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DSP toxin profile in the coastal waters of the central Adriatic Sea
Toxicon 40 (2002) 1601–1607 www.elsevier.com/locate/toxicon
DSP toxin profile in the coastal waters of the central Adriatic Sea M. Pavela-Vrancˇicˇa,*, V. Mesˇtrovic´a, I. Marasovic´b, M. Gillmanc, A. Fureyc, K.J. Jamesc a
Department of Chemistry, Faculty of Natural Sciences, Mathematics and Education, University of Split, N. Tesle 12, 21000 Split, Croatia b Institute of Oceanography and Fisheries, Sˇetalisˇte Ivana Mesˇtrovic´a 63, 21000 Split, Croatia c Department of Chemistry, Cork Institute of Technology, Bishopstown, Cork, Ireland Received 28 January 2002; accepted 17 June 2002
Abstract A monitoring program, carried out in 1996 and 1997, has confirmed that toxic compounds, other than the most frequently detected toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1), are involved in DSP phenomena in the Adriatic Sea. Toxicity was assessed by the mouse bioassay; the content and the nature of the toxic components were established through fluorometric HPLC analysis combined with mass spectrometry. A rare pectenotoxin-2 (PTX-2) derivative, 7-epi-pectenotoxin2 seco acid (7-epi-PTX-2SA), was the exclusive contaminant of samples collected from the central Adriatic in 1996. Contrary to its marked oral toxicity, intraperitoneally 7-epi-PTX-2SA displayed no toxic effects, hampering its detection by the mouse bioassay. In 1997, its concentration and frequency of appearance were lower than in 1996, with concomitant occurrence of OA, DTX-2, and a new unidentified component related to the DSP toxic group of compounds. This is the first report on the occurrence of DTX-2 in Adriatic mussels. A survey of the phytoplankton community in the surrounding seawater has established the presence of Prorocentrum micans and several potentially toxic species from the Dinophysis genus. A case of unexplained toxicity, associated with the occurrence of Gonyaulax polyedra, suggested possible shellfish contamination with yessotoxin (YTX). q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Central Adriatic Sea; Phytoplankton toxins; Diarrhoeic shellfish poisoning; Okadaic acid; Dinophysistoxin-2; 7-epi-pectenotoxin-2 seco acid
1. Introduction Red tide blooms and ‘mucilagine’ were known to occur in the central Adriatic Sea, however the incidence of toxic blooms, associated with shellfish intoxication in this area, is a newly recognized phenomenon (Orhanovic´ et al., 1996; Marasovic´ et al., 1998; Pavela-Vrancˇicˇ et al., 2001). Mussel contamination by toxic compounds for several consecutive periods indicates that this is not an occasional occurrence, implying serious health and economic consequences. Diarrhoeic toxins are secondary metabolites produced by toxigenic dinoflagellates of the Dinophysis and Prorocentrum species. Shellfish accumulate these toxic substances in their digestive glands, thereby causing diarrhoeic shellfish poisoning (DSP) in humans, an intoxication characterized by severe gastro* Corresponding author. Fax: þ 385-21-385-431. E-mail address: [email protected] (M. Pavela-Vrancˇicˇ).
intestinal disturbances. DSP toxins are classified into three groups of related polyether compounds: the acidic compound okadaic acid (OA) and its derivatives named dinophysistoxins (DTX-1,2); the neutral polyetherlactones of the pectenotoxin group PTX-1,2,3,6; yessotoxin (YTX) and its analogue 45-hydroxyyessotoxin (45-OH YTX) (Yasumoto et al., 1985). DTX-3 is a complex mixture of 7-O-acyl derivatives of OA and DTX-1,2, produced by bioconversion in the digestive glands of shellfish. OA and DTX-1 have been considered as the principal causative agents of DSP outbreaks in the Adriatic region (Fattorusso et al., 1992; Draisci et al., 1995, 1996) The first occurrence of PTX-2 was reported in 1996, and associated with contamination of mussels from the coastal area of northern Italy (Draisci et al., 1996). YTX, so far known to occur in shellfish from Norway (Lee et al., 1988; Ramstad et al., 2001), and its analogue homoYTX have been recently found in the northern Adriatic following a Gonyaulax
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polyedra bloom (Tubaro et al., 1998). Several new compounds related to okadaic acid and DTX-1 have been detected in mussels from the coastal areas of both the northern and the southern Adriatic (Draisci et al., 1995). In the central Adriatic, shellfish toxicity was first monitored in 1993, in the eastern part of the Kasˇtela Bay, during an intensive red tide bloom of G. polyedra (Orhanovic´ et al., 1996). Shellfish samples, harvested during massive fish kills after a Gonyaulax bloom in 1994, revealed the occurrence of both OA and DTX-1, though at levels not endangering human health (Orhanovic´ et al., 1996). In 1995, incidents of toxicity in the central Adriatic were associated with the occurrence of new analogues related to the DSP family of compounds (Marasovic´ et al., 1998). The characterization of two acidic PTX-2 analogues, pectenotoxin-2 seco acid (PTX-2SA) and its epimer 7-epi-pectenotoxin-2 seco acid (7-epi-PTX-2SA), isolated from the dinoflagellate D. acuta from Ireland (Daiguji et al., 1998; James et al., 1999), had enabled the recent identification of 7-epi-PTX-2SA in shellfish from the central Adriatic (Pavela-Vrancˇicˇ et al., 2001). The wide diffusion of DSP toxins throughout the Adriatic and the frequency of their appearance have emphasized the necessity of exploring the variability of toxin composition in the central Adriatic, essential in evaluating the risk of human poisoning by consumption of DSP-contaminated seafood.
2. Materials and methods 2.1. Sampling Wild populations of the mussel Mytilus galloprovincialis were harvested at regular intervals in 1996 and 1997 from a localized site in the eastern part of the Kasˇtela Bay, Central Adriatic Sea (FI: 438290 – 438330 , LA: 168150 – 168290 ). The mussels were collected at a depth of 50 cm, and stored at 2 20 8C. 2.2. Phytoplankton analysis The composition of the phytoplankton community in waters surrounding the sampling site was determined according to Utermo¨hl, 1958. Seawater samples were preserved in formaldehyde (2%, v/v) solution. Counting and identification of the organisms was conducted using an Opton inverted microscope. The live material was analysed using an Olympus IMT-2 microscope with a Nomarski difference contrast attachment. 2.3. Mouse bioassay Analysis of DSP toxicity by mouse bioassay was performed according to Yasumoto et al., 1985. The
hepatopancreases of the mussels were extracted with acetone at room temperature. After evaporation of acetone, the residue was partitioned between diethyl ether and water. The organic fraction was evaporated to dryness and the residue dissolved in 1% (v/v) Tween 60, followed by intraperitoneal injection of the diluted sample into the mouse (strain BALB/C, weight limits 18 – 20 g). Three parallel tests were performed, and the reaction of the mice was observed over 24 h after treatment or until death.
