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Mattioli 1885

SEA-on-a-CHIP 2nd PROGRESS WORKSHOP

MONITORING FOR A SUSTAINABLE MANAGEMENT OF MARINE RESOURCESEditors: G. Sacchettini, M. Giulivo

euro 10,00

Chemical contamination of estuarine and coastal areas is a highly complex issue with ne-gative implications for the environment and human health (through the food chain) andrelated coastal industries such as fisheries. To face this issue, a series of workshops havebeen organized in the frame of the European project Sea-on-a-Chip with the main pur-pose of promoting constructive multidisciplinary interactions among scientists and mainstakeholders. The implementation of integrated approaches with the inclusion of a widerange of stakeholders into the debate appear the only path to achieve a sustainable use ofmarine resources.

Ettore Capri13th April 2016, Ferrara (Italy)

SEA-on-a-CHIP has received founfing from the European Union’sSeventh Programme for research, technological development and

demonstration under grant agreement No 614168

00-copertina ok.qxp_copertina 04/04/16 17:04 Pagina 1

SEA-on-a-CHIP 2nd Progress Workshop

MonItorIng for A SuStAInAblE MAnAgEMEnt of MArInE rESourCES

editors: Gabriele sacchettini, Monica GiUliVo

01-Prime pagine.indd 1 04/04/16 17:07

editorsgabriele Sacchettini, Monica giulivo

titoloSEA-on-a-CHIP 2nd Progress WorkshopMonitoring for a sustainable management of marine resources

isbn978-88-6261-556-3

pubblicazionefidenza, 2016

© Mattioli 1885

www.mattioli1885.com

© Mattioli 1885. All rights reserved. No part may be reproduced, stored in a retrieval systemor transmitted in any form or by any means without the prior permissionof both the author and the publisher.


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I N D E X

IntroductionGabriele Sacchettini, Monica Giulivo ..........................................................................................5

Session 1: Seawater pollution – Oral presentations

Insights from an international stakeholder consultation to identify informational needs related to seafood safety Alice Tediosi, Silke Jacobs, Wim Verbeke, Diana Álvarez-Muñoz, Jorge Diogene, Marieke Reuver, António Marques, Ettore Capri ...................................................................................................9

Impact of Megalopolis on the Adjacent Marine Environments: PAHs flow rates and nutrients export Angela Wagener, Leticia Lazzari, Cristiane Mauad, Carlos Mazzoni .........................................11

Occurrence of marine biotoxins in the western Mediterranean Sea Cristina Bosch-Orea, Josep Sanchís, Marinella Farré, Damià Barceló ..........................................13

Analytical characterization of microplastics and assessment of their adsorption capacity for organic contaminants Gabriella Schirinzi, Marta Llorca, Josep Sanchís, Marinella Farré, Damià Barceló .....................15

Insights on domoic acid production by 3 Pseudo-nitzschia diatom species and filtration rates of 4 shellfish species exposed to Pseudo-nitzschia diatoms Lia Godinho, Alexandra Silva, Ana Gamboa, Maria Ana de Castelo Branco, Maria Emília Cunha, Pedro Pousão-Ferreira, António Marques, Pedro Reis Costa ...............................19

Halogenated flame retardants in fish from European river basins Monica Giulivo, Ethel Eljarrat, Ettore Capri, Damià Barceló ....................................................21Novel passive sampling to assess stratification of contaminants in coastal watersYann Aminot, A. Belles, C. Alary, J.W. Readman .......................................................................23

Session 1: Seawater pollution – Poster presentations

Analysis of antibiotics by LC-MS2 in aquatic ecosystems Alejandro Margareto Mato, M. Silvia Díaz-Cruz, Damià Barceló Culleres ................................27

Economic Sustainability and Competitiveness of European Traditional Seafood Market Giovanni Sogari, C. Mora, D. Ottaviano, D. Menozzi ..............................................................30

Monitoring pesticide residues in a large tropical bay, Bahia de Todos os Santos, Brazil Fernando da Piedade Carvalho, C. Cattini, J.P. Villeneuve, I. Tolosa, J.M.Oliveira .....................32

Assessment of saxitoxin levels in Mediterranean Coastal Waters: a preliminary studyTheodoti Papadimitriou, Nikolaos Mellios, Chrysi Laspidou .......................................................35

Development of a biodiversity indicator as a tool for marine environment managementElena Botteon, Ana Cristina Costa, Andrea Zita Botelho. ..........................................................37


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Legally and non-legally binding legislation on sustainable aquaculture: the code of conduct for responsible fisheriesPierluigi Monticini, Ettore Capri..............................................................................................40

The millennium declaration (mdgs) and the new sustainable development goals Pierluigi Monticini, Ettore Capri..............................................................................................41

Trout, Seabass and Seabream Value Chain Overview in Italy Giovanni Sogari, C. Mora, G. Morelli, D. Menozzi ...................................................................42

Contaminants in the marine environment: What do weface?D. Alvarez-Muñoz, M. Llorca, J. Blasco, D. Barceló ..................................................................44

Session 2: Marine biosensor & Microelectronics in biosensing applications – Oral presentations

Biosensors for real time monitoring of biohazard and man made chemical contaminants in the marine environmentKonstantinos Petropoulos,Arben Merkoci, Aziz Amine,Bengt Danielsson, Chrysi Laspidou, Elise Regairaz, Linda Medlin, Luca Sanfilippo, Nives Kovač, Salvatore Chiavarini, Giuseppe Palleschi .....................................................................................................................47

MARIABOX an autonomous sea water pollution monitoring device for natural and man-made pollutants: challenging user requirements and tackling design criticalitiesPaolo Barattini, Silvia Mier, Matteo Bonasso, Esther Garcés, Panayiotis Philimis, Alessandro Giusti, Kevin Thomas, Antonio Varriale, Sabato D’Auria ..........................................49

Portable acquisition electronic systems applied to electrochemical biosensorsRaquel Pruna, Francisco Palacio, Monica Martinez, Manel López .............................................50

Novel strategy for detection of Sulfapyridine using a fully integrated Hybrid Lab-on-a-chipNadia El Alami El Hassani, Abdoullatif Baraket, Ernandes Taveira Tenório Neto, Michael Lee, Juan Pablo Salvador, Maria pilar Marco Colas, Marinella Farré, Damià Barceló, Joan Bausells, Nezha El Bari, Benachir Bouchikhi, Abdelhamid Elaissari, Abdelhamid Errachid, Nadia Zine ..............................................................................................................................52

A new competitive BioLab-on-a-chip for the detection of Irgarol 1051 for seawater monitoringTalha Jamshaid, Abdoullatif Baraket, Michael Lee, Juan Pablo Salvador, Maria Pilar Marco Colas, Joan Bausells, Marinella Farré, Damià Barceló, Damià Barceló, Nadia Zine, Abdelhamid Elaissari, Abdelhamid Errachid ..........................................................54

Session 2: Marine biosensor & Microelectronics in biosensing applications – Poster presentations

Determination of pesticides used in marine aquaculture by LC-High Resolution Orbitrap MSV.I. Boti, Ch.I. Nannou, M.G. Kapsi, T.A. Albanis ....................................................................59

Sensing devices for marine algal toxins detectionMarianna Rossetti, Alessandro Porchetta, Konstantinos Petropoulos, Francesco Ricci, Laura Micheli, Giulia Volpe, Giuseppe Palleschi .........................................................................61


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I N T R O D U C T I O N

Seas and oceans have a huge impact on our daily lives as critical source of different kind of important resources, goods and services (e.g. energy, food, tourism). However, these are under continuous threat from a large number of anthropogenic factors (e.g. maritime transport, coastal development, resource extraction, fisheries, aquaculture) which lead to a series of negative im-pact into the water quality and the marine environment (e.g. loss of species, damage of habitats, nutrient and chemical pollution).

To achieve a sustainable use of marine resources, the European Union has developed a series of new policies aiming to find the right balance between economic, social and environmental aspects. The most important recent initiative is the European Marine Strategy Framework Di-rective (2008/56/EC) where, for the first time, European Member States are meant to formulate appropriate strategies to protect and preserve the marine environment taking measures to reach and maintain good marine environmental status. Member States were required to develop their own national strategies by 2015, implement their programs of measures until 2016 and, finally, reach the objectives of the Directive by 2020.

In this view appear quite clear how cost-effective monitoring, automatic detections and real-time measurements are of fundamental importance to ensure compliance with new marine legislation. However, it is important to take into consideration that marine sciences and technologies are by their nature cross-cutting and involve many disciplines which are interconnected and need to be addressed in parallel in a wide sense of sustainability. For this reason, the implementation of integrated approaches with the inclusion of a wide range of stakeholders into the debate appear critical. This is especially true in the aquaculture sector where quality of the marine ecosystems and sea-food safety concerns are strongly correlated requiring the involvement of a wide range of stakeholders along the entire food chain until the final consumer.

To contribute in this direction, a series of workshops have been organized in the frame of the European project Sea-on-a-Chip (www.sea-on-a-chip.eu) with the main purpose of developing a scientific platform to stimulate the exchange of the most recent information in the various fields of marine science promoting constructive interactions between them and among the main stakeholders involved (especially scientific community, policy makers and private bodies).

In this sense, this workshop has been structured around three main complementary and in-terconnected topics (Seawater pollution, Marine biosensor and Microelectronics in biosensing applications) considering Monitoring as main horizontal drivers for a consistent measurement of the performance and validation of the related phenomena.

An Opening session has been organized in a round-table format involving representativeness of main stakeholders to identify key needs and challenges that scientist needs to take into account in their studies. On the other hand, following core sessions have been developed with oral and poster presentations to share main recent scientific discoveries. Moreover, plenary discussions were included to identify future directions for advancing research and regulation to guarantee an effective protection of marine environment.

Valuable scientific contributions has been received from our call showing the high interest among scientists on these topics and on the approach proposed. For this reason, it is important to see


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this event not only as a simple occasion to exchange experiences among colleagues but also as a real opportunity that we need to take together to build and reinforce a network among experts from different disciplines who share similar views and objectives.

Last but not least, we would like to take the occasion to thank all the people that have acti-vely contributed to the development of such important event with particular emphasis to all the members of the Scientific Committees such as Damià Barceló, Marinella Farré, Marianne Köck, Federico Ferrari, Ettore Capri, Edoardo Turolla and Graziano Caramori. Furthermore, we would like also to thank all partners of the European project Sea-on-a-Chip in the frame of which it was organized this workshop as well as all participants willing to share their valuable research experiences.

Gabriele Sacchettini

Monica Giulivo


S E S S I O N IS E a w at E r p O l l u t I O N O r a l p r E S E N tat I O N S

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S E A - o n - a - C H I P 2 n d P R O G R E S S WO R K S H O P

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Insights from an international stakeholder con-sultation to identify informational needs related to seafood safetyAlice Tediosi1, Silke Jacobs2,3, Wim Verbeke3, Diana Álvarez-Muñoz4, Jorge Diogene5, Marieke Reuver6, António Marques7, Ettore Capri8

1Aeiforia Srl, 29027 Gariga di Podenzano (PC), Italy2 Department of Public Health, Ghent University, 9000 Ghent, Belgium3 Department of Agricultural Economics, Ghent University, 9000 Ghent, Belgium4 Catalan Institute for Water Research (ICRA), Parc Científic i Tecnològic de la Universitat de Girona, 17003 Girona, Spain5 IRTA, 43540 Sant Carles de la Ràpita, Spain6 AquaTT, Dublin 2, Ireland7 Division of Aquaculture and Upgrading (DivAV), Portuguese Institute for the Sea and Atmosphere (IPMA), 1449-006 Lisbon, Portugal8 OPERA Research Center, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy E-mail contact: [email protected]

Highlights • Weconsultedstakeholderstoidentifytheirneedsaboutseafoodsafety• Anon-linesurveywaspreparedfollowingtheDelphimethodologyandsenttogather

stakeholders’opinions• Communicationamongstakeholdersneedstobeimproved• Thereisadeficitofinformationanddatainthefieldofseafoodsafety• On-linetoolsareperceivedtobethemostusefulcommunicationchannel.

Foodsafetyassessmentandcommunicationhasastrongimportanceinreducingrisksfor human health related to food consumption. The research carried out within theECsafeSEAFOODprojectaimstoassessseafoodsafetyissues,mainlyrelatedtonon-regulatedprioritycontaminants,andtoevaluatetheirimpactonpublichealth.Inordertomaketheresearchresultsaccessibleandexploitable,andrespondingtorealstakeholders’demands,aconsultationwithstakeholderswasperformedbymeansofa2-roundDelphi survey, focusingonpolicyanddecisionmakers, foodproducersandprocessors,andagenciesandconsumerorganisations.Thesurveyconsideredquestionsrelatedto:seafoodsafetyassessmentandmitigationstrategies,dataavailability(asle-vel of information on different contaminants), and communication among differentstakeholder groups. The first-round questionnaire was sent to 531 key stakeholdersand91responseswerereceivedfrom30Europeanandnon-Europeancountries.Thesecond-roundquestionnairewassenttothefirst-roundrespondentsand37responseswerereceived.Themainresultsshowthatcommunicationbetweendifferentgroupsofstakeholdersneedstobeimprovedandthatthereisadeficitofinformationanddatainthefieldofseafoodsafetyandregardingenvironmentalcontaminants.On-linetoolsareperceivedtobethemostusefulcommunicationchannel.

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AcknowledgementTheresearchleadingtotheseresultshasreceivedfundingfromtheEuropeanCommu-nity’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no311820.Thispublicationreflectstheviewsonlyoftheauthor,andtheEuropeanUnioncannotbeheldresponsibleforanyusewhichmaybemadeoftheinformationcontainedtherein.

