DSP COMPLEX TOXIN PROFILES RELATION WITH DINOPHYSIS SPP. OCCURRENCE AND DOMOIC. ACID CONFIRMATION BY LC-MS IN PORTUGUESE ...
503 DSP COMPLEX TOXIN PROFILES RELATION WITH DINOPHYSIS SPP. OCCURRENCE AND DOMOIC ACID CONFIRMATION BY LC-MS IN PORTUGUESE BIVALVES P. Vale*, M.A. de M. Sampayo* and M.A. Quilliam** * Instituto de Investigação das Pescas e do Mar, Av. Brasília, 1400-Lisboa, Portugal **Institute for Marine Biosciences, National Research Council, 1411 Oxford St., Halifax, N.S. B3H 3Z1, Canada. ABSTRACT While the occurrence of OA has been reported worldwide, the occurrence of DTX2 has until now been reported only in mussels from Ireland and the Atlantic coast of the Iberian Peninsula. Our findings here reported suggest that Dinophysis acuta is the main responsible for DTX2 contamination of mussels from the Portuguese coast, while D. acuminata mainly accounts for OA contamination. ASP monitoring was introduced on a regular basis in 1996. Domoic acid (DA) contamination was detected in 1996 and 1997, occurring in low levels in a variety of species and with a wide temporal and geographical dispersion. A few values close to but less than the 20 µg/g level were found. Confirmation of DA in a sample harvested in 1995 was done by LC-MS. The presence of isodomoic acids D, E and A was also determined by LCMS and LC with gradient elution and diode array detection. INTRODUCTION In 1994 the HPLC method of Lee et al. [1] was introduced and applied to all the samples of our DSP monitoring program for confirming okadaic acid and analogues presence and determining toxin profiles. It was possible to identify the presence of two diarrhetic toxins: okadaic acid (OA) and dinophysistoxin-2 (DTX2) [2]. The pursuing of the researches led to the identification of acyl derivatives of both OA and DTX2 [3]. The confirmation of all these toxins was done by LC-MS (manuscript in preparation). The chemical results from 1995 and 1996 have been compared with those from the phytoplankton survey and the mouse bioassay (MBA) and reported here. Following a preliminary study carried out in 1995 that detected the presence of domoic acid (DA) in Portuguese shellfish [3], amnesic shellfish poisoning (ASP) monitoring was introduced on a regular basis in May 1996. Since 1986 there is in Portugal a program for monitoring bivalve molluscs for the presence of paralytic and diarrhetic shellfish poisoning toxins (PSP and DSP) using mouse bioassays. Based on this pre-existent monitoring program, the shellfish extracts tested for PSP toxins were also screened for domoic acid on a weekly or fortnightly basis. The AOAC first action procedure [4], based on acid extraction and liquid chromatography (LC) with UV absorbance detection, was chosen because it
provides an extract that is also suitable for simultaneous testing of PSP toxins. MATERIALS & METHODS DSP MBA were carried out according to the modifications proposed by Le Baut et al. [5,6], which includes after the acetone extraction a resuspension of the extract in methanol and a hexane washing step that prevents interference from the excess of certain free fatty acids that can be lethal to mice. HPLC analysis for OA and DTX2 was performed as described by Lee et al. [1] with minor modifications, using 1g of the same hepatopancreas mixture prepared for the MBA. ASP DA analysis was carried out according to [4] with the main modifications cited below. The acid extracts for PSP determination were prepared according to [7], but scaled down to use only 50 g of meat and a final pH adjustment of 2.0. After centrifugation the supernatants were kept in 20 mL bottles at -20 ºC until further processing. Immediately before the LC analysis each extract was thawed and a 250 µL aliquot was diluted with 750 µL of aqueous ammonia 0.05 M in Eppendorf tubes, and centrifuged at 15,000 x g for 10 min. The supernatants were filtered into autosampler vials using a 0.22 µm nylon disposable syringe filter. Samples where some turbidity still remained (mostly oyster extracts), were first passed through a 0.45 µm filter into a test tube, and transferred to the vial through a 0.22 µm filter in order to better protect the column. The equivalent of 1.25 mg extract (10 µL, 1:800 dilution) were injected on the column. The LC-MS and LC-DAD gradient elution analyses for DA were performed as described by Quilliam et al. [8,9] on a sample of smooth callista (Callista chione) harvested at Setúbal (Atlantic coast) in June 1995 and extracted with aqueous 50 % methanol. RESULTS DSP In one semi-closed area, Aveiro Ria, where mussel sampling is quite regular, generally on a weekly (fortnightly sometimes) basis, and the presence of toxins is recurrent, it was possible not just to relate toxicity with the normally implicated microalgae, but also to have a relation of toxin profile variation associated with phytoplankton succession. The species implicated in DSP in Portugal are mainly Dinophysis acuminata and D. acuta, and these both occur often at Aveiro Ria. In the
