Italian Journal of Food Safety 2017; volume 6:7015

Yessotoxin detection in bivalve The toxins can be accumulated by filtering molluscs: A case study from within the edible bivalve molluscs, like Correspondence: Alessandro Graziano mussels, scallops, and clams; this permits Mudadu, Veterinary Public Health Institute of coastal mussel farms (Sardinia, their entry into the food chain. YTXs were Sardinia, Via Duca degli Abruzzi 8, 07100 Italy) initially included in the Diarrhetic Shellfish Sassari (SS), Italy. Tel.: +39.0792892348 - Fax: +39.0792892324. Poisoning (DSP) toxin group due to their E-mail: [email protected] Alessandro Graziano Mudadu, occurrence with okadaic acid (OA) and Giuseppa Lorenzoni, Anna Maria other lipophilic toxins, but it was demon- Key words: Lipophilic toxins, Mediterranean, strated that they have a different mechanism Bazzoni, Riccardo Bazzardi, Giuseppe Mussels, Shellfish farms, YTXs. of action and not produce diarrheal neither Tedde, Igor Arras, Giovanna Sanna, in humans nor in mice (Satake et al., 1997). Contributions: the authors contributed equally. Cinzia Santucciu, Edoardo Marongiu, For these reasons, nowadays they are con- Sebastiano Virgilio sidered in different groups (Alfonso et al., Conflict of interest: the authors declare no potential conflict of interest. Veterinary Public Health Institute of 2016) and are independently regulated Sardinia, Sassari, Italy (European Commission, EC 2004; 2013). Received for publication: 23 August 2017. The toxicological information for YTXs is Revision received: 30 October 2017. limited and includes mainly studies on their Accepted for publication: 30 October 2017. acute toxicity in mice (Rodrìguez et al., 2015), even because no human cases of This work is licensed under a Creative intoxication have been reported to date. Commons Attribution-NonCommercial 4.0 Abstract International License (CC BY-NC 4.0). Related YTX symptoms and toxicity effects This work reports the first communica- after oral assumption are rather unknown in tion relating to the presence of yessotoxins ©Copyright A.G. Mudadu et al., 2017 humans, so consumption of YTX-contami- in Mytilus galloprovincialis from coastal Licensee PAGEPress, Italy nated shellfish still remains an unidentified mussel farms (Sardinia, western Italian Journal of Food Safety 2017; 6:7015 health risk (Tubaro et al., 2010). In contrast, doi:10.4081/ijfs.2017.7015 Mediterranean) detected during 2008 and only mice showed discoordination described by 2013 through a monitoring programme. The jumping and restlessness, due to cerebellar paper emphasizes how the changes both in cortical alterations as a result of damaged yessotoxin permitted limits and used meth- indicated in the Official Bulletin of Italian motor function (Wolf et al., 1996). ods, established by legislation, have influ- Republic (Gazzetta Ufficiale della The Regulation EC No 853/2004use estab- enced the interpretation of the obtained Repubblica Italiana, G.U.R.I.) n. 165, 16 lished the maximum levels of some marine results. Consequently, the samples that July 2002. Between 2011 and 2014, sam- biotoxins in molluscs for human consump- resulted negative during 2008 would have ples which provoked mice death within the tion. Successively, the Regulation EC No been positive until August 2013 and nega- 24 hours were subsequently confirmed by 15/2011 of 10 January 2011 amended the tive from September 2013 up to now, and LC-MS/MS. From 2015 the MBA has been previous Regulation EC No 2074/2005 as the samples that were positive in 2013 replaced by the official LC-MS/MS proto- regards recognised testing methods for would have been positive in 2008 and neg- col. detecting marine biotoxins in live bivalve ative nowadays, according to Regulation This study shows the first evidence molluscs and established that Liquid currently in force. Regular monitoring of relating to the presence of YTXs in shellfish Chromatography-Mass Spectrometry (LC- biotoxins demonstrated that, although from Sardinia (Italy, western MS/MS) method will be used as the refer- yessotoxins have been rarely present in the Mediterranean), occurred during 2008 and ence method instead of mouse bioassay past in Sardinia, they may cause toxicity in 2013. Furthermore, it also emphasizes how (MBA). However, to allow Member States shellfish. So, it’s important to keep up on the changes established by legislations, may to adapt their methods to the chemical one, legislation’s changing and laboratory meth- have influenced the interpretation of the the MBA could be used until 31 December ods. obtained results, both in YTX permitted 2014. Furthermore, Commission limits and in used methods. Non-commercialRegulation No 786/2013 of 16 August 2013 amending Annex III to Regulation EC No 853/2004, as regards the permitted limits of Introduction YTXs in live bivalve molluscs, established Yessotoxins (YTXs) are sulphated the increasing from 1 to 3.75 mg eq Materials and Methods polyethers belonging to lipophilic phyco- YTX/Kg e.p.. The selective procedure for Shellfish were collected fortnightly toxins family. They were first isolated in detection of YTXs defined from Spanish (Mytilus galloprovincialis, Tapes decussat- Japan in 1986 and afterward, production of agency for food security (Agencia Española us, Crassostea gigas, Ostrea edulis, YTXs has been demonstrated worldwide de Seguridad Alimentaria y Nutrición, Veneroidae) in 2008 and in 2013 in breed- (Tubaro et al., 2010). In particular, in Italy AESAN) in the Annex 2009, didn’t allow to ing areas located along coastline of Sardinia the presence of YTXs in mussel was report- evaluate this amount, so the extraction was (Figure 1), where a plan of periodic moni- ed for the first time in hepatopancreas of performed with the non-selective method toring and of sanitary control for the pro- mussels breeded in northern Adriatic Sea for lipophilic toxins. The mice death was duction and marketing of molluscs has been (Ciminiello et al., 1997). Potentially YTX followed by LC-MS/MS method confirma- in force since 1988. A total of 834 bivalve producers are the dinoflagellates tion, that identifies and quantifies the tox- mollusc samples were analysed in 2008 and Protoceratium reticulatum (Claparède & ins. a total of 892 during 2013. Lachmann) Bütschli, Lingulodinium polye- Following European legislation, toxici- For the research of lipophilic toxins dra (F. Stein) J.D. Dodge and Gonyaulax ty analyses were carry out until 31 using the MBA, lipid extract preparation spinifera (Claparède & Lachmann) Diesing. December 2014 by means of the MBA, as involved 100 g of mussels mixed with 300

[Italian Journal of Food Safety 2017; 6:7015] [page 213] Short Communication ml of acetone (VWR CHEMICALS, was recovered with 30 ml of re-extracted twice with 60 mL of methanol. Fontenay-sous-Bois, France) in Ultra- dichloromethane (SIGMA-ALDRICH, St For both extracts, solvent was evaporated Turrax (T25 basic IKA_WERKE), filtered Luis USA) and 60 ml of methanol (VWR and the resultant residue suspended in aque- and re-extracted with 200 ml of acetone. PROLABO, Fontenay-sous-Bois, France) ous solution of 1% Tween 60 (SIGMA- The acetone supernatant was mixed and (60% v/v) and subjected to partition by the ALDRICH, St Luis USA). For the investi- evaporated with a rotavapor (Büchi separating funnel. The methanol phase was gation of OA-toxin group, Pectenotoxin Rotavapor R-200), yielding a residue which stored and the dichloromethane phase was group (PTXs) and Azaspiracid group

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Figure 1. Monitoring network and changes of result interpretation based on Regulation update from 2008 to 2013. G.U. Gazzetta Ufficiale; EC: European Commission; EU: European Union; AESAN: Agencia Española de Seguridad Alimentaria y Nutrición.

