HRMSwide-scopetargetandsuspectscreeningmethodologiesforthemonitoringofemergingcontaminantsinraptors

Dr.NikolaosS.Thomaidis,ProfessorofAnalyticalChemistry,NationalandKapodistrianUniversityofAthens(NKUA)

NationalandKapodistrianUniversityofAthensSchoolofScienceDepartmentofChemistryLaboratoryofAnalyticalChemistry

ERBFacilityWG3Collections-VirtualMeeting

TheroleofcollectionsforcontaminantmonitoringinraptorsacrossEurope:Stateofplayandnextsteps

11February2021

@ThomaidisLab

OurResearchTeam

NationalandKapodistrianUniversityofAthensLaboratoryofAnalyticalChemistry

TraceAnalysisandMassSpectrometry(Tr.A.M.S.)group

7post-doctoralresearchers

15PhDCandidates

10MasterStudents

http://trams.chem.uoa.gr

Athens

AdvancedanalyticalmethodologiesCutting-edgeinstrumentaltechniques

EnvironmentalChemistryFoodChemistryPharmaceuticalChemistryIndustrialChemistry

Ourmission

Developmentofadvancedchemometrictoolsanddata

processingmethods(supportingnon-targettools)

Answers in environmental problems on emerging contaminants andtheir (bio)transformation products (holistic research approach;monitoring,fatestudiesandecotoxicology)

State-of-the-artmassspectrometricinstrumentation

Developmentofnovelanalyticalmethodologiesonsamplepreparation

Instrumentation

timsTOFflex

Experience&ExpertiseLaboratoryofAnalyticalChemistry(LAC)àgreatexperienceinthefieldofemergingcontaminants’identificationinbiota

developed&validatedmultianalyte,multiresiduesensitiveanalyticalmethodologies

pharmaceuticals&veterinarydrugs

per-andpolyfluoroalkylsubstances(PFAS)

polycyclicaromatichydrocarbon(PAHs)

organochlorinepesticides(OCPs)

organotins

metals

analyticalmethodologiesforthedeterminationofWFDcompounds

ApplicationinnumerousnationalandEuropeanfundedprojects

2013-todayàHRMS

developedandvalidatedHRMSanalyticalmethodologiesforthesimultaneousdeterminationofthousandsofchemicals

LC-MS/MS

GC-MS/MS

ETAAS,ICP-MS

smartvalidationàrepresentativemixofcompounds

Theanalyticalproblem

Whyraptorspecimens?ü  uppertrophiclevelpredatorü  relativelylonglifespanàaccumulationofECsü measurableresponsestoECs(e.g.populationdeclineduetoDDE)ü  catholicdiet(avianpreyfromtheaquatic&terrestrialenvironments)ü  globaldistributionü  diversehabitattypes

Long-termmonitoringstudiesofECsusingwildlife:ü  effectivenessofchemicalsriskmitigationmeasuresinreducingexposureü  betterchemicalsmanagement(prioritizationofsubstancesforPBTassessment)ü  possiblecandidatesforfutureEUlegislation

EmergingContaminants(ECs)…

…Bioaccumulatedthroughthefoodwebs

…end-upintheenvironment…

?

ThousandsofECsØ  DiverseclassesofcompoundsØ  DifferentphysicochemicalpropertiesØ  Multilevelconcentrations

KnownECs

UnknownECs

WhyHRMS?HRMSgivesaccesstoalargernumberofDiagnosticToolsØ  AccuratemassmeasurementsfromfullscanmeasurementsØ  Isotopicinformation

•  elementalcompositionofbothprecursorandfragmentionsØ  DataDependentMSnorDataIndependentMS/MSfeatures

•  excellenttoolsfortheidentificationofcompounds•  allowdifferentiatingisobariccompounds

Ø  RetrospectiveAnalysisØ  Quantitativeapplications

•  advancesregardingmatrixeffect,sensitivityanddynamicrangeØ  Target,Suspect&Non-targetScreening

screeningofthousandsofchemicals

MolecularFingerprint

HRMSScreeningworkflows

GenericSamplePreparation

Pre-treatment Extraction Cleanup

SPE

Severaldevelopedandvalidatedgenericsamplepreparationprotocols(polar&non-polarchemicals)

Wide-scopetargetanalysis

NKUADatabases

>2,400compoundsin4datasets; LC-ESI(+/-)-QToFMS&GC-APCI(+/-)-QToFMS

ü  PAHsü  OCPsü  PCBsü  PlantProtectionProductsü  PCNsü  PBDEsü  FAMEs

Thedatabasesarecontinuouslybeingupdated!