2.4. Isolation of polyether acid Homogenised shellfish hepatopancreases (1 g) were extracted with 4 ml MeOH/H2O (80/20) containing 0.3% (v/v) acetic acid. Following centrifugation (15 min, 1000 g), an aliquot (3 ml) of the supernatant was extracted with 11 ml H2O/isopropyl acetate (3/8) by agitating on a Vortex mixer for 1 min. The aqueous phase was extracted again with 8 ml isopropyl acetate, and the combined organic fractions were evaporated to dryness. Isolation, purification and identification of the polyether acids was performed as previously described (James et al., 1999; Pavela-Vrancˇicˇ et al., 2001). Derivatisation was carried out using 9-anthryldiazomethane (ADAM) in methanol, followed by separation on HPLC using fluorometric detection at lex 365 nm and lem 412 nm. The toxin profile was examined in comparison to standard samples of OA (Rt 15.50 min), DTX-1 (Rt 22.34 min) and DTX-2 (Rt 16.70 min), and a reference material obtained from algae extracts of Dinophysis acuta, containing pectenotoxin-2 seco acid (PTX-2SA, Rt 12.967 min), OA (Rt 15.483 min), 7-epi-pectenotoxin-2 seco acid (7-epi-PTX-2SA, R t 16.233 min), and DTX-2 (Rt 16.883 min) (James et al., 1999). The toxin concentration was estimated from the peak area with reference to a calibration sample containing 3.8 ng OA. Separation and purification of the toxins was carried out on a micro column packed with Vydac 218TP51 (250 mm £ 1 mm, 5 mm).
2.5. Liquid chromatography – mass spectrometry The LC system (HP 1100, Agilent, Cheshire, UK) was linked to a Finnigan MAT LCQ Classic ion-trap mass spectrometer (Thermo-Finnigan, San Jose, CA, USA). Isocratic chromatography was performed using acetonitrile – water (60:40) containing 0.5 mM ammonium acetate, at a flow rate of 200 ml/min, with an analytical column (Luna-2, 5 mm, 30 £ 2.0 mm and 150 £ 2.0 mm, Phenomenex, Macclesfield, UK) at 40 8C. Mass spectrometric analysis was carried out in atmospheric pressure ionisation using an electronspray source in positive mode.
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Table 1 Toxicity data, estimated by the DSP mouse bio-assay, the toxin content and its nature, and the composition of the corresponding phytoplankton community. Analyses were performed using hepatopancreas extracts of mussels collected from the Kasˇtela Bay, Central Adriatic Sea, in 1996 and 1997 Time of sampling
Medial mouse survival time
Toxin composition
Toxin concentration (mg/g HP)
Phytoplankton composition
Abundance (cells/dm3)
February 18, 1996
.24 h
7-epi-PTX-2SA
1.140
June 19, 1996a
3 h 58 min
7-epi-PTX-2SA
0.195
July 29, 1996
.24 h
7-epi-PTX-2SA
0.187
August 23, 1996
.24 h
7-epi-PTX-2SA
0.632
May 16, 1997
7 h 10 min
DTX analogue DTX-2
nd 0.105
June 16, 1997a
16 h
OA 7-epi-PTX-2SA
0.133 0.090
7.2 £ 104 1.2 £ 105 8.3 £ 103 2.7 £ 105 4.1 £ 104 5.0 £ 105 4.3 £ 104 5.3 £ 105 1.6 £ 105 5.0 £ 104 6.6 £ 104 3.3 £ 104 6.7 £ 105 2.6 £ 105
June 25, 1997
16 h
DTX-2
0.147
Dominating diatoms P. micans P. micans G. polyedra C. marina D. sacculus P. micans D. sacculus P. micans P. micans D. sacculus G. polyedra G. polyedra C. marina P. micans nd
a
HP, hepatopancreas; nd, not detected. The toxicity and the toxin composition data have been previously reported by Pavela-Vrancˇicˇ et al., 2001.
3. Results Analyses of shellfish samples collected in 1996 demonstrate a predominant presence of 7-epi-PTX-2SA at concentrations up to 1.140 mg/g hepatopancreas (HP) (Table 1). Mice treated with an extract from mussels collected on February 18, 1996 did not reveal shellfish intoxication, based on an average mouse survival time of over 24 h. Although HPLC analysis confirmed the absence of the common DSP compounds, OA and DTXs, it established shellfish contamination with a markedly high
concentration of 7-epi-PTX-2SA. A representative chromatogram of the mussel extract is shown in Fig. 1; the signal with the retention time Rt 16.342 min matches well with that of the 7-epi-PTX-2SA standard sample (Rt 16.233 min). Structural information, confirming that the structure of the identified compound corresponds to that of 7-epi-PTX-2SA, was obtained by mass spectrometric analysis. A molecular mass of 876 was determined (Fig. 2), the same as of PTX-2SAs, based on molecular mass signals at m=z 899 [M þ Na]þ (876 þ 23) and m=z 894 [M þ NH4]þ (876 þ 18). Collision-induced dissociation
Fig. 1. The HPLC profile of a shellfish sample collected from the Kasˇtela Bay (Central Adriatic Sea) on February 18, 1996. The sample was analysed with reference to an algae extract of Dinophysis acuta containing PTX-2SA (Rt 12.967 min), OA (Rt 15.484 min), 7-epi-PTX-2SA (Rt 16.233 min), and DTX-2 (Rt 16.883 min) after derivatisation with ADAM.
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Fig. 2. The mass spectrum of 7-epi-PTX-2SA. A molecular mass of 876 was calculated from molecular mass signals at m=z 899 [M þ Na]þ and m=z 894 [M þ NH4]þ.
(CID), obtained by positive and negative mass spectrometry (MS – MS), showed a fragmentation pattern characteristic of 7-epi-PTX-2SA, as previously established by James et al., (1999). Mussels from June 19, 1996 showed a greater toxicity than expected, considering that the only identified contaminant was 7-epi-PTX-2SA. Again, samples from July 29 and August 23, 1996 demonstrated no toxicity, relating to the occurrence of 7-epi-PTX-2SA as the only contaminant. In 1997, the toxin profile demonstrates the increased predominant role of toxins from the DTX toxin group. HPLC analysis of the sample from May 16, 1997 revealed the presence of DTX-2 at a concentration estimated to 0.105 mg/g HP (Fig. 3). Its identity was confirmed by comparison of the retention time (Rt 16.683 min) with that of the standard sample of DTX-2 (Rt 16.700 min). However, the chromatogram displayed another prominent peak at Rt 20.583 min, that did not match the retention time of any so far known DSP compound. Unfortunately, not enough material was available for structural analysis. The ratio of the peak area compared to that of DTX-2 suggests a significant contribution to the toxin content in the shellfish sample. Mice inoculated with an extract of this sample displayed a survival time of 7 h 10 min. On the other hand, a
survival time of 16 h was obtained with the sample from June 25, 1997 revealing only the occurrence of DTX-2 at a concentration of 0.147 mg/g HP. The same survival time was recorded with the sample from June 16, 1997, containing OA (0.133 mg/g HP), and a relatively minor amount of 7-epi-PTX-2SA. Along with shellfish sampling, seawater samples were taken for analysis of the phytoplankton community and cell abundance (Table 1). Although in the winter of 1996 diatoms dominated the phytoplankton community, the sample collected on February 18, 1996 contained P. micans at an abundance of 7.2 £ 104 cells/dm3. In June 1996, when high DSP toxicity in shellfish was measured, an elevated abundance of P. micans and G. polyedra was recorded. In the following period from July to September, P. micans, D. sacculus and Chatonella marina were the principal components of the respective phytoplankton community. In the summer of 1997 very low concentrations of D. fortii, D. caudata and D. acuta, well known progenitors of OA and its analogues, were found. However, in May of the same year, three potentially toxic species were detected in the seawater sample, D. sacculus (acuminata?), G. polyedra and P. micans. Again, P. micans was present in the phytoplankton community during the whole monitoring period. In
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Fig. 3. The HPLC profile of a shellfish sample collected from the Kasˇtela Bay (Central Adriatic Sea) on May 16, 1997, analysed with reference to a standard sample of DTX-1 (Rt 22.34 min) and DTX-2 (Rt 16.70 min) after derivatisation with ADAM.
