Standard Operating Procedures for Measuring Bulk Stable

NOAA Processed Report NMFS-NWFSC-PR-2020-04

https://doi.org/10.25923/3mwp-ce02

Standard Operating Procedures for Measuring Bulk Stable Isotope Values of Nitrogen and Carbon in Marine Biota by Isotope Ratio Mass Spectrometry (IRMS)

February 2020

U.S. DEPARTMENT OF COMMERCE

National Oceanic and Atmospheric AdministrationNational Marine Fisheries ServiceNorthwest Fisheries Science Center


NOAA Processed Report Series NMFS-NWFSC-PR

The Northwest Fisheries Science Center of NOAA’s National Marine Fisheries Service uses the NOAA Processed Report NMFS-NWFSC-PR series to disseminate information only. Manuscripts have not been peer-reviewed and may be unedited. Documents within this series represent sound professional work, but do not constitute formal publications. They should only be footnoted as a source of information, and may not be cited as formal scientific literature. The data and any conclusions herein are provisional, and may be formally published elsewhere after appropriate review, augmentation, and editing.

NWFSC Processed Reports are available from the NOAA Institutional Repository, https://repository.library.noaa.gov.

Mention throughout this document of trade names or commercial companies is for identification purposes only and does not imply endorsement by the National Marine Fisheries Service, NOAA.

Cover image: Reactor reagents used for bulk carbon and nitrogen analysis. Clockwise from the top: silvered cobaltous, reduced copper, chromium oxide. Also depicted is an outline of nitrogen and carbon dioxide peaks; from left to right: N2 sample peak, CO2 sample peak, and two CO2 reference gas peaks. Photograph by J. Gates, NMFS/NWFSC.

Recommended citation:

(Gates et al. 2020)1

1 Gates, J. B., P. M. Chittaro, and K. B. Veggerby. 2020. Standard Operating Procedures for Measuring Bulk Stable Isotope Values of Nitrogen and Carbon in Marine Biota by Isotope Ratio Mass Spectrometry (IRMS). U.S. Department of Commerce, NOAA Processed Report NMFS-NWFSC-PR-2020-04.


https://repository.library.noaa.gov


U.S. DEPARTMENT OF COMMERCE

National Oceanic and Atmospheric AdministrationNational Marine Fisheries ServiceNorthwest Fisheries Science Center

Standard Operating Procedures for Measuring Bulk Stable Isotope Values of Nitrogen and Carbon in Marine Biota by Isotope Ratio Mass Spectrometry (IRMS)Jonelle B. Gates,1 Paul M. Chittaro,1 and Karl B. Veggerby2


February 2020

Environmental and Fisheries Sciences DivisionNorthwest Fisheries Science Center2725 Montlake Boulevard EastSeattle, Washington 98112

Fish Ecology DivisionNorthwest Fisheries Science Center2725 Montlake Boulevard EastSeattle, Washington 98112

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Contents

1.0 Introduction...................................................................................................................................................................... 1

1.1 ScopeandApplicability................................................................................................................................... 2

1.2 SummaryofProcedure.................................................................................................................................... 2

1.3 Definitions............................................................................................................................................................. 2

1.4 Safety....................................................................................................................................................................... 2

2.0 DocumentationandRecordkeeping...................................................................................................................... 3

3.0 EquipmentList................................................................................................................................................................ 3

3.1 Materials,PerSample....................................................................................................................................... 3

3.2 SolventsandCleansers.................................................................................................................................... 3

3.3 OtherMaterialsandApparatus.................................................................................................................... 4

4.0 SamplePreparation/Dissection............................................................................................................................. 4

4.1 LabelingSampleVials...................................................................................................................................... 4

4.2 PreparingDissectionEquipment(ifapplicable).................................................................................. 5

4.3 DissectingTissuefromaSingleSpecimen.............................................................................................. 5

4.4 SubsamplingfromaHomogenizedSampleorSampleComposite............................................... 5

4.5 CleaningtheDirtySpatulas........................................................................................................................... 6

5.0 FreezeDrying.................................................................................................................................................................. 6

5.1 PreparingtheFreezeDryer........................................................................................................................... 7

