Research on HAB microalgae and toxins at IRTA, 2014: scientific challenges and interaction with the monitoring programme.

Transcription

1 Research on HAB microalgae and toxins at IRTA, 2014: scientific challenges and interaction with the monitoring programme. Jorge Diogène Pablo de la Iglesia, Margarita Fernández, Mònica Campàs, Amandine Caillaud, María García Altares, Olga Carnicer, Lucía Soliño, Diana Garibo, Gemma Giménez, Vanessa del Río, Alexis Casanova IRTA - Sant Carles de la Ràpita Tarragona, España. Nantes GR Phycotox 4-6 Février 2014

2 The Catalan Shellfish Marine Monitoring Program in Shellfish harvesting areas Protect the consumer in front of environmental hazards Allow the shellfish industry to develop their work Environmental Parameters Phytoplankton and Marine Toxins Microbiology parameters Chemical pollutants ISO quality standards Legal requirement for official controls Awareness and suveillance of non-regulated hazards: e.g. emerging toxins through research programmes

3 Marine phytoplankton taxonomy, culturing, population dynamics, modeling. Phytoplankton cultures: we isolate single cells of target species to start clonal cultures which we will grow in order to conduct further studies on taxonomy or toxicity. Studies on population dynamics are constrained by the quality on phytoplankton identification. In depth taxonomic studies are needed to differentiate between species with similar morphologies as for examples species from the genera Karlodinium and Pseudo-nitzschia. Population dynamics and modeling take advantage of time series on phytoplankton species abundance and environmental parameters.

4 Toxins: Multi-disciplinary approaches for qualitative and quantitative determination. Potential application to new bio-active compounds from the sea. Mouse bioassay Instrumental analytical chemistry Evaluation of toxicity in vivo only whether strictly required, external to IRTA analytical and semi-prep scale Spectroscopy and mass spectrometry detection Functional and structural recognition methods Cell-based assays α-naphthyl phosphate α-naphthol NO OA PP Naphtoquinone e - Differential Pulse Voltammetry α-naphthyl phosphate OA!!! PP Differential Pulse Voltammetry No toxicity Toxicity Enzymes, antibodies, aptamers aimed at specific/selective molecule recognition Ethically-approved in vitro approaches for toxicity assessment.

5 Impact of the Marine Monitoring and Food Safety Sub Program activities (some examples) Ciguatoxin-like toxicity detection in fish from the Canary Islands (Seriola spp.) by cell-based assays * * * * * Discovery of acyl * * ester metabolites of * gymnodimines in shellfish using LC-MS * * * (A) (B) First domoic acid detection in clams from the Catalan coast Homoyessotoxin standard production The Marine Monitoring program guarantees safe shellfish, protects human health, supports the aquaculture production sector in Catalonia and preserves the marine environment. * * * * * * * * * Magnetic particles as antibody and enzyme supports for biosensor development Molecular probes for Pseudo-nitzschia spp.

6 The 3 main ENVIRONMENTAL HAZARDS Background that can affect the SAFETY of shellfish products Microbiological contamination Toxins Chemical pollutants LEGISLATION EU: REGs 852, 853, 854/2004 Protect the consumer Facilitate the activity PREVENTIVE measures! rather than remediation actions Dynamic MONITORING PROGRAMS under CURRENT LEGISLATION RESEARCH Respond to limitations of legislation Need to go beyond to predict HOT issues

7 Endusers and Impact The administration: - EU legislation bodies Requirement of toxicological and toxicity data to establish Maximum Permitted Levels for Marine Toxins in food Determine official methods - Food safety authorities Require risk assessment and expertise Require implementing methods for non legislated hazards or for unexplained events - Regional and national fisheries and environmental departments Execution of monitoring programs to cover legal requirements Use of long-term data for descriptive studies

8 Endusers and Impact The aquaculture and fisheries sector: - Execution of the monitoring program CSMM Allows activity of the shellfish aquaculture sector according to legal requirements Allows identification of optimal aquaculture sites according to eventual hazards - Environmental data Optimization of practices and identification of hazards to their product (Chlorophyll, high temperatures, hypoxias, etc ) Consumers and retailers: Long-term data associated to food safety assure confidence of clients (supermarkets, consumers) on marine products Trust the administration regarding its responsibility on public health issues

9 Endusers and Impact The scientific and technological communities : Coastal oceanography and environment Analytical chemistry Biosensors Toxicology Harmful Algal Blooms Food Safety Risk assessment Natural substances

10 Facilities: Oceanography, laboratories, vivarium, equipment

11 Spirolides Mediterranean

12 SPXs have a wide distribution Distribution Map Fitoplancton Marisco contaminado 1

13 LC-MS/MS determination of SPXs using alcaline conditions Mytilus galloprovincialis January 2011 (Fangar)

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15 First domoic acid detection in clams from the Catalan coast

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18 Gymnodimines Mediterranean

19 Jan-09 Feb-09 Mar-09 Apr-09 May-09 Jun-09 Jul-09 Aug-09 Sep-09 Oct-09 Nov-09 Dec-09 Jan-10 Feb-10 Mar-10 Apr-10 May-10 Jun-10 Jul-10 Aug-10 Sep-10 GYMs concentration By LC-MS/MS (µg kg -1 ) TUNISIA Sampling site M2 S M2 Sampling site MBA (mice died from a total of three) 0 0 M2 Concentration GYM-A Concentration GYM-B and/or GYM-C LD50 reported by Kharrat et al., 2008 MBA (mice died from a total of three) Only GYMs were found in samples. Max. sample February 2009 with 2,136 µg GYM-A/kg. Good agreement among MBA positive results and quantitative results obtained by LC-MS/MS. Much higher concentrations of GYMs in M2 than in S6. GYM-B/-C is present in all samples containing GYM-A (on contrary of previous report of Biré et al., 2002; in agreement with Kharrat et al., 2008)

20 Gymnodimines Mediterranean

21 Results Precursor ion scan for acyl esters of GYM-B/-C TIC for (A) the precursors of m/z 506 LC-MS trace from product (C) ion spectrum on m/z showing 18:0 esters of GYM-B and GYM-C * ** m/z (B) Contour plot with potential fatty acid esters of GYM-B/-C. 22-C 20-C 19-C 18-C 17-C 16-C 15-C 14-C Time (min) Relative intensity (%) Relative intensity (%) Time (min) (D) Product ion spectra of 18:0 fatty acid esters of of GYM-B and -C (E) m/z Identification (Prec scan) and confirmation (Product ion scan) of mono-substituted acyl ester derivatives of GYM-B/-C

22 Ostreopsis Mediterranean

23 Cultures of Ostreopsis sp. Problemas de deformaciones Ostreopsis ovata -28 o C -Medium f/10 -Salinity 36 -Photoperiod 12:12 h -90 µmol m -2 s -1

24 23/05 24/05 25/05 26/05 27/05 28/05 29/05 30/05 31/05 01/06 02/06 03/06 04/06 05/06 06/06 Células/litro Cultures of Ostreopsis sp. Problems due to aggregates Growth curve Fecha without treatment HCl treatment EDTA treatment ,5 1,8-15,4-21,3 8,9 9,0 15,9-30,3 4,7-0,9 1-12,2-11,7-7,3-5,2-9,1 13,5 1,2-12,3 3,8 3-2,8-0,1-6,0 0,8 3,6-9,8 3,2 6,4-0,9 5 1,9 1,3-4,2-1,8 2,2-7,3 0,9 5,6 4,9 12 9,0 6,8 0,3 6,8-2,5 1,4 2,5 5,8-1,0 20 5,9-0,2 2,0 5,8 10,5 9,2 9,5 2,5 6,0 Better results after treatments with EDTA

25 Ciguatoxins Macaronesia-Canary Islands

26 Optimization of the Neuro-2a cell based assay for CTXs Aplication of cell-based assays (CBA) with Neuro-2a cells for ciguatoxins for the determination of CTX sin fish form the Canary Islands. Determine the maximum carriing capacity for fish matrix to eliminate interferences 20 mg/ml-1 (Lewis 2003) Limit of quantification established a to ppb(ngg-1) CTX-1B (lc20). MaximumLevelproposed: Pacific CTX-1 (CTX-1B) : 0.01 ppb Caribbean CTX-1 (C-CTX-1): 0.1 ppb Neuro-2a CBA is very sensitive And applicable to determine CTXs in fish From the Canary Islands(Seriola sp) Dose response curves in control Seriola samples piked within creasing concentrations of P-CTX-1 (= CTX-1B)

27 The Neuro-2a CBA applied to determine CTXs in fish from the Canary Islands (Seriola spp) No muestra Especie pescado Morfología Especie pescado Genetica Intoxicación Cigua-Chek Ensayo celular (eq. CTX-1B) CRLMB/038 Seriola dumerili Seriola dumerili CRLMB/039 Seriola fasciata Seriola dumerili * 6.23 CRLMB/040 Seriola fasciata Seriola dumerili * 4.59 CRLMB/041 Seriola fasciata Seriola rivoliana - CRLMB/042 Seriola fasciata Seriola dumerili * CRLMB/050 Acanthocybium solandri - No concluyente - CRLMB/051 Acanthocybium solandri - No concluyente - CRLMB/052 Seriola rivoliana - + < LQ CRLMB/053 Seriola sp. Seriola dumerili + - CRLMB/054 Seriola sp. Seriola dumerili + - CRLMB/055 Seriola sp. Seriola dumerili + - CRLMB/056 Seriola sp. CRLMB/062 Seriola fasciata Seriola dumerili + - Seriola dumerili + -

28 Biosensors There is a need for functional methods for fast screening and detection of emerging toxins. METHODS: - Assays (colorimetric, fluorescence..) - Biosensors, arrays and compact analysis devices (electrochemical, optical ) RECOGNITION MOLECULES: - Enzymes (including recombinant enzymes) - Antibodies (including antibody fragments) - Aptamers - Receptors - MIPs ADVANTAGES: - Specificity - Sensitivity - Short analysis times - High throughput - Low cost - Possibility to measure in real time - Possibility to operate in situ ALREADY: - Abs/ELISAs for AZAs - AbS/SPR for PlTXs - Receptors/assays for SPXs and GYMs - CBAs for PlTX and CTXs. and more NEAR FUTURE WORK: electrochemical immunosensors for AZAs, CTXs and PlTXs

29 Homo-YESSOTOXIN CRM

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31 GLOBAL FOCUS Complex Landscape EVIDENCE PREDICTION SALINITY SHELLFISH METEOROLOGY Tº TX CURRENTS X STRATIFICATION ALEXANDRIUM Dissolved Toxins Y NUTRIENTS Z P

32 Acknowledgments

33 Foto grupo

34 Redibal