Toxicon Vol. 30, No. 5/6, pp. 673-676, 1992. Printed in Great Britain.
00414)101/92 $5.00+ .00 © 1992 Pergamon Press Ltd
D I A R R H O E I C SHELLFISH TOXINS IN A D R I A T I C SEA M U S S E L S E V A L U A T E D BY A N ELISA M E T H O D AURELIA TUBARO,I SILVIOSOSA, 1 MILENA BRUNO,2 PAOLA MARGHERITA BIANCA GUCCI,2 LAURA VOLTERRA2 and ROBERTODELLA LOGGIAt* qnstitute of Pharmacologyand Pharmacognosy,Universityof Trieste, Via A. Valerio 32, 34100 Trieste, Italy; and 2Departmentof EnvironmentalHygiene,Istituto Superioredi Sanitfi, Viale Regina Elena 299, 00161 Rome, Italy
(Received 28 August 1991;accepted 6 December 1991) A. TUBARO, S. SOSA, M. BRUNO, P. M. B. GUCCt, L. VOLTERRAand R. DELLA LOGGIA. Diarrhoeic shellfish toxins in Adriatic Sea mussels evaluated by an ELISA method. Toxicon 30, 673-676, 1992.--A competitive enzyme immunoassay (ELISA) was used to determine the presence of okadaic acid (OA) and/or dinophysistoxin-1 (DTX-1) in mussels. The accuracy and sensitivity of the ELISA method has been checked. The sensitivity of the method (100 ng/g of hepatopancreas) makes it possible to determine OA concentrations ten times lower than the tolerance limits established by the Health Authorities of many countries. For the first time, OA and/or DTX-1 were detected in mussels (Mytilus galloprovincialis) collected in different stations along the Adriatic Sea coasts. DIARRHOEIC shellfish poisoning (DSP) is a gastroenteritis caused by ingestion of shellfish contaminated by toxin-producing organisms of the Dinophlagellatae order. Okadaic acid (OA) has been reported to be the principal DSP toxin of European mussels, growing along the Atlantic and North Sea coasts (KUMAGAIet al., 1986). In 1985 about 400 cases of DSP were registered as a consequence of shellfish consumption along the French Mediterranean coast (VOLTERRAet al., 1990); in 1989 cases of diarrhoea were reported in Italy and were related to mussels collected along the Italian coast of the upper and middle Adriatic Sea (MOLTERRAet al., 1990). The development of the pathology and the presence of dinoflagellates belonging to the genus Dinophysis (VOLTERRA e t al., 1990) suggested DSP. A monitoring programme over the Italian shellfish banks started in 1989 using the Yasumoto's mouse bioassay (YASUMOTOel al., 1978). A death time of 5 hr was selected as a criterium for shellfish interdiction. This limit provoked long banning periods and complaints from fishermen in 1989 and 1990. A question arose about the specificity of Yasumoto's bioassay, which could be influenced also by unsaturated fatty acids (HAMANO et al., 1985). The aim of the present work was to verify whether okadaic acid is present in mussels of the Adriatic Sea and whether it may be involved in the observed effects on human health. *Author to whom correspondenceshould be addressed. 673
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TABLE 1. OKADAIC ACID (OA) IN ADRIATIC SEA MUSSELS(M. galloprovincialis) Sample
Sampling site
Sampling date
OA*
1 2
Muggia (Trieste) Grignano (Trieste)
August 1990 October 1990
0.37 + 0.04 0.69+0.05
3 4 5 6 7
Duino (Trieste) Passetto (Rovigo) Porto Garibaldi (Ferrara) Cesenatico (Forli) Fermo (Ascoli Piceno)
December 1990 July 1990 July 1990 March 1990 November1990
< 0.10 0.33+0.04 0.56+0.05 0.23_ 0.02 0.87+0.05
*Mixture of OA+dinophysistoxin-I expressed as #g of OA per g of hepatopancreas
(mean + S.D. of three analyses). Since more than 50% of toxins concentrate in the digestive glands of shellfish (YAsuMOTO et al., 1978; EDEBO et al., 1988a), the hepatopancreas of mussels (Mytilus galloprovincialis) collected in seven different stations along the Adriatic Sea coasts were examined. The toxin contents of the hepatopancreas were measured with a commercially available competitive enzyme immunoassay kit ('DSP-Check' by UBE Industries Ltd, Tokyo, Japan), following the procedure described by UDA et aL (1989). The method is responsive both to OA and to dinophysistoxin-1 (DTX-1) (USAGAWAe t al., 1989), but the separate determination of the two toxins appears to be of little importance, since they possess similar toxicity (HAMANO et al., 1985; LEE et al., 1987). Therefore, the concentrations of the toxins found in mussels are cumulatively expressed as/~g