5.2 LoadingtheSamplesintotheFreezeDryerChamber....................................................................... 7

5.3 RemovingtheSamplesfromtheFreezeDryer...................................................................................... 7

6.0 LipidExtractionontheAutomatedSolventExtractor(ASE)...................................................................... 8

6.1 PrepareASECells.............................................................................................................................................. 8

6.2 PrepareSamples................................................................................................................................................ 8

6.3 Loading/OperatingtheASE.......................................................................................................................... 9

6.4 UnloadingSamplesfromtheASEPost-LipidExtraction.................................................................. 9

6.5 CleaningtheASECells.................................................................................................................................... 10

7.0 Homogenization........................................................................................................................................................... 10

7.1 HomogenizingSamplesPost-LipidExtraction..................................................................................... 11

7.2 HomogenizingSampleswithoutLipidExtraction.............................................................................. 11

7.3 CleaningtheCapsules,Spatulas,andForceps......................................................................................12

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8.0 WeighingSamples.........................................................................................................................................................13

8.1 BuildingaLoadingSheet...............................................................................................................................13

8.2 PillingofSamplesforAnalysis....................................................................................................................13

8.3 FinalizingtheLoadingSheet........................................................................................................................15

9.0 LoadingtheSamplesontheElementalAnalyzer(EA).................................................................................15

9.1 InstrumentMaintenance...............................................................................................................................15

9.2 RunningLinearityandOn/Offs.................................................................................................................. 16

9.3 CheckingtheOn/OffsandLinearityChecks......................................................................................... 16

9.4 CheckingtheBackgroundintheIRMS.................................................................................................... 18

9.5 BuildingaSequencein Isodat ..........................................................................................................18

9.6 LoadingtheEAAutosampler...................................................................................................................... 19

9.7 StartingtheRun................................................................................................................................................ 19

9.8 Post-Run............................................................................................................................................................... 19

10.0 ExcessSampleArchival.............................................................................................................................................20

11.0 ProcessingtheData.....................................................................................................................................................20

11.1 ManipulatingtheRawData.........................................................................................................................20

11.2 ProcessingtheDatainR ....................................................................................................................20

12.0 Data.....................................................................................................................................................................................21

12.1 DataReview.........................................................................................................................................................21

12.2 DataStorage........................................................................................................................................................21

13.0 QualityAssurance.........................................................................................................................................................21

13.1 QuantitationRange..........................................................................................................................................21

13.2 InstrumentCalibration.................................................................................................................................. 22

13.3 ContinuingCalibrationVerification(CCV)............................................................................................ 22

13.4 ReferenceMaterials........................................................................................................................................ 23

13.5 MethodBlanks................................................................................................................................................... 23

13.6 SampleReplicates............................................................................................................................................24

13.7 ReportedResults..............................................................................................................................................24

References................................................................................................................................................................................... 25

AppendixA:InstructionsforSubmissionofSamplesforStableIsotopesAnalyses...................................26

AppendixB:SubmissionFormsforStableIsotopesAnalyses..............................................................................30

ProjectInformation.....................................................................................................................................................30

SampleInformation.....................................................................................................................................................31

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1.0 Introduction

Theisotoperatiomassspectrometer(IRMS)housedattheNorthwestFisheriesScienceCenter(NWFSC)isusedtoconductaccurateandprecisedeterminationsofisotoperatiosofcarbonandnitrogen.NWFSC’sEnvironmentalChemistryProgramlabfacilityincludesaThermoFisherScientific’sFlash2000ElementalAnalyzercoupledwiththeConfloIVinterfaceandtheDeltaVAdvantageIRMS.Thesesystemsarecomplexandsensitiveandrequiredetailedcareandregularmaintenancetoensureahighlevelofperformance.Thisprocessedreportiswrittentobeusedinconjunctionwithhands-ontraining.Itismeanttobetreatedasalivingdocumentandwillbeupdatedaschangesaremadeintheprocedures.

Thisreportprovidesstep-by-stepinstructionsforsamplepreparation,instrumentoperation,maintenanceoftheinstruments,samplearchival,qualityassurance(QA)andqualitycontrol(QC)measuresandcriteria,anddatareporting.TheQuality Assurance Plan for Analyses of Environmental Samples(Sloanetal.2019)wasreferencedthroughoutSection13.Referenceinformationcanbefoundattheendofthisreport.