August, the G. polyedra bloom developed to an abundance of 2.0 £ 106 cells/dm3.
4. Discussion Until 1996, the most frequently detected DSP phycotoxins in the Adriatic area were OA and DTX-1, OA being the major component. More recent analysis of phytoplankton and shellfish samples collected in European waters, revealed a frequent occurrence of intoxication with DSP compounds other than the OA group, as well as with novel DSP analogues. Analysis of the toxin profile in mussels from the central Adriatic Sea demonstrates a similar trend substantiated by the occurrence of a new and rare class of PTX compounds, the pectenotoxin-2 seco acids. Although the sample from February 28, 1996 contained a significant quantity of 7-epi-PTX-2SA, the negative mouse bioassay response is indicative of its low intraperitoneal (i.p.) toxicity. Incidents of acute human intoxication related to PTX-2SAs, accompanied by a negative mouse bioassay, were documented in 1997 in Australia confirming its oral toxicity, however no data are available on the quantity of the ingested toxin. So far there is no reliable explanation for the lack of agreement between the i.p. and oral toxicity of PTX-2SAs. The potential cause has been attributed to the open chain structure of the two stereoisomers. PTX-2SAs are closely related to PTX-2, containing an open chain carboxylic acid instead of a lactone ring. Under the influence of the metabolic processes, it may through chain closure adopt the structure exerting toxicity. Recent comparison of PTX profiles of the toxic dinoflagellate D. acuta, Greenshell mussel Perna canaliculus, and Blue
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mussel Mytilus galloprovincialis collected in New Zealand has shown that, although the major PTX analogue in D. acuta was PTX-2, both mussel cultures contained PTX-2SA as the predominant toxin (Suzuki et al., 2001). PTX-2 has never been found in shellfish, generating by bioconversion a variety of toxic analogues including the highly hepatotoxic derivatives PTX-1, 3 and 6, which are produced by oxidation at C43 in the shellfish hepatopancreas (Yasumoto et al., 1989; Zhou et al., 1994; Jung et al., 1995). The mechanism of action and the toxicological effects of pectenotoxins are not fully understood. Severe injuries of the small intestine were detected upon oral injection of mice with OA and PTX-2 (Ishige et al., 1988). PTX-1 damages the liver in the same manner as toxins from mushrooms and moulds (Terao et al., 1986; Zhou et al., 1994). Since standard samples are not commercially available for these toxins, routine methods for their determination have not been established. Hence, the presence of PTX and related compounds in the phytoplankton community may indicate a new and severe risk of shellfish contamination and consequently human poisoning. To overcome the discrepancies observed between the findings of the mouse bioassay for the sample from June 19, 1996, displaying high i.p. toxicity, and the results of the HPLC analysis, the presence of a toxin other than 7-epiPTX-2SA was hypothesized. YTX is not detectable by the respective HPLC method, and injected intraperitoneally into mice displays the highest toxicity level (lethal dose 0.100 mg/g edible tissue) (Daiguji et al., 1998). The hypothesis is strengthened by the fact that G. polyedra was found in the accompanying seawater sample at an abundance of 8.3 £ 103 cells/dm3. The first occurrence of YTX in Europe has been established in Italian mussels, following a bloom of G. polyedra in 1996 (Draisci et al., 1996). Likewise, a new analogue homoyessotoxin has been recently identified in mussels from the northern Adriatic collected during a G. polyedra bloom (Tubaro et al., 1998). In 1997 the major toxic components were OA and DTX2, however well bellow the legal tolerance level. This is the first report on the occurrence of DTX-2 in Adriatic mussels. DTX-2 is the main causative toxin of DSP intoxication in Ireland (Marr et al., 1992; Carmody et al., 1996; James et al., 1997a), however its presence has also been recorded in dinoflagellates from Spain (Blanco et al., 1995) and in shellfish from Portugal (Vale and Sampayo, 2000). The toxicity of the sample from May 16, 1997 is likely to be due in part to the presence of a DTX-related substance. An OA concentration of 0.200 mg/g mussel meat, approximately equivalent to 0.8 mg/g HP, causes mouse death within 5 h (Carmody et al., 1996). Although no toxicity data are available for DTX-2, it is expected that they could be similar to that of OA and DTX-1. Hence, the concentration of DTX2 in the sample from May 16, 1997 is not sufficient to account for the recorded toxicity. This strongly suggests that still unidentified compounds could be involved in DSP intoxication of shellfish from the central Adriatic.
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Previous episodes of DSP toxicity in the central Adriatic have been associated with G. polyedra and the Dinophysis species (Orhanovic´ et al., 1996), whereas D. fortii was found to be responsible for the production of PTX-2 in the northern Adriatic region (Lee et al., 1988; Draisci et al., 1996). In Europe, the most frequent DSP causative organism is D. acuminata, however shellfish intoxication in the Netherlands and in Scandinavia has also been associated with P. micans. D. sacculus has often been associated with D. acuminata, establishing a belief they may represent different morphotypes of the same species, the occurrence of a specific morphotype being determined by the diversity of the environmental conditions. An explanation for the relatively high winter shellfish toxicity may lie in the occurrence of various species of the Dinophysis genus (D. fortii, D. sacculus, and D. caudata ) during the autumn phytoplankton bloom, preceded by a very strong red tide bloom of G. polyedra. Concentrations as low as 200 cells/dm3 of toxic phytoplankton such as D. sacculus, D. acuta, D. caudata, and D. fortii may cause severe intoxication of shellfish (Viviani et al., 1995). In Irish waters and in Spain, D. acuta was shown to be the progenitor of DTX-2 (Blanco et al., 1995; James et al., 1997b; Draisci et al., 1998). In June 1997, when shellfish intoxication with OA and DTX-2 was registered, the phytoplankton analysis had not detected the presence of species from the Dinophysis genus (Pavela-Vrancˇicˇ et al., 2001). However, the appearance of OA and DTX-2 followed shortly after the occurrence of D. sacculus in May, implying its possible involvement as the source of DSP toxicity. The presence of multiple diarrhoeic toxins in shellfish creates complications due to lack of toxicity data for some toxins and the absence of analytical reference compounds. The frequent discovery of new and related compounds with variable substitution patterns, and the metabolic activity observed in shellfish digestive glands, shown to modify the algal toxin structure and consequently its toxicity level, requires further toxicological and chemical studies to gain knowledge on their biological and structural properties.
Acknowledgments This work was supported by grants from the Croatian Ministry of Science and Technology No. 177050 and No. 000100101.