of OA per g of hepatopancreas (#g/g). The range of linearity of the response, the recovery of the toxin and the reproducibility of the measures were checked using toxin-free samples added with known amounts of OA (Sigma Chemicals Co., MO, U.S.A.). The calibration curve for OA was linear (r -- 0.995) in the range 0.01-0.10 #g/ml, corresponding to 0. l-1.0 pg/g of OA in the hepatopancreas. Within the linearity range of the method, the recovery was almost complete, ranging from 99-109%. The reproducibility of measures depended on the OA content of the samples. Using data from four separate extractions of the same sample, a coefficient of variation (CV) of 20% was found for 0.1 #g/g of OA. The CV values for 0.40 and 0.70 #g/g were 14% and 7%, respectively. Table 1 reports the amount of OA found in the samples of mussels from the seven stations, together with the date o f collection. The reported values represent the m e a n _ S.D. of three separate determinations, each made in duplicate. Okadaic acid was detected in most of the tested samples, one of them (no. 3) being below the sensitivity limit o f the method. The lowest detectable amount of toxins was 0.10 #g/g, which represents a sensitivity four times higher than that of the H P L C method (LEE et al., 1987). It should be noted that the tolerance levels officially adopted in various countries such as Japan, France, Norway and Sweden correspond to about 1 #g/g of OA or more. The consumption of 200 g of edible tissues of mussels (a large meal) containing 0.87 #g of toxins per g of hepatopancreas (the highest toxins concentration measured) represents a total intake of about 26 #g of toxins, since the toxin accumulates in the hepatopancreas, which represents about 15% of the edible tissues of mussels. From the available epidemiological data it has been estimated that at least 40-60 #g o f toxins is needed to induce gastro-intestinal symptoms in adult humans (YASUMOTO et al., 1984; UNDERDAL et al., 1985). None of the tested samples showed a concentration of toxins sufficient to cause gastro-intestinal effects in
Short Communications
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humans and all values found were lower than the official tolerance limits established by the Health Authorities of various countries. Therefore, all o f the examined mussels could be considered safe. Although the method does not require clean-up steps, the preparation of samples is critical, since recovery and reproducibility are linked to the extraction procedure. The homogenization of mussel hepatopancreas by Ultra-Turrax in the presence of 90% aqueous methanol gives satisfactory results, in agreement with the observations of other authors (EDEBO et al., 1988b). The time interval between the preparation of the sample and the E L I S A assay is also critical: if samples are tested again l hr after the extraction procedure, a decrease of 25% in OA concentration is observed, compared with the values obtained immediately after extraction. This can be due to an oxidative alteration of the OA in the presence of methanol and/or to the progressive absorption of the toxin onto/into the colloids that slowly separate from the solution. Our data demonstrate, for the first time to our knowledge, the presence of OA and/or DTX-1 in M y t i l u s galloprovincialis, the mussels o f the Adriatic Sea. In none of the analyzed samples were the OA + DTX-1 titres dangerous to human health, whereas most of them were positive in the Yasumoto bioassay in mice (VOLTERRA, personal communication). It has to be noted that the mouse bioassay demonstrates not only the diarrhoegenic toxins (OA and derivatives), but also other lipophilic compounds that are toxic for mice after i.p. administration, such as other toxins (yessotoxins, pectenotoxins) and certain fatty acids (HAMANO et al., 1985). It remains to be shown whether these substances are present in the Adriatic Sea mussels and whether they represent a hazard for humans, after oral consumption. The evaluation of the safety of the mussels by means of chemical analysis will be possible only after any hypothesized new toxins have been isolated and suitable methods are available for their determination, together with appropriate standards.