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1.1 Scope and Applicability

Thismethodwasdevelopedusingapproximately1gramofmarinebiotatissueandproducesahomogenized,stablesamplethatcanbestoredatroomtemperatureindefinitely.Thishomogenatecanbesampledmultipletimesforbulkcarbonandnitrogenstableisotopeanalysisaswellascompound-specific15Nanalysisofaminoacids.

Ittakesapproximately3–7daystoprocessasamplesetsubmittedforbulkstableisotopeanalysis,dependingonsamplesetsizeandwhetherornotthesethastobelipidextracted.Ittakesabout8minutespersampletorunontheEA-IRMSforbulkcarbonandnitrogenstableisotoperatios.

1.2 Summary of Procedure

1. Tissuesamplesaresubsampledandplacedinalabeledscintillationvialwithcap.Samplesareplacedina–80°Cfreezerforatleastanhouroruntiltheyarefrozensolid,andthenplacedinafreezedryerfor24hourstoremovewater.

2. Iflipidremovalisrequired,thensamplesareloadedontoanacceleratedsolventextractor(ASE)350toobtainlipid-freesamples.Formorein-depthdetailsoftheASE350extractionmethod,seeHermanetal.2005.

3. Samplesthatdonotrequirelipidremovalorpost-lipidremovalsamples,arehomogenizedusingaMixerMill(SPEXSamplePrep5100).

4. Homogenizedsamplesandstandardsarethenweighedintoindividualtincapsules,andthesearerunontheIRMS.

5. DatafromtheIRMSarerunthroughastandardQA/QCanddataprocessingprocedure,andfinalizeddataarereported.

1.3 Definitions

ASE: acceleratedsolventextractor IRM: internalreferencematerialCSIA:compound-specificisotopeanalysis IRMS:isotoperatiomassspectrometerDQO:dataqualityobjective SOP:standardoperatingprocedureEA:elementalanalyzer SRM:standardreferencematerial

1.4 Safety

Thismethodusesnumerouschemicalsandreagentsthatareconsideredhazardousforflammabilityandtoxicity.Therefore,afumehoodisusedwhenworkingwithhazardousmaterialstominimizeexposureoftheanalysttosolventvapors.Whenworkingwithallchemicals,aminimumofnitrileglovesandeyeprotectionareworn.AdditionallaboratorysafetypracticesarefollowedaccordingtotheNWFSCChemical Hygiene PlanandtheNWFSCChemical Waste Management Guide.TheManageMaterialSafetyDataSheets(MSDS)shouldbereviewedforallchemicalsandreagentspriortostartingthisprocedure.

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2.0 Documentation and Recordkeeping

Itisimperativethatallstepsthroughouttheprocessprocedure(e.g.,samplepreparation,maintenanceoftheinstrumentation,andoperationoftheinstrumentation)arerecordedintheappropriatelogbookswithcorrespondingsamplesetnumber,dates,andsamplepreparer/instrumentoperatorinitials.ItisalsoimportanttoupdatethestatusofthesamplesetinEnvironmentalChemistry’sGoogleDriveunderSI Current Set Status.

3.0 Equipment List

Itiscrucialthatallscalpels,spatulas,forceps,pulverizationchambers,andsteelbearingsarecleanedwithoil-removingdetergent(Micro-90detergent),anappropriatescrubbrush,amplewaterrinses,followedbysonicationinanappropriatesolvent.Thesestepswillhelppreventpotentialcontaminationthatcouldaffecttheisotopemeasurements.