References Blanco, J., Ferna´ndez, M., Marino, J., Reguera, B., Miguez, A., Maneiro, J., Cacho, E., Martinez, A., 1995. From Dinophysis spp. toxicity to DSP outbreaks: a preliminary model of toxin accumulation in mussels. In: Lassus, P., Arzul, G., Erard, E., Gentien, P., Marcaillou-LeBaut, C. (Eds.), Harmful Marine
Algal Blooms, Lavoisier Science Publishers, Paris, pp. 777 –782. Carmody, E.P., James, K.J., Kelly, S.S., 1996. Dinophysistoxin-2: the predominant diarrhoetic shellfish toxin in Ireland. Toxicon 34, 351–359. Daiguji, M., Sataka, M., James, K.J., Bishop, A., Makenzie, L., Naoki, H., Yasumoto, T., 1998. Structures of new pectenotoxin analogs, pectenotoxin-2 seco acid and 7-epi-pectenotoxin-2 seco acid, isolated from a dinoflagellate and greenshell mussels. Chem. Lett. 7, 653–654. Draisci, R., Lucentini, L., Giannetti, L., Boria, P., Stacchini, A., 1995. Detection of diarrhoetic shellfish toxins in mussels from Italy by ionspray liquid chromatography–mass spectrometry. Toxicon 33, 1591–1603. Draisci, R., Lucentini, L., Giannetti, L., Boria, P., Poletti, R., 1996. First report of pectenotoxin-2 (PTX-2) in algae (Dinophysis fortii ) related to seafood poisoning in Europe. Toxicon 34, 923 –935. Draisci, R., Giannetti, L., Lucentini, L., Marchiafava, C., James, K.J., Bishop, A.G., Healy, B.M., Kelly, S.S., 1998. Isolation of a new okadaic acid analogue from phytoplankton implicated in diarrhetic shellfish poisoning. J. Chromatogr. 798, 137–145. Fattorusso, E., Ciminiello, P., Costantino, V., Magno, S., Mangoni, A., Milandri, A., Poletti, R., Pompei, M., Viviani, R., 1992. Okadaic acid in mussels of Adriatic Sea. Mar. Pollut. Bull. 24, 234 –237. Ishige, M., Satoh, N., Yasumoto, T., 1988. Pathological studies on mice administered with the causative agent of diarrhetic shellfish poisoning (ocadaic acid and pectenotoxin-2). Hakkaidoritsu Eisei Kenkyushoho 18, 15 –18. James, K.J., Bishop, A.G., Gillman, M., Kelly, S.S., Roden, C., Draisci, R., Lucentini, L., Giannetti, L., Boria, P., 1997a. Liquid chromatography with fluorometric, mass spectrometric and tandem mass spectrometric detection for the investigation of the seafood-toxin-producing phytoplankton, Dinophysis acuta. J. Chromatogr. 777, 213 –221. James, K.J., Carmody, E.P., Gillman, M., Kelly, S.S., Draisci, R., Lucentini, L., Giannetti, L., 1997b. Identification of a new diarrhoetic toxin in shellfish using liquid chromatography with fluorometric and mass spectrometric detection. Toxicon 35, 973 –978. James, K.J., Bishop, A.G., Draisci, R., Palleschi, L., Marchiafava, C., Ferretti, E., Satake, M., Yasumoto, T., 1999. Liquid chromatographic methods for the isolation and identification of new pectenotoxin-2 analogues from marine phytoplankton and shellfish. J. Chromatogr. A 844, 53–65. Jung, J.F.H., Sim, C.S., Lee, C.O., 1995. Cytotoxic compounds from a two-sponge association. J. Nat. Prod. 58, 1722–1726. Lee, J.S., Tangen, K., Dahl, E., Hovgaard, P., Yasumoto, T., 1988. Diarrhoetic shellfish toxins in Norwegian mussels. Bull. Jpn. Soc. Sci. Fish. 54, 1953–1957. Marasovic´, I., Nincˇevic´, Zˇ., Pavela-Vrancˇicˇ, M., Orhanovic´, S., 1998. A survey of shellfish toxicity in the central Adriatic Sea. J. Mar. Biol. Assoc., UK 78, 745– 754. Marr, J.C., Hu, T., Pleasance, S., Quilliam, M.A., Wright, J.L.C., 1992. Detection of new 7-O-acyl derivatives of diarrhetic shellfish poisoning toxins by liquid chromatography–mass spectrometry. Toxicon 30, 1621–1630. Orhanovic´, S., Nincˇevic´, Zˇ., Marasovic´, I., Pavela-Vrancˇicˇ, M., 1996. Phytoplankton toxins in the central Adriatic Sea. Croat. Chem. Acta 69, 291–303.
M. Pavela-Vrancˇicˇ et al. / Toxicon 40 (2002) 1601–1607 Pavela-Vrancˇicˇ, M., Mesˇtrovic´, V., Marasovic´, I., Gillman, M., Furey, A., James, K.J., 2001. The occurrence of 7-epipectenotoxin-2 seco acid in the coastal waters of the Central Adriatic (Kasˇtela Bay). Toxicon 39, 771–779. Ramstad, H., Hovgaard, P., Yasumoto, T., Larsen, S., Aune, T., 2001. Monthly variations in diarrhetic toxins and yessotoxins in shellfish from the coast to the inner part of Sognefjord, Norway. Toxicon 39, 1035–1043. Suzuki, T., Mackenzie, L., Stirling, D., Adamson, J., 2001. Pectenotoxin-2 seco acid: a toxin converted from pectenotoxin-2 by the New Zealand Greenshell mussel, Perna caniculus. Toxicon 39, 507 –514. Terao, K., Ito, E., Yanagi, T., Yasumoto, T., 1986. Histopathological studies on experimental marine toxin poisoning. 1. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin-1 and pectenotoxin1. Toxicon 24, 1141–1151. Tubaro, A., Sidari, L., Della Loggia, R., Yasumoto, T., 1998. Occurrence of yessotoxin-like toxins in phytoplankton and mussels from northern Adriatic Sea. In: Reguera, B., Blanco, J., Ferna´ndez, M.L., Wyatt, T. (Eds.), Harmful Algae, Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, pp. 470–472.
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Utermo¨hl, H., 1958. Zur Vervollkommung der Quantitativen Phytoplankton Methodik. “Mitteilungen-Communications” Internationale Vereinigung fu¨r Theoretische und Angewandte Limnologie 9, 1–38. Vale, P., Sampayo, M.A., 2000. Dinophysistoxin-2: a rare diarrhoeic toxin associated with Dinophysis acuta. Toxicon 38, 1599–1606. Viviani, R., Boni, L., Cattani, O., Milandri, A., Polleti, R., Pompei, M., 1995. ASP, DSP, NSP and PSP monitoring in mucilaginous aggregates and in mussels in a coastal area of the Northern Adriatic Sea facing Emilia-Romagna in 1988, 1989 and 1991. Sci. Total. Environ. 165, 203. Yasumoto, T., Murata, M., Oshima, Y., Sano, M., Matsumoto, G.K., Clardy, J., 1985. Diarrhetic shellfish toxins. Tetrahedron 41, 1019–1025. Yasumoto, T., Murata, M., Lee, J.S., Torigoe, K., 1989. Polyether toxins produced by dinoflagellates. In: Natori, S., Hashimoto, K., Ueno, T. (Eds.), Mycrotoxins and phycotoxins’88, Elsevier, Amsterdam, pp. 375–382. Zhou, Z.H., Komiyana, M.K., Teran, K., Shimada, Y., 1994. Effects of pectenotoxin-1 on liver cells in vitro. Nat. Toxins 2, 132 –135.