Harmful Algae Reguera, B., Blanco, J., Fernández, M.L. and Wyatt, T. Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO 1998
504
10
5000
AVEIRO RIA - 1995 Toxicity in blue mussel (Mytilus edulis )
9
8
DTX2
4500
OA D. acuminata
4000
10
5000
AVEIRO RIA - 1996 Toxicity in blue mussel (Mytilus edulis )
8
DTX2
4500
OA 4000
D. acuminata D. acuta
7
3500
17-dez
03-dez
18-nov
04-nov
22-out
08-out
24-set
09-set
0 27-ago
0 06-ago
500
22-jul
1
08-jul
1000
24-jun
2
03-jun
1500
20-mai
3
06-mai
2000
08-abr
4
11-mar
2500
12-fev
5
15-jan
3000
03-jan
6
cells/L
9
g/g hepatopâncreas
cells/L
12-dez
28-nov
13-nov
30-out
17-out
0 04-out
0 18-set
500
06-set
1
25-ago
1000
09-ago
2
25-jul
1500
12-jul
3
26-jun
2000
05-jun
4
25-mai
2500
07-mai
5
10-abr
3000
20-mar
6
01-fev
3500
02-jan
g/g hepatopâncreas
D. acuta 7
Fig. 1. Evolution of the annual content of DSP toxins in the Aveiro Ria blue mussel in 1995 and 1996, superposed with the annual variation of two Dinophysys species. The presence of D. acuta does seem to be responsible for contamination of mussels with DTX-2. These graphs also ilustrate the large difference between a very DSP toxic year (1995) with a mild toxic one (1996).
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ASP Domoic acid was detected again in Portuguese bivalve molluscs in 1996 and 1997. DA contamination occurred at low levels in a variety of species and with a wide temporal and geographical dispersion. Up until now, this toxin has been detected in shellfish from the south coast (Algarve) and the Northwest and Southwest coast of Lisbon. As to the temporal distribution, it has been found in several months of the year related to Pseudo-Nitszchia spp. blooms (Fig. 2). A few values around the 20 µg/g level were found. However, there were never levels of contamination that required the stopping of harvesting of any bivalve. Almost half of the screened samples were mussels, the other half consisted of clams, razor clams, cockles, oysters, etc. When in one area DA was detected for the first time and mussels were monitored simultaneously with other shellfish from the same area, the other species contained more domoic acid than mussels. In the next week mussels had cleared this toxin faster than the other species (Tables I and II).
Table I. Discrepancies found between domoic acid content of three different species harvested 2 weeks apart at Mondego Estuary. Mussels (M. edulis) present the lowest content. Values are in µg/g. Species
25-March-97
8-April-97
0.9 2.0 10.4
n.d.* 1.5 1.5
Mytilus edulis Cerastoderma edule Scrobicularia plana * n.d. = non detectable.
Table II. Similarities found between domoic acid content of three mussels samples harvested at different locations of Aveiro Ria. For comparison are the contents of the common cockle (C. edule) from the same area, that presents the double of DA. Values are in µg/g. Species
Location
Mytilus edulis Mytilus edulis Mytilus edulis Cerastoderma edule
São Jacinto Costa Nova Triângulo R. Rebocho
24-Feb.-97
4-March97
1.7 1.7 1.6 3.7
n.a.* 0.3 0.2 0.9
* n.a. = not available
2 Absorvance at 242nm (mAU)
1995 DSP outbreaks the first seemed to be responsible for okadaic acid contamination in mussels and, the second, with occurrence of DTX2. In 1995 there was first a bloom of D. acuminata in late spring and OA was then the dominant toxin (Fig. 1). Later in the summer, D. acuta bloomed and DTX2 became the dominant toxin. In 1996, when only D. acuminata bloomed, the mussels were contaminated only with okadaic acid (Fig.1). The LC results from 1995 and 1996 were again compared with the MBA. Levels of OA+DTX2 > 2 µg/g corresponded to mouse survival times inferior to 5 hours, our safety criteria by MBA, like reported previously for 1994 [2]. However there were several cases where measured OA+DTX2 levels could not account for the 5 h mouse survival limit. In 1996 this problem was very persistent in the south coast at Sagres. Also Dinophysis counts throughout the coast that year were low; 1996 was in overall a characteristically "non DSP-toxic" year, in contrast with 1994 [2] and 1995 (Fig. 1, and data not shown). In 1997 this problem become more notorious, starting very early in the year, while normally DSP episodes start in late spring. When partitioning between aqueous methanol and chloroform was carried out toxic compounds were found on both phases. A wide span of mouse survival times and toxic bivalve species have been found. Mouse symptoms were neurologic and can be classified in several apparently distinct groups representing different toxins.
domoic acid
1
(TRP)
0 0
4
8
12
Time (min)
Care has been taken to analyse the AOAC extracts within no longer than a week to prevent extensive DA decomposition in acidic media that was observed to occur despite the extracts being kept frozen. Generally all the samples that arrived at the laboratory during the week were analysed altogether at the end of that week or immediately at the beginning of the next.