3.1 Materials, Per Sample

Non-LipidExtractionSamples• 1ea.20-mLscintillationvialwithpolyethylenelinedcap• 1ea.cleanstainless-steelpulverizationchamberwithbearing• 1ea.4×6tincapsule

LipidExtractionSamples• 2ea.20-mLscintillationvialwithpolyethylenelinedcap• 5.5-cmroundglass-fiberfilter—VWR696• 250-mLAcceleratedSolventExtractor(ASE)collectionbottle• 3glass-fiberfiltersperASEcell,firedovernightat400°Cinamufflefurnace• 1cleanASEcellpersample(seeSection6.5)• 1ea.4×6tincapsule

3.2 Solvents and Cleansers

• methylenechloride(forASE)• acetone(forcleaningsuppliesviasonication)• Micro-90detergent

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3.3 Other Materials and Apparatus

• AcceleratedSolventExtractorASE350—DionexCorpASE• desiccator• VirtisFreezemobile12XL• MixerMill—SPEXSamplePrep5100• ultrasoniccleaner—Branson5800• stainlesssteelscalpelwithblades• stainlesssteelspatulas• forceps• paperclips,rinsedwithacetone• 96-welltrays• six-placebalance—MettlerToledoExcellencePlus• two-placebalance—AdventurerOhausAR3130• –20°Cfreezerfortemporarysamplestorage• –80°Cfreezerforlong-termsamplestorageandpre-freezedry• pillingtools• frozeniceblock• DeltaVAdvantageIRMS• ThermoScientificFlash2000OrganicElementalAnalyzerFlashHTPlus• ThermoScientificConfloIV

4.0 Sample Preparation/Dissection

Priortosamplepreparation,refertotheInstructionsforSubmissionofSamplesforStableIsotopesAnalyses(AppendixA)aswellastheSubmissionFormforStableIsotopesAnalyses(AppendixB).Oncethesamplesethasbeenscheduled,itisbesttolocatethesamplesandensuretheyarereadytobesubsampled.Ifthesamplesneedtobehomogenized,doso.Makesuretokeepallpaperworkthatisassociatedwiththesamplesettogetherinthecorrespondingfolder.

4.1 Labeling Sample Vials

Beginbypreparingclothlabelsfortheentiresetbywritingtheassignedlabnumber,fieldID,andthesetnumberonthelabelforeachsample(setnumberisfoundonwhitesheet).Attacheachlabeltoanewglassscintillationvialandrecordthelabnumberontheviallidusingafeltpen.Ifthesetrequireslipidextraction,theinitialscintillationvialonlyrequiresthelabnumberwrittenwithafeltpenonthecapandvial.Post-lipidextraction,thesamplewillbetransferredtoanewscintillationviallabeledwithclothlabelsthatincludethelabnumber,fieldID,andthesetnumberonboththecapandvial.

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4.2 Preparing Dissection Equipment (if applicable)

Cutaluminumfoilsquaresforeachsample.Inafumehood,rinsethefoilsurfacewithacetoneandletitdry.Pullthefrozeniceblockfromthefreezerandplaceinthehood.Covertheworkingsurfaceoftheblockwithnewacetone-rinsedaluminumfoil.Afterassemblingthebladesontothescalpelhandles,acetone-rinsethebladesinafumehoodandsetthemonacleanpieceofaluminumfoil.PlaceaQuickSmartautomaticscalpelbladeremovernexttoyourdissectionsurface.Locatecleanforcepsandplacethemnearyourworkspace.

4.3 Dissecting Tissue from a Single Specimen

Removethetissuefromthesamplecontainerusingforcepsandplaceitonthecleanaluminumfoilatopoftheiceblock.Fortheskinanalysis,carefullycuttheskinfromthefieldsampleusingacleanscalpel,beingcarefultoremovealloftheblubberandmuscle.Forblubberanalysis,carefullycuttheblubberfromthefieldsampleusingascalpel,beingcarefultoremovealloftheskin.Forallothertissues,removeasmallpiecefromthemiddlesection,toavoidtheareasofthesamplethatmighthavebeenexposedtofreezerfrost.Cutthesampleintosmallpieces(~0.1gorsmaller)usingthescalpelandplacethemseparatelyinthelabeledscintillationvial,placingthembackinthefreezerbetweeneachsample.

4.4 Subsampling from a Homogenized Sample or Sample Composite

Placeallofthesamplejarsouttothawforatleast45minutespriortosubsampling.Afterthesampleshavethawed,scoopoutasmallamountofthesampleusingacleanspatula,tryingtoavoidtheverytoplayer.Useanew,cleanspatulaforeachsample.Placethelabeled20-mLscintillationvialonthetwo-placebalanceandweighoutasmallamountofthesample.Theweightsdonotneedtoberecordedandareonlyusedtomakesureenoughsamplehasbeensubsampledandthatsamplematerialisnotwasted.Approximatesubsamplemassesarelistedbelow:

• Forwholebodyfishsamples,itisbesttoweighoutatleast0.5goftissue,keepinginmindthattheremightbepiecesofskinorboneinthesubsamplethatcannotbegroundtoapowder.