DSP toxin profile in the coastal waters of the central Adriatic Sea M. Pavela-Vrancˇicˇa,*, V. Mesˇtrovic´a, I. Marasovic´b, M. Gillmanc, A. Fureyc, K.J. Jamesc a
Department of Chemistry, Faculty of Natural Sciences, Mathematics and Education, University of Split, N. Tesle 12, 21000 Split, Croatia b Institute of Oceanography and Fisheries, Sˇetalisˇte Ivana Mesˇtrovic´a 63, 21000 Split, Croatia c Department of Chemistry, Cork Institute of Technology, Bishopstown, Cork, Ireland Received 28 January 2002; accepted 17 June 2002
Abstract A monitoring program, carried out in 1996 and 1997, has confirmed that toxic compounds, other than the most frequently detected toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1), are involved in DSP phenomena in the Adriatic Sea. Toxicity was assessed by the mouse bioassay; the content and the nature of the toxic components were established through fluorometric HPLC analysis combined with mass spectrometry. A rare pectenotoxin-2 (PTX-2) derivative, 7-epi-pectenotoxin2 seco acid (7-epi-PTX-2SA), was the exclusive contaminant of samples collected from the central Adriatic in 1996. Contrary to its marked oral toxicity, intraperitoneally 7-epi-PTX-2SA displayed no toxic effects, hampering its detection by the mouse bioassay. In 1997, its concentration and frequency of appearance were lower than in 1996, with concomitant occurrence of OA, DTX-2, and a new unidentified component related to the DSP toxic group of compounds. This is the first report on the occurrence of DTX-2 in Adriatic mussels. A survey of the phytoplankton community in the surrounding seawater has established the presence of Prorocentrum micans and several potentially toxic species from the Dinophysis genus. A case of unexplained toxicity, associated with the occurrence of Gonyaulax polyedra, suggested possible shellfish contamination with yessotoxin (YTX). q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Central Adriatic Sea; Phytoplankton toxins; Diarrhoeic shellfish poisoning; Okadaic acid; Dinophysistoxin-2; 7-epi-pectenotoxin-2 seco acid
1. Introduction Red tide blooms and ‘mucilagine’ were known to occur in the central Adriatic Sea, however the incidence of toxic blooms, associated with shellfish intoxication in this area, is a newly recognized phenomenon (Orhanovic´ et al., 1996; Marasovic´ et al., 1998; Pavela-Vrancˇicˇ et al., 2001). Mussel contamination by toxic compounds for several consecutive periods indicates that this is not an occasional occurrence, implying serious health and economic consequences. Diarrhoeic toxins are secondary metabolites produced by toxigenic dinoflagellates of the Dinophysis and Prorocentrum species. Shellfish accumulate these toxic substances in their digestive glands, thereby causing diarrhoeic shellfish poisoning (DSP) in humans, an intoxication characterized by severe gastro* Corresponding author. Fax: þ 385-21-385-431. E-mail address: [email protected] (M. Pavela-Vrancˇicˇ).
intestinal disturbances. DSP toxins are classified into three groups of related polyether compounds: the acidic compound okadaic acid (OA) and its derivatives named dinophysistoxins (DTX-1,2); the neutral polyetherlactones of the pectenotoxin group PTX-1,2,3,6; yessotoxin (YTX) and its analogue 45-hydroxyyessotoxin (45-OH YTX) (Yasumoto et al., 1985). DTX-3 is a complex mixture of 7-O-acyl derivatives of OA and DTX-1,2, produced by bioconversion in the digestive glands of shellfish. OA and DTX-1 have been considered as the principal causative agents of DSP outbreaks in the Adriatic region (Fattorusso et al., 1992; Draisci et al., 1995, 1996) The first occurrence of PTX-2 was reported in 1996, and associated with contamination of mussels from the coastal area of northern Italy (Draisci et al., 1996). YTX, so far known to occur in shellfish from Norway (Lee et al., 1988; Ramstad et al., 2001), and its analogue homoYTX have been recently found in the northern Adriatic following a Gonyaulax
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polyedra bloom (Tubaro et al., 1998). Several new compounds related to okadaic acid and DTX-1 have been detected in mussels from the coastal areas of both the northern and the southern Adriatic (Draisci et al., 1995). In the central Adriatic, shellfish toxicity was first monitored in 1993, in the eastern part of the Kasˇtela Bay, during an intensive red tide bloom of G. polyedra (Orhanovic´ et al., 1996). Shellfish samples, harvested during massive fish kills after a Gonyaulax bloom in 1994, revealed the occurrence of both OA and DTX-1, though at levels not endangering human health (Orhanovic´ et al., 1996). In 1995, incidents of toxicity in the central Adriatic were associated with the occurrence of new analogues related to the DSP family of compounds (Marasovic´ et al., 1998). The characterization of two acidic PTX-2 analogues, pectenotoxin-2 seco acid (PTX-2SA) and its epimer 7-epi-pectenotoxin-2 seco acid (7-epi-PTX-2SA), isolated from the dinoflagellate D. acuta from Ireland (Daiguji et al., 1998; James et al., 1999), had enabled the recent identification of 7-epi-PTX-2SA in shellfish from the central Adriatic (Pavela-Vrancˇicˇ et al., 2001). The wide diffusion of DSP toxins throughout the Adriatic and the frequency of their appearance have emphasized the necessity of exploring the variability of toxin composition in the central Adriatic, essential in evaluating the risk of human poisoning by consumption of DSP-contaminated seafood.
2. Materials and methods 2.1. Sampling Wild populations of the mussel Mytilus galloprovincialis were harvested at regular intervals in 1996 and 1997 from a localized site in the eastern part of the Kasˇtela Bay, Central Adriatic Sea (FI: 438290 – 438330 , LA: 168150 – 168290 ). The mussels were collected at a depth of 50 cm, and stored at 2 20 8C. 2.2. Phytoplankton analysis The composition of the phytoplankton community in waters surrounding the sampling site was determined according to Utermo¨hl, 1958. Seawater samples were preserved in formaldehyde (2%, v/v) solution. Counting and identification of the organisms was conducted using an Opton inverted microscope. The live material was analysed using an Olympus IMT-2 microscope with a Nomarski difference contrast attachment. 2.3. Mouse bioassay Analysis of DSP toxicity by mouse bioassay was performed according to Yasumoto et al., 1985. The
hepatopancreases of the mussels were extracted with acetone at room temperature. After evaporation of acetone, the residue was partitioned between diethyl ether and water. The organic fraction was evaporated to dryness and the residue dissolved in 1% (v/v) Tween 60, followed by intraperitoneal injection of the diluted sample into the mouse (strain BALB/C, weight limits 18 – 20 g). Three parallel tests were performed, and the reaction of the mice was observed over 24 h after treatment or until death.