ReferencesTediosi,A.,Fait,G.,Jacobs,S.,Verbeke,W.,Álvarez-Muñoz,D.,Diogene,J.,Reuver,M.,Marques,A.,Capri,E.Insightsfromaninternationalstakeholderconsultationtoidentifyinformationalneedsrelatedtoseafoodsafety

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Impact of Megalopolis on the Adjacent Marine En-vironments: PAHs flow rates and nutrients exportAngela Wagener1, Leticia Lazzari1, Cristiane Mauad1, Carlos Mazzoni1

1Department of Chemistry, Pontifical Catholic University of Rio de Janeiro, 22453-900 Rio de Janeiro, Brazil E-mail contact: [email protected]

Highlights • EstimatetheinputsofPAHsthoughriversintoGuanabaraBayandtheoutputof

nutrientsandcarbonintocoastalareas• TargetingthemajorriversforPAHandtheexchangesatthebaymouthfornutrients• Samplingatsitesandtimeintervalsrepresentativeofspace-timevariations• LoadsforPAHswereof3.64±2.4tyear-1accountingforabout30%ofthedeposi-

tedannuallyinthereceivingbody• Theestimatedannualcarbonexportshowthebaycontributeswith0.01%ofthetotal

globalcarboninfluxtotheocean

TheGuanabaraBayBasininRiodeJaneirohousesmorethan10106inhabitantsandthesecondmostrelevantindustrialdistrictinBrazil.Becauseemissionsofhydrocarbonsfrommobileandstationarysourcesmayposearisktohumansandtothemarinelifeastudywasperformedtoquantifytheannualinputsthroughthemajorriverscrisscrossingthebasin.Forthis,particulatematterwassampledmonthlyoveroneyearinsixmajorriversandhydrocarbonsweredeterminedtoassessconcentrationlevelsand,mostimportant,

sourceidentificationand quantification.Forthemassbalancesedimentation ratesand concentrationinsurfacesedimentsof the receivingbodywerealsoused.Resulting loads forPAHs were of 3.64± 2.4 t year-1 ac-counting for about30%oftheinvento-rydepositedannual-ly in the receivingbody which surpassthe reported for theSusquehanna(2.37t

PAHs mass load deposited on annual basis in sediments

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year-1)river,oneofthefivemostimportantriversoftheChesapeakeBayinUSA,andthatfortheEbroRiver(14±2tonano-1)intheWesternMediterraneanSea.Mosthydrocarboninsedimentsandcarriedbytheriversderivefromvehicularemissions.Watersampleswerecollectedfromdifferentdepthsover25hintwoseasonsatthebayentrance.Measurementsincludedphysicochemicalparameters,nutrients,chlorophylls,dissolvedorganiccarbon(DOC),particulateorganiccarbon(POC),particulatenitro-gen(PN),carbon(d13C)andnitrogen(d15N)isotopiccomposition.Mostvariablesshowedhighervaluesinebbtideevents.Theflowratescalculatedondailybasisandestimatedonannualbasisrevealedtheexportationtothecontinentalshelfof1.27×104kmolyear-1dissolvedinorganicnitrogen(DIN),9.52×102kmolyear-1dissolvedinorganicphosphorus(DIP),2.65×104tyear-1DOC,1.96×104tyear-1POC,and2.96×104tyear-1PN.Theestimatesshowthebaycontributeswith0.01%ofthetotalglobalcarboninfluxtotheocean.

AcknowledgementCAPES,CNPqandFAPERJforthefinancialsupport.

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Occurrence of marine biotoxins in the western Mediterranean SeaCristina Bosch-Orea1, Josep Sanchís1, Marinella Farré1,*, Damià Barceló1,2

1 Water and Soil Quality Research Group, Department Of Environmental Chemistry (IDAEA -CSIC), Barcelona,Spain 2 Catalan Institute for Water Research (ICRA), H2O Building, Girona,Spain - *E-mail contact: [email protected]

Highlights • Inthisstudy,amethodologybasedinsolidphaseextractionfollowedbyhighperfor-

manceliquidchromatographycoupledtohigh-resolutionmassspectrometryhasbeendevelopedandapplied.

• 18marinebiotoxinspertainingto4groups;paralyticshellfishpoisoning(PSP),diarrheicshellfishpoisoning(DSP),amnesicshellfishpoisoning(ASP)andazaspiracidshellfishpoisoning(AZP)weresimultaneouslyanalysed.

• Thenewmethodspresentedlimitsofdetectionnintherangeof0.52to0.14µg/l.• 40sampleswerecollectedfromtheCatalancoast(NEofSpain)• Okadaicacidwaspresentinthe70%ofthesamplesinconcentrationsrangingfrom

2.10to1778.10ng/l.

Introduction and objectivesMarinebiotoxinsaresecondarymetabolitessynthetizedbyphytoplanktonandbacteriaasadefensivestrategyagainstotherorganisms.Thetoxicpropertiesofthesecompoundshaveanegativeinfluenceinthegrowth,reproductionandsurvivalofdeterminedspecies.Moreover,ithasbeenevidencedthatcanbioaccumulatealongthefoodchain1.Therefore,thepresenceofthesesubstancesisanissueofconcern,especiallywhenaharmfulalgalbloom(HAB)takesplace.Duringthisphenomenon,highamountsoftoxinsaregenera-tedaffectingtheecosystemandconsequentlysupposinganimportantriskforthehumanhealth.Theobjectiveofthisstudyistoassesstheoccurrenceanddistributionof18toxinsper-tainingto4groupsofmarinebiotoxinsintheseawateralongtheCatalancoast.Forthis,thetoxinsstudiedwere:Azaspiroacids(azaspiracid-1,AZA1;azaspiracid-2,AZA2;aza-spiracid-3,AZA3;azaspiracid-4,AZA4;andazaspiracid-5,AZA5);andrelatedpecte-notoxins (pectenotoxin2, PTX2), yessotoxins (yessotoxin, YTX; and homoyessotoxin(hYTX); okadaic acid (OA) and related dinophysistoxins (dinophysistoxin-1, DTX1);domoic acid, DA; saxitoxins (neosaxitoxin, NEO) and related gonyautoxins (gonyau-toxin-2,GTX2;gonayutoxin-3,GTX3;gonyautoxin-5,GTX5;decarbamoylgonyauto-xin-2,dcGTX2;decarbamoylgonyautoxin-3;dcGTX3;N-sulfocarbamoylgonyautoxin-2,C1;N-sulfocarbamoylgonyautoxin-3,C2);andtetrodotoxin,TTX.Forthisreasonthefirstobjectivewastodevelopandvalidateamultiresidueanalyticalmethodbasedonsolidphaseextraction(SPE)followedbyHighperformanceliquidchromatographywascou-pledtohigh-resolutionmassspectrometry(HPLC-HRMS).

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MethodologySurfaceseawaterwascollectedindifferentcoastalareasalongtheCatalancoast insidesportiveportsandinpublicaccessbeachesduringthespringof2015.Sampleswerecol-lectedinglasscontainerandpreservedincoolconditionsduringtheirtransporttothelaboratory.Seawatersampleswerefilteredwithnylon(0.45µmmeshsize)filters.Theparticulateretainedbythefilterwasextractedbyultrasoundsassistedsolid-liquidextraction(UAE)withmethanol.Then,theextractswerepurifiedbysolidphaseextraction(SPE)usingOasisHLBcartridges.Chromatographicseparationwasachievedonahydrophilicinteractionliquidchromato-graphy(HILIC)(150×2.0mm,particlesize3µm)analyticalcolumnfromPhenomenex,Themobilephasecompositionconsistedin90%acetonitrileand10%of10mMammo-niumacetateattheflowrateof0.300ml/min.TheLCsystemwascoupledtoaQ-Exac-tive(ThermoFisherScientific)hybridquadrupole–Orbitrapmassspectrometerwithanelectrosprayionizationsourceoperatedmodeinpositiveandnegative.Quantificationwasperformedusingmatrix-matchedcalibrationcurveofallcompounds.StandardsofeachcompoundwerepurchasedfromCifgawithapurityrangingfromthe≥96%tothe≥99%.

Results and conclusionsThemarinebiotoxinmostpresentinthesampleswasOkadaicacidthatappearedinthe90%ofthesampleswitnconcentrationsrangingfrom2.10to1778.10ng/.Concentrationsoftoxinsfoundinsidesportiveportswerehigherofthosedeterminedinsamplesfrompublicaccessbeaches.Thisfactisduetothehighturbidityandlowcircula-tionofthewaterinsidethesportiveports.Inconclusion,ananalyticalmethodhasbeensuccessfullyappliedfortheanalysisofmorethanadozenofmarinebiotoxinsinseawater.ThemethodwasappliedtocharacterizetheoccurrenceoftheseemergingcontaminantsinsamplesfromtheWesternMediterraneanCoast.Okadaicacidwasdetectedintherealenvironmentattraceconcentrationsinnonalgalbloomconditions.

AcknowledgementThisworkwassupportedbythebytheEuropeanUnionthroughtheprojectsSea-on-a-chip(FP7-OCEAN-2013-614168)andBraavoo(FP7-OCEAN-2013-614010),andbytheGeneralitatdeCatalunya(ConsolidatedResearchGroups“2014SGR418”WaterandSoilQualityUnit”).TheauthorswouldliketoexpresstheirdeepestgratitudetotoRoserChalerandDoriFanjul,becauseoftheirtechnicalsupportduringtheLC-HRMSanalyses.

References 1)Buratti,S.etal.,Bioaccumulationofalgaltoxinsandchangesinphysiologicalparame-

ters in Mediterranean mussels from the North Adriatic Sea (Italy). Environmentaltoxicology,2013.28(8):p.451-470.

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Analytical characterization of microplastics and assessment of their adsorption capacity for organic contaminantsGabriella Schirinzi1, Marta Llorca1, Josep Sanchís1, Marinella Farré1,*, Damià Barceló1,2

1 Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Barcelona, (Catalonia, Spain) 2 Catalan Institute for Water Research (ICRA), Girona (Catalonia, Spain) - *E-mail contact: [email protected]

Highlights• Thepotencyofmicroplastics (MPLs) tostabilizeandtransportotherco-contami-

nantsinsameaquaticenvironmentswasstudied.• Asthefirststep,analyticalapproachestoidentify,quantifyandcharacterizeMPLs,

agedinrealenvironmentalwatersampleswereexplored,andacombinedanalyticalapproachisproposed.

• FortheanalysisofMPLs,theuseoftwodimensionsgaschromatographycoupledto time-of-flight spectrometry (GC x GC-TOF), matrix-assisted laser-desorptionionization(MALDI)coupledtoTOFwithoutstepof separationandLCcoupledtohigh-resolutionmassspectrometry(HRMS)werecomparedregardingionizationandquantification.

• Theuseofinfrared(IR)spectroscopyandopticalmicroscopywereaswellassessedforidentificationandcharacterizationofMPLsagedinrealsamples.

• Secondadsorption/desorptioncapabilitywasstudiedfortwogroupsoforganiccon-taminants,polycyclicaromatichydrocarbons (PAHs)andperfluorinatedalkyl sub-stances(PFASs)asrepresentativesoflegacypersistentorganicpollutants(POPs)andemergingcontaminants,respectively.

• MPLsshowedthecapacitytoadsorb,stabilizeandtransportotherorganicco-conta-minants,whichcaninfluencetheirtoxicologicalbehaviour(Trojanhorseeffect).

Microplastics(MPLs)havebeendefinedassmallparticlesofplasticinthemillimetretosub-millimetresizerange(<5mm)andhighdensities(e.g.,100000itemsperm3).Intheaquaticenvironments,plasticresiduescanbeaprominentsourceofemergingcontaminants,whichimpliesdamagesfortheenvironmentandpotentialproblemforthehumanhealth.Duetotheirsmallsize,MPLsaresuspectedtobebioaccumulatedandtheycanenterthefoodchain.Inaddition,theyareasocio-economicimpactfortheaffectedareasascoastalenvironments(1,2).MPLshavelowdegradabilityandtheirsizehamperstheoxygenexchangeandlight,whichleadtoeutrophicationprocesses(2).Furthermore,theadsorption/desorptioncapabilitiesofthesecompoundscanleadthestabilizationandtransportofothertoxiccontaminantsinthesamecompartments(3,4).Theyaresuspectedtomodulatethetoxicityofotherco-contaminants,whichisknownasTrojan-horseeffect.Inthiscontext,themainobjectiveofthisworkwastoassessthepotencyofMPLstostabili-zeandtransportotherco-contaminantsinaquaticenvironments.Therefore,thefirstspecific

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objectivewastodevelopacombinedanalyticalapproachtoanalyseMPLs.GCxGC-TOF/MS,MALDI-TOFandLC-HRMSusing anOrbitrap instrumentswere compared intermsofionizationandquantification.However,thisinformationshouldbecomplementedintermsofpolymersidentificationandcharacterization.ThepotencyIRspectroscopyandmicroscopyforthecharacterizationofsyntheticpolymershavebeenevaluated.Finally,themechanismsofadsorptionofpolycyclicaromatichydrocarbons(PAHs)andperfluoroalkylsubstances(PFASs)ontohighdensitypolyethylene(HDPE),polystyre-ne(PS)andpolystyrenecarboxylated(PS-COOH)werestudied.

Material and methodsHighDensityPolyethylene(HDPE)microspheres(3-16µm),whichwerepurchasedfromCospheric(SantaBarbara,California,USA);andpolystyreneandfunctionalizedpolystyrene-COOH (10 µm), supplied by Phosphorex (Hopkinton, Massachussets,USA).16PAHscompoundsweresuppliedbyDr.Ehrenstorfer-Schäfers(Augsburg,Germany)and18PFASswerefromWellingtonLabs(Ontario,Canada).

Synthetic samplesSyntheticwatersamplespreparedaccordingtotheproceduredescribedbytheEUde-scriptor(6).WatersampleswerefortifiedwiththedifferentpolymericMPLs,thentheywerefilteredthrough0.75µmfibreglassfilters,driedandstoreduntilanalysis.

Quantitative and qualitative analysisTheanalysisofplasticizersandadditiveswereperformedinthefiltratedwatersamples.Avolumeof500mLofthemwereextractedbyliquid-liquidextractionwithdichloro-methaneandhexane(1:1).Then,theextractsweredriedandreconstitutedusing1mLofisooctane.Forgas-chromatographicseparation,twocolumnsinserieswereused.ThefirstwasHP-5MS(50mx0.25mmx0.25µm,AgilentTechnology)andthesecondonewasTRB-50HT(2mx0.1mmx0.1µm,Teknokroma).FortheanalysisofpolymersbyLC-HRMS,thefiltratedresiduesweredriedovernightat60ºC.Theresidueswerereconstitutedwith10mLoftolueneusingultrasonicas-sistedextraction.Successively,theextractsweredriedunderagentleN2currentandreconstitutedtill1mLoftoluene.ThechromatographicseparationwasachievedusingPhenogel10µm10E5Acolumnandtoluene100%asmobilephase.Inparallel,thesameextractsweredissolvedindihydroxyacetophenoneandthenwereanalyzedbyMALDI-TOF.Finally,thefilteredresiduesweredirectlyanalyzedbyIRspectroscopyandcharacterizedbymicroscopy.