• Forfishmusclesamples,subsample0.2–0.4goftissue.• Formusselorshellfishsamples,weighoutatleasta1-gsampleduetothe

increasedamountofwaterineachsamplecomparedtoblubberorfishmuscle.

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4.5 Cleaning the Dirty Spatulas

Aftercompletingthesubsamplingstep,takethedirtyspatulastothesinkandscruboffanyresidualtissueusingMicro-90soap.Itisbesttoworkwithgloves,asthedetergentisconcentratedandcorrosive.Oncethespatulasarecleanoftissue,placethemintheperforatedstainlesssteelinsertinsideofthesonicatorandfillwithwaterandMicro-90detergenttothefillline.Neveroperatethesonicatorwithoutaninsert.

Afterthesonicationstepiscompleted,drainthewaterfromtheunitusingthevalvelocatedontheleftside.Openthevalveandletthewaterflowdownthedrainlocatedinthefumehood.Removetheinsertandrinseeverythingunderwater.Laythespatulasoutonabsorbentpapernexttothesinktodryovernight.

Whenthespatulashavecompletelydried,placetheminthesolidsonicatortrayinsertandsubmergewithacetoneinafumehood.Makesuretousetheappropriatenitrilegloveswhileworkingwiththeacetone.Fillthesonicatorwith1,650mLofwaterbeforeinsertingthesolidtrayinsertintotheunit.Settheunittosonicatefor30minutes.

Afterthesonicationstepiscompleted,drainthewaterfromtheunitusingthevalvelocatedontheleftsideoftheunit.Openthevalveandletthewaterflowdownthedrainlocatedinthefumehood.Wearingnitrilegloves,removethesolidtrayinsertanddraintheacetoneintoawastebucket.Pourtheacetonewasteinthewastecontainerlabeled100% acetone,locatedinthefumehood.Leavetheinserttositinthefumehooduntilallofthesolventhascompletelyevaporated(atleast30minutes).Returnthecleanspatulasfromtheinsert,usinggloves,totheglassjarlabeledSpatulas.

5.0 Freeze Drying

Afterallofthesampleshavebeensubsampled,loosenallofthecapsofthesamplesandplacethemina–80°Cfreezerforatleastanhourpriortofreezedrying.

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Open. Closed.

5.1 Preparing the Freeze Dryer

Beginbycheckingtheleveloftheoilintheroughpumptomakesurethattherewerenoleaksbetweenruns.Theroughpumpcanbeaccessedbyopeningthedoorlocatedtotherightofthecondensingunit.Checktheleveloftheoilinthesightglassandactaccordingly.IftheoillevelisOKtooperate,latchtherefrigeratordoorallthewayshut(itshouldbeopenfromthelastrunsothatthechambercandryout).UsingtheRefrigerateswitch,turntherefrigeratoronandletitcoolforthehourthatyoursamplesareinthe–80°Cfreezer(untilthefreezedryerreachesatleast–30°C).

5.2 Loading the Samples into the Freeze Dryer Chamber

Removethelidtothechamberandplaceitonitssideonthetable.Removethetrayfrominsideofthechambersothatthesamplescanbeplacedontheshelveseasily.Placethesamplevialsonthefreezedryertraysandbesurethecapshavebeenslightlyloosenedsothatmoisturecanescape.Onlyplacesamplesonthemiddleandbottomshelves(notontopoftherack).Duringthevacuumchange,samplesmaycomeoutoftheirvialsandcontaminatenearbysamples,soitisadvisabletokeepthecapsonbutloosened.