REFERENCES EDEBO, L., LANGE, S., LI, X. P. and ALLENMARK,S. (1988a) Toxic mussels and okadaic acid induce rapid hypersecretion in the rat small intestine. Acta path. microbiol, immun, scand. 96, 1029-1035. EDEBO,L., LANGE,S., LI, X. P., ALLENMARK,S., LINDGREN,K. and THOMPSON,R. (1988b) Seasonal, geographic and individual variation of okadaic acid content in cultivated mussels in Sweden. Acta path. microbiol, immun. scand. 96, 1036-1042. HAMANO,Y., KINOSHITA,Y. and YASUMOTO,T. (1985) Suckling mice assay for diarrhetic shellfish toxins. In: Toxic Dinoflagellates, pp. 383-388 (ANDERSON,D. M., WHITE,A. W. and BADEN,D.G., Eds). New York: Elsevier Science. KUMAGAI,M., YANAGI,T., MURATA,M., YASUMOTO,T., KAT,M., LASSUS,P. and RODRIGUEZ-VAZQUEZ,J. A. (1986) Okadaic acid as the causative toxin of diarrhetic shellfish poisoning in Europe. Agric. biol. Chem. 50, 2853-2857. LEE,J. S., YANAGI,T., KENMA,R. and YASUMOTO,T. (1987) Fluorometric determination of diarrhetic shellfish toxins by high performance liquid chromatography. Agric. biol. Chem. 51,877-881. UDA,T., ITOH,Y., NISHIMURA,m., USAGAWA,T., MURATA,i . and YASUMOTO,T. (1989) Enzymeimmunoassay using monoclonal antibody specific for diarrhetic shellfish poisons. In: Mycotoxins and Phycotoxins "88, pp. 335-342 (NATORI,S., HASHIMOTO,K. and UENO,Y., Eds). Amsterdam: Elsevier Science. UNDERDAL, B., YNDESTAD, i . and AUNE, T. (1985) DSP Intoxication in Norway and Sweden, Autumn 1984-Spring 1985. In: Toxic Dinoflagellates, pp. 489-494 (ANDERSON,D. M., WHITE, A. W. and BADEN, D. G., Eds). New York: Elsevier Science. USAGAWA,T., NISHIMURA,i . , ITOH, Y., UDA, T. and YASUMOTO,T. (1989) Preparation of monoclonal antibodies against okadaic acid prepared from the sponge Halichondria okadai. Toxicon 27, 1323-1330. VOLTERRA,L., Guccl, P. i . B. and BRUNO, i . (1990) Problemi tecnici connessi con il fenomeno delle DSP (diarrhoeic shellfish poisoning). Ambiente Risorse Salute 5, 12-17.
676
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YASUMOTO, T., OSmMA, Y. and YAMAGUCm, M. (1978) Occurrence of a new type of shellfish poisoning in the Tohoku district. Bull. Jpn. Soc. scient. Fish. 44, 1249-1255. YASUMOTO, T., MURATA, M., OSHIMA, Y., MATSUMOTO, G. K. and CLARDY, J. (1984) Diarrhetic shellfish poisoning. A.C.S. Syrup. S. 262, 207-214.
00414)101/92 $5.00+ .00 © 1992 Pergamon Press Ltd
D I A R R H O E I C SHELLFISH TOXINS IN A D R I A T I C SEA M U S S E L S E V A L U A T E D BY A N ELISA M E T H O D AURELIA TUBARO,I SILVIOSOSA, 1 MILENA BRUNO,2 PAOLA MARGHERITA BIANCA GUCCI,2 LAURA VOLTERRA2 and ROBERTODELLA LOGGIAt* qnstitute of Pharmacologyand Pharmacognosy,Universityof Trieste, Via A. Valerio 32, 34100 Trieste, Italy; and 2Departmentof EnvironmentalHygiene,Istituto Superioredi Sanitfi, Viale Regina Elena 299, 00161 Rome, Italy
(Received 28 August 1991;accepted 6 December 1991) A. TUBARO, S. SOSA, M. BRUNO, P. M. B. GUCCt, L. VOLTERRAand R. DELLA LOGGIA. Diarrhoeic shellfish toxins in Adriatic Sea mussels evaluated by an ELISA method. Toxicon 30, 673-676, 1992.--A competitive enzyme immunoassay (ELISA) was used to determine the presence of okadaic acid (OA) and/or dinophysistoxin-1 (DTX-1) in mussels. The accuracy and sensitivity of the ELISA method has been checked. The sensitivity of the method (100 ng/g of hepatopancreas) makes it possible to determine OA concentrations ten times lower than the tolerance limits established by the Health Authorities of many countries. For the first time, OA and/or DTX-1 were detected in mussels (Mytilus galloprovincialis) collected in different stations along the Adriatic Sea coasts. DIARRHOEIC shellfish poisoning (DSP) is a gastroenteritis caused by ingestion of shellfish contaminated by toxin-producing organisms of the Dinophlagellatae order. Okadaic acid (OA) has been reported to be the principal DSP toxin of European mussels, growing along the Atlantic and North Sea coasts (KUMAGAIet al., 1986). In 1985 about 400 cases of DSP were registered as a consequence of shellfish consumption along the French Mediterranean coast (VOLTERRAet al., 1990); in 1989 cases of diarrhoea were reported in Italy and were related to mussels collected along the Italian coast of the upper and middle Adriatic Sea (MOLTERRAet al., 1990). The development of the pathology and the presence of dinoflagellates