Adsorption/desorption experimentsThe adsorption/desorptioncapabilities of MPLswere carried out. In this experimentseawatersampleswerefortifiedatenvironmentalrelevantconcentrationsof10µg/LofPAHsandPFASsinpresenceofMPLs(PE,PSandPS-COOH)atdifferentconcen-

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trationsbetween2and5mg/L.Then,thesampleswereagitatedfor4,7and46dayswithturbulence,emulatingrealconditionsandcontrollingthattheoxygenpresentinwaterwassimilarthatinacostalareassituation.Day/nightperiodswereaswellemu-lated.Afterexposure,watersampleswereanalyzedbygaschromatographycoupledtotandemmassspectrometry(GC-MS/MS)equippedwithanelectronimpact.Whereas,for the analysis ofPFASs, liquid chromatograph coupled to amass spectrometer intandem(LC-MS/MS),equippedwithanelectrosprayionizationsourceoperatedinne-gativemode,wasused.TheresultsofmodelcontaminantsinwatersamplesinpresenceandwithoutMPLswerecompared.

Results and conclusionsRegarding the different analytical procedures to quantify, identify and characterizethe different MPLs, the different approaches have presented combined capabilities.IR spectroscopy was the technique showing less sensitivity, especially for real envi-ronmentalsamples.RegardingtoMALDI-TOF,semi-quantitativemeasurementswereachievedbutwithlimitsofquantification(LOQ),whichwerehigherthanmg/L.Thatmeans that this couldbe an excellent technique after an improved extractionor forbiotasampleswithhighconcentrations.Ontheotherhand,LC-HRMSshowedgoodperformanceforquantitativeandsemiquantitativemeasurements,inparticularusingAPPI/APCIasionizationsource.TheLOQwasmuchbetterthaninMALDI-TOF.Besides,GCxGC-TOF/MSpresentedexcellentcapabilitiesfornon-targetscreeningofpolymericresiduesinrealenvironmentalsamplesduetothefullscananalysis.Intermsofadsorption/desorptionexperiments,MPLswereshowntobeabletosta-bilizeandtransportco-contaminates.Forexample,till35%ofshortchainPFASwereadsorbedinPSandPS-COOHintheseawaterexperiments.The8-carbonchaincom-poundssuchasPFOSandPFOAwereadsorbedinthesametypeofMPLsreachinghigherpercentages(50%)after46daysofexposureunderambientconditions.LongchaincompoundswereshowntobestabilizedinalltypesofMPLsreachingupto80%.Itshouldbehighlightedthatdifferentambientbehaviorwasobserveddependingonwater type. Ingeneral, the stabilizationoforganiccontaminatesontoMPLssurfaceinseawaterwasfavored.TheseresultsunderpintherequirementtostudyotherwaterconditionsinwhichorganicmaterialsarepresentandthepotentialtoxicitymodulationofmixturesofcontaminantsinthepresenceofMPLs.

AcknowledgementThisworkwassupportedbytheSpanishMinistryofEconomyandCompetitivenessthroughtheprojectIntegra-Coast(CGL-2014-56530-C4-1-R)andbytheGenera-litatdeCatalunya (ConsolidatedResearchGroups“2014SGR418”WaterandSoilQualityUnit).MLacknowledgesJuandelaCierva–Incorporaciónresearchfellowship( JCI-2014-21736)fromtheSpanishMinistryofEconomyandCompetitiveness.TheauthorswouldliketoexpresstheirdeepestgratitudetotoRoserChalerandDoriFanjul,becauseoftheirtechnicalsupportduringtheLC-HRMSanalyses.

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References

1)ColeM.etal.Microplasticsascontaminantsinthemarineenvironment:Areview.Ma-rinePollutionBulletin62(2011)2588–2597.

2)HarrisonJ.P.etal.Interactionsbetweenmicroorganismsandmarinemicroplastics:ACallforResearch.MarineTechnologySocietyJournal45(2011)12-20.

3)ColeM.etal.Microplasticingestionbyzooplankton.EnvironmentalScience&Techno-logy47(2013)6646-55.

4)LlorcaM.etal.LevelsandfateofperfluoroalkylsubstancesinbeachedplasticpelletsandsedimentscollectedfromGreece.MarinePollutionBulletin87(2014)286–291

5)RiosaL.M.et al.Persistentorganicpollutants carriedby syntheticpolymers in theoceanenvironment.MarinePollutionBulletin54(2007)1230–1237.

6)Songetal.Acomparisonofmicroscopicandspectroscopicidentificationmethodsforanalysis of microplastics in environmental samples. Marine PollutionBulletin 2015,93(1):202-209.

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Insights on domoic acid production by 3 Pseudo-nitzschia diatom species and filtration rates of 4 shellfish species exposed to Pseudo-nitzschia diatomsLia Godinho1, Alexandra Silva1, Ana Gamboa1, Maria Ana de Castelo Branco1, Maria Emília Cunha1,Pedro Pousão-Ferreira1, António Marques1, Pedro Reis Costa1

1Portuguese Institute for the Sea and Atmosphere (IPMA, I.P.), Av. Brasilia, s/n, 1449-006 Lisbon, Portugal - E-mail Contact: [email protected]

Highlights• DomoicacidwasdetectedinculturesofP. multiseriesbutnotinP. fraudulenta,andP.

pungensvar. aveirensis.• Intracellularandextracellularlevelsofdomoicacidwerecharacterizedunder3light

conditions (15 µmol.m-2.s-1, 120 µmol.m-2.s-1 and 560 µmol.m-2.s-1). Higher levelswerefoundduringthestationaryphaseat120µmol.m-2.s-1.ArelationshipbetweenDAandgrowthratewasobserved.

• FiltrationrateswerecalculatedforMytilus galloprovincialis, Cerastoderma edule, Do-nax sp.andSpisulasolidafeedwithP. multiseriesandfeedwith P. multiseries andSkele-tonema costatum.HigherfiltrationrateswereobservedforM. galloprovincialis and C. eduleinalltestingtemperatures.

ThePseudo-nitzschiadiatomgenuscomprises12speciesassociatedwiththeproductionofdomoicacid(DA).ThisneurotoxinisresponsiblefortheAmnesicShellfishPoisoningsyndrome(ASP),firstdiscoveredasaresultofhumanconsumptionofcontaminatedbluemusselsinCanadain1989(Bates,etal.,1995).Theworld-widedistributionofthisgenusposes a threat to human health and environment integrity, making bivalve harvestingunpredictableandcreatingsignificantlossestotheaquacultureindustry.Thereisaneedtounderstandbloomdynamicsandecophysiological factors thatcontrol toxicdiatomsgrowthrates,DAproductionandfinallyDAaccumulationinshellfish.TheproductionofDAbyPseudo-nitzschiavariesaccordingwithspecies,growthphaseandenvironmentalconditions.TheaimofthisworkwastodeterminethegrowthratesandDAproduction,both intracellular and extracellular, ofPseudo-nitzschia multiseries, P. fraudulenta andP. pungensvar. aveirensisisolatedfromCascaisbayinOctober2014.Thestrainsweregrownunderthreedifferentlightsettings(15,120and560µmol.m-2.s-1).DAwasdetectedinalllightsettingsforP. multiseries,ranginginconcentrationbetween10and1625fgDA.cell-1,anreachinghigherlevelsduringthestationaryphase.Theoptimalconditionsforcellgrowthanddomoicacidproductionwereobservedat120and560µmol.m-2.s-1.Aten-dencyfornegativecorrelationbetweendiatomculturegrowthrateanddomoicacidpro-ductionwasobserved.Thistendencywasparticularlyevidentathigherlightconditions(560µmol.m-2.s-1).ExtracellularDA(dissolvedinculturemedium)wasdetectedattheveryendofthestationaryphase,beingassociatedwithbloomsdecayandcellssenescence.

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ThesecondobjectiveofthisstudywastodeterminethefiltrationratesforfourshellfishspeciesfeedwithP. multiseries.ThefiltrationratesofMytilus galloprovincialis,Cerasto-derma edule,Donaxsp.andSpisula solida wereestimatedinthreedifferenttemperatures(14,19and25ºC)withtwodifferentfeedingpatterns(P. multiseries onlyoramixtureofP. multiseries and Skeletonema costatum).Shellfishwerefeedduring60minandcellswerecountedevery10min.HigherfiltrationrateswereobservedforM. galloprovincialis andC. edule inalltreatments.TheresultsevidencethatlightintensityanddiatomstrainplayanimportantroleinDAproductionandthatDAisonlydetectedinthemediuminalatterphaseofthegrowthcurve.Thisaspectcombinedwiththedistinctfiltrationratesofbivalvesfosterfurtherinvestigationontheuptakeandeliminationkineticsofdomoicacidinbothshellfishspecies.Suchinformationisofcrucialimportancefortheimplementationofearlywar-ningsystemstohelpshelfishmanagerstobettercontroltheharvestingprocedures.

AcknowledgmentsThis studywas supportedby theSea-on-a-Chipproject (EuropeanUnion’sSeventhFrameworkProgramme-FP7/2007-2013).AntónioMarquesandPedroR.CostaaresupportedthroughtheFCTInvestigatorprogram(IF).

ReferencesBates,S.S.,Douglas,D.J.,Doucette,G.J.andLeger,C.(1995).EnhancementofDo-moicAcidProductionbyReintroducingBacteria toAxenicCulturesof theDiatomPseudo-nitzschia multiseries.Naturaltoxins3:428-435.

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Halogenated flame retardants in fish from Euro-pean river basinsMonica Giulivo1, Ethel Eljarrat2*, Ettore Capri1, Damià Barceló2,3

1Institute of Agricultural and EnvironmentalChemistry, UniversitàCattolica del Sacro Cuore di Piacenza, Via Emilia Parmense 84, 29100 Piacenza, Italy2Dep. of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18-26, 08014 Barcelona, Spain3ICRA, Parc Científic i Tecnològic de la Univ. de Girona, Emili Grahit, 101, E-17003 Girona, Spain - E-mail contact: [email protected]

Highlights• HFRs,widelyused,havebeenprovedtobetoxicfortheenvironmentandhumans.• HFRshavehighlipophilicity,andtheybioaccumulatedinfattissuesoffoodchains.• HFRanalyseswereperformedwithaselectiveandsensitivetechniquebasedonthe

useofGC-MS/MS.• HFRsweredetectedinalltheanalysedfishspecies,atconcentrationlevelsupto661

ng/glw,beingPBDElevelshigherthanEQS.• HFRcontamination found inAdige river basinwashigher than that observed in

Evrotasriverbasin.

1. IntroductionHalogenated flame retardants (HFRs) are a diverse group of chemicals widely usedin industrialandincorporatedinvariousconsumerproductssuchaselectronics,clo-thes,toys,motorvehicles,furniture,plastics,paintsandtextilesinordertoreducethelikelihoodofignitionoftheflammablematerials.Inthepastpolybrominateddiphenylethers(PBDEs)andMirex(organochlorinepesticide)wereextensivelyusedbutdueto their persistence, bioaccumulation, long-range transport, toxicity andbiomagnifi-cationthroughfoodwebstheuseofthesecompoundswerebanned.Thus,theusageofalternativeFRssuchashexabromobenzene(HBB),pentabromoethyl(DBDPE),pen-tabromoethylbenzene(PBEB)anddechlorans(Dec602,603,604,syn-DP,anti-DP)issignificantlyincreased.TheaimofthisworkistodeterminethepresenceofPBDEsandalternativeFRsindifferentfishsamples.

2. Materials and methodsThesecompoundsweredetectedbygaschromatographycoupledtotandemmassspec-trometry (GC-MS/MS).Analyzed samples are: brown trout,marble trout, grayling,bullhead and chub collected from the Adige river basin (Italy), and menida (Squa-liuskeadicus)fromEvrotasriverbasin(Greece).Theparametersoftheanalyticalme-thodare:recoveriesrangedbetween57-99%,methodlimitsofdetection(MDLs)from0.002-10.6ng/glipidweight(lw)andmethodlimitsofquantification(MQLs)from0.008-35.4ng/glw.

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3. Results and discussionTable1reportedlevelsobtainedinfishcollectedfromAdigeriverbasin.PBDEsanddechloransweredetectedinallthesamplesatconcentrationlevelsrangingfrom6.07to652,andfromnqto55.9ng/glw,respectively.Comparingresultsobtainedforthesamefishspeciesatdifferentlocationsites,weobservedthatsite7A1isthemostconta-minated.Ontheotherhand,ifwecompareresultsobtainedfordifferentfishspeciesatthesamesamplingpoint,wecanconcludethatcontaminationdecreaseinthefollowingorder:marbletrout>grayling>chub.ConcerningtheEvrotasriverbasin,PBDEswerealsodetectedinthefourcollectedsamples:9.32,13.2,30.8and355ng/glw.However,dechloransweredetectedbutalwaysatconcentrationlevelsbelowtheMQLs.

4. ConclusionsAlthoughfishspeciesarenotthesame,itseemsthatHFRcontaminationwashigherintheAdige.Thiscouldberelatedtothedifferentimpact:theAdigeundergoesanurbancontaminationwhilethedominantpressuresinEvrotasderivemainlyfromagriculturalactivities.ItisimportanttonotealsothatalltheanalysedfishpresentedPBDElevelshigherthanestablishedenvironmentalqualitystandards(EQS).

Acknowledgement This work was funded by the European Union Seventh Framework Programme(FP7/2007-2013)undertheGlobaquaproject(No.603629)andtheSEA-on-a-CHIPproject (No. 614168), and by the Generalitat de Catalunya (Consolidated ResearchGroups2014SGR418—WaterandSoilQualityUnit).MonicaGiulivothanksCA-RIPLOforthepartialsupporttoherPhDgrantreceveidinthBIOSAFEframework.”