Next,placethevacuumchamberlidon,makingsureitiscenteredaroundthelipofthechamber,andclosethewhitevalvelocatedonthetopofthelid.Thevalveisclosedwhenthehandleisinthebackandthesmalldimpleisvisibleinthevalvewindow.SwitchonthevacuumusingtheswitchlabeledVacuum.Oncethefreezedryerissealed,startthevacuumpump.Afterabout15minutesitisbesttocheckbackandmakesurethatthevacuumsensorisreadingalowvacuum(approximately15Millitorr).Forsufficientsamplelyophilization(waterremoval),lettheunitrunforapproximately24hours.

Recordthesamplesetinformation,thedate,yourinitials,thematrixaswellasthenumberofsamplesinthefreezedryerinthefreezedryerlogbook.BesuretoupdatethefileentitledSI Current Set Status,whichisfoundinEnvironmentalChemistry’sGoogleDrive.

5.3 Removing the Samples from the Freeze Dryer

Whenthesamplesaredry,stopthevacuumpumpbyswitchingtheVacuumswitchoff.SwitchoffRefrigerateaswell.Slowlyopenthewhitevalvelocatedatopthevacuumchamberlidandlettheairout—slowly,sothattheairsurgedoesnotdisruptthesamples.Oncethereisnolongervacuum,removethechamberlid,settingitonitsside,andremovethetrayfrominsidethechamber.Removethesamples,tightenthecaps,andplacetheminorderinthevialtray.

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Leavethecondenserdoorcrackedopen,withafewpapertowelsinthechambertohelpspeedupthedryingprocess.ExaminetheO-ringslocatedonthecondenserdoorandvacuumchamberforanytearsordebris,andmakenoteiftheyneedtobelubricatedorreplaced.Placethevacuumchamberlidbackontopoftheunitsothatdustanddebrisdonotcollectintheunitwhileidle.

If the samples do not require lipid extraction, skip Sections 6 and 7. Proceed to Section 8.

6.0 Lipid Extraction on the Automated Solvent Extractor (ASE)

TheASEcellsthatareusedtolipid-extractsamplestoberunforstableisotopeanalysisarekeptseparatefromtheothers(theyareonlysonicatedinAcetonebetweenruns).

6.1 Prepare ASE Cells

SetoutallofthecleanASEcellsthatyouplantouseand,usingafeltpen,labelthebodyofeachcellwiththesamplenumber.Unscrewthetopcapofthecelland,usingforceps,placetwofiredglass-fiberfiltersinside.Usingtheappropriateblackrod,pushthefiltersdowntothebottomofthecell.Makesurethatthebottomcapisontight.

6.2 Prepare Samples

Wearinggloves,takea5.5-cmdiameterglass-fiberfilter,folditinhalftwice,andopenonesidetomakeafiltercone.Becarefulwhenhandlingtheglass-fiberfilters;theyarefragile.Clipthreeofthefoldedsidesofthefilterconetogetherwithacleanpaperclipandattachthecliptotheedgeofaclean,labeledASEcellinordertoholdtheconeopen.

Usingacleanspatula,scoopthesampleoutofthevialandplaceitinthefiltercone.Becausethesamplesaredry,youcanuseonespatulaforthewholeset,makingsuretowipeitoffbetweensampleswithapapertowel.Wearinggloves,foldtheconeinhalfandpapercliptheconeclosed.Placethesamplepocketinsideoftheappropriatelabeledcell.Usingforceps,putoneglass-fiberfilterontopofthesampleandpushitdowngentlywiththeblackrod.PlacethecellcapontightlyandloadthesampleontheASE.Repeatthisprocessuntilallofthecellshavebeenused.

Ifthereistoomuchsampletofitintoonefiltercone,anotherconeshouldbemadeortherestofthesampleshouldbediscarded.Thevialthatthesamplewasfreeze-driedinwillbediscardedintheBroken GlassboxafterthesamplehasbeenloadedintheASEcell.

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MostSIsetscontaintoomanysamplestolipid-extractinasinglerunontheASE,sotheywillrequiremultiplerunsontheASEandpropercleaningbetweensamplesets.SeeSection6.5forhowtocleantheASEcells.