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(AZAs), one ml of dichloromethane extract six and eight hours). maximum level has been amended from 1 was injected intraperitonelly into albino During April 2013, YTXs were again to 3.75 mg YTX/Kg e. p., and it could be mice Swiss strain weighing between 19 g detected in mussels, once more in M. gallo- still reassessed if were new scientific evi- and 21 g. The death of 2 out of 3 injected provincialis (Table 1). Based on the applica- dence available. If we tried to re-evaluate mice, within a 24 hours observation period, ble regulation (Figure 1), which established our data following the legislation evolution: constituted a positive result. For the specific the death time within six hours, the biolog- i) the samples that resulted negative during determination of YTXs, the methanolic ical test detected the presence of YTXs in 2008 would have been positive in 2013 and extract was injected in mice. The death of 2 five samples, two at GOL1, two at Cirdu negative again from September 2013 up to out of 3 mice, within a 5 hours (with a 6 (CIR) and one at Porto Pozzo (PPZ). The now; ii) the samples that resulted positive in hours observation period), constituted a following LC-MS/MS-based approach con- April 2013 would have been negative both positive result (G.U.R.I. 165/2002). firmed the toxin presence over the legal in 2008 and nowadays, with the legislation In the use of Harmonised Standard limit (1 mg YTX/Kg e. p.) only in three currently in force (Figure 1). This shows Operating Procedure by MBA (AESAN, shellfish samples that tested positive to the that there is still considerable uncertainty 2009), the death of 2 out of 3 injected mice MBA, two from GOL1 and one from CIR about threat for humans who have generat- within 6 hours observation, with a YTX (Table 1). The YTX contamination levels in ed a series of legislative changes in recent specific symptomatology, constituted a pos- shellfish from both studied areas were quite decades, but also a constant attention to itive result. The positive samples were sub- low (maximum 1.53 mg YTX/Kg e.p. in what derives from different European stud- jected to LC-MS/MS method. It was based GOL1) (Table 1) and subsequently ies and researches. on the extraction of OA toxin group, PTXs, decreased gradually in about two weeks. AZAs and YTXs by homogenized mussel Since 2013 to now in no one shellfish tissues with methanol 90%. Extract was samples, all analysed by LC/MS/MS, YTX then filtered and directly analyzed by LC- evidence was found. Conclusions MS/MS (AB SCIEX QTRAP 4500) Both on 2008 and 2013 the presence of In recent years, various scientists have (AESAN, 2015). To determine the total YTXs was not linked to the occurrence of published about the YTX detection and content of lipophilic toxins, an alkaline harmful microalgae potentially producing quantizationonly in shellfish samples (Alvez-de hydrolysis was necessary. After hydrolysis, YTX and no other lipophilic toxins current- Souza et al., 2013; Li et al., 2012; Paz et al., extract was filtered and analysed by LC- ly legislated in the Europe was isolated at 2013). This highlights the widespread dis- MS/MS. Chromatographic separation was the same time. tribution of YTXs along coasts and their performed by gradient elution. The lack of chemical, physicaluse and increasing occurrence in edible bivalve environmental data related to YTXs did not molluscs. Our study is placed in the context allow us to identify a seasonal trend, any- to make data available for the existing way as in 2008 as in 2013 all positive sam- database to build up information for the Results and Discussion ples were observed between late March and management of the risk of these toxins in During 2008, symptoms attributable July. This is in contrasts with general trend shellfish. YTXs were seen in mice injected with reported in several areas (e.g. French coast- extract of Mytilus galloprovincialis line, Amzil et al., 2008; north-central (Lamarck) from the eastern and western Adriatic Sea, Bacchiocchi et al., 2015) Sardinian coasts, in particular in March at where it seems that autumn-winter is the References Gulf of Olbia (in five stations, named most critical period of the year for YTX AESAN, 2009. EU-harmonised standard GOL1 to GOL5) and Tortolì (TOR), and in contamination. operating procedure for detection of July at Gulf of Oristano (GOR) and Foce In the last fifteen years, legislation lipophilic toxins by mouse bioassay. del Tirso (FTR). The mice died between about YTXs has changed several times both European Union Reference Laboratory five and six hours. It declared no shellfish in reference methods and in the regulatory for Marine Biotoxins, Agencia intoxication, in according to regulations in limits. New regulations (EC No 15/2011) Española de Consumo, Seguridad force that defined the death time within five have imposed to replace the