2.4. Isolation of polyether acid Homogenised shellfish hepatopancreases (1 g) were extracted with 4 ml MeOH/H2O (80/20) containing 0.3% (v/v) acetic acid. Following centrifugation (15 min, 1000 g), an aliquot (3 ml) of the supernatant was extracted with 11 ml H2O/isopropyl acetate (3/8) by agitating on a Vortex mixer for 1 min. The aqueous phase was extracted again with 8 ml isopropyl acetate, and the combined organic fractions were evaporated to dryness. Isolation, purification and identification of the polyether acids was performed as previously described (James et al., 1999; Pavela-Vrancˇicˇ et al., 2001). Derivatisation was carried out using 9-anthryldiazomethane (ADAM) in methanol, followed by separation on HPLC using fluorometric detection at lex 365 nm and lem 412 nm. The toxin profile was examined in comparison to standard samples of OA (Rt 15.50 min), DTX-1 (Rt 22.34 min) and DTX-2 (Rt 16.70 min), and a reference material obtained from algae extracts of Dinophysis acuta, containing pectenotoxin-2 seco acid (PTX-2SA, Rt 12.967 min), OA (Rt 15.483 min), 7-epi-pectenotoxin-2 seco acid (7-epi-PTX-2SA, R t 16.233 min), and DTX-2 (Rt 16.883 min) (James et al., 1999). The toxin concentration was estimated from the peak area with reference to a calibration sample containing 3.8 ng OA. Separation and purification of the toxins was carried out on a micro column packed with Vydac 218TP51 (250 mm £ 1 mm, 5 mm).
2.5. Liquid chromatography – mass spectrometry The LC system (HP 1100, Agilent, Cheshire, UK) was linked to a Finnigan MAT LCQ Classic ion-trap mass spectrometer (Thermo-Finnigan, San Jose, CA, USA). Isocratic chromatography was performed using acetonitrile – water (60:40) containing 0.5 mM ammonium acetate, at a flow rate of 200 ml/min, with an analytical column (Luna-2, 5 mm, 30 £ 2.0 mm and 150 £ 2.0 mm, Phenomenex, Macclesfield, UK) at 40 8C. Mass spectrometric analysis was carried out in atmospheric pressure ionisation using an electronspray source in positive mode.
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Table 1 Toxicity data, estimated by the DSP mouse bio-assay, the toxin content and its nature, and the composition of the corresponding phytoplankton community. Analyses were performed using hepatopancreas extracts of mussels collected from the Kasˇtela Bay, Central Adriatic Sea, in 1996 and 1997 Time of sampling
Medial mouse survival time
Toxin composition
Toxin concentration (mg/g HP)
Phytoplankton composition
Abundance (cells/dm3)
February 18, 1996
.24 h
7-epi-PTX-2SA
1.140
June 19, 1996a
3 h 58 min
7-epi-PTX-2SA
0.195
July 29, 1996
.24 h
7-epi-PTX-2SA
0.187
August 23, 1996
.24 h
7-epi-PTX-2SA
0.632
May 16, 1997
7 h 10 min
DTX analogue DTX-2
nd 0.105
June 16, 1997a
16 h
OA 7-epi-PTX-2SA
0.133 0.090
7.2 £ 104 1.2 £ 105 8.3 £ 103 2.7 £ 105 4.1 £ 104 5.0 £ 105 4.3 £ 104 5.3 £ 105 1.6 £ 105 5.0 £ 104 6.6 £ 104 3.3 £ 104 6.7 £ 105 2.6 £ 105
June 25, 1997
16 h
DTX-2
0.147
Dominating diatoms P. micans P. micans G. polyedra C. marina D. sacculus P. micans D. sacculus P. micans P. micans D. sacculus G. polyedra G. polyedra C. marina P. micans nd
a
HP, hepatopancreas; nd, not detected. The toxicity and the toxin composition data have been previously reported by Pavela-Vrancˇicˇ et al., 2001.
3. Results Analyses of shellfish samples collected in 1996 demonstrate a predominant presence of 7-epi-PTX-2SA at concentrations up to 1.140 mg/g hepatopancreas (HP) (Table 1). Mice treated with an extract from mussels collected on February 18, 1996 did not reveal shellfish intoxication, based on an average mouse survival time of over 24 h. Although HPLC analysis confirmed the absence of the common DSP compounds, OA and DTXs, it established shellfish contamination with a markedly high
concentration of 7-epi-PTX-2SA. A representative chromatogram of the mussel extract is shown in Fig. 1; the signal with the retention time Rt 16.342 min matches well with that of the 7-epi-PTX-2SA standard sample (Rt 16.233 min). Structural information, confirming that the structure of the identified compound corresponds to that of 7-epi-PTX-2SA, was obtained by mass spectrometric analysis. A molecular mass of 876 was determined (Fig. 2), the same as of PTX-2SAs, based on molecular mass signals at m=z 899 [M þ Na]þ (876 þ 23) and m=z 894 [M þ NH4]þ (876 þ 18). Collision-induced dissociation
Fig. 1. The HPLC profile of a shellfish sample collected from the Kasˇtela Bay (Central Adriatic Sea) on February 18, 1996. The sample was analysed with reference to an algae extract of Dinophysis acuta containing PTX-2SA (Rt 12.967 min), OA (Rt 15.484 min), 7-epi-PTX-2SA (Rt 16.233 min), and DTX-2 (Rt 16.883 min) after derivatisation with ADAM.
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Fig. 2. The mass spectrum of 7-epi-PTX-2SA. A molecular mass of 876 was calculated from molecular mass signals at m=z 899 [M þ Na]þ and m=z 894 [M þ NH4]þ.
(CID), obtained by positive and negative mass spectrometry (MS – MS), showed a fragmentation pattern characteristic of 7-epi-PTX-2SA, as previously established by James et al., (1999). Mussels from June 19, 1996 showed a greater toxicity than expected, considering that the only identified contaminant was 7-epi-PTX-2SA. Again, samples from July 29 and August 23, 1996 demonstrated no toxicity, relating to the occurrence of 7-epi-PTX-2SA as the only contaminant. In 1997, the toxin profile demonstrates the increased predominant role of toxins from the DTX toxin group. HPLC analysis of the sample from May 16, 1997 revealed the presence of DTX-2 at a concentration estimated to 0.105 mg/g HP (Fig. 3). Its identity was confirmed by comparison of the retention time (Rt 16.683 min) with that of the standard sample of DTX-2 (Rt 16.700 min). However, the chromatogram displayed another prominent peak at Rt 20.583 min, that did not match the retention time of any so far known DSP compound. Unfortunately, not enough material was available for structural analysis. The ratio of the peak area compared to that of DTX-2 suggests a significant contribution to the toxin content in the shellfish sample. Mice inoculated with an extract of this sample displayed a survival time of 7 h 10 min. On the other hand, a
survival time of 16 h was obtained with the sample from June 25, 1997 revealing only the occurrence of DTX-2 at a concentration of 0.147 mg/g HP. The same survival time was recorded with the sample from June 16, 1997, containing OA (0.133 mg/g HP), and a relatively minor amount of 7-epi-PTX-2SA. Along with shellfish sampling, seawater samples were taken for analysis of the phytoplankton community and cell abundance (Table 1). Although in the winter of 1996 diatoms dominated the phytoplankton community, the sample collected on February 18, 1996 contained P. micans at an abundance of 7.2 £ 104 cells/dm3. In June 1996, when high DSP toxicity in shellfish was measured, an elevated abundance of P. micans and G. polyedra was recorded. In the following period from July to September, P. micans, D. sacculus and Chatonella marina were the principal components of the respective phytoplankton community. In the summer of 1997 very low concentrations of D. fortii, D. caudata and D. acuta, well known progenitors of OA and its analogues, were found. However, in May of the same year, three potentially toxic species were detected in the seawater sample, D. sacculus (acuminata?), G. polyedra and P. micans. Again, P. micans was present in the phytoplankton community during the whole monitoring period. In
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Fig. 3. The HPLC profile of a shellfish sample collected from the Kasˇtela Bay (Central Adriatic Sea) on May 16, 1997, analysed with reference to a standard sample of DTX-1 (Rt 22.34 min) and DTX-2 (Rt 16.70 min) after derivatisation with ADAM.