6.3 Loading/Operating the ASE

Beginbyopeningthevalveonthenitrogentanksothatthesystemhasgasflow.LoadallofthecellsonthetopcarouseloftheASEinorderofsamplenumber.Makesurethereisawastebottle/vialforeachcellthathasbeenloadedaswellasarinsebottle/vialintraypositionsR1andR2.Removethesolventreservoir,placingthespargeinacleanASEvialtemporarily,andcapthebottle.Workinginthehoodwiththeappropriatenitrilegloves,fillthesolventreservoirwithmethylenechloride.Hookthesolventreservoirbackuptotheinstrument.

Usingthekeypadontheinstrument,locateMethod 3,themethodusedforlipidextraction.Theparametersshouldmatch:

Cycles:2Purge:60secondsStatic:5minutesHeat:0minutesRinseVolume:80%Temperature:0°CCellType:SSTSolventSaver:OffSolventA:1MethyleneChloride

Double-checkthatyouhavethesamenumberofcellsaswastebottles,aswellasrinsebottlesintraypositionsR1andR2.PresstheRinsebuttontorunamanualrinse,andrepeatthatsteptwomoretimes.Afterthesystemhasbeenthoroughlyrinsed,presstheStartbuttonandallowthesystemtolipid-extractthesampleset.

6.4 Unloading Samples from the ASE Post-Lipid Extraction

Afterallofthesampleshavebeenlipid-extracted,presstheTraybuttonuntilthegreenindicatorlightmovestotheleftsideofthebuttonwhichreleasesthesampletrays.Workingwithnitrilegloves,removeallofthewastebottlesandpourthemethylenechlorideintothelabeledmethylenechloridewastebottlestoredinafumehood.Tosstheperforatedcapsawayandcompletelydrythebottlesinthefumehoodforseveralhoursbeforerecappingthemforthenextset.Thebottlesarereusedforlipidextractingstableisotopesamplesorrinsingcells.Asnotedpreviously,itmaytakemorethanoneroundofASEextractionstolipid-extractanentireSIsampleset.

RemovetheASEcellsfromtheuppertrayandplacetheminafumehood.Removethecellcapand,usingforceps,removethesamplefromthecellandplaceitinanewlylabeledscintillationvial.Looselyplacethecaponthescintillationvialsothatthesolventcan

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fullyevaporatefromthesample.Repeatthesestepsuntilallsampleshavebeenremovedfromthecells.Oncethesolventhasevaporatedfromthesamples,tightenthecapsandremovethemfromunderthefumehood.Leavethefiltersinthehooduntilthesolventhasevaporatedfromthemandthendisposeofthem.

6.5 Cleaning the ASE Cells

AftertheASEcellshavebeendismantled,placeallofthecapsinacylindricalsteelsonicatortub.Workingwithnitrilegloves,removethesamplenumbersfromthecellbodieswithacetoneandapapertowelandplacethebodiesinanothercylindricalsteelsonicatortub.Fillbothtubswithacetoneuntilallofthecapsandbodiesaresubmerged.Placethetubsinsidetheperforatedsonicatorinserttrayandfillthetrayaroundthetubswithwatertothefillline.SonicatetheASEcellpartsfor30minutes.Afterthesonicatorhasfinished,pourtheacetoneintotheappropriatewastecontainerandtransferittotheflammablewastecontainerunderthefumehood.Layallofthecellpartsoutonacleanpieceoftinfoiltodryinthefumehood.Oncethesolventhasfullyevaporatedfromthecellparts,reassemblethecellsandstorethemintheirassignedspace.

7.0 Homogenization

Inordertoachievesamplehomogeneity,samplesmustberunthroughaMixerMillandgroundforapproximatelythreeminutesusingasteelballbearinginasteelcapsule.

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7.1 Homogenizing Samples Post-Lipid Extraction

Beginbycarefullyopeningthebundlewithcleanforcepsorcleangloves.Unfoldthefilterconeand,usingacleanspatula,transferthedriedsampleintoacleanpulverizationcapsuleequippedwithasteelballbearing.Placethelidontothecapsulebody,makingsurethatitisequippedwithanO-ring.ClampthechamberintotheMixerMill,makingsurethatthereisrubbercushioningoneithersideofthecapsuleinsideoftheclamp.Setthetimerfor3minutesandpushthewhitebuttonlocatedinthemiddleofthetimerdialtopulverizethesample.

WhentheMixerMillhasfinished,removethechamberfromtheMixerMillandremovethelidfