ORGANIZING COMMITTEE ORGANIZING SUBCOMMITTEE

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2 2 10 TH INTERNATIONAL CONFERENCE ON MOLLUSCAN SHELLFISH SAFETY ORGANIZING COMMITTEE Viviana Cachicas Carlos Aranda Enrico Buenaventura Anthony Zammit Claudia Rozas Dinorah Medina ORGANIZING SUBCOMMITTEE SCIENTIFIC COORDINATOR Alejandra Goya Veronica Jurquiza Carmen Lopez Orialis Villarroel Rita Orozco Jorge Mardonez M Angelica Larrain Jaminton Ramirez Monica Jara Cristina Martinez Gonzalo Diaz Andrea Rivera

3 Puerto Varas / Chile March INTERNATIONAL ADVISORY COMMITTEE IAC Ron Lee Iddya Karunasagar Gary Richards William Burkhardt Jesus Romalde Francesca Leoni Marcela Costagliola Michelle Price-Haywart Jessica Jones Stephen Jones Miguel O Ryan Philipp Hess Soizick Le Guyader Elisabetta Suffredini Dr. Andrew Turner Ana Gago Martinez Rachell Hartnell Angelo De Paola Irma Rivera Jaime Martinez Urtaza Veronica Madrid Elsa Quiñones Heriberto Fernandez

4 4 10 TH INTERNATIONAL CONFERENCE ON MOLLUSCAN SHELLFISH SAFETY BIOTOXIN AND PHYTOPLANKTON 1. Oral Presentation SOMETHING IS HAPPENING IN FJORDS AND CHANNELS OF SOUTHERN CHILE (41-55 ): The case of Alexandrium catenella and Paralytic Shellfish Poison for the past 40 years. Guzmán, L 1., 1 Departamento de Medio Ambiente, División de Investigación en Acuicultura, Instituto de Fomento Pesquero. The first record of Alexandrium catenella and Paralytic Shellfish Poison (PSP) was in 1972, then blooms occurred in 1981 and 1989, but restricted to the Magellan region. Since 1991, recurrent A. catenella blooms and PSP outbreaks are annually occurring, expanding gradually its distribution to the North. Records of this expansion dating from 1992 (45 S) in the Aysén region, 1998 in the Southeastern sector of Chiloé Island (43 S) and recently was detected in the Northern area of the inner sea of Chiloé (41 48 ºS) during the Spring of A. catenella motile stage presence, preceded at least two years, PSP detection in shellfish in its geographic expansion. Today its distribution encompasses from to S. The PSP outbreaks are associated to 35 fatal cases and about 5 hundreds of intoxicated people, most in the Magellan region (23), situation that has changed since year 2000, since no fatal cases have occurred and undesirable intoxications have decreased to a few cases. Besides the clear expansion of the species in its geographic distribution, different studies realize interannual variability in the relative abundance and density of A. catenella, and toxicity in shellfish; also clear differences between macro-regions and within these regions in the temporal and spatial distribution of A. catenella and PSP levels. Furthermore, the highest records of PSP toxicity show a clear latitudinally increase, being the highest, recurrently detected in specific localities of the Magellan region. In addition, there are clear differences, between and within geographic macro-regions, in the periods with higher probabilities when A. catenella presents density increases and PSP outbreaks. A. catenella, as an exception may be the numerical dominant species of phytoplankton assemblages, as occurred in the exceptional 2009 bloom, but usually the species represents a low proportion of total phytoplankton (<1%). The climatic oceanographic hypothesis would explain this phenomenon in Chilean fjords and channels.

5 Puerto Varas / Chile March Oral Presentation Mussel long-line culture and the risks of toxic Dinophysis in the water column. Roughan, B 1., 1 Verification Services Ministry for Primary Industries. The New Zealand BMS aquaculture industry is heavily reliant on GreenShell mussels (Perna canaliculus). Approximately 90,000 tonnes mussels are exported annually worth approximately NZD$200 million per annum. The majority of the production is in the sheltered waters of the Marlborough Sounds. The mussels grow on a series of dropper ropes hanging down to 15 metres from a sturdy backbone rope that is held up by a row of buoys. Where BMS flesh samples are not tested for marine biotoxins on a weekly basis, the New Zealand programme requires at least weekly samples of water to be analysed for phytoplankton counts. Seawater samples are taken using an integrated tube method with flesh taken at the same time should they need to be analysed at the laboratory. Based on the risk profile, many areas routinely test the flesh weekly for the at-risk toxins. New Zealand has conservative phytoplankton trigger levels which require flesh testing for the relevant toxin should they be exceeded. D. acuminata and D. acuta blooms occur annually in some areas of the Marlborough Sounds. Experience from the routine marine biotoxin monitoring programme has identified significant difference in flesh toxin levels depending on the depth of flesh sampling from the dropper rope. During 2010 and 2014 bloom events DSP toxins levels varied from nil detect to over the regulatory level of 0.16mg Okadaic acid equivalents/kg. This paper will highlight the importance of knowing the growing area hydrodynamics, phytoplankton risk profile and adapting sampling during risk periods to ensure consumers are protected.

6 6 10 TH INTERNATIONAL CONFERENCE ON MOLLUSCAN SHELLFISH SAFETY 3. Oral Presentation Operational Forecasting of Harmful Algal Blooms and Toxicity in Ireland Silke, Joe 1., Cusack, Caroline 1.,Glenn, Nolan 2., 1 Marine Environment and Food Safety Services Irish Marine Institute. 2 Ocean Science and Information Services Irish Marine Institute. The shellfish industry depend on having a robust biotoxin programme and appropriate regulations to ensure produce is safe to place on the market. In Ireland, results from the national biotoxin monitoring programme are distributed direct to the aquaculture industry, regulatory agencies and public within hours of laboratory sample receipt. To ensure that fish farmers can harvest and market their product in a sustainable manner, they require advance knowledge of impending HAB events. An ideal solution would be an alert system that would enable farms to improve harvesting practices (eg. get bivalve products onto market prior to intoxication event) and ensure that protective measures are implemented on time when a HAB is imminent. Such improved decision on risk and crisis management will encourage industry growth. The EC FP7 funded project ASIMUTH developed a short term HAB forecasting (2-3 day) system for Scotland, Ireland, France, Spain and Portugal. In this presentation we discuss the implementation of this system in Ireland. The system uses a HAB Decision Support System designed to assimilate information (satellite images, model forecasts, ground data) and via expert interpretation of results reports back to end users. The 2012 and 2013 Irish field campaign will be examined. This includes collaboration with the Irish coast guard to detect high biomass blooms in Irish shelf waters and model their movements. Regular reports to predict HABs were prepared and published to inform the industry. The feedback from this exercise demonstrated the value to the industry and provided extra assurance regarding food safety of bivalves placed on the market.

7 Puerto Varas / Chile March Oral Presentation New insights on the ecophysiology of Alexandrium catenella from Chilean fjords Mardones, Jorge 1., Dorantes-Aranda, Juan Jose 1.,Müller, Marius 2.,Bolch, Chris 1.,Varela, Daniel 3.,Hallegraeff, Gustaaf 1., 1 Institute for Marine and Antarctic Studies (IMAS) University of Tasmania. 2 Instituto Oceanográfico University of São Paulo. 3 i-mar Universidad De Los Lagos. (Sponsored by BECAS CHILE Program Of The National Commission For Scientific And Technological Research (CONICYT)) Harmful Algal Blooms (HABs) are common natural phenomena that occur along the Chilean coast. In the southern fjords, the dinoflagellate Alexandrium catenella is the most studied species because of its production of saxitoxin and analogues responsible for Paralytic Shellfish Poisoning (PSP). Despite the deleterious effects on the local aquaculture, little is known about its ecology in this complex ecosystem. In vitro experiments were conducted to determine the role of cyst in bloom dynamics, the physiological response against pco 2 /ph variations and the potency of toxic compounds with cytolytic properties in A. catenella. Cyst experiments using pairwise crosses showed highly variable reproductive compatibility among 10 Chilean Group 1 A. catenella strains; synchronized encystment with cyst production starting 26 days and terminating 45 days after inoculation; a dormancy period between 80 and 120 days and excystment rate stimulated by low temperature (10-12 C), high salinity (30 psu), N and/or P depletion, and low irradiance (20 μ photons m -2 s -1 ). The broad natural pco 2 /ph fluctuations in the southern fjords play an important role in the growth response of A. catenella. Optimum growth and dissolved inorganic carbon (DIC) uptake occurred around ph =8.0, but was negatively affected by lower and higher ph. Chain-formation (and hence swimming speed) was enhanced under low pco 2 /high ph conditions while cell size (ESD) was negatively correlated with increasing ph. Finally, our results on the ichthyotoxic potency of A. catenella assessed by a fish gill cell line assay, showed that pure PST fractions (C1&C2, STX, GTX 1&4) had an insignificant role in lytic activity compared to the synergistic interaction between reactive oxygen species (superoxide anion) and polyunsaturated fatty acids such as DHA.

8 8 10 TH INTERNATIONAL CONFERENCE ON MOLLUSCAN SHELLFISH SAFETY 5. Oral Presentation The Red Tide Project as a health monitoring system for the shellfish exposed to Harmful Algal Blooms (HABs) Castillo, M 1., 1 ExecutiveDirection of SpecialPrograms Ministry of Health, COFEPRIS, Mexico. In 2001, thefederalcommission for Protectionagainst Sanitary Risks(COFEPRIS) was created by a governmental decree, as a decentralized technical, administrativeand operational autonomousbodywithin the Ministryof Health. It is responsible forregulation, controlandpromotion of health.in 2004, the COFEPRIS created The Red Tide Project. This project its main purpose is the sanitary control of shellfish exposed to Harmful Algal Blooms (HABs) in the Mexican coasts, through a program of ongoing monitoring which detectsphytoplankton in seawater, complementary to the sampling of biotoxins in shellfish, and this functions as an early warning system for detecting HABs. Its objective is to avoid the human consumption of shellfish exposed to such events. For this purpose, a set of Guidelines have been established to support the Sanitary Authorities of States in Mexico with coastlines to perform a permanent application of monitoring programs and proceed with actions in the event where HABs are detected. As a result of the application of the phytoplankton sampling Plan, from 2005 to HABs were identified, 55 of them were potentially toxic for humans, 12 wereichthyotoxic,and 31 werenon-toxic.

9 Puerto Varas / Chile March Oral Presentation Occurrence of phycotoxins in the coast of Santa Catarina, the largest aquaculture mollusks producer in Brazil. Schramm, Mathias 1., Proença, Luis Antonio 1.,Alves, Thiago 1., 1 LAQUA/MPA-Itajaí/SC Instituto Federal de Santa Catarina - Campus Itajaí. Analysis of marine biotoxins focusing mollusks safety started in Brazil in mid 90ies with the implemantation of HPLC-DAD and HPLC-FLD analytical methods and mice bioassay in Santa Catarina. These methods were set in order to provide support for the new growing mussel and oyster aquaculture in the region. In this first period were detected toxins from groups of okadaic acid, saxitoxin and domoic acid, in different sources. From 2009, a second period of analysis started with LC-MS/MS methods which enabled the detection of several lipophilic toxins either in algae or in mollusks extracts. In this paper, we list all the toxins found in the coast of Santa Catarina until now, from both in phytoplankton and/or mollusks, including: OA, DTX1, DTX2, AZA-1, AZA-2, AZA-7/37, YTX, 45-hyd-YTX, PTX-2, DA, neo-stx, dc-stx, GTX1,2,3,4,5, dc-gtx-2, dc-gtx-3, SPX-1, OVA-A. These findings were considered to set the toxins scope to be monitored in the Brazilian legislation regarding mussel sanitation control. At this moment, based on these findings, the groups of toxins officially monitored are: a) PSP - saxitoxin and congeners; b) DSP - okadaic acid and dinophysistoxins; c) DSP - yessotoxins; d) AZP azaspiracids; and e) ASP - domoic acid and congeners, mainly associated to Gymnodinium catenatum, Dinophysis spp., Azadinium spp. and Pseudonitzschia spp., respectively.

