Marine Biotoxins and Food Safety

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1 Marine Biotoxins and Food Safety Ana Gago-Martínez 1.- University of Vigo, Faculty of Chemistry, Dept. of Analytical and Food Chemistry, Campus Universitario de Vigo, Vigo, Spain 2.- EU Reference Laboratory for Marine Biotoxins, Campus Universitario de Vigo, Vigo, Spain

2 UTLINE Marine Biotoxins: Introduction Transition from animal tests to chemistry Current situation on the control of marine biotoxins in the EU Challenges and future perspectives Emerging toxins in the EU

3 EURL FR MARINE BITXINS EURPEAN REFERENCE appointed by EU Commision (DGSANTE) SPANIS REFERENCE appointed by Spanish Competent Authority (AECSAN) Vigo

4 armful algal blooms and marine biotoxins Natural contamination : Proliferation of toxic phytoplankton BILGICAL RISK

5 Phytoplankton control

6 Shellfish Toxins 2 N N N + 2 N N N Cl - N 2 + Cl - C Saxitoxin Yessotoxins and Adriatoxin 3 C C C 3 Domoic Acid N C C R 2 3 S Pectenotoxin R C Azaspiracid kadaic Acid C 3 N R1 Yessotoxin MW R2

7 Marine toxins/seafood Safety/ uman ealth Phytoplankton & Bacteria Birds Bivalves Finfish Marine Mammals uman Crustaceans

8 2 N Biological risk Cl - N N + 2 N N N Chemical Risk N 2 + Cl - Difficulties in implementing alternative non-animal methods: 1. Long time success of animal methods. 2. Ease of use of animals- no need for highly trained staff. 3. Chemical/ instrumental methods can be expensive especially to set up.

9 The Mouse Bioassay The long-standing reference method for shellfish toxins Mouse Bioassay for PSP Toxins An AAC method that has been used over 50 years for protection against PSP Intraperitoneal (IP) injection of 0.1M Cl extract of shellfish tissues into 20 g mouse Measurement: time to death Disadvantages Poor detection limit Poor precision (± 20-30%) Poor accuracy at low levels No information on toxin(s) present Subject to false positives Animal rights concerns (banned in some countries) Mouse Bioassay for DSP Toxins The mouse bioassay for DSP has never been very successful. Several protocols exist and none of these have ever been validated. As new lipophilic toxins appeared, attempts were made to extend the method with more modifications. MBA for DSP suffers from: serious false positive and false negative problems. poor sensitivity poor correlation with human oral toxicity

10 ALTERNATIVE METDS Based on mechanism of action

11 PYSIC- CEMICAL METDS LIQUID CRMATGRAPY/ DETECTIN MDES LC/UV LC/FLD LC-RMS LC-MS/MS IDENTIFICATIN/QUANTITATIN/CNFIRMATIN

12 Current Situation in the EU Legislation PLC /UV Ref. method for ASP PLC /FLD fficial method for PSP mau AD I* I8* min. LC-MS/MS Reference method for LPTs (Dec. 2014)

13 The transition from MBA to Chemistry Most recent changes ip. injection PLC injection Absence of CDEX Criteria (Type IV ) CDEX criteria Type III (Reference)

14 Analytical challenges of the transition armonised Criteria armonised accreditations From LC-MS/MS to RMS

15 Measurement of the Uncertainty of the analytical result EURLMB Working group harmonization of the measurement of the uncertainty among EU-NRLs

16 issues Toxicological Issues EU LEGISLATIN MARINE BITXIN REGULATRY LIMIT REGULATIN (EC) No 853/2004 REGULATIN (EU) No 786/2013 kadaic Acid, dinophysistoxins and pectenotoxins together Azaspiracids Yessotoxins 160 micrograms of okadaic acid equivalents per kilogram 160 micrograms of azaspiracid equivalents per kilogram 3,75 milligram of yessotoxin equivalent per kilogram A equivalents?? TEF Toxicity Equivalent Factor IP/RAL MBA inheritance ow to express results? Are the TEFs adequate?

17 Future perspectives armonization of phytoplanton control PCR

18 Future Perspectives Detecting new or unknown toxins Reg (EU) 15/2011. After the period established in point B(1) of this Chapter (31 December 2014), the mouse bioassay shall be used only during the periodic monitoring of production areas and relaying areas for detecting new or unknown marine toxins on the basis of the national control programmes elaborated by the Member States Ciguatoxin Tetrodotoxin

19 EMERGING TXINS IN TE EU Climatic changes Fisheries activity International trade Emerging toxins versea Traffic increase Scientific pinion on marine biotoxins in shellfish Emerging toxins: Ciguatoxin group (July 2010)

20 Identification and confirmation of Ciguatoxins in the EU Ciguatoxin Seriola * Cell Assay LC-MS/MS and RMS Development of standards and reference materials Development of sample pretreatment protocols Development and optimization of screening and confirmation methods

21 Citotoxicity assay/ LC-MS/MS Canary Islands Unknown source STANDARD C-CTX1 100 pg 10 pg 1 pg 0.1 pg 0.01 pg 100 pg 10 pg 1 pg 0.1 pg 0.01 pg SCREENING Me FRACTIN CCTX [M+Na] + CTX1B [M+Na ]+ CTX1B P-CTX3C CNFIRMATIN P-CTX1B [M+Na ]+

22 SUMMARY New and Emerging toxins Reference materials phytoplankton TEFs ciguatoxin tetrodotoxin palitoxin armonization

23 Acknowledgements EURLMB Staff UNIVERSITY F VIG STAFF EU RLMB NETWRK SPANIS NRL NETWRK Collaborators worlwide

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