Fig. 2. LC chromatogram of a phytoplankton sample from Costa Nova location in Aveiro Lagoon (collected in 20/May/97). Pseudo-nitzschia spp. concentration was 3x105 cells/L and DA was found at 149 ng/L level.
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Domoic Acid
Isodomoic Acids D E A
0
5
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Time (min)
Fig. 3. LC-MS analysis of Callista chione. The separation was performed on a 5 µm Vydac 201TP column (250 x 2.1 mm) using 0.2 mL/min of 10% acetonitrile with 0.1% TFA as mobile phase. Ion-spray ionization and selected ion monitoring of 3 ions (m/z 312 (M+H), 266 (M+H-HCOOH), and 220 (M+H-2HCOOH)) was used. The trace shows the sum of the three signals. Ammonia was used to raise the pH for better efficiency of the derivatization of PSP toxins with periodic acid under alkaline conditions, by the highly sensitive precolumn derivatization method of Lawrence and Menard [10]. The use of a common HPLC sample preparation procedure for ASP and PSP toxins (and also PSP by MBA) is very practical, for the laboratory work, and it is also an economical solution. One shellfish (Callista chione) harvested in 1995 and contaminated with DA at a 30 µg/g level was checked by liquid chromatography-mass spectroscopy (LC-MS). This technique allowed the confirmation of DA and isodomoic acids D, E and A (Fig. 2). LC gradient elution with diode array detection (LC-DAD) analyses was also used to confirm these results (chromatogram not shown). DISCUSSION While the occurrence of OA has now been almost reported world-wide, the occurrence of DTX-2 has been until now reported only in mussels from Ireland and the Atlantic coast of the Iberian Peninsula. Although Dinophysis acuminata and D. acuta are the species responsible for DSP in these areas, only D. acuta has been implicated with shellfish contamination with high levels of DTX2 in Ireland [11] and Spain [12]. This is in accordance with our findings reported here. The toxin
profiles of the acetonic extracts described up until now do not still account for all the toxicity found with the MBA. Pursuing of research is needed to fully understand the implications of MBA results for risk assessment of consumer's safety. The detection of DA in Portuguese bivalves again in 1996 and 1997 justifies the need to have a monitoring program for ASP. Although mussels are often appointed as sentinel species for marine toxins, care should be taken with other species of commercial importance in regard to DA accumulation. We have found that from the same area they can present higher values for this toxin than mussels. REFERENCES 1. Lee, J.S., Yanagi, T., Kenma, R. & Yasumoto, T. (1987) Agric. Biol. Chem., 51: 877-881 2. Vale, P. and Sampayo, M.A.M. (1996) In: Yasumoto, T., Oshima, Y. and Fukuyo, Y. (Eds) Harmful and Toxic Algal Blooms. I.O.C. of UNESCO: 539-542. 3. Vale, P. and Sampayo, M.A.M. (1996) "IX International IUPAC Symposium on Mycotoxins and Phycotoxins". Abstract Book. pp. 274. 4. Lawrence, J.F.; Charbonneau, C.F., and Ménard, C. (1991) J. Assoc. Off. Anal. Chem., 74, 68-72. 5. Le Baut, C., Bardin, B., Bardouil, M., Bohec, M., Masselin, P. & Truquet, P. (1990) Rapport IFREMER DERO-90-02 MR, 21 pp. 6. Marcaillou-Le Baut, C., Lucas, D. and Le Dean, L. (1985) In: Anderson, D.M., White, A.W. and Baden, D.G. (eds) Toxic Dinoflagellates. Elsevier, New York: 485-488. 7. Official Methods of analysis (1984) 14th Ed., AOAC, Arlington, VA, secs. 18.086-18.092; (1990) 15th Ed., 959.08. 8. Quilliam, M.A., Xie, M. and Hardstaff, W.R. (1995) J. AOAC Internat., 78 (2), 543-554. 9. Quilliam, M.A., Sim, P.G., McCulloch, A.W. and McInnes A.G. (1989) Intern. J. Environ. Anal. Chem., 36, 139-154. 10.Lawrence, J.F., Ménard, C. and Cleroux, C. (1995) J. AOAC Internat., 78 (2), 514-520. 11.Carmody, E.P., James, K.J. and Kelly, S.S. (1996) Toxicon, 34 (3), 351-359. 12. Blanco, J., Fernández, M., Mariño, J., Reguera, B., Miguez, A., Maneiro, J., Cacho, E. & Martínez, A. (1995) In: Lassus, P., Arzul, G., Erard, E., Gentien, P., & Marcaillou, C. (eds.), Harmuful Marine Algal Blooms. Lavoisier Science Publishers, Paris: 777-782. ACKNOWLEDGEMENTS This work was partially supported by JNICT with grants nº PBIC/C/MAR/1292/95 and BIC-854, and by IPIMAR through the Programme PROPESCAS.
