INRH, N8102.

Taleb, H., Idrissi, H., Blaghen, M., 1998. Seasonality of PSP toxicity in shellfish from the Atlantic and Mediterranean coasts of Morocco. In: Reguera, B., Blanco, J., Ferna´ndez, M.L., Wyatt, T. (Eds.), Harmful Algae, Xunta de Galicia and IOC of UNESCO, Spain, pp. 68 – 69.

Taleb, H., Vale, P., Jaime, E., Blaghen, M., 2001. Study of paralytic shellfish poisoning toxin profile in shellfish from the Mediterra- nean shore of Morocco. Toxicon 39 (12), 1855 – 1861.

Tber, A., 1983. Personal communication. Laboratoire d’analyses des services ve´te´rinaires. Casablanca, Maroc. 5p.

Vale, P., Sampayo, M.A.M., 2001. Determination of paralytic shellfish toxins in Portuguese shellfish by automated pre-column oxidation. Toxicon 39 (4), 561 – 571.

Vale, P., Sampayo, M.A.M., 2002. Evaluation of marine biotoxin’s accumulation by Acanthocardia tuberculatum from Algarve, Portugal. Toxicon 40 (5), 511 – 517.