Table 1. HFR concentration (ng/g lw) in fish samples collected in Adige river basin.

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Novel passive sampling to assess stratification of contaminants in coastal watersY. Aminot 1, A. Belles2, C. Alary2, J.W. Readman1

1Biogeochemistry Research Centre, Plymouth University, Plymouth, UK 2Department of Civil and Environmental Engineering, Mines Douai, LGCGE-GCE, F-59508, Douai, France - E-mail contact: [email protected]

Highlights• Verticaldistributionoforganiccontaminantsinwaterbodiesprovidesinformationon

theirsourcesandfate• Alowcostandeasilydeployablemethodbasedonpassivesamplingwasdevelopedto

evaluatethestratificationofhydrophobicpollutants• Polyethylenestrips(3mlong)weredeployedinPlymouthharbour,UK,over3weeks• PAHs,organophosphorousflameretardantsandpersonalcareproductswerescree-

ned• Amolecule-dependentdistributionwasobserved

Coastalandestuarineareasreceivecontaminantsfromwastewaterdischargesandlit-toralactivitiessuchasboating,seatradeandfisheriesaswellasfromupstreaminputsthroughriverinedischargesandsoilwashout.Assessingthedistributionofthesecon-taminantsinwaterbodiesprovidesinsightsfortheunderstandingoftheirsourcesandfate,henceallowingabettermanagementofourwaters.Inparticular,averticalprofileofpollutantscanindicatesourcesoriginatingfromthewatersurface,freshwater-deri-vedmaterialifcorrelatedtosalinityorsurfaceprocessessuchasphotodegradationorevaporationattheair-seainterface.For thispurpose,passivesampling isamethodofchoice for themonitoringofmo-deratelyhydrophobicorganiccontaminantsatmarine trace levelsas theyareable toconcentratetheanalytesin-situ,thusprovidinglimitsofdetectionthatcanbeaslowasthepg.L-1.Inthisstudy,verticaldistributionsofselectedorganiccontaminantswereassessedinanEnglishmarinafromthesurfacetomorethan3mdepth.Polyethylenestripswereheldverticallyandimmersedfor18daysfromfloatingpontoons.Followingdeployment,thestripswereslicedtoprovide46depthrelatedsub-samples.Asectionofapproximatively1cmatthesurfaceallowedprofilingofthesea-surfacemicrolayer.Time-weightedave-rageconcentrationswerecalculatedforPolycyclicAromaticHydrocarbons(PAH)byusingpassivesamplingandtheperformanceandreferencecompoundapproach.Asanexample,resultsrevealedasurfaceenrichmentforphenanthreneandchrysenewith a 5-fold increase in the first 50 cm, whilst fluoranthene and pyrene exhibiteddecreasingconcentrationsclosertothesurface.SourcetrackingofthePAHsindicated

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anoil-derivedcontaminationatthesurfacewithenhancedcombustionderivedPAHconcentrationsatlowerdepths.Measurementswerefurtherextendedtoselectedantifoulingcompounds,organopho-sphorousflameretardantsandpersonalcareproducts.Theverticalprofilesofdetectedcompounds exhibited comparable distributions to the model PAH’s which aided ininterpretationoftheirrelativesourcesandfates.Thisstudydemonstratesthatpassivesamplingcanprovideacost-effectiveandnovelmonitoringtoolto investigatethesourcesandfateofcontaminantswhilstprovidingimportantinformationoncompounddependentverticalprofilingincontaminatedwa-ters.

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S E S S I O N IS E a w at E r p O l l u t I O N p O S t E r p r E S E N tat I O N S

10-front Seawater pollution-poster presentations.indd 25 04/04/16 15:01

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Analysis of antibiotics by LC-MS2 in aquatic ecosystemsAlejandro Margareto Mato1, M. Silvia Díaz-Cruz1, Damià Barceló Culleres1, 2

1Water and Soil Quality Research Group, Dep. of Environmental Chemistry (IDAEA-CSIC) c/JordiGi-rona 18-26, Barcelona, Spain 2Catalan Institute for Water Research (ICRA), c/EmiliGrahit 101, ParcCientífic i Tecnològic, Universitat de Girona, Girona, Spain - E-mail contact: [email protected] / [email protected]

Highlights• Occurrenceofmulticlassantibioticsintheaquaticenvironmentduetoconstantanduncon-

trolledantibioticdischargeviawastewater,farmingandaquaculturehaspotentialhumanandecosystemhealthimplications.

• Developmentandvalidationofquantitativeanalyticalmethodsfortheanalysisofmulticlassantibioticsinaquaticecosystemmatricesbasedinliquidchromatographycoupledtomassspectrometry(LC-MS2).

• Twoquantitativemethodsweredeveloped,validatedandappliedsuccessfullytorealwatersamplesfor34antibioticsofdifferenttherapeuticgroups.

• Effluentandinfluentwastewateranalysedsamplesshowedthatwastewatertreatmentshavealimitedremovalefficiencyofantibiotics.

• Lower concentration and detection rate of antibiotics in the analysed seawater samplesevidencedaquacultureantibioticcontributionandthedilutioneffectofthemarineenvi-ronment.

IntroductionAntibioticsconstituteoneofthemostusedpharmacotherapeuticgroupsappliedtobothhu-manandveterinary(farmingandaquaculture)medicine.Theyaremainlyemployedfortrea-tingandpreventinginfectionsofbacterialorigin.Reportshaveshownthatmanyantibioticsandtheirmetabolitesdonotdegradeduringconventionalwastewatertreatment,being,there-fore,constantlyreleasedintoenvironmentalwaters(1).Sofar,thereisnolegislationregardingthepresenceofantibioticsoranyotherpharmaceuticalingroundwateroranyenvironmentalcompartment.However,despiteantibioticsareusuallydetectedatlowconcentrations,theyarebeingcontinuouslyintroducedintotheenvironment,andconcentrationsthatwerepreviouslyconsideredasharmlessareleadingtotheemergenceofantibiotic-resistantbacteriastrains,andpotentialimplicationsforhumanandtheecosy-stemshealthshouldbeconsidered(2).Antibiotic-resistantbacteriaare foundinthenaturalenvironment,butsignificantlyhighernumbersofthesebacteriaarepresentinwastewaterorevenintreatedwastewater.Inadditiontocontinentalwaters,marineecosystemsarealsoimpactedbyantibioticresidues,mainlyasaconsequenceofthewastedischargesintotheseaandthroughaquaculture,whereantibioticsareextensivelyused(seeFigure1).

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Theobjectiveof thisworkwastodevelopandvalidateanalyti-calmethodology for thedeter-minationof34multiclassanti-bioticsandmetabolitesinwatersamples.

Materials and MethodsTwo analytical methods weredeveloped to analyze the se-lectedantibioticsi)incontinen-tal waters (groundwater, riverwater and wastewater) and ii)in seawater.For theanalysisofcontinental water samples, themethodwasbasedonanauto-mated on-line solid phase ex-traction(SPE)followedbyhighperformance liquid chromato-graphy coupled toquadrupole-

lineariontrap-tandemmassspectrometry(HPLC-QqLIT-MS/MS).Seawatersampleswereanalysedusingoff-lineSPEinsteadofanon-linecouplingduetotheprecipitationintheon-linesystemofthesaltspresentinseawater.OASIS®HLBSPEcartridgeswereusedforboththeon-lineandoff-lineSPEQuantificationoftargetantibioticswasperformedbytheinter-nalstandardandmatrix-matchedcalibrationapproach,usingisotopiclabelledcompoundsforthedifferentantibioticgroups.Theselectionoftheappropriateinternalstandardusedforthequantificationofeachanalytewasdecidedbasedonchromatographicretentiontimeproximityandinternalstandardcalibrationcurvecorrelationcoefficient.Theapplicabilityofthedevelopedmethodswastestedthroughtheanalysisof8wastewatersamplesfromwastewatertreatmentplants(WWTPs)fromCatalonia(Spain),6seawatersamplesfromsixsitesalongtheTarragonacoast(Catalonia,Spain)and3samplesnearafishfarmfacilityfromIoánnina(Greece).

Results and DiscussionOn-lineandoff-lineSPEfollowedbyHPLC-(ESI)-MS/MSwassuccessfullyappliedtothesimultaneousanalysisof34multiclassantibioticsandmetabolitesinwastewaterandseawa-ter.Someanalytescouldnotbedeterminedinseawaterduetothenotoriouslyloworhighrecoveryratesachievedintherecoveryexperiments,likelyasresultsofco-precipitationorre-dissolutionofthesaltsduringtheextractionprocess.Resultsindicatedthatawiderangeofantibioticswerepresentinbothinfluentandeffluentwastewater,whichrevealedthewidespreadoccurrenceofthesepharmaceuticalsinCataloniaandthelimitedremovalachievedformanyofthemintheselectedWWTPs.

Figure 1. Pathway of antibiotics for human and veterinary use into the environment. The antibiotic residues are symboli-cally represented as red dots. Picture extracted from (3)

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Someofthemostfrequentlyantibioticsfoundinwastewaterinfluentsandeffluentssamplesanalysed were Azithromycin, Ofloxacin, Norfloxacin andTrimethoprim. The higher con-centrationcorrespondedtoOfloxacin inLesFonts influentwithaconcentrationof32.25ng·mL-1.SeawatersamplesfromtheTarragonacoastportswerealmostfreeofantibiotics,withpositivedetectionsonlyforSulfapyridineinthreesamplingsites.IntheanalysedseawatersamplesfromGreece,11antibioticsweredetectedintheconcentrationrange0.2–24ng·L-1.Sul-fadiazineandSulfapyridinewereubiquitous.Theoccurrenceofthese11antibioticscouldbeexplainedbytheproximitytoanaquaculturefacility.

ConclusionsThedevelopment,validationandapplicationoftheon-lineanalyticalmethodwereaccompli-shedforwastewater.Seawaterdissolvedsaltscausedaseriousproblemfortheon-linemetho-dologydevelopedforcontinentalwater,thusadifferentoff-linemethodologywasdevelopedandsuccessfullyapplied.ApplicationofthedevelopedmethodsshowedthewidespreadoccurrenceofantibioticsintheaquaticenvironmentandthelimitedeliminationofthembyWWTPs.Thedilutioneffect in themarine environment is evidentdue to the lower concentrationsobservedinallseawatersamples.

AcknowledgementsThisworkwasfundedbytheEUthroughtheProjectSeaonaChip(Contract614168)andbytheGeneralitatdeCatalunya(ConsolidatedResearchGroup“2014SGR418-WaterandSoilQualityUnit”)

References1)SKim,DSAga.Potentialecologicalandhumanhealthimpactsofantibioticsandantibio-

tic-resistantbacteriafromwastewatertreatmentplants.JToxicologyEnvironmentalHealthB:Cri.Rev.2007;10:559–73

2)MJGarcía-Galán,TGarrido,JFraile,AGinebreda,MSDíaz-Cruz,DBarceló.Applica-tionoffullyautomatedonlinesolidphaseextraction-liquidchromatography-electrospray-tandemmassspectrometryforthedeterminationofsulfonamidesandtheiracetylatedme-tabolitesingroundwater.AnalBioanalChem2011;399:795–806

3)SBerkeretal.,EMBOreports2014:15:740–44

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Economic Sustainability and Competitiveness of European Traditional Seafood MarketG. Sogari, C. Mora, D. Ottaviano, D. Menozzi

Department of Food Science,University of Parma (IT) - Email- contact: [email protected]

EU is one of the largest and most valuable seafood markets in the world. Consumption in Europe is rising (23.1 kg per person per year). Capture fisheries have declined. Most of the increased EU consumption come from outside EU. The two most consumed aquaculture spe-cies in the EU are salmon and mussel. EU aquaculture provides a fresh, local supply of healthy seafood and follows strict rules to protect the consumer, the fish and the environment Prime-Fish project(http://primefish.eu) is a H2020 project aims to develop an innovative market orientated prediction toolbox. The overall aim is to strengthen the economic sustainability of European fisheries and aquaculture sectors on Local and Global markets. So far, EU aquacul-ture has shown little growth or stagnated in the last few years (only 24% of EU consumption comes from aquaculture). However, aquaculture will probably surpass wild fisheries as the main source of seafood in the near future. The main research question which we are trying to address is: What are the drivers which will influence consumer’s demand for farmed seafood products in European Union?

Procedures/methodologies/approachesThe effectiveness of demand stimulation through health, label and certification claims will be evaluated and compared with actual consumer behaviour. After literature review to un-derstand the state of the art we are carrying out: Qualitative methods (in-depth interviews, brainstorming and focus groups) and Quantitative methods(questionnaire and choice mo-delling) At the beginning, motivations and barriers to purchase a seafood product have been investigated as well as habits of consumption (frequency, price, place of purchase, occasion of consumption). Researchers have been also shownimages of different seafood products (whole fish, ready to cook, boneless, …) to the participants in order to explore different perception and It will follow a choice experiment to assess profile attributes derived from previous tasks and relative willingness to pay.

ResultsPreliminary results of the qualitative research have highlighted motivations and barriers of the purchase of fish products. The results suggest that the main motivations for the purchase of fish products are the taste (“pleasure of eating fish”), health benefit (“related to the presence of Omega-3”), substitute of meat (“importance in a varied diet”); whereas the barriers are the price, preparation (“perceived to be difficult and time consuming”), safety (“concerns about

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origin and pollution”). On the other hand, also motivations and barriers for the purchase of ready-to-cook fish products are presented.

ConclusionsSustainable aquaculture (from an economic, social and environmental point of view) is needed because fisheries alone will not meet the growing global demand for seafood. At the same time local, tradition and small enterprises need support and help from Academic Institutions, Research centres and Policy makers in order to be competitive in this fragmented and “boom-and-bust” sector. The commercialisation potentialis to consolidate the economic sustainability, performance and competitiveness of European fisheries and aquaculture sectors. The results of this research will help to develop strategies to analyse: (1) the impact of consumer behavior, (2) recent market trends and (3) successful product innovation (i.e. packaging, recipes, labels).

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Monitoring pesticide residues in a large tropical bay, Bahia de Todos os Santos, BrazilFernando da Piedade Carvalho1, C. Cattini2, JP Villeneuve2,I. Tolosa2, J.M.Oliveira1

1Laboratório de Protecção e Segurança Radiológica. Instituto Superior Técnico/ Universidade de Lisboa. Estrada Nacional 10, km 139, 2695-066 Bobadela LRS, Portugal 2Environment Laboratories – Monaco, International Atomic Energy Agency, 4, Quai Antoine 1er, 98000 Monaco, Principauté de Monaco - E-mail contact: [email protected]

Highlights• ContaminationoftheBahiadeTodos-os-Santosbyorganiccontaminantswasinvestigated

toassesstheenvironmentalhealthofthisecosystem• Water,sediment,andoystersampleswerecollectedaroundthebayandanalyzedbyGC-

MSandothertechniques• ContaminationbyresiduesofpolarcompoundsfromagriculturewasnegligibleandDDTs

andPCBswerethemajorOCcontaminantsinsedimentsandoysters• BesidesDDTs,severalbannedOCcompoundsarestillinuseasdieldrinandlindane• ThemainsourcesofOCcontaminantswerenotintheagricultureareas,buttheurbanareas

forPCBs,andurbanandleisurecoastalareasforDTTs

Lagoonsandbaysarerichcoastalmarineecosystems,generallyusedintensivelybysurroun-dinghumansettlementsandthusreceivingurbanandindustrialresidues.Coastallagoonsbe-sidesincreasinguseforaquaculture,generallyarealsocrucialsystemsforthereproductionandgrowthofwildmarinefauna.ThecoastalsystemofBaiadeTodos-os-Santos,Brazil,alargebaywithasurfaceareaof1100km2,ishomeformarinelifeandsupportsfisheries,aquacultureandtourismindustries.Thebayreceivesriverdischargesfromagricultureareas,particularlylocatedinthewatershedofriverParaguaçu,anditissurroundedbytheverylargecityofSal-vadorandseveralsmallertowns,aswellasindustrialareas,harborsandnavalbases,andhostedformeroilriggs.Theenvironmentalhealthofthebayecosystemandsafetyofmarinefoodfromthebayaremajorconcerns.TheBaíadeTodos-os-Santoswasmonitoredforthecontaminationbyagrochemicalandin-dustrialcompoundsincludingherbicides,organophosphorus,organonitrogenandorganoch-lorinecompoundsinwater,sedimentandoysters.Itwashypothesizedthat,ononehand,polarcompoundssuchasherbicidesappliedintheagricultureareasinthecatchementofParaguaçurivercouldbeeasilytransporteddownstreamandcontaminatethebaywaterand,ontheotherhand,chlorinatedcompoundsusedtocontrolpests(e.g.,DDTformalariavectorcontrol)andOCindustrialcompounds(e.g.,PCBs)fromurbanrunoffandindustrialdischargescouldenterthebayaquaticsystem,accumulateinmarineorganismsandbetransferredinthefoodchaintohumans.Theassessmentofcontaminationwasmadethroughsamplecollection(water,sediments,oy-sters),immediatepre-treatmentonsite,andanalysesinthelaboratory.Watersampleswere

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pre-filtered through GF/F precom-busted filters; passed through SPEcartridgesandlaterelutedforanalysisbyGC-MSandGC-APCI-MS.Thetechniqueusedwasprevioulyvalida-tedfor72compounds.Samplesofse-dimentandbiota(oyster’ssofttissues)weresoxhletextractedwithn-hexaneansextractsanalyzedbyGCandGC-MS. A strict QA/QC was appliedwithanalysisofIAEACRMs.Watersolublepolarcompoundssuchas atrazine, simazine, alachlor, andother common herbicides were notdetectedinwater;theirconcentrationswerealwayslessthan10-35ng/L,de-pending on the compound. On theother hand, non-polar chlorinatedcompounds were present in higherconcentrations in bottom sediments

andinbiota.ResultsshowedthatthemorewidespreadcontaminantswerethechlorinatedhydrocarbonssuchasDDTs,PCBs,lindaneandafewotherlipophiliccompounds,suchaschlorpyrifosandendosulfan(Figure1).Concentrationsofpp´DDE,themainmetaboliteofDDT,inthesofttissuesofoystersrangedfrom2.1to28.9ng/g(dw).Theresultsforsofttissuesofoystersindicatedthatt-DDTswerealsothemorewidespreadcontaminants.ConcentrationsofDDEinoystersgenerallyreflectedtheconcentrationsinsedimentsandthehighestvaluesweredetectednearSalvadorandothertownsaroundthebay.Similardistributionwasfoundforaroclors(PCBs), fromindustrialdischarges.ItwasconcludethatherbicidesfromtheagricultureinParaguaçurivercatchmentwerenotreachingtheaquaticsysteminthebay,atleastinmeasurableconcentrations.Dilutionandrapiddegradationofherbicidesmaypreventthebuildupofthesepolarresiduesinthebay.Hu-manexposuretoresiduesofpolarcompounds(herbicides)throughbathinginthebaywaterseemsthereforeunlikelyanddonotposeanenvironmentalhealthrisk.Resultsshowedalsothat, inspiteofthebanonpersistentorganiccompounds,DDTandotherOCsstillareinuseinurbanareasaroundthebayandresiduesendupintheaquaticenvironment.ProbablytheneedforvectorcontrolinmalariapreventionwasstillencouragingtheuseofDDT.Recurrentmalariaupsurges,andnowtheZikaepidemics,mayfosterfurtheruseofDDTsandotherOCs.Chlorpyrifosandendosulfanareinuseaswellandaremoretoxictoaquaticfauna,inparticulartocrustacea.IndustrialOCchemicalsofthePCBgroup,werebannedanddischargesintotheaquaticenvironmentseemcurrentlyundercontrol.ThePCBresiduesmeasuredinthebaymightbefrompastdischargesandconfirmthepersistenceof

Figure 1. Distribution of DDTs and PCBs (sum of 19 congeners) concentrations in the sediments of Baía de Todos-os Santos and watershed of Paraguaçu river.

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thesecompoundsintheaquaticenvironmentinthetropicalenvironment.AnalysesofPCBsandotherpersistentchlorinatedcompundsinlayersofsedimentcoresdatedby210Pbgeo-chronologyindicatedthattheywereinuseanddepositinginthebaysedimentscontinuouslyduringthelastseveraldecades.Growingconcernswithseafoodsafetyadvisetoimplementatightermonitoringofbaywaterquality.Particularlyagrochemicalresiduesandemergingcontaminants,amongstmanyotherchemicalsandtoxins,shouldbetargetted.Insuchavastecosystem,currentlyitisdifficulttoensuresuchcontrolthroughtheusualsamplecollectioninthefieldandchemicalanalysisinthelaboratory.Fastermethods,suchasin-situandrealtimeresponsemethodswouldbewelcome.

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Assessment of saxitoxin levels in Mediterranean Co-astal Waters: a preliminary study Theodoti Papadimitriou, Nikolaos Mellios, Chrysi Laspidou

Civil Engineering Department, University of Thessaly, Pedion Areos, Volos, Greece - E-mail contact: [email protected]

Highlights• Thefrequency,intensity,andgeographicdistributionoftoxiccompoundsincoastalwaters

increased• AssessmentofsaxitoxinconcentrationbyELISA• SignificantsaxitoxinslevelsinE.MediterraneanSea• Needforefficientmonitoringtools

Marineneurotoxinsarenaturalproductsproducedbyphytoplanktonandvariousspeciesofinvertebratesandfish.Theneurotoxinsproducedbyphytoplankton—algaltoxins—encom-passarangeofmolecularstructures,withdifferentstructureandbiologicalactivity.Amongmarineneurotoxins,saxitoxinisuniqueinthatitisproducedbyorganismsencompassingtwokingdomsoflifeinhabitingdifferentaquaticsystems.Eukaryoticdinoflagellatesarethepre-dominantproducersinmarinesystems,whilefivegeneraofcyanobacteriaarethesourceinfreshwatersystems.Saxitoxinsbindtothevoltage-gatedsodiumchannel,blockingthepas-sageofnerveimpulsesandleadingtoparalyticshellfishpoisoningandsaxitoxinpufferfishpoisoning.Severalstudiesexistonsaxitoxininfreshwatersystems;however,researchontheoccurrenceofsaxitoxinsinmarinesystemsandespeciallyintheMediterraneanregionisscarce.ThepresentworkaimstoassesssanitoxinconcentrationsintheEasternMediterraneanSea.TheinvestigatedareapresentsthesamplingregionsalongthecoastlinesoftheNorthAegeanSea(ThermaikosGulf,S1),theWesternAegeanSea(PagassitikosGulf,S2,S3,S4)andtheIoanianSea(AmvrakikosGulf,S5).SampleswerecollectedinMarch2016.Seawater(1.5L)wasobtainedfromadepthof0.5meter.Sampleswerefilteredon47mmdiameter(0.45μmporesize)GF/Cmembranefiltersandthefilterswerefrozen.Frozenfilterswereextractedwith5ml0.05Maceticacid.TwomLofthesamplewerecentrifugedat10000xgby5minutes,thesupernatantwaspassedthroughacartridgecolumn(C-18Sep-Packcartridges,Millipore),previouslyregeneratedwith10mLmethanolandequilibratedwith10mLwater.Thesaxitoxinwascollectedimmediatelyand50μLofthisfiltratewasusedforsaxitoxinquan-tificationbyenzyme-linkedimmunosorbentassay(ELISA).ASaxitoxin(PSP)ELISAkit(Product52255B,Abraxis,Inc.,USA)wasusedfollowingtheinstructionsofthemanufacturer.Tworeplicatesweremeasuredforeachsample.Detec-tionlimitoftheassaywas0.015μgSaxitoxins/LandtheresultsareexpressedasSaxitoxinequivalents.Accordingtoresults,allthesamplesexamined,withtheexceptionofthesampleoriginatedfromAmvrakikosGulf,containedsignificantconcentrationsofsaxitoxins.Maximumsaxito-

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xinvaluesreached257.3ngSa-xitoxinequiv./LinThermaikosGulf.Minimum saxitoxin values rea-ched76.29ngSaxitoxinequiv./LinPagassitikosGulf.All basins examined are eutro-phic since they receive watersand fertile material from largeriversand/orsmallerwaterout-falls derived from agriculturalandindustrialactivities.Thein-tensive growth of certain toxinproducingspecies(HarmfulAl-galBlooms)isaconsequenceofeutrophication and creates pro-blemsinecosystemstructureandpublic health. Concerns aboutharmful algae in Greek coastalwaters has been growing sincethe late 1970s, when the firstsymptomsoffishkillsledtothefactthatHABs-oftenquotedas

thephenomenonofredtides-acquiredtheattentionofscientistsandthepublic.Accordingtorecentstudies,61algaespecies(toxic,potentiallytoxicandhighbiomassproducingalgae)havespreadacrosstheGreekcoastlineduringthelast30years.Amongthese,certainalgae(16)wereassociatedwiththeoccurrenceofimportantHABincidentscausingdamageinmarinebiotaandwaterquality.Alexandriumcatenella,apotentialsaxitoxinproducingspeciesofDi-noflagellates,hasbeenfoundinThermaikosandPagassitkosGulfs.Potentialtoxicspecies(e.g.Prorocentrumemarginatum,KareniamikimotoiandAmphidiniumcarteraeHulburt)knowntoproducetoxinsotherthansaxitoxins(e.g.Gymnocin-A,Maitotoxin)havealsobeenfoundinAmvrakikosGulf.BloomsofsaxitoxinproducingalgaeintheGulfsofGreecehavebeenassociatedwithmassivefishkillsandextensiveshellfishdeaths.Duetotheobviousrisksofsaxitoxins,theintroductionofacontinuousonlinedetectionmethodofsaxitoxins,suchastheonebeingdevelopedinSMSproject,wouldbehighlyvaluable.

AcknowledgementThisworkwas supportedby theprojectSMS-Sensing toxicants inMarinewatersmakesSenseusingbiosensors-whichisimplementedintheframeworkoftheEU7thFrameworkProgrammeforResearch,TechnologicalDevelopmentandDemonstrationunderGrantA-greementNo.613844

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Development of a biodiversityindicator as a toolfor marine environmentmanagementElena Botteon1, Ana Cristina Costa2, Andrea Zita Botelho2

1 Aeiforia Srl, Loc. Faggiola 12-16, 29027 Gariga di Podenzano (PC), Italy. 2 CIBIO - Research Centre in Biodiversity and Genetic Resources, InBIO Associate Laboratory, University of the Azores, Rua Mãe de Deus 9501-801, Ponta Delgada, Azores, Portugal. - E-mail contact: [email protected]

Highlights• Effectivedecision-supporttoolsformarineareasmanagerandpolicymakersarestill

lackingornotapplicableduetolackofinformation• ThisworkfocusedontheassessmentofMarineBiologicalValue• WeuseddatasetfromUVCsurveys,whichisawidelyusedandnot-destructiveme-

thod,quitecheapandreplicableelsewhere.• TheBVIdevelopedshowntobeavalidalternativetospeciesrichness,integratingmore

informationabouttherealqualityoftheoverallecosystemforthesameresearcheffort.

TheaimoftheMarineStrategyFrameworkDirectivetoreachGoodEnvironmentalStatus(GES)ofUE’smarinewatersleadstotheneedofassessmarineareasintermsoftheirecologicalandbiodiversityfeaturesinordertoprioritizebiologicalimportantare-asforprotectionandtomonitortheirstatusimprovementordeterioration(2008/56/ECDirective).TheBiologicalValueIndicator(BVI)developedinthisstudycanbeaneasy,cheapandobjectivetool,apllicableinmarineprotectedareasdesign,managementandmonitoring.TheBVIisasemi-quantitativerepresentationoftheintrinsicbiodiver-sityvalueofthemarineecosysteminaparticularmarinecoastalarea,excludinganthro-pogenicprovidedbenefits.AccordingtoDerousetal.(2007a,b),biologicalevaluationisaprocesswiththepurposeofassignalabeloftherespectivebiologicalvaluetosubzoneswithinthetargetstudyarea.TheBVIwastestedtofivesitesintotheResourcesMa-nagementProtectedAreaofCaloura-IlhéudeVilaFranca(RMPACaloura-VFC)onSãoMiguelIsland,Azores,Portugal.TwositesoutoftheRMPAwerealsoconsideredascontrolsitesforcomparison.Thestudyareawaschoosenbasedontheavailabilityofanunderwatervisualcensus(UVC)monitoringsurveycarriedoutsinceSeptember2010toJune2012.Thespecieslist(109recordedspecies)resultingfromthemonito-ring surveywas cross-checkedwith literature, databases and legislation information.Everysubzoneofthestudyareahasbeenassessedunder4evaluationcriteriaidentifiedthroughaliteraturereview:rarity,aggregation/fitness,naturalnessandlocalimportance(Derousetal.2007a,b;Robertsetal.2003a,b).Byidentifyingasetofassessmentque-stionsforeachevaluationcriterionanddevelopingspecificmathematicalalgorithmsforeachassessmentquestion,thetotalscoreforeverysubzonehasbeenfinallycalculatedbysumminguptheindividualvaluesrelatedtoeveryassessmentquestionrelatedtothesamesubzone.Subzonesofthestudyareahavebeenweightedagainsteachotherwithasemi-quantitativescoringsystemof5classes,fromverylow(1)toveryhigh(5),Figure

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1.ThefoursubzonesinsidetheRMPCaloura-VFCresulttohaveahigherBVI(from3to5)thantherestofthemarineareaunderstudy.Arawindexofdatareliabilitybasedonthenumberofunderwatervisualcensus(UVC)carriedoutineverysubzonehavebeenalsocalculated.TheoptimalnumberofUVC,inordertoachieveacomprehensiveBVIwiththeminimumresearcheffort,is7.TheBiologicalValueIndicator(BVI)thatwehavedevelopedcanbeaneasy,cheapandobjectivetoolformarineprotectedareasdesign,managementandmonitoringof themarinecoastal environment.Finally thebiologicalevaluationmap(BEM)thatgraphicallyrepresentstheBVIofthedifferentsitesprovidesabaselinemapformarinespatialplanningandausefulinstrumentfordecision-makersingeneral.InspiteofbeingdevelopedintheAzoreswebelieveBVIcanalsobeadaptedandappliedtoothermarineareaswithsimilardataavailability.

References• ECMarineStrategyFrameworkDirective.CouncilDirective2008/56/ECof17June

2008establishingaframeworkforcommunityactioninthefieldofmarineenvironmen-talpolicy(MarineStrategyFrameworkDirective),OJL164,25.06.2008,p.19.

• DerousS,AgardyT,HillewaertH,HostensK,JamiesonG,LieberknechtL,MeesJ,MoulaertI,OleninS,PaelinckxD,RabautM,RachorE,RoffJ,StienenEWM,vanderWalJT,VanLanckerV,VerfaillieE,VincxM,WeslawskiJM,DegraerS.Aconceptforbiologicalvaluationinthemarineenvironment.Oceanologia2007a;49(1):99-128.

• DerousS,AustenM,ClausS,DaanN,DauvinJC,DepesteleJ,DesroyN,HeessenH,HostensK,MarboeAH,LescrauwaetAK,MorenoM,MoulaertI,PaelinckxD,RabautM,ReesH,RessurreiçãoA,RoffJ,SantosPT,SpeybroeckJ,StirnnenEWM,TakarekA,HofstedeRT,VincxM,ZarzyckiT,DegraerS.Buildingona the con-ceptofmarinebiologicalvaluationwithrespecttotranslatingittoapraticalprotocol:ViewpointsderivedfromajointENCORA-MARBEFinitiative.Oceanologia2007b;

Subzone Species richness Biological value

BaixadaLagoa 25 1Lagoaparede 35 1BaixadaAreia 60 4ArcosdoHotel 55 3Calouracerco 65 5Calouraporto 70 4BaixadoGaroupa 37 1

r= 0.940855359p= 0.001582491 (α =0,01)

Table 1. Species richness and biological value for every subzones.Pearson correlation coefficient (r), signifi-cant limit (α), student’s t-test result (p).

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49(4):579-586.• RobertsCM,AndelmanS,BranchG,BustamanteRH,CastillaJC,DuganJ,Halpern

BS,LaffertyKD,LeslieH,LubchencoJ,McArdleD,PossinghamHP,RuckelshausM,WarnerRR.Ecologicalcriteriaforevaluatingcandidatesitesformarinereserves.EcologicalApplications2003a;13(1):S199–S214.

• RobertsCM,BranchJ,BustamanteRH,CastillaJC,DuganJ,HalpernBS,LaffertyKD,LeslieH,LubchencoJ,McArdleD,RuckelshausM,WarnerRR.Applicationofecologicalcriteriainselectingmarinereservesanddevelopingreservenetworks.Ecolo-gicalApplications2003b;13(1):S215-S228.

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Legally and non-legally binding legislation on sustainable aquaculture: the code of conduct for responsible fisheriesPierluigi Monticini, Ettore Capri

OPERA Research Center, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy E-mail contact: [email protected]

Soft law tools are not legally binding documents, which constitute an important in-strument for aquaculture activities; these include the Kyoto Declaration (Conference on Aquaculture held in Kyoto, Japan in 1976), the Bangkok Declaration and Stra-tegy (Aquaculture Development beyond 2000) and the Agenda 21. Rio Declaration and Agenda 21 have also provided the basis for the implementation of the 1995 Code of Conduct for Responsible Fisheries (Code of Conduct or Code). On 31 October 1995, during the Twenty-eighth Session of the FAO Conference, more than 170 FAO Member parties adopted unanimously the Code of Conduct of Responsible Fisheries (CCRF). The Code is organized in 12 Articles, and 2 Annexes (all referring to the ori-ginal elaboration of the Code and Resolution 4/95), however is a non-legally binding, voluntary set of principles. Although it is not legally binding, FAO has been disseminating the Code in various languages in order to promote its implementation as widely as possible by States, in-dustry, NGOs and other concerned organizations. UN Resolution 4/95, contained in Annex 2 of the Code, urges developing countries to upgrade their fishing capabilities in order to meet the Code’s provisions.

CCRF remains the main overarching document on Responsible Fisheries at a global le-vel. Its effectiveness would be much greater not only if it were a legally-binding instru-ment applied by all FAO member states, but also if it were combined with education and diffusion of culture on sustainable fishing practices.

The main obstacles to its effective application contributing to a weaker control over fishing practices identified in our studies were:- the cultural differences among the countries;- diversity of their short-term economic interests;- difficulties of monitoring illegal fishing and poaching, especially in developing and in

politically unstable countries;- scarcity of data provided by some States.

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The millennium declaration (MDGs) and the new sustainable development goalsPierluigi Monticini, Ettore Capri

OPERA Research Center, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy E-mail contact: [email protected]

Any veritable perspective on Sustainable Development in Aquaculture and Fisheries is to be integrated in an international context that needs to fulfill the Millennium De-velopment Goals by the end of 2015. Although a few of the goals have been at least partially achieved, most of them are far from being fulfilled.

The achievement of the Millennium Goals was the core of our work. We have closely examined Goal 7: Ensure environmental sustainability; we have then focused on its targets, and in particular on Target 7B, concerned with the reduction of biodiversity loss by 2010.

In our work we applied the indicators for monitoring the progress of the Target 7BIn different case studies. Results demonstrate that Target 7B is far from being achieved, but it also won’t be achieved in the short or medium period.After Milan Expo 2015 the MDGs are been exceeded by the new 17 Sustainable Deve-lopment Goals (and its 169 Targets and a set of global indicators currently under deve-lopment) were the main Goals is “to eradicate the hunger by 2030, the focus is not only in Developing Countries but also in developed Countries. As regards Aquaculture, Fi-sheries and Environmental safeguard the SDG 14 – Oceans, Seas and Marine resources (Life below water) cover all the areas of interests. FAO says, “A comprehensive approach to fisheries and aquaculture targeting small-scale artisanal fishers is well captured in SDG14. Sustainable management of ocean ecosystems is imperative for ensuring sustainable fisheries. Stewardship must balance priorities between growth and conservation, and between indu-strial and artisanal fisheries and aquaculture, ensuring equitable benefits for communities”.

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Trout, Seabass and Seabream Value Chain Over-view in ItalyG. Sogari, C. Mora, G. Morelli, D. Menozzi

Department of Food Science, University of Parma (IT) E-mail: [email protected]

In 2014 aquaculture production overtook for the first time the fishing and 50% of aquatic products consumed by world population is bred. This growth trend in aquaculture is the highest in the agricultural sector, with an increase of + 8.6% in the period 2002-2012.The European Union is the world’s largest market of aquatic origin products (12.3 mil-lion tons in 2012), but more than 65% of products consumed is imported. In Italy, as in Europe, aquaculture in the decade 2003-2013 is stagnant (-1.3%). In 2013, Italian production counted 140,846 tons, with an increase in volume compared to 2012, (+ 2.6%), for a recovery in the production of molluscs (+ 5.7%). Italy has an important role in the European aquaculture: contributes to 13% of the volume in EU 27 (it is at fourth place after Spain, France and the UK) and 10.7% of production value (FAO, 2014).

Procedures/methodologies/approachesThis report is completely based on secondary data. Sources of data and information are from Italian governmental statistics (ISTAT, ISMEA), European database (EUMO-FA), and other organizations (e.g. FAO). This study is part of H2020 project called Pri-meFish (http://primefish.eu) whit the aims to develop an innovative market orientated prediction toolbox to strengthen the economic sustainability of European fisheries and aquaculture sectors.

ResultsFarmed fish products in Italy are mainly addressed to the market of live and fresh fishes (Table 1). Only for the trout sector is common to process/transform the fish, offering a wider range of products to final consumers: smoked products, hamburgers, skewers, etc.One key factor that makes the supply chain of aquaculture shorter than the fishing sector is the direct contact between producers and the large retail. For the seabass and seabream production, the larger share of the production is originated from few large businesses, managed professionally, selling both in Italy and abroad. Although supermarkets represent the largest share of retail sales, traditional channels such as fishmongers and municipal retail markets are more present in Italy than in other European countries.

ConclusionsCurrently, in Italy there seems to be a low chance to see new big companies entering in the aquaculture sector of seabream, seabass and trout. It seems not attractive (from a

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profitability point of view) and requires significant investments. Within the new CMO (Common Market Organisation) for fish (Regulation 1380/2013 / EU), the European Commission redefined the strategic guidelines for European aqua-culture for the period 2014-2020, which aims to promote growth and to increase the production of aquaculture for the Member States, reduce dependence on imports and encourage development in coastal and rural areas.Another interesting point regards organic aquaculture which, in the last years, is growing, although it remains a niche market on the total production. The number of consumers who pay attention to sustainable and healthier food is increasing.

FAO (2014). The State of World Fisheries and Aquaculture. FAO Fisheries and Aqua-culture Department Food and Agriculture Organization of the United Nations, Rome, 2014.

Common name Scientific Production (t) *Production name 2013 Units (n) 2013

Seabass Dicentrarchus labrax 6,330.5 78Seabream Sparus aurata 6,184.3 78Trouts n.i Salmo spp. 1,140.3 54Brown Trout Salmo trutta 152.8 18Rainbow Trout Oncorhynchus mykiss 35,058.5 213

Source: Elaboration ISPRA on data MiPAAF - UNIMAR*Note: facilities might have one or more production units (based on technology and farmed species).

Table 1.Main fish species farmed in Italy

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Contaminants in the marine environment: What do weface?D. Alvarez-Muñoz1, M. Llorca2, J. Blasco3, D. Barceló1,2

1Catalan Institute for Water Research (ICRA), Science and Technology Park of the University of Girona, Girona, Spain 2Water and Soil Quality Research Group, Department of Environmental Chemistry (IDAEA-CSIC), Barcelon, Spain 3Instituto de Ciencias Marinas de Andalucia (ICMAN-CSIC), Campus Rio San Pedro, Cádiz, Spain E-mail contact: [email protected]

Highlights• Thereisawideandcomplexmixtureofhazardouschemicalssimultaneouslypresentinthe

marineenvironment• Anoverviewofthesecontaminantsispresentedincludingmetals,persistentorganicconta-

minantsandemergingorganiccontaminants.• Theyposeapotentialrisktobothwildlifeandhumanhealth• Theirmainsourcesofcontaminationandlevelsinmarineenvironmentarereported.

Chemicalcontaminationofthemarineenvironment(bothestuarineandcoastalareas)isahi-ghlycomplexissue.Upto15differentgroupsofpollutantsincludingmetals,persistentorganiccontaminantsandemergingorganiccontaminantscanbesimultaneouslypresentatdifferentlevelsinmarineecosystems.Allofthemmakea“cocktail”ofhazardoussubstancesthatmayposesnegativeimplicationsfortheaquaticenvironment,humanhealth(troughthepossibleingestionofcontaminatedseafood),andrelatedcoastalactivitiessuchasfishing,aquaculture,orrecreationalactivities.Anoverviewofthechemicalsubstancesthatpollutethemarineenvironmentwillbepresen-tedincludingcontaminantsgroupssuchasmetals,polycyclicaromatichydrocarbons(PAHs),polychlorinatedbiphenyl(PCBs),surfactants,pesticides,dioxins,pharmaceuticalsandperso-nalcareproducts(PPCPs),endocrinedisruptingcompounds(EDCs),per-andpoly-fluori-natedalkylsubstances(PFASs),brominatedflameretardants(BFRs),nanomaterials,marinebiotoxins,polydimethylsiloxanes(PDMS),andmicroplastics.Theirmainsourcesofcontamination,themostusedanalyticaltechniquesfortheirdetectionandquantificationinthemarineenvironmentandtheirusuallevelsfoundinseawater,sedi-ments,andmarineorganismswillbereportedprovidinganexposurescenariotowildlife.