10 10 10 TH INTERNATIONAL CONFERENCE ON MOLLUSCAN SHELLFISH SAFETY 7.Oral Presentation Review of three years of monitoring of palytoxin-group toxins in different marine organisms in Villefranche bay on the French Mediterranean coast Biré, R 1., Amzil, Z 2.,Lemée, R 3,4.,Trotereau, S 1.,Hess, P 2.,Delpont, C 1.,Brissard, C 2.,Krys, S 1., 1 Department of Chemical contaminants in food, Pesticides and marine biotoxins unit ANSES, Maisons-Alfort laboratory for food safety. 2 Phycotoxins Laboratory, Atlantic Centre IFREMER. 3 UPMC Univ Paris 06, UMR 7093, LOV, Observatoire Océanologique Villefranche/mer Sorbonne Universités. 4 LOV, Groupe Biodiversité et Biogéochimie, Observatoire océanologique Villefranche/mer CNRS, UMR Palytoxin (PLTX) and analogues are potent non-protein marine compounds produced by corals of the genus Palythoa and by dinoflagellates of the genus Ostreopsis. Since the beginning of the 2000s, Ostreopsis blooms have been reported in the Mediterranean Sea with increasing frequency. In September 2006, divers and swimmers around the Frioul Island, offshore of Marseilles, reported various symptoms related to the presence of Ostreopsis in the water. Since then, a monitoring system has been set up by the competent authority along the French Mediterranean coast; when Ostreopsis abundances exceed 4,000 cells/l in the water column, shellfish are analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for the presence of PLTX-group toxins. Currently no regulations exist for these toxins in the European Union or elsewhere, however the European Food Safety Authority (EFSA) recommends a threshold of 30 µg/kg applicable to shellfish. Other marine organisms were not considered due to the lack of occurrence data. In 2009, 2010 and 2012, various marine organisms including fishes, echinoderms, gastropods, crustaceans and cephalopods were sampled in Villefranche bay, on the French Mediterranean coast and analysed by the hemolytic assay and LC-MS/ MS for the presence of PLTX-group toxins. The toxin levels in sea urchins (Paracentrotus lividus), fishes (Sarpa salpa, Mugil cephalus), crustaceans (Maja squinado) and gastropods (Hexaplex trunculus) were above the EFSA threshold of 30 µg/kg, either once or repeatedly during the sampling periods. The omnivorous and herbivorous organisms were most contaminated, showing the influence of the diet in the contamination process. Toxins accumulated almost exclusively in digestive tube for all species, with the exception of octopus (Octopus vulgaris) which exhibited detectable toxin amounts also in remaining tissues. The toxin profile determined by LC-MS/MS identified ovatoxin-a as the major compound (90 to 100%).

11 Puerto Varas / Chile March Oral Presentation Communicating with stakeholders: new tools to meet challenges Mellano, Grace 1., Edwards, Robyn 2., 1 Aquaculture Management Directorate Fisheries and Oceans Canada. 2 Domestic Food Safety Systems Canadian Food Inspection Agency. The Canadian Shellfish Sanitation Program (CSSP) is a federal food safety program developed to minimize health risks from the consumption of bivalve molluscan shellfish. The CSSP is administered by the Canadian Food Inspection Agency, Fisheries and Oceans Canada, and Environment Canada. The three partners collaborate to jointly deliver the program, manage risk and communicate with stakeholders. Over the years, we have recognized various challenges in providing the public with precise and timely information of whether areas are open or closed to harvesting. Specifically, interviews with stakeholders such as federal and provincial government authorities not directly involved in CSSP delivery, industry and the general public, determined that they are looking for a web-based solution that allows them to quickly and easily access locations and real-time information about where shellfish are safe to harvest. As a result, the CSSP partners are developing a geospatial web mapping application using ArcGIS and Geocortex Essentials. Led by Fisheries and Oceans Canada, the application will target distinct end-users: commercial harvesters, the general public, and the CSSP partners. The application is being designed to comply with Government of Canada accessibility guidelines and will be hosted on DFO s network. Official launch is expected in in This tool will provide real-time information about harvest area openings and closures and detailed information about each area, such as biotoxin closures. The layered structure of the application will allow for different degrees of data accessibility depending on the constituent from the general public to the regulators. Ultimately, this mapping tool will improve commercial and recreational harvester understanding about where they can safely harvest. Industry will be provided with enhanced ability to make informed business decisions about harvesting and purchasing product, thus providing both food safety and economic benefits.

12 12 10 TH INTERNATIONAL CONFERENCE ON MOLLUSCAN SHELLFISH SAFETY 9. Oral Presentation THE TRANSITION FROM MOUSE BIOASSAY TO CHEMICAL METHODS FOR THE DETERMINATION OF SAXITOXIN GROUP TOXINS: THE EURLMB EXPERIENCE Ben-Gigirey, B. 1., Rossignoli, A. 1.,Gago-Martinez, A. 1,2., 1 EURLMB European Union Reference Laboratory for Marine Biotoxins-Spanish Consumers Affairs, Food Safety and Nutrition Agency. Ministry of Health, Social Affairs and Equality. 2 Department of Analytical and Food Chemistry, Chemistry Faculty., Universidad de Vigo. The drawbacks of the MBA for marine biotoxins control have been the main reason for the search of analytical alternatives for these compounds. Three AOAC Official Methods, including two Liquid Chromatographic methods with Fluorescence Detection (LC-FLD) and a Receptor Binding Assay, have been accepted as alternative to the MBA for the control of STX toxins group (STXs). The two methods based on a chromatographic separation prior to the FLD of the STXs derivatives differ in the STXs precolumn or postcolumn derivatisation. Significant efforts have been devoted to the extension and implementation of both methods among scientists. In particular, the EU Reference Laboratory for Marine Biotoxins (EURLMB) has made considerable efforts on these implementations, not only through research but also through the organisation and/or participation in different collaborative studies. The suitability of the AOAC OMA for Official Control prior to its inclusion in the E.U. legislation was evaluated by the EURLMB in Since then many attempts have been made among the EURLMB Working Group (WG) experts to identify specific method drawbacks and to find solutions to overcome them. The WG tasks included, among others, a study for the quantification of GTX6 after hydrolysis, an interlaboratory study for the extension of the method validation for dcgtx2,3, and the evaluation of the method recovery following an harmonised procedure. The EURLMB has been also working on the setting up of AOAC OMA and participated in its international AOAC validation, as well as in the certification study of the first tissue reference material containing STXs. Present efforts are focused on the implementation of the chromatographic resolution for certain STXs and GTXs. The EURLMB experience on the coordination of Proficiency Testing Schemes, trainings and validations allowed concluding the importance of such efforts to a reliable and efficient application of both methods for STXs analysis.