August, the G. polyedra bloom developed to an abundance of 2.0 £ 106 cells/dm3.
4. Discussion Until 1996, the most frequently detected DSP phycotoxins in the Adriatic area were OA and DTX-1, OA being the major component. More recent analysis of phytoplankton and shellfish samples collected in European waters, revealed a frequent occurrence of intoxication with DSP compounds other than the OA group, as well as with novel DSP analogues. Analysis of the toxin profile in mussels from the central Adriatic Sea demonstrates a similar trend substantiated by the occurrence of a new and rare class of PTX compounds, the pectenotoxin-2 seco acids. Although the sample from February 28, 1996 contained a significant quantity of 7-epi-PTX-2SA, the negative mouse bioassay response is indicative of its low intraperitoneal (i.p.) toxicity. Incidents of acute human intoxication related to PTX-2SAs, accompanied by a negative mouse bioassay, were documented in 1997 in Australia confirming its oral toxicity, however no data are available on the quantity of the ingested toxin. So far there is no reliable explanation for the lack of agreement between the i.p. and oral toxicity of PTX-2SAs. The potential cause has been attributed to the open chain structure of the two stereoisomers. PTX-2SAs are closely related to PTX-2, containing an open chain carboxylic acid instead of a lactone ring. Under the influence of the metabolic processes, it may through chain closure adopt the structure exerting toxicity. Recent comparison of PTX profiles of the toxic dinoflagellate D. acuta, Greenshell mussel Perna canaliculus, and Blue
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mussel Mytilus galloprovincialis collected in New Zealand has shown that, although the major PTX analogue in D. acuta was PTX-2, both mussel cultures contained PTX-2SA as the predominant toxin (Suzuki et al., 2001). PTX-2 has never been found in shellfish, generating by bioconversion a variety of toxic analogues including the highly hepatotoxic derivatives PTX-1, 3 and 6, which are produced by oxidation at C43 in the shellfish hepatopancreas (Yasumoto et al., 1989; Zhou et al., 1994; Jung et al., 1995). The mechanism of action and the toxicological effects of pectenotoxins are not fully understood. Severe injuries of the small intestine were detected upon oral injection of mice with OA and PTX-2 (Ishige et al., 1988). PTX-1 damages the liver in the same manner as toxins from mushrooms and moulds (Terao et al., 1986; Zhou et al., 1994). Since standard samples are not commercially available for these toxins, routine methods for their determination have not been established. Hence, the presence of PTX and related compounds in the phytoplankton community may indicate a new and severe risk of shellfish contamination and consequently human poisoning. To overcome the discrepancies observed between the findings of the mouse bioassay for the sample from June 19, 1996, displaying high i.p. toxicity, and the results of the HPLC analysis, the presence of a toxin other than 7-epiPTX-2SA was hypothesized. YTX is not detectable by the respective HPLC method, and injected intraperitoneally into mice displays the highest toxicity level (lethal dose 0.100 mg/g edible tissue) (Daiguji et al., 1998). The hypothesis is strengthened by the fact that G. polyedra was found in the accompanying seawater sample at an abundance of 8.3 £ 103 cells/dm3. The first occurrence of YTX in Europe has been established in Italian mussels, following a bloom of G. polyedra in 1996 (Draisci et al., 1996). Likewise, a new analogue homoyessotoxin has been recently identified in mussels from the northern Adriatic collected during a G. polyedra bloom (Tubaro et al., 1998). In 1997 the major toxic components were OA and DTX2, however well bellow the legal tolerance level. This is the first report on the occurrence of DTX-2 in Adriatic mussels. DTX-2 is the main causative toxin of DSP intoxication in Ireland (Marr et al., 1992; Carmody et al., 1996; James et al., 1997a), however its presence has also been recorded in dinoflagellates from Spain (Blanco et al., 1995) and in shellfish from Portugal (Vale and Sampayo, 2000). The toxicity of the sample from May 16, 1997 is likely to be due in part to the presence of a DTX-related substance. An OA concentration of 0.200 mg/g mussel meat, approximately equivalent to 0.8 mg/g HP, causes mouse death within 5 h (Carmody et al., 1996). Although no toxicity data are available for DTX-2, it is expected that they could be similar to that of OA and DTX-1. Hence, the concentration of DTX2 in the sample from May 16, 1997 is not sufficient to account for the recorded toxicity. This strongly suggests that still unidentified compounds could be involved in DSP intoxication of shellfish from the central Adriatic.
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Previous episodes of DSP toxicity in the central Adriatic have been associated with G. polyedra and the Dinophysis species (Orhanovic´ et al., 1996), whereas D. fortii was found to be responsible for the production of PTX-2 in the northern Adriatic region (Lee et al., 1988; Draisci et al., 1996). In Europe, the most frequent DSP causative organism is D. acuminata, however shellfish intoxication in the Netherlands and in Scandinavia has also been associated with P. micans. D. sacculus has often been associated with D. acuminata, establishing a belief they may represent different morphotypes of the same species, the occurrence of a specific morphotype being determined by the diversity of the environmental conditions. An explanation for the relatively high winter shellfish toxicity may lie in the occurrence of various species of the Dinophysis genus (D. fortii, D. sacculus, and D. caudata ) during the autumn phytoplankton bloom, preceded by a very strong red tide bloom of G. polyedra. Concentrations as low as 200 cells/dm3 of toxic phytoplankton such as D. sacculus, D. acuta, D. caudata, and D. fortii may cause severe intoxication of shellfish (Viviani et al., 1995). In Irish waters and in Spain, D. acuta was shown to be the progenitor of DTX-2 (Blanco et al., 1995; James et al., 1997b; Draisci et al., 1998). In June 1997, when shellfish intoxication with OA and DTX-2 was registered, the phytoplankton analysis had not detected the presence of species from the Dinophysis genus (Pavela-Vrancˇicˇ et al., 2001). However, the appearance of OA and DTX-2 followed shortly after the occurrence of D. sacculus in May, implying its possible involvement as the source of DSP toxicity. The presence of multiple diarrhoeic toxins in shellfish creates complications due to lack of toxicity data for some toxins and the absence of analytical reference compounds. The frequent discovery of new and related compounds with variable substitution patterns, and the metabolic activity observed in shellfish digestive glands, shown to modify the algal toxin structure and consequently its toxicity level, requires further toxicological and chemical studies to gain knowledge on their biological and structural properties.
Acknowledgments This work was supported by grants from the Croatian Ministry of Science and Technology No. 177050 and No. 000100101.