belonging to the genus Dinophysis (VOLTERRA e t al., 1990) suggested DSP. A monitoring programme over the Italian shellfish banks started in 1989 using the Yasumoto's mouse bioassay (YASUMOTOel al., 1978). A death time of 5 hr was selected as a criterium for shellfish interdiction. This limit provoked long banning periods and complaints from fishermen in 1989 and 1990. A question arose about the specificity of Yasumoto's bioassay, which could be influenced also by unsaturated fatty acids (HAMANO et al., 1985). The aim of the present work was to verify whether okadaic acid is present in mussels of the Adriatic Sea and whether it may be involved in the observed effects on human health. *Author to whom correspondenceshould be addressed. 673
Short Communications
674
TABLE 1. OKADAIC ACID (OA) IN ADRIATIC SEA MUSSELS(M. galloprovincialis) Sample
Sampling site
Sampling date
OA*
1 2
Muggia (Trieste) Grignano (Trieste)
August 1990 October 1990
0.37 + 0.04 0.69+0.05
3 4 5 6 7
Duino (Trieste) Passetto (Rovigo) Porto Garibaldi (Ferrara) Cesenatico (Forli) Fermo (Ascoli Piceno)
December 1990 July 1990 July 1990 March 1990 November1990
< 0.10 0.33+0.04 0.56+0.05 0.23_ 0.02 0.87+0.05
*Mixture of OA+dinophysistoxin-I expressed as #g of OA per g of hepatopancreas
(mean + S.D. of three analyses). Since more than 50% of toxins concentrate in the digestive glands of shellfish (YAsuMOTO et al., 1978; EDEBO et al., 1988a), the hepatopancreas of mussels (Mytilus galloprovincialis) collected in seven different stations along the Adriatic Sea coasts were examined. The toxin contents of the hepatopancreas were measured with a commercially available competitive enzyme immunoassay kit ('DSP-Check' by UBE Industries Ltd, Tokyo, Japan), following the procedure described by UDA et aL (1989). The method is responsive both to OA and to dinophysistoxin-1 (DTX-1) (USAGAWAe t al., 1989), but the separate determination of the two toxins appears to be of little importance, since they possess similar toxicity (HAMANO et al., 1985; LEE et al., 1987). Therefore, the concentrations of the toxins found in mussels are cumulatively expressed as/~g of OA per g of hepatopancreas (#g/g). The range of linearity of the response, the recovery of the toxin and the reproducibility of the measures were checked using toxin-free samples added with known amounts of OA (Sigma Chemicals Co., MO, U.S.A.). The calibration curve for OA was linear (r -- 0.995) in the range 0.01-0.10 #g/ml, corresponding to 0. l-1.0 pg/g of OA in the hepatopancreas. Within the linearity range of the method, the recovery was almost complete, ranging from 99-109%. The reproducibility of measures depended on the OA content of the samples. Using data from four separate extractions of the same sample, a coefficient of variation (CV) of 20% was found for 0.1 #g/g of OA. The CV values for 0.40 and 0.70 #g/g were 14% and 7%, respectively. Table 1 reports the amount of OA found in the samples of mussels from the seven stations, together with the date o f collection. The reported values represent the m e a n _ S.D. of three separate determinations, each made in duplicate. Okadaic acid was detected in most of the tested samples, one of them (no. 3) being below the sensitivity limit o f the method. The lowest detectable amount of toxins was 0.10 #g/g, which represents a sensitivity four times higher than that of the H P L C method (LEE et al., 1987). It should be noted that the tolerance levels officially adopted in various countries such as Japan, France, Norway and Sweden correspond to about 1 #g/g of OA or more. The consumption of 200 g of edible tissues of mussels (a large meal) containing 0.87 #g of toxins per g of hepatopancreas (the highest toxins concentration measured) represents a total intake of about 26 #g of toxins, since the toxin accumulates in the hepatopancreas, which represents about 15% of the edible tissues of mussels. From the available epidemiological data it has been estimated that at least 40-60 #g o f toxins is needed to induce gastro-intestinal symptoms in adult humans (YASUMOTO et al., 1984; UNDERDAL et al., 1985). None of the tested samples showed a concentration of toxins sufficient to cause gastro-intestinal effects in
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humans and all values found were lower than the official tolerance limits established by the Health Authorities of various countries. Therefore, all o f the examined mussels could be considered safe. Although the method does not require clean-up steps, the preparation of samples is critical, since recovery and reproducibility are linked to the extraction procedure. The homogenization of mussel hepatopancreas by Ultra-Turrax in the presence of 90% aqueous methanol gives satisfactory results, in agreement with the observations of other authors (EDEBO et al., 1988b). The time interval between the preparation of the sample and the E L I S A assay is also critical: if samples are tested again l hr after the extraction procedure, a decrease of 25% in OA concentration is observed, compared with the values obtained immediately after extraction. This can be due to an oxidative alteration of the OA in the presence of methanol and/or to the progressive absorption of the toxin onto/into the colloids that slowly separate from the solution. Our data demonstrate, for the first time to our knowledge, the presence of OA and/or DTX-1 in M y t i l u s galloprovincialis, the mussels o f the Adriatic Sea. In none of the analyzed samples were the OA + DTX-1 titres dangerous to human health, whereas most of them were positive in the Yasumoto bioassay in mice (VOLTERRA, personal communication). It has to be noted that the mouse bioassay demonstrates not only the diarrhoegenic toxins (OA and derivatives), but also other lipophilic compounds that are toxic for mice after i.p. administration, such as other toxins (yessotoxins, pectenotoxins) and certain fatty acids (HAMANO et al., 1985). It remains to be shown whether these substances are present in the Adriatic Sea mussels and whether they represent a hazard for humans, after oral consumption. The evaluation of the safety of the mussels by means of chemical analysis will be possible only after any hypothesized new toxins have been isolated and suitable methods are available for their determination, together with appropriate standards.
REFERENCES EDEBO, L., LANGE, S., LI, X. P. and ALLENMARK,S. (1988a) Toxic mussels and okadaic acid induce rapid hypersecretion in the rat small intestine. Acta path. microbiol, immun, scand. 96, 1029-1035. EDEBO,L., LANGE,S., LI, X. P., ALLENMARK,S., LINDGREN,K. and THOMPSON,R. (1988b) Seasonal, geographic and individual variation of okadaic acid content in cultivated mussels in Sweden. Acta path. microbiol, immun. scand. 96, 1036-1042. HAMANO,Y., KINOSHITA,Y. and YASUMOTO,T. (1985) Suckling mice assay for diarrhetic shellfish toxins. In: Toxic Dinoflagellates, pp. 383-388 (ANDERSON,D. M., WHITE,A. W. and BADEN,D.G., Eds). New York: Elsevier Science. KUMAGAI,M., YANAGI,T., MURATA,M., YASUMOTO,T., KAT,M., LASSUS,P. and RODRIGUEZ-VAZQUEZ,J. A. (1986) Okadaic acid as the causative toxin of diarrhetic shellfish poisoning in Europe. Agric. biol. Chem. 50, 2853-2857. LEE,J. S., YANAGI,T., KENMA,R. and YASUMOTO,T. (1987) Fluorometric determination of diarrhetic shellfish toxins by high performance liquid chromatography. Agric. biol. Chem. 51,877-881. UDA,T., ITOH,Y., NISHIMURA,m., USAGAWA,T., MURATA,i . and YASUMOTO,T. (1989) Enzymeimmunoassay using monoclonal antibody specific for diarrhetic shellfish poisons. In: Mycotoxins and Phycotoxins "88, pp. 335-342 (NATORI,S., HASHIMOTO,K. and UENO,Y., Eds). Amsterdam: Elsevier Science. UNDERDAL, B., YNDESTAD, i . and AUNE, T. (1985) DSP Intoxication in Norway and Sweden, Autumn 1984-Spring 1985. In: Toxic Dinoflagellates, pp. 489-494 (ANDERSON,D. M., WHITE, A. W. and BADEN, D. G., Eds). New York: Elsevier Science. USAGAWA,T., NISHIMURA,i . , ITOH, Y., UDA, T. and YASUMOTO,T. (1989) Preparation of monoclonal antibodies against okadaic acid prepared from the sponge Halichondria okadai. Toxicon 27, 1323-1330. VOLTERRA,L., Guccl, P. i . B. and BRUNO, i . (1990) Problemi tecnici connessi con il fenomeno delle DSP (diarrhoeic shellfish poisoning). Ambiente Risorse Salute 5, 12-17.
676
Short Communications
YASUMOTO, T., OSmMA, Y. and YAMAGUCm, M. (1978) Occurrence of a new type of shellfish poisoning in the Tohoku district. Bull. Jpn. Soc. scient. Fish. 44, 1249-1255. YASUMOTO, T., MURATA, M., OSHIMA, Y., MATSUMOTO, G. K. and CLARDY, J. (1984) Diarrhetic shellfish poisoning. A.C.S. Syrup. S. 262, 207-214.