ü  Pharmaceuticals(>450)Antibiotics(>50)

ü  Personalcareproductsü  Illicitdrugs&NewPsychoactiveSubstances(>500)ü  IndustrialChemicals(>100)ü  PlantProtectionProducts(>900)ü  Sweetenersü  Surfactantsü  Steroids&hormonesü  TransformationProducts/Metabolites

https://zenodo.org/record/3753372

Gago-Ferreroetal.2020,https://doi.org/10.1016/j.jhazmat.2019.121712

Suspectscreeningworkflows

DigitalSampleFreezingPlatform(DSFP)

Alygizakisetal.2019,https://doi.org/10.1016/j.trac.2019.04.008

ü  Automatedtoolü  Digitalarchivingü  Retrospectivesuspectscreening

Suspectscree

ningof>111,

000chemical

s

(NORMANSus

Dat)

EnvironmentalAnalysis–Projects

EMBLASproject(2016-2018,2019-2020)I.  EQS2013/39/ECtargetscreening(LC-QqQ,GC-QqQ)II.  Wide-scopetargetscreeningof(>2,200chemicals,LC-QToFMS)III.  Non-targetscreening(LC-QToFMS,GC-APCI-QToFMS,GC-EI-

MS)

JointDanubeSurvey4(JDS4)(2019-2020)I.  Targetanalysisofdrugsofabuse(LC-QqQ)II.  Wide-scopetargetscreeningof(>2,200chemicals,LC-QToFMS)III.  Non-targetscreening(LC-QToFMS)

LIFE17ENV/SK/000355(LIFEAPEX)“Systematicuseofcontaminantdatafromapexpredatorsandtheirpreyinchemicalsmanagement”

(2018-2022)I.  Samples’pre-treatment(lyophilization,homogenization)anddistributionII.  Wide-scopetargetscreeningof(>2,200chemicals,LC-QToFMS)III.  Non-targetscreening(LC-QToFMS,GC-APCI-QToFMS)

Biomonito

ringEurope

anProjects

(aquaticorg

anismsfrom

differenttro

phiclevels)

SamplesØ  Wastewater(influent&effluent)Ø  SewagesludgeØ  SurfacewaterØ  Sediment&SoilØ  Biota(mussels,fish,apexpredators)

+raptors(

commonBu

zzard)

àterrestr

ialecosyst

em

EnvironmentalAnalysis–Projects

•  LeibnizInstituteforZooandWildlifeResearch(IZW),Germany

white-tailedseaeagle(liver)

•  NaturalisBiodiversityCenter&DutchPeregrineWorkingGroup,theNetherlands

peregrinefalcon(liver)

•  LandesanstaltfürUmwelt,MessungenundNaturschutzBaden-Württemberg(LUBW),Germany

littleowl,greatcurlew,peregrinefalcon,eagleowl(egg)

Biomonitoringstudies(raptorspecimens)

CONNECTproject(2020-2021)

•  40organismsfromthelowertrophiclevels(mussels,oysters,flounders)andsediments,gatheredfromtheNorth-EastAtlanticecosystem

•  Wide-scope target analysis (>2,400 substances) and suspect screening (>111,000NORMANSusDatsubstances)byLC-HRMSandGC-APCI-HRMS

HELCOMproject(2020-2021)

•  Marinemammals,gatheredfromtheBalticSea•  Wide-scope target analysis (>2,400 substances) and suspect screening

(>111,000NORMANSusDatsubstances)byLC-HRMSandGC-APCI-HRMS

PrioritypollutantsandChemicalsofemergingconcerninpolarregions

PhaseI(2019-2020):Organismsfromthelowertrophiclevels(macrophytes,seastar,seaurchin,fishspecimens)

PhaseII(2020-2021):ApexPredatorsàdifferentmatrices(e.g.placenta,eggs)

SurveysintheframeworkofNORMANnetworkactivities

LIFEAPEXTheAIMofLIFEAPEXistoimprovethesystematicuseofchemicalmonitoringdatafromapexpredatorsandpreyforprotectinghumanhealthandtheenvironment.

Tier1:Wide-scopescreening12/67;commonBuzzardspecimensà3countriesofNorthernEurope(Germany,

UnitedKingdom,Netherlands)

Tier2:Timetrendanalyses19/65;commonBuzzards(UnitedKingdom)àSamplingYears:2002-2018

Tier3:ReplicationwithR&TpartnersApexpredatorspecimensfromdifferentEuropeancountries

ReplicationoftheTier1approachinapan-Europeanlevel(e.g.,Buzzardsamples)

widerEuropeanpictureofoccurrenceofECs

ProjectCoordinator:EnvironmentalInstitute(DrSlobodnikJ.)ProjectDuration:September2018–August2022

Projectbudget:€3.35M(ofwhichEULIFEfunding60%)

LIFEAPEX

Tier1(commonBuzzards)DetectedECsinTier1à164

DetectedECsà86(Buzzards)

Pharmaceuticals&TPsà28%

Caffeine,NicotineandtheirTPs(Theobromine,Theophylline,Cotinine,Hydroxy-Cotinine,Nor-Nicotine)

Surfactantsàhighestconcentrationsamongthedetectedcompounds

PFOSandits’branchedisomersà100%FoA,highconcentrations(upto953μg/Kgw.w.)