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S E S S I O N I IM a r I N E b I O S E N S O r & M I c r O E l E c t r O N I c S I N b I O S E N S I N g a p p l I c at I O N S O r a l p r E S E N tat I O N S

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Biosensors for real time monitoring of biohazard and man made chemical contaminants in the mari-ne environmentKonstantinos Petropoulos1,Arben Merkoci3, Aziz Amine4,Bengt Danielsson2, Chrysi Laspidou10,Elise Regairaz9,Linda Medlin6,Luca Sanfilippo7,Nives Kovač5,Salvatore Chiavarini8,Giuseppe Palleschi1

1Dipartimento di Scienze e TecnologieChimiche, Università di Roma TorVergata, ViadellaRicercaScienti-fica 1-00133 Roma. 2Acromed Invest AB, Magistratsvägen 10, S-226 43 Lund, Sweden. 3Nanobioelectronics & Biosensors Group Catalan Institute of Nanoscience and Nanotechnology (ICN2)Campus de la UAB 08193 Bellaterra (Barcelona) Spain. 4Université Hassan II de Casablanca Faculté des Sciences et TechniquesLaboratoireGénie des Procédés et Environnement B.P. 146-20800 Mohammedia, Morocco. 5Nacionalni InštitutzaBiologijo, Morskabiološkapostaja Piran Fornače 41-6330 Piran, Slovenija. 6Microbia Environment, University of Pierre and Marie Curie, ObservatoireOcéanologique de Banyuls sur mer, Avenue du Fontaulé 66650 Banyuls-sur-Mer France. 7Systea S.p.A, Analytical Technologies, ViaPaduni 2A-03012 Anagni-Roma. 8ENEA, Agenzianazionale per le nuovetecnologie, l ’energia e lo sviluppoeconomicosostenibile, Casaccia, viaAnguillarese 301-00123 Roma. 9AlienorEU, 11 rue de l’industrie 1000 Brussels-Belgium. 10University of Thessaly, Civil Engineering Department, PedionAreos 38334 Volos-Greece. E-mail contact:[email protected]

Highlights• DevelopmentofaEuropeanopendatabase,accessibletoend-users,regardingconta-

minantsofemergingconcerninmarineenvironment.• Theuseofnanotechnologyforenhancingboththedetectionlimitsandthesensitivity

oftheproposedelectrochemicaland/oropticalbiosensorshasbeenadopted.• Good sensitivity and lowdetection limits have been achieved for themost of the

contaminants.• Themaincontaminantsarealgaltoxinsandtheirassociatedalgalspecies,pesticides,

flameretardants,antibioticsandaseriesofstandardwaterqualityparameters.

ThemaingoalofSMS(SensingtoxicantsinMarinewatersmakesSenseusingbiosensors)projectistodeliveranovelautomatednetworkedsystemthatwillenablereal-timeandinsitumonitoringofmarinewaterincoastalareasbythedetectionofaseriesofcontaminants.TheteamworkofSMSisengagedinthedesigningofamulti-modularapparatusthatwill host in a single unit, named the Main Box (MB), a Sampling Module and anAnalysis Module. The former will contain sample collection and treatment compo-nents,whereasthelatterwillincludefourbiosensorsub-modulesthatwillenablede-tectionandmeasurementofmarinealgaltoxins(e.g.Okadaicacid,Saxitoxin,Palyto-xinandDomoicacid)andtheirassociatedalgalspecies,severalhazardouscompoundssuchastributyltin(TBT),3-(3,4-Dichlorophenyl)-1,1-dimethylurea(Diuron),penta-

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PolybrominatedDiphenyl Ethers(BDPEs), as also sulphonamidesandaseriesofstandardwaterqua-lityparameters.The MB will be equipped with acommunication module for real-timedatatransfertoacontrolcen-ter,wheredataprocessingwilltakeplace,enablingalarmfunctionalitytoHealthWarningSystems,whe-neversomecriticalvalueexceedsapre-definedthreshold.TheMBwillbe placed on a floating platformorbuoypositioned in loco, at th-ree different demonstration sites(e.g. La Spezia-Italy, SlovenianAdriatic Sea and Alonissos mari-nepark-Greece).Thesethreesiteshavebeenproperlychosenbecausetheyofferawiderangeoftestcon-ditions,intermsofcontamination,formarinewatermonitoring.

AcknowledgementTheauthorswouldliketoacknowledgethefinancialsupportfromtheEuropeanUnion’sSeventhFrameworkProgrammeforresearch,technologicaldevelopmentanddemon-strationundergrantagreementno613844.

Figure 1. Schematic representation of the proposed au-tomated networked system for the monitoring of marine water’s chemical status in coastal areas.

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MARIABOX an autonomous sea water pollution monitoring device for natural and man-made pol-lutants: challenging user requirements and tac-kling design criticalitiesPaolo Barattini1, Silvia Mier2, Matteo Bonasso1, Esther Garcés3, Panayiotis Philimis4, Alessandro Giusti4, Kevin Thomas5, Antonio Varriale6, Sabato D’Auria6

1Kontor 46, Turin, Italy. 2Acorde SA, Santander, Spain. 3Institut de Ciènciesdel Mar, CSIC, Spain 4Cyprus Research and Innovation Center-Cyric, Cyprus. 5Norwegian Institute for Water research-NIVA, Norway. 6ISA- Consiglio Nazionale delle Ricerche, Italy. * E-mail Contact: [email protected], Tel +39 328-1250084.

Abstract

The MariaBox project, funded by the European Commission (Contract 614088), is develo-ping an autonomous, analytical device, based on novel biosensors, for monitoring chemical and biological pollutants in sea water. The device will be suitable for installation in free flo-ating devices, buoys, ships, or to be used as a portable instrument. The main, high-level user requirements for the system are for the device to be of high-sensitivity, portable and capable of repeating measurements over a long time, allowing permanent deployment at sea.All of these requirements are demanding in terms of design choices that must weighed and balanced against the different technical trade-offs necessary to find a viable implementation solution.

Key words: biosensor, algal toxin; heavy metals; wireless analytical device.

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Portable acquisition electronic systems applied to electrochemical biosensorsRaquel Pruna1, Francisco Palacio1, Monica Martinez2, Manel López1

1Departament d’Electrònica, Universitat de Barcelona, Martí i Franquès 1, E-0828 Barcelona, Spain 2Departament de Ciència de Materials i Química Física, Universitat de Barcelona, Martí i Franquès 1, E-0828 Barcelona, Spain - E-mail contact: [email protected]

Highlights • Portable,miniaturizedandlow-powerconsumingelectronicdevicesareofincreasingimpor-

tanceformeasuringsensorsinlab-on-a-chipenvironments• Lowdetectionlimitscanbeachievedbymultipleelectronicconfigurations,thechoicewill

onlydependontheapplicationandthetransductionmechanism• Softwaredataprocessingimplementationsimprovethesignal-to-noiseratio,stillmaintai-

ningthedeviceminiaturization.

Theincreasingneedfornovelandsophisticatedsensingmethodsinthebiomedicalengineeringandbiotechnologyfieldshasyieldedthepolepositiontoelectrochemistry,duetoitsreliability,relativeease-of-useandalsoofunderstanding.Outofalltheavailablemeasurementtechni-quesinthiskindofexperiments,potentiostatshavecatchedscientists’attentionandhavebeenwidelydevelopedinthepastyears1.Theirimplementationallowshavingperfectcontrolofthepotentialappliedtothesensorwhilstsimultaneouslyregisteringthecurrentattheworkingelectrode.Theincreasingattempttoworkatlow-voltageandlow-currentconfigurationshasledtoawidevarietyofpotentiostatdesignsintermsofinstrumentation,someofwhichhavebeenreportedduringthelastyears2,3.Theseimplementationsareallbasedonsiliconelectro-

nics,as thispermitscheapmassive production andgood compatibility withthestandardelectronicde-vices.

Intermsofdesignandim-plementation of a poten-tiostatamplifier,thediscre-te implementation is theoptimal solution for por-table systems for standardelectrochemical assays,although the increasingdemandofreducedsystemandelectrodessize,aswellasultra-lowcurrentdetec-

Figure 1. Block diagram representing the mode of operation of a potentiostat

Figure 2. Competition detection of Ab-155 coupled to SP by the coating antigen

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tion,isleadingtoimplementationofintegratedpotentiostatamplifiersbasedoncomplemen-tarymetaloxidesemiconductor(CMOS)processes.Takingforgrantedthatapotentiostat’smainfunctionalitiesare1)ononeside,tostablishtheelectrodesatadesiredandinvariable-inthecaseofamperometry-voltageand2)ontheotherhand,toextractanoutputsignalofthecurrentflowingthroughtheelectrochemicalcell,ablockdiagramofthemainstagesisshowninFig.1.Alongourtalk,wewillreviewsomeofthemostimportantpotentiostaticcircuitsbasedonoperationalamplifiersthathavebeendevelopedduringthelastyears,togetherwithconditio-ningelectronicsforlowpowerandportablesystems.Wewillalsofocusonsignalprocessingelectronics.AlltheconceptswillbeendorsedinthecontextofthedevelopedelectronicsfortheSea-on-a-Chipprototypes.

AcknowledgementsTheauthorswishtothankthe7thFrameprogramEuropeanProject“Biosensorsforrealtimemonitoringofbiohazardandman-madechemicalcontaminantsinthemarineenvironment(Sea-on-a-Chip)”,614168,andalsotheUniversitatdeBarcelonatheaidtopromotethepar-ticipationininternationalresearchprojects(2015).

References1.BardAJ,FaulknerLR.ElectrochemicalMethods:FundamentalsandApplications.Wiley

NewYork;1980.2.KimuraM,BundoK,ImuroY,SagawaY,SetsuK.ChronoamperometryUsingIntegra-

tedPotentiostatConsistingofPoly-SiThin-FilmTransistors.ElectronDeviceLett.IEEE2011;32(2):212-214.

3.LiL,LiuX,QureshiWA,MasonAJ.CMOSamperometricinstrumentationandpacka-gingforbiosensorarrayapplications.Biomed.CircuitsSyst.IEEETrans.2011;5(5):439-448.

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Novel strategy for detection of Sulfapyridine using a fully integrated Hybrid Lab-on-a-chipNadia El Alami El Hassani1,2,3, Abdoullatif Baraket3, Ernandes Taveira Tenório Neto4, Michael Lee3, Juan Pablo Salvador5, Maria pilar Marco Colas5, Marinella Farré7,8, Damià Barceló7,8, Joan Bausells6, Nezha El Bari1, Benachir Bouchikhi2, Abdelhamid Elaissari4, Abdelhamid Errachid3, Nadia Zine3

1Biotechnology Agroalimentary and Biomedical Analysis Group , Department of Biology, Faculty of Sciences, Moulay Ismail University, BP 11201, Zitoune, Meknes, Morocco 2Sensor Electronic and Instrumentation Group, Department of Physics, Faculty of Sciences, Moulay Ismail University, BP 11201, Zitoune, Meknes, Morocco 3University of Lyon, Institute of Analytical Sciences, UMR 5280, Claude Bernard Lyon 1 University, 5, Rue de la Doua, 69100, Villeurbanne, France 4LAGEP, University of Lyon, Claude Bernard Lyon 1 University, Villeurbanne, France 5IQAC Department of Chemical and Biomolecular Nanotechnology, C/ Jordi Girona 18-26 08034, Barcelo-na, Spain. 6 CSIC Campus UAB, 08193, Bellaterra, Barcelona, Spain. 7Department of Environmental Chemistry, IDAEA-CSIC, c/Jordi Girona 18-26, E-08034 Barcelona, Spain 8Catalan Institute for Water Research (ICRA), Emili Grahit 101, E-17003 Girona, Spain *E-mail contact: [email protected]

Highlights.• ElectrochemicalbioLab-on-a-chipforSulfpyridine(SP)detectioninseawater.• Studyofthecontaminationofseawater.• CompetitivedetectionbetweenSPandSA2BSAbyusingpolypyrrole-2-carboxylicacid

(Ppy-COOH)modifiedmagneticnanoparticles(MNP).

Sulfpyridine(SP)asacategoryofsulfonamides,havebeenwidelyusedasveterinarydrugsforprophylacticandtherapeuticpurposes.However,severalclassesofbacteriahavebecomeresi-stanttothisdrugsduetotheirabusiveuses.Thispracticecanalsosignifyapotentialrisktohu-manhealthsincemanydiseasesmayemerge,suchasthyroidcancerandanaphylacticreactions.WereportinthepresentworkthedevelopmentofahighlysensitiveBioLab-on-a-chipsy-stems(Fig.1-a)forthedetectionofsulfapyridineantibioticresiduesatthelowppbconcen-trationlevel.QuantificationisachievedthroughcompetitionprocedurebetweenSA2-BSAimmobilizedontheworkingmicroelectrodesurfaceandSPforbindingsitesofthecapturepolyclonalantibody.Therefore,goldWEswerepreviouslyfunctionalizedwithpolypyrrole-2-carboxylicacid(Ppy-COOH) modified magnetic nanoparticles (MNP) (Fig.1-b). Pulsed chronoamperometrywasusedforPpy-COOHdeposition.Cyclicvoltammetry(CV)wasappliedtocharacterizeeachstepsofthedeposition.Afterward,SA2-BSAantigenswerecovalentlybondedtothePpy-COOH/MNPmodifiedgoldWEsthroughamidebonding.Thedetectionanalytewasmadebyamixtureofafixedconcentrationofantibody(Ab-155at10µg/mL)anddecreasing

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concentrationsofSP.Therangeofthislatterwasfrom50µg/mLto0ng/mL.Thebio-reco-gnitionofSA2-BSAandAb-155wereanalysedbyelectrochemicalimpedancespectroscopy(EIS)(Fig.2).ThedetectionofSPbythismethodishighlypromisingtogiveastablemeasu-rementandarapidresponse.AcknowledgementThe authors acknowledge the financial support from the projects NATO (CBP.NUKR.SFP 984173) and SEA-on-a-CHIP (FP7-OCEAN-2013) under the grant agreementNo.614168.

Figure 1. a) BioLab-on-a-chip system; b) Microelectrodes surface taken by a stereo microscope after deposition of Ppy-COOH/MNP


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A new competitive BioLab-on-a-chip for the de-tection of Irgarol 1051 for seawater monitoringTalha Jamshaid1,2, Abdoullatif Baraket1, Michael Lee1, Juan Pablo Salvador3, Maria Pilar Marco Colas3, Joan Bausells4, Marinella Farré5,6, Damià Barceló5,6, Damià Bar-celó5,6, Nadia Zine1, Abdelhamid Elaissari2, Abdelhamid Errachid1

1 Institut des Sciences Analytiques (ISA), Université de Lyon, Université Claude Bernard Lyon-1, Villeur-banne, France. 2Laboratoire d’Automatique et de Génie des Procédés (LAGEP), University of Lyon, Université Claude Bernard Lyon-1, Villeurbanne, France. 3Institute of Advanced Chemistry of Catalonia (IQAC) Department of Chemical and Biomolecular Nano-technology, Barcelona, Spain. 4Instituto de Microelectronica de Barcelona, IMB-CNM (CSIC) Campus UAB, 08193, Bellaterra, Barcelo-na, Spain. 5Department of Environmental Chemistry, IDAEA-CSIC, c/Jordi Girona 18-26, E-08034 Barcelona, Spain 6Catalan Institute for Water Research (ICRA), Emili Grahit 101, E-17003 Girona, Spain. E-mail contact: [email protected]

Highlights• ElectrochemicalbioLab-on-a-chipforIrgaroldetectioninseawater.• Studyofthecontaminationofseawater.• Competitivedetectionbetween Irgarol and4eCona antigensusingmagneticnano

particles(MNP).

Irgarol1051isas-triazineherbicideusedinpopularslime–resistantanti-foulingpaints[1].Ithasbeenshowntobeacutelytoxictocoralsandseagrassesthusinhibitingphotosynthesisatlowconcentrations(>50ng/L-1)[2].Inthepresentwork,wereportonthedevelopmentofahighlysensitiveBioLab-on-a-chipusinggoldtransducersastheworkingmicroelectrodes(WEs).ThedetectionofIrgarol1051wasappliedasacompetitiveassaywiththeantibody(As87)vsthecoatingantigen(4e-CONA).Therefore,the4e-CONAcoatingantigen(50µg/mL)wascovalentlybondedtothegoldWEsthroughelectroaddressingwithdiazoniumsalt(CMA)[3].Cyclicvoltammetry(CV)wasusedfortheCMAdeposition(Figure1b)andforallstepsofthegoldWEscharacterization.Theanalytefordetectionwasmadebyamixtureofafixedconcentrationofantibody(As87at10µg/mLthatwaspreviouslyoptimizedbyELISA)anddecreasingconcentrationsofIrgarol1051.Thebio-recognitionof4e-CONAandAs87wereanalyzedbyelectrochemical impedancespectroscopy(EIS).ThedetectionrangeofIrgarol1051wasmadefrom0µg/mLto2µg/mL.InordertoincreasethesensitivityoftheBio-MEMS,thesameprincipleofthecompetitiveassaywasappliedusingfunctionalizedma-gneticnanoparticles(MNPs)withtheimmobilizedAs87antibodythroughamidebonding(MNP-COOH).Here,thegoldtransducerscontainedthecoatingantigen4e-CONAandthe3DcompetitiveassaywasanalyzedwithAs87vsIrgarol1051(Figure1c).


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Finally,fromthesetwodevelopedandnovelstrategiesforthedetectionofIrgarol1051,weobservedthattheresultsusingMNPsareverypromisingfortheintegrationofthepresentedBioLab-on-Chip.Thispre-concentrationprocedurewasshowntoavoidthematrixeffectandthedetectionofIrgarol1051atweakconcentrationsforenvironmentalapplication.

AcknowledgementTheauthorsacknowledgethefinancialsupportfromtheprojectsNATO(CBP.NUKR.SFP984173)andEuropeanCommunitiesSeventhFrameworkProgrammes:SEA-on-a-CHIP(FP7-OCEAN-2013)underthegrantagreementNo.614168,andfundingfromtheEuro-peanUnion’sHorizon2020researchandinnovationprogrammeentitledHEARTENundergrantagreementNo643694.

References1)P.B.Carrasco,S.Dı́ ez,J.Jiménez,M-PMarco,J.M.Bayona,WaterResearch,37,Issue15,

(2003)3658-36652)K.Carbery,R.Owen,T.Frickers,E.Otero,J.Readman,MarinePollutionBulletin52

(2006)635–6443)A.Baraket,M.Lee,N.Zine,M.Sigaud,N.Yaakoubi,M.G.Trivella,M.Zabala,J.Bau-

sells,N.Jaffrezic-Renault,A.Errachid,SensorsandActuatorsB:Chemical,189(2013)165-172.

Figure 1. (a) Lab-on-a-chip passivated with an epoxy resin, (b) CV for the electrodeposition of CMA onto the gold WEs, (c) schematic illustration of MNPs functionalized with As87 antibodies in order to detect 4e-CONA (•) or Irgarol 1051 (•).

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S E S S I O N I IM a r I N E b I O S E N S O r & M I c r O E l E c t r O N I c S I N b I O S E N S I N g a p p l I c at I O N S p O S t E r p r E S E N tat I O N S

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Determination of pesticides used in marine aqua-culture by LC-High Resolution Orbitrap MSV.I. Boti, Ch.I. Nannou, M.G. Kapsi, T.A. Albanis

Lab of Analytical Chemistry, Department of Chemistry, University of Ioannina, Ioannina 45110, Greece - E-mail contact: [email protected]

Highlights• Aquacultureexpansion–extendeduseofbiocides-insecticides• DeterminationbymeansofSPE/LC-LTQ-Orbitrap-MS• Accurateandrobustmethod• Applicationtomonitorcontaminationlevelsinaquacultures1. IntroductionThemassiveexpansionofaquaculturehasagreatimpactonwaterqualityandaquaticlife.Thisexpansion leads to therequirementofwideruseofdrugs,disinfectants,antifoulingcompounds(biocides)andinsecticidestoeliminatethemicroorganismsintheaquaculturefacilities.Numerousstudieshaveshownthatthesecompoundsaretoxictoawidevarietyofaquaticorganisms.Theirsystematicmonitoringmaycontributetothepreservationandsustainabilityofmarineenvironment.Inaddition,manyofthesecompoundsmayoccurinmarineenvironmentduetoothersourcessuchasagriculturalactivitiesormorespecificusa-ges.Forinstance,antifoulingcompoundsarealsoappliedtothehullsofshipsandboatstopreventadhesionandgrowthofbacteria,macroalgae,musselsandinvertebratesintheaqua-ticenvironment.Thus,itismorethannecessarytofocusonthestudyoftheirconcentrationlevelsintheareasclosetofishfarms.Inthisstudy,seawatersamplescollectedfromSagiadaarea(N.W.Greece)wereusedtovalidateananalyticalmethodwiththeaimtoassessthedistributionofazamethiphos,irgarol1051anddeltamethrininthepollutantload.SampleswereextractedbySolidPhaseExtraction(SPE)andanalysedwithultra-highperformanceliquidchromatography(UHPLC)high-resolutionOrbitrapmassspectrometry.

2. Materials and methodsAnalyticalstandards(purity>98.4%)werepurchasedbyFisherScientific(Leicestershire,UK).Stockstandardsolutionswerepreparedataconcentrationof1–2μg/Linmethanolandwerestoredat−20°C.Thesolventsused,includingmethanolandwaterLC-MSgrade,weresupplied by Merck (Darmstadt, Germany). OasisTMHLB cartridges (divinylbenzene/N-vinylpyrrolidonecopolymer,200mg,6cm3)werepurchasedfromWaters(Mildford,MA,USA).Formicacid(purity,98%)wasobtainedfromFluka(Buchs,Germany).Theextractionoftargetanalytesfromseawatersampleswascarriedoutinanoff-line,16-portSPEmanifoldconnectedtoavacuumpump.HLBcartridgeswerefirstconditionedwith6mLmethanoland6mLdistilledwater(withoutlettingthedisksbecomedry).Af-terwards,avolumeof250mLofwatersamplewaspercolated.Theanalyteswereelutedwith

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3mLofdichloromethane,hexaneandacetone,consecutively.TheextractwasevaporatedtodrynessunderagentlestreamofN2andreconstitutedintothedesirablevolumeofmetha-nol.Thefinalsamplewasfiltereddirectlyintoananalysisvialusinga0.45μmPVDFsyringefilter(Millex-HV,Millipore,Cork,Ireland)andinjectedtoaliquidchromatography-LTQOrbitrapMSsystem.TheLCLTQ-FTOrbitrapMSsystemconsistedofanAccelaASau-tosampler,anAccelaquaternarygradientU-HPLC-pumpandanLTQOrbitrapXL2.5.5SP1massspectrometer(ThermoFisherScientific,Inc.GmbH,Bremen,Germany).Thelineariontrap(LTQ)partofthehybridMSsystemwasequippedwithanIonMaxElec-trosprayIonization(ESI)probe.Full-scanaccuratemassspectrawereobtainedathighreso-lution(60000FWHM).Theanalyteswereelutedona50mmx2.1mmi.d.HypersilGOLDcolumn,1.9μm,usingagradientelutionsystemconsistingofwater(A)andmethanol(B)bothcontaining0.1%formicacidand5mMammoniumformateat300μL/minflowrate.

3. Results and discussionThetargetanalytesweredetectedas[M+H]+adductionsforirgarolandazamethiphoswhi-ledeltamethrinwasdetectedas[M+NH4]+,allunderpositiveionmode.Massaccuracyofmeasuredionswascalculatedbelow3ppmat50ppb,inallcases.Thevariablesinvolvedinthechromatographicprocesswereoptimizedininstrumentautotunesections.Theselectedconditionsprovedtobeexcellentwithin10minelutionwiththequantificationLTQ-Orbi-trapLC-MSlimitdowntoapproximately0.25ppb.Underoptimizedconditions,thefrag-mentationprocessintheLTQsystem,takingtheabove-mentionedadductionsasprecursoronesandusingCIDenergyof35%yieldsmorethanenoughstructuralinformationforpo-sitiveidentificationbasedonmonitoringthebasicfragmention.SPEextractionconditionswerevalidatedusingspikedseawatersamples(previouslyensuredtobefreeoftheanalyteofinterest)fortifiedwiththetargetanalytesattwoconcentrationlevels,20ng/Land500ng/Lrespectively.Theanalyticalmethodprovedtobelinearoverawiderangeofconcentration,exhibitedsatisfactoryrepeatabilityandreachedtracelevelintheorderoflowng/L.

4. ConclusionsTheanalyticalmethodologypresentsexhibitedexcellentanalyticalcharacteristicsandpro-vedtobereliablefortheestimationofthepollutantloadinseawatersfrommarineaquacul-ture.Theresultsobtainedconfirmedthathigh-resolutionmassspectrometryisahelpfulandrobusttoolfortheidentificationandquantificationofsuchcontaminantsresidues,provi-dingatthesametimehighaccuracy.

AcknowledgmentsThisprojecthasreceivedfundingfromEuropeanUnion’sSeventhFrameworkProgramme(FP7-OCEAN-2013)undergrantagreementNo.614168.TheauthorswouldalsoliketothanktheUnitofEnvironmental,OrganicandBiochemicalhighresolutionanalysis-OR-BITRAP-LC-MSoftheUniversityofIoanninaforprovidingaccesstothefacilities.

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Development of novel sensing devices for marine algal toxins detectionMarianna Rossetti1, Alessandro Porchetta1, Konstantinos Petropoulos1, Francesco Ricci1, Laura Micheli1, Giulia Volpe1, Giuseppe Palleschi1

1Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata, Via della Ricerca Scienti-fica 1, 00133 Roma - E-mail contact: [email protected]

Highlights• Developmentofaptamerbasedsensorsforsinglestepmeasurements,rapidandreagentless.• Developmentofaflow-injectionimmunoassay(FI-IA)methodforadetectionthatcombi-

nestherapidityandreproducibilityoftheflow-injectiontechniquewiththehighselectivityandsensitivityofimmunochemicalreactions.

• Developmentofaninhibitionassaysensibleandautomated.

Aptamersaresingle-strandedDNAorRNAselectedinvitro,capableofbindingtoitstargetantigenwithhighaffinityandspecificity.Bylabellingaptamer,throughtheintroductionofafluorophore/quenchercouple,aptamersignalsthetargetpresencethroughabinding-inducedconformationalchangewhichbringsthefluorophoreclosetothequencherthusdecreasingthefluorescencesignal.Accordingwiththisapproach,wehavedevelopedasensortodetectSaxitoxin(STX),atoxinresponsibleforcausingparalyticshellfishPoisoning.AptamerscanalsobejoinedtonucleicacidenzymestocreateallostericRNAzymes.Byintroducinganap-tamerdomaininanhammerheadribozyme,RNAself-cleavageisdependentonthepresenceofeffector.Bymodifyingthestructureofhammerheadwithafluorofore/quencercouple,uponbindingofeffector,theribozymeundergoesself-cleavageandreleasesashortflorescein-labe-ledproductoligonucleotide.WeareusingthisstrategytodetectPalytoxin,anintensevasocon-strictor,andDomoicAcid,responsibleforcausingamnesicshellfishpoisoning.RegardingSTX,alternativelytotheuseofaptamers,wearealsoworkingonaflow-injectionimmunoassay(FI-IA)methodwithamperometricorcolorimetricdetection.Thissystemcombinestherapidityandreproducibilityoftheflow-injectiontechniquewiththehighselec-tivityandsensitivityofimmunochemicalreactions.Finally,wehavedevelopedacolorimetricassayforthedetectionofOkadaicAcid,basedontheinhibitionofproteinphosphatasetype2A(PP2A)bythetoxin.WeproposeacolorimetricassayinwhichtheactivityofPP2Aisdeterminedbymeasuringtherateofcolorproductionfromtheproductionofyellowp-nitro-phenolusingp-nitrophenylphosphateasthesubstrate.Theassayhasgoodsensibilityandhasbeenautomatizedbydevelopingamodularautomatedprototype.

Acknowledgement

TheauthorswouldliketoacknowledgethefinancialsupportfromtheEuropeanUnion’sSe-venthFrameworkProgrammefor research, technologicaldevelopmentanddemonstrationundergrantagreementno613844.

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finito di stampare a Fidenza (PR - Italy)nel mese di aprile 2016presso Mattioli 1885

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