13 Puerto Varas / Chile March Oral Presentation Single laboratory validation of a UPLC-HILIC-MS/MS method for quantitation of paralytic shellfish toxins in twelve commercially produced bivalve shellfish species Turner, Andrew 1.,Harwood, Tim 2., Selwood, Andrew 2.,McNabb, Paul 2.,Boundy, Michael 2., 1 Food Safety Centre for Environment, Fisheries and Aquaculture Science (UK). 2 Analytical Services Cawthron Institute (NZ). (Sponsored by Queen Elizabeth II Technicians Study Award) Routine regulatory monitoring of paralytic shellfish toxins (PST) in bivalve shellfish can prove difficult using instrumental techniques. There is a need for alternative methods that provide faster turnaround times and improved detection limits. A sensitive quantitative hydrophilic interaction liquid chromatography (HILIC) UPLC-MS/MS method has been developed as a simpler alternative to the precolumn oxidation AOAC and post-column oxidation AOAC liquid chromatography with fluorescence detection methods, as well as the biological assay AOAC This new method employs a novel desalting clean-up procedure with inexpensive carbon solid phase extraction cartridges. HILIC HPLC-MS/MS has been used previously for PST research purposes, but substantial sample matrix issues and high detection limits have to date prevented it from being a viable alternative for routine shellfish monitoring purposes. A single-laboratory validation study was conducted using the new method for the analysis of PST in 12 commercially produced bivalve shellfish species. PST assessed included saxitoxin (STX), neosaxitoxin (NEO), decarbamoylsaxitoxin (dcstx), decarbamoylneosaxitoxin (dcneo), deoxydecarbamoylsaxitoxin (dostx), gonyautoxins 1-6 (GTX1-6), decarbamoylgonyautoxin 1-4 (dcgtx1-4), N-sulfocarbamoyl gonyautoxin 2&3 (C1&2) and N-sulfocarbamoyl gonyautoxin 1&4 (C3&4). Twelve commercially produced bivalve species from both New Zealand and the United Kingdom were assessed, including mussels, oysters, scallops and clams. Validation studies demonstrated acceptable method performance characteristics for specificity, linearity, recovery, repeatability and within-laboratory reproducibility. Limits of detection and quantitation were significantly improved in comparison to current fluorescence-based detection methods and the method was shown to be robust. The method performed well in comparison with AOAC , with evidence obtained from both comparative analysis of 1,141 PST contaminated samples and successful participation in proficiency testing schemes.