Bioaccumulation?àsupportinginformationfromprey(terrestrialecosystem)

PlantProtectionProductsàAldicarb-sulfoxide,Dichlorobenzamide,Ethiofencarb-sulfone

OutcomeofDSFPSuspectscreeninginapexpredators&theirpreyspecimens,gatheredfrom4countriesinNorthernEurope(LIFEAPEX,Tier1)

LIFEAPEX

Tier2:time-trendanalysis

ThecaseofPerfluorooctanesulfonicacid(PFOS)2010-2018àtrendtolowerconcentrationsofPFOS

AttributedtoPFOSreplacementcompounds(GenX,ADONA)?

wide-scopetargetanalysis

LIFEAPEX

Tier2:time-trendanalysissuspect&non-targetscreeningØ Monotonictrenddetection(Spearmanrank,Mann-Kendall,Hirsch-Slack)Ø Non-randomnessdetection(Cox-Stuart,Wallis-Moorephasefrequencytest,BartelsrankvonNeumann'sratiotest)

time-trendanalysisMultivariateTrendAnalysisbyTrendTrAMS

Surveyswithraptorspecimens

Wide-scopetargetandsuspectscreeningofemergingcontaminantsandtheirtransformationproductsinPeregrineFalconsamplesgatheredfromtheNetherlands

8liversamples(2015&2017)Adults(Males&Females)

7differentsamplingregions

•  43ECsweredetectedthroughwide-scopetargetanalysis•  Pharmaceuticals&TPs;35%,PFAS;26%•  PFOS&its’branchedisomersàhigh%FoA•  Antibiotics (Enrofloxacin, Ciprofloxacin, Ofloxacin)à high concentrations in one specimen,

(foundaliveinpoorcondition,diedbyTrichomonasàtreatedinarecoverycenter)•  PlantProtectionProducts[Myclobutanil,Aldicarb-sulfone(Aldoxycarb)andAldicarb-sulfoxide]

ü  posterpresentationinSETACSciCon2020(virtuallymeeting)ü  articleisunderpreparation •  54emergingcontaminantswereidentifiedthroughsuspectscreening

•  mainlyindustrialchemicals,pharmaceutical,personalcareproducts•  acquiredHRMSchromatogramsàfutureretrospectivescreening(NORMANDSFP)

SurveyswithraptorspecimensDeterminationofemergingcontaminantsinliversofwhite-tailedseaeaglefromGermanyusing

novelandcomplementaryHighResolutionMassSpectrometrytechniques

white-tailedseaeagle(Haliaeetusalbicilla)specimens30liversamples(2015–2018)3differentfederalstatesinNorthernGermany

•  87emergingcontaminantsweredetectedthroughwide-scopetargetanalysis•  mainlyPlantProtectionProducts&TPs(25%),Pharmaceuticals&TPs(25%)•  PFOSandits’branchedisomers(sum)à100%FoA•  detectionofmany(bio)TPsàshouldbeincludedinthefuturemonitoringprogrammes

•  30emergingcontaminantswereidentifiedthroughsuspectscreening•  IndustrialChemicals(53%),Pharmaceuticals,PlantProtectionProducts•  mostabundant:N-Benzylformamide

•  acquiredHRMSchromatogramsàfutureretrospectivescreening(NORMANDSFP)

DeterminationofemergingcontaminantsineggsofraptorsfromGermanyusingnovelandcomplementaryHRMStechniques

27raptoreggsamplesSamplingyears:2005-2020

wide-scopetargetanalysis(>2,400substances)suspectscreening(>111,000NORMANSusDatsubstances)

byLC-HRMSandGC-APCI-HRMS

mainlyAntibiotics(l

ikeNalidixicacid&C

inoxacin),

Analgesics(suchasPa

racetamol&Tramad

ol)

FutureObjectives

CollaborationwithNHMsandESBsàEuropeanRaptorBiomonitoringFacility-Applicationofthedevelopedanalyticalprotocolsinraptorspecimens(liver,muscles,eggs)-InvestigationofthepresenceanddistributionofECsindifferentmatrices(blood,feathers,…)ofraptorspecimens-Collectionofpreysamples(terrestrialecosystem)àsupportinginformationonthebioaccumulationofECs

InvestigationofthepresenceofECsinfeathers(lackofknowledge)collectedfromraptors(theNetherlands)collaborationwithNaturalisBiodiversityCenterandPeregrineandDutchPeregrineWorkingGroup

Collaboration within NORMAN network through the Joint Activity “Contaminants of Emerging Concern (CECs) in terrestrialorganismsfromdifferenttrophiclevels”

EnrichmentofNORMANDatabaseswithmonitoringdatafromraptors

CollaborationforthedevelopmentofchemometrictoolsforthetoxicitypredictionofECsadaptedtoraptors(ToxTrAMS).

EnrichmentofNORMANEcotoxicologicaldatabasewithPNECsforterrestrialbiota

NationalandKapodistrianUniversityofAthensSchoolofScienceDepartmentofChemistryLaboratoryofAnalyticalChemistry

Acknowledgements

Dr.NikaMaria-Christina

Dr.AlygizakisNikiforos

Dr.AalizadehReza

M.Sc.NikolopoulouVarvara

M.Sc.GkotsisGeorgios

OrfaniotiAnastasia