MBA with the Alimentaria y Nutrición, Vigo, Spain. hours of observation. Furthermore,Non-commercial on other LC-MS/MS method. Moreover, on basis of AESAN, 2015. EU-harmonised standard occasions, in the same areas, YTXs caused provisional data which indicate that YTXs operating procedure for determination the mice’s death in a longer time (between pose a modest risk for European population, of lipophilic marine biotoxins in mol- luscs by LCMS/MS. Version 5. European Union Reference Laboratory for Marine Biotoxins, Agencia Table 1. Mice death time and amount of yessotoxins found in 2013 by LC-MS/MS-based Española de Consumo, Seguridad approach. Alimentaria y Nutrición, Vigo, Spain. Site Site Code Data Mice death time YTXs Alfonso A, Vieytes MR, Botana LM, 2016. (h) (mg YTX/Kg e. p.) Yessotoxin, a promising therapeutic Gulf of Olbia GOL1 03/04/2013 <5 1.53 tool. Mar Drugs 14:30. Alves-de-Souza C, Varela D, Contreras C, Gulf of Olbia GOL1 08/04/2013 <3 1.03 de La Iglesia P, Fernandez P , Hipp B, Cirdu CIR 03/04/2013 <3 1.37 Hernandez C, Riobò P, Reguera B, Cirdu CIR 17/04/2013 <3 0.97* Franco JM, Diogene J, Garcıa C, Lagos Porto Pozzo PPZ 22/04/2013 <4 0.77* N, 2014. Seasonal variability of *Toxin presence without the overcoming of the legal limit of 1 mg YTX/Kg e. p. (EC No 853/2004, AESAN 2009). GOL1: Station 1 of Gulf of Dinophysis spp. and Protoceratium Olbia. reticulatum associated tolipophilic

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shellfish toxins in a strongly stratified Regulation (EC) No 854/2004 of the 16/07/2002, p. 15. Chilean fjord. Deep-Sea Res 101:152- European Parliament and of the Council Li A, Ma J, Cao J, McCarron P, 2012. 62. and Regulation (EC) No 882/2004 of Toxins in mussels (Mytilus galloprovin- Amzil Z, Sibat M, Royer F, Savar V, 2008. the European Parliament and of the cialis) associated with diarrhetic shell- First report on azaspiracid and yesso- Council, derogating from Regulation fish poisoning episodes in China. toxin groups detection in French shell- (EC) No 852/2004 of the European Toxicon 60:420-5. fish. Toxicon 52:39-48. Parliament and of the Council and Paz B, Blanco J, Franco JM, 2013. Bacchiocchi S, Siracusa M, Ruzzi A, Gorbi amending Regulations (EC) No Yessotoxins production during the cul- S, Ercolessi M, Cosentino MA, 853/2004 and (EC) No 854/2004. In: ture of Protoceratium reticulatum Ammazzalorso P, Orletti R, 2015. Two- Official Journal, L 338, 22/12/2005. strains isolated from Galician Rias year study of lipophilic marine toxin European Commission, 2011. Commission Baixas (NW Spain). Harmful Algae 21- profile in mussels of the North-central Regulation (EU) No 15/2011 of 10 22:13-9. Adriatic Sea: First report of aza- January 2011 amending Regulation Rodríguez LP, González V, Martínez A, Paz spiracids in Mediterranean seafood. (EC) No 2074/2005 as regards recog- B, Lago J, Cordeiro V, Blanco L, Vieites Toxicon 108:115-25. nised testing methods for detecting JM, Cabado A. G, 2015. Occurrence of Ciminiello P, Fattorusso E, Forino M, marine biotoxins in live bivalve mol- lipophilic marine toxins in shellfish Magno S, Poletti R, Yasumato T, 1997. luscs. In: Official Journal, L 6/3, from Galicia (NW of Spain) and syner- Yessotoxin in mussel of the northern 11/01/2011. gies among them. Mar Drugs 13:1666- Adriatic Sea. Toxicon 35:177-83. European Commission, 2013. Commission European Commission, 2004. Regulation of Regulation (EU) No 786/2013 of 16 87. the European Parliament and of the August 2013 amending Annex III to Satake M, Tubaro A, Lee JS, Yasumoto T, Council of 29 April 2004 laying down Regulation (EC) No 853/2004 of the 1997. Two new analogs of yessotoxin, specific hygiene rules for on the European Parliament and of the Council homoyessotoxin and 45-hydroxyho- hygiene of foodstuffs, 853/2004/CE. In: as regards the permitted limits of yesso- moyessotoxin, isolated from mussels of Official Journal, L 139/55, 30/04/2004. toxins in live bivalve molluscs. In: the Adriatic Sea. Nat Toxins 5:107-10. European Commission, 2005. Regulation of Official Journal, L 220/14, 17/08/2013. Tubaroonly A, Dell’Ovo V, Sosa S, Florio C, the European Parliament and of the Gazzetta Ufficiale della Repubblica 2010. Yessotoxins: A toxicological Council of 5 December 2005 laying Italiana, 2002. Regulation of 16 May overview. Toxicon 56:163-72. down implementing measures for cer- 2002 as regards the maximum levels Wolf LW, LaRegina MC, Tolbert DL, 1996. tain products under Regulation (EC) No and analytic methods for algal biotoxinsuse A behavioral study of the development 853/2004 of the European Parliament in live bivalve molluscs, echinoderms, of hereditary cerebellar ataxia in the and of the Council and for the organisa- tunicates and marine gasteropods (in shaker rat mutant. Behav Brain Res tion of official controls under Italian). Serie Generale, 165, 75:67-81.

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