14 14 10 TH INTERNATIONAL CONFERENCE ON MOLLUSCAN SHELLFISH SAFETY 11. Oral Presentation Screening of a wide variety of fycotoxins in different matrices using liquid chromatography high resolution mass spectrometry Gerssen, A 1., Klijnstra, Mirjam 1., 1 Natural Toxins and Pesticides RIKILT - Institute of Food Safety - Wageningen UR. Phycotoxins such as azaspiracids, microcystins, saxitoxins and ciguatoxins are produced by algae which are naturally occurring in marine and fresh water. Different types of phycotoxins can end up in matrices like shellfish, water or food supplements. Analytical methods are available to analyse regulated phycotoxins, however these methods are only suitable for a small specific group of toxins and/or a specific matrix (mainly shellfish). Therefore, a screening method is developed for all kinds of phycotoxins in different matrices such as shellfish, fresh and sea water and food supplements. To analyse lipophilic phycotoxins, water samples are cleaned and toxins are concentrated with use of solid phase extraction (SPE). Due to the chemical properties of hydrophilic phycotoxins, these toxins are not retained on a C18 SPE cartridge. Therefor the hydrophilic phycotoxins another type of clean-up and concentration step is required. For shellfish (tissue) samples and food supplements (solids) a different approach is needed. To extract the phycotoxins out of tissue (shellfish) and food supplements a two-step extraction is used. First methanol is added (lipophilic phycotoxins) followed by extraction using an ultrasonic disruptor. Second, a mixture of acetonitrile, water, ammonium formate and formic acid is used (hydrophilic phycotoxins) and vortex mixing. Both extracts are combined. Subsequently, food supplement extracts are cleaned with use of C18 SPE for lipophilic phycotoxins. Shellfish extracts are analysed with LC-hrMS directly after extraction. For separation, liquid chromatography methods are developed for lipophilic and hydrophilic phycotoxins. Lipophilic phycotoxins are separated by reversed phased chromatography (C18) as opposed to the hydrophilic phycotoxins, which are separated with use of a HILIC LC-column. For both methods a gradient with a runtime of 20 minutes is used with the same mobile phase composition, water, acetonitrile, ammonium formate and formic acid. Subsequently, the samples are measured with use of a full scan high resolution mass spectrometer (Orbitrap) at a resolution of and in combination with HCD fragmentation. For the developed extraction procedures acceptable recoveries for the known phycotoxins are obtained between 70% and 150%. Compared to current available methods toxins are well retained and produce acceptable peak shapes on the LC-column. By using an in house created library and developed software (MetAlign) we were able to screen various sample types for over 300 different phycotoxins. During the presentation the development and the application of the method will be presented.

15 Puerto Varas / Chile March Oral Presentation Volunteer-based harmful algae monitoring networks with rapid toxin detection as an added safety layer and managing option for aquaculture Cassis, D 1., 1 Centro de Investigación e Innovación para el Cambio Climático Universidad Santo Tomás / Jellett Rapid Testing. Shellfish aquaculture operations are affected by extraction closures, harvest delays and loss of consumer confidence produced by toxic harmful algal blooms, causing upwards of US80 million in the US. These blooms depend on environmental variables and follow relatively predictable seasonal trends. Climate change seems to be affecting these cycles as unseasonal conditions may lead to the appearance of species, blooms and toxins not observed before. In 2011 DSP was detected for the first time in British Columbia, and despite the ongoing toxin monitoring program, 62 people resulted intoxicated. As a result of the negative effects on public health the BC shellfish industry with support from government agencies (BCCDC, CFIA) requested the creation of the capabilities to confront these threat through the formation of a volunteer-based harmful algae monitoring program to complement the biweekly toxin monitoring program. It was based on in situ phytoplankton monitoring supported by on-line taxonomical guides, networking capabilities, and an event response protocol. This protocol included the use of rapid qualitative tests to confirm the presence of toxins in phytoplankton and shellfish as a precautionary measure. Soon after its deployment in 2013, this monitoring network scored its first success by preventing PSP-contaminated shellfish from being harvested in the Strait of Georgia after an Alexandrium bloom was observed soon after samples were obtained for the regulatory toxin monitoring program. Toxicity in phytoplankton and shellfish samples was detected through rapid tests and confirmed as above the legal limit several days later though quantitative analysis. These efforts resulted in the grower s increased awareness of the safety of their products, an early warning system to prevent harvest of toxic products, and a proactive and more detailed layer to current toxin monitoring programs that can help manage their harvests. The design and initial results of this network are hereby presented.

16 16 10 TH INTERNATIONAL CONFERENCE ON MOLLUSCAN SHELLFISH SAFETY 14. Oral Presentation Mode of action-based bioassays for marine biotoxins causing gastrointestinal disturbances Bodero, M 1., Bovee, Toine 2.,Hoogenboom, Ron 1.,Hendriksen, Peter 1., 1 Toxicology-RIKILT Wageningen. 2 Toxicology and Bioassays -RIKILT Wageningen. The presence of marine biotoxins in shellfish is a risk for consumers and requires constant monitoring of production areas and products. In several countries (including Chile), the method of detection is the Mouse Bioassay (MBA), where mice are injected with an extract of the sample, and the occurrence of death or other signs and symptoms are measured. Chemical analytical methods were developed, allowing the proper identification and quantification of the toxins. However, most groups of toxins can comprise different analogues and the identity of many of these toxins may still be unknown or proper standards are missing. For this reason the application of the MBA is still allowed for testing of production areas after However, the final aim is to replace these tests completely. In vitro bioassays based on the mode of action (MOA) offer the advantage over analytical methods for being capable to detect known and unknown compounds with a similar MOA. For other toxic endpoints MOA-based bioassays have already been shown to allow characterization, selection, purification and identification of unknown compounds in an approach called bioassay-directed identification. The aim of this project is to develop such in vitro bioassays for the detection of marine biotoxins. We first investigated the effects of three shellfish poisons on the whole genome mrna expression of the intestinal cell line in Caco-2 cells exposed to two diarrheic shellfish poisons (okadaic acid and dinophysistoxin-1) and one azaspiracid shellfish poison (azaspiracid-1). Bioinformatics analysis tools were used to identify genes potentially useful as marker genes, and generate hypotheses on MOA. Our analysis revealed many genes that were either toxin type-specific or affected by both types of toxins. Our hypothesis on mode of action and selection of biomarkers to be used as detection method will be presented in the conference.

17 Puerto Varas / Chile March Oral Presentation Development and validation of a range of lateral flow immunoassays for the rapid screening of marine biotoxins (ASP, DSP, PSP) in shellfish Jawaid, W 1., Melville, K 1.,Meneely, J 2.,Campbell, K 2.,Rice, J 3.,Holmes, S 1.,Elliott, C 2., 1 Research and Development Neogen Europe Ltd. 2 School of Biological Sciences Queens University Belfast. 3 Research and Development Neogen Corporation. The presence of harmful algal blooms in seawater can result in the accumulation of biotoxins in shellfish such as mussels, scallops, oysters, clams and cockles. Dependent on the particular toxins present, consumption of contaminated shellfish could lead to illness in humans including amnesic, diarrheic or paralytic shellfish poisoning (ASP, DSP or PSP respectively). There has been a lack of suitable rapid screening tools to complement accepted reference methods such as LC-FD, LC-MS/MS and MBA that are used in national monitoring programs. Our aims were to develop a range of simple and accurate lateral flow immunoassays (LFIA) for the rapid screening of key phycotoxins from shellfish extracts, which could be performed either in a laboratory or in the field. Three competitive LFIAs were developed and validated: Reveal 2.0 ASP (DA), Reveal 2.0 DSP (OA & DTXs) & Reveal 2.0 PSP (STX suite). One of the challenges was to ensure that the thresholds for tests were based at concentrations relevant to regulations, whilst minimizing the risks of generating positive results from samples deemed compliant to regulations. Qualitative results (negative/positive) were generated using a portable reader (AccuScan ) to remove interpretation subjectivity. Rapid and simple procedures were devised whereby samples could be screened in parallel for key phycotoxins (DA, OA, DTX1, DTX2, STX, NEO, GTX1&4, GTX2&3, dcstx, dcneo, dcgtx2&3, C1&C2 and GTX5) in 20 min, including sample extractions. To include detection of DTX3 toxins, a hydrolysis step is incorporated into the procedure. Validation consisted of evaluating various performance characteristics such as accuracy, sensitivity, cross reactivity, matrix effects, stability, robustness and ruggedness. Inter-laboratory and intra-laboratory evaluations demonstrated that key toxins could be detected from a variety of spiked and naturally contaminated shellfish samples and included comparisons with reference methods. Further data will be presented at the conference.

18 18 10 TH INTERNATIONAL CONFERENCE ON MOLLUSCAN SHELLFISH SAFETY 16. Oral Presentation High throughput assays for sensitive screening of marine neurotoxins in seafood Nicolas, J 1., Hendriksen, Peter 2.,Rietjens, Ivonne 1.,Bovee, Toine 2., 1 Toxicology Wageningen UR. 2 Toxicology RIKILT Institute of Food Safety. The current techniques for the detection of marine biotoxins in seafood are the in vivo mouse bioassay and LC/MS-MS based analysis. The mouse bioassay is highly unethical and the LC/MS-MS technique is expensive and does not allow for the detection of unknown marine biotoxins. Therefore, there is an urgent need for the development of in vitro assays with high sensitivity, enabling the detection of marine biotoxins below current regulatory levels. The present study investigated the suitability of the multielectrode array (MEA) using rat cortical neurons and the mouse neuroblastoma neuro-2a assay with measurement of changes in membrane potential for rapid screening of marine neurotoxins in seafood. Model neurotoxic compounds were selected that affect either Na +, Ca 2+, K + channels or the Na + /K + ATPase pump. In addition to these model compounds, pure marine neurotoxins commercially available and seafood extracts were tested as a first proof of principle. The MEA platform allowed for the detection of most model compounds, pure marine neurotoxins and neurotoxins present in contaminated extracts. All model compounds (except K + channel blockers), pure marine neurotoxins and two extracts contaminated with saxitoxin and tetrodotoxin elicited changes in membrane potential in neuro-2a cells detected by fluorescent probes. The data obtained with both techniques suggest that these models represent promising alternatives for reducing and ultimately replacing the unethical in vivo assays currently used for the screening of both known and unknown marine biotoxins in seafood.

19 Puerto Varas / Chile March Oral Presentation Cell based bioassays for the detection of marine toxins in fish and shell fish for replacing animal testing Bovee, T 1., 1 Toxicology and Bioassays & Biosensors RIKILT-Wageningen University and Research Centre. Marine toxins are a worldwide threat to consumers. Most counties rely on in vivo assays and chemical analytical methods to detect marine toxins. The main in vivo assays used for the detection of marine biotoxins in seafood are the mouse and rat bioassay, respectively MBA and RBA. The MBA suffers from false positives and the RBA does not allow the detection of neurotoxic marine biotoxins.at the moment the LC-MS/MS technique developed by Gerssen et al. in 2010 and the Lawrence method have been validated as an alternative routine method to the in vivo assays for the detection of lipophilic marine biotoxins in seafood at the European level (European Commission, 2005; 2011). However, there is no LC-MS/MS or other chemical analytical method that is routinely applicable for the broad detection of marine biotoxins, i.e. ciguatera toxins (CTXs) and neurotoxic brevetoxins (PbTxs) for example are missed.there is thus an urgent need for the development of cheap animal friendly high-throughputin vitro tests that would allow the detection of marine biotoxins in seafood products, both toxins that are currently known, but also unknown toxins and those which might emerge, e.g. due to climate changes. Recently a broad in vitro test strategy in combination with analytical methods was set-up at our institute. This multi-disciplinary approach includesseveral bioassays, i.e., biomarker detection in human Caco-2 intestinal cells with qpcr and Luminex technology for DSPs, the Neuro-2a MTT tests for both DSPs and NSPs, detection of NSPs with beating cardiomyocytes anddetection of NSPs in neural cells with a multi electrode array (MEA). Over 100 samples from Chile have been analysed by the bioassays, showing very good agreement with chemical analysis, indicating that the test strategy will allow a replacement of the MBA.

20 20 10 TH INTERNATIONAL CONFERENCE ON MOLLUSCAN SHELLFISH SAFETY 18. Oral Presentation Temperature effects on kinetics of paralytic shellfish toxin elimination in the Atlantic surfclam Spisula solidissima Bricelj, V. Monica 1.,Cembella, Allan 2.,Laby, David 3., 1 Institute of Marine and Coastal Sciences Rutgers University. 2 Ecological Chemistry, Biological Sciences, Alfred-Wegener-Institut. 3 Biosciences NRC Institute for Marine Biosciences. The Atlantic surfclam Spisula solidissimais characterized by accumulation of extremely high levels of paralytic shellfish poisoning (PSP) toxins, slow toxin elimination rates and an extremely high post-ingestive capacity for toxin bioconversion. Surfclam populations experience a wide range of temperatures along the NW Atlantic continental shelf, and are undergoing range contraction attributed to global warming. In this study we determined the influence of temperature on detoxification kinetics of individual PSP toxins in two tissue compartments of juvenile surfclams under controlled laboratory conditions, over prolonged (2.4 months) depuration. Clams were toxified with an isolate of the dinoflagellate Alexandriumfundyense from the Gulf of Maine, allowing tracking of toxin composition and calculated toxicity in surfclam tissues. The viscera detoxified at all temperatures, although toxin loss rate increased with increasing temperature. In contrast, total toxin content and calculated toxicities in other tissues remained constant or even increased during depuration, suggesting physiological or biochemical toxin-retention mechanisms and temperature-independent detoxification. In vivo toxin compositional changes in surfclam tissues provide evidence of specific toxin conversion pathways, involving both reductive and decarbamoylation pathways. We conclude that such toxin biotransformations, especially in non-visceral tissues, may introduce a discrepancy in describing kinetics of total toxicity (in saxitoxin equivalents [STXeq]) over prolonged detoxification. Nevertheless, total toxicity values generated by routine regulatory monitoring based upon mouse bioassays or calculated from chemical analytical determination of molar toxin concentrations is adequate for first-order modeling of toxin kinetics in this species. Furthermore, the differential detoxification response of viscera and other tissues in relation to temperature emphasizes the need for two-compartment modeling to describe the fate of PSP toxins. Finally, we identified key parameters that may prove useful in hindcasting the timing of toxic blooms or predicting new toxin input to deep offshore waters where routine monitoring of toxic phytoplankton is impractical.