References Blanco, J., Ferna´ndez, M., Marino, J., Reguera, B., Miguez, A., Maneiro, J., Cacho, E., Martinez, A., 1995. From Dinophysis spp. toxicity to DSP outbreaks: a preliminary model of toxin accumulation in mussels. In: Lassus, P., Arzul, G., Erard, E., Gentien, P., Marcaillou-LeBaut, C. (Eds.), Harmful Marine
Algal Blooms, Lavoisier Science Publishers, Paris, pp. 777 –782. Carmody, E.P., James, K.J., Kelly, S.S., 1996. Dinophysistoxin-2: the predominant diarrhoetic shellfish toxin in Ireland. Toxicon 34, 351–359. Daiguji, M., Sataka, M., James, K.J., Bishop, A., Makenzie, L., Naoki, H., Yasumoto, T., 1998. Structures of new pectenotoxin analogs, pectenotoxin-2 seco acid and 7-epi-pectenotoxin-2 seco acid, isolated from a dinoflagellate and greenshell mussels. Chem. Lett. 7, 653–654. Draisci, R., Lucentini, L., Giannetti, L., Boria, P., Stacchini, A., 1995. Detection of diarrhoetic shellfish toxins in mussels from Italy by ionspray liquid chromatography–mass spectrometry. Toxicon 33, 1591–1603. Draisci, R., Lucentini, L., Giannetti, L., Boria, P., Poletti, R., 1996. First report of pectenotoxin-2 (PTX-2) in algae (Dinophysis fortii ) related to seafood poisoning in Europe. Toxicon 34, 923 –935. Draisci, R., Giannetti, L., Lucentini, L., Marchiafava, C., James, K.J., Bishop, A.G., Healy, B.M., Kelly, S.S., 1998. Isolation of a new okadaic acid analogue from phytoplankton implicated in diarrhetic shellfish poisoning. J. Chromatogr. 798, 137–145. Fattorusso, E., Ciminiello, P., Costantino, V., Magno, S., Mangoni, A., Milandri, A., Poletti, R., Pompei, M., Viviani, R., 1992. Okadaic acid in mussels of Adriatic Sea. Mar. Pollut. Bull. 24, 234 –237. Ishige, M., Satoh, N., Yasumoto, T., 1988. Pathological studies on mice administered with the causative agent of diarrhetic shellfish poisoning (ocadaic acid and pectenotoxin-2). Hakkaidoritsu Eisei Kenkyushoho 18, 15 –18. James, K.J., Bishop, A.G., Gillman, M., Kelly, S.S., Roden, C., Draisci, R., Lucentini, L., Giannetti, L., Boria, P., 1997a. Liquid chromatography with fluorometric, mass spectrometric and tandem mass spectrometric detection for the investigation of the seafood-toxin-producing phytoplankton, Dinophysis acuta. J. Chromatogr. 777, 213 –221. James, K.J., Carmody, E.P., Gillman, M., Kelly, S.S., Draisci, R., Lucentini, L., Giannetti, L., 1997b. Identification of a new diarrhoetic toxin in shellfish using liquid chromatography with fluorometric and mass spectrometric detection. Toxicon 35, 973 –978. James, K.J., Bishop, A.G., Draisci, R., Palleschi, L., Marchiafava, C., Ferretti, E., Satake, M., Yasumoto, T., 1999. Liquid chromatographic methods for the isolation and identification of new pectenotoxin-2 analogues from marine phytoplankton and shellfish. J. Chromatogr. A 844, 53–65. Jung, J.F.H., Sim, C.S., Lee, C.O., 1995. Cytotoxic compounds from a two-sponge association. J. Nat. Prod. 58, 1722–1726. Lee, J.S., Tangen, K., Dahl, E., Hovgaard, P., Yasumoto, T., 1988. Diarrhoetic shellfish toxins in Norwegian mussels. Bull. Jpn. Soc. Sci. Fish. 54, 1953–1957. Marasovic´, I., Nincˇevic´, Zˇ., Pavela-Vrancˇicˇ, M., Orhanovic´, S., 1998. A survey of shellfish toxicity in the central Adriatic Sea. J. Mar. Biol. Assoc., UK 78, 745– 754. Marr, J.C., Hu, T., Pleasance, S., Quilliam, M.A., Wright, J.L.C., 1992. Detection of new 7-O-acyl derivatives of diarrhetic shellfish poisoning toxins by liquid chromatography–mass spectrometry. Toxicon 30, 1621–1630. Orhanovic´, S., Nincˇevic´, Zˇ., Marasovic´, I., Pavela-Vrancˇicˇ, M., 1996. Phytoplankton toxins in the central Adriatic Sea. Croat. Chem. Acta 69, 291–303.
M. Pavela-Vrancˇicˇ et al. / Toxicon 40 (2002) 1601–1607 Pavela-Vrancˇicˇ, M., Mesˇtrovic´, V., Marasovic´, I., Gillman, M., Furey, A., James, K.J., 2001. The occurrence of 7-epipectenotoxin-2 seco acid in the coastal waters of the Central Adriatic (Kasˇtela Bay). Toxicon 39, 771–779. Ramstad, H., Hovgaard, P., Yasumoto, T., Larsen, S., Aune, T., 2001. Monthly variations in diarrhetic toxins and yessotoxins in shellfish from the coast to the inner part of Sognefjord, Norway. Toxicon 39, 1035–1043. Suzuki, T., Mackenzie, L., Stirling, D., Adamson, J., 2001. Pectenotoxin-2 seco acid: a toxin converted from pectenotoxin-2 by the New Zealand Greenshell mussel, Perna caniculus. Toxicon 39, 507 –514. Terao, K., Ito, E., Yanagi, T., Yasumoto, T., 1986. Histopathological studies on experimental marine toxin poisoning. 1. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin-1 and pectenotoxin1. Toxicon 24, 1141–1151. Tubaro, A., Sidari, L., Della Loggia, R., Yasumoto, T., 1998. Occurrence of yessotoxin-like toxins in phytoplankton and mussels from northern Adriatic Sea. In: Reguera, B., Blanco, J., Ferna´ndez, M.L., Wyatt, T. (Eds.), Harmful Algae, Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, pp. 470–472.
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Utermo¨hl, H., 1958. Zur Vervollkommung der Quantitativen Phytoplankton Methodik. “Mitteilungen-Communications” Internationale Vereinigung fu¨r Theoretische und Angewandte Limnologie 9, 1–38. Vale, P., Sampayo, M.A., 2000. Dinophysistoxin-2: a rare diarrhoeic toxin associated with Dinophysis acuta. Toxicon 38, 1599–1606. Viviani, R., Boni, L., Cattani, O., Milandri, A., Polleti, R., Pompei, M., 1995. ASP, DSP, NSP and PSP monitoring in mucilaginous aggregates and in mussels in a coastal area of the Northern Adriatic Sea facing Emilia-Romagna in 1988, 1989 and 1991. Sci. Total. Environ. 165, 203. Yasumoto, T., Murata, M., Oshima, Y., Sano, M., Matsumoto, G.K., Clardy, J., 1985. Diarrhetic shellfish toxins. Tetrahedron 41, 1019–1025. Yasumoto, T., Murata, M., Lee, J.S., Torigoe, K., 1989. Polyether toxins produced by dinoflagellates. In: Natori, S., Hashimoto, K., Ueno, T. (Eds.), Mycrotoxins and phycotoxins’88, Elsevier, Amsterdam, pp. 375–382. Zhou, Z.H., Komiyana, M.K., Teran, K., Shimada, Y., 1994. Effects of pectenotoxin-1 on liver cells in vitro. Nat. Toxins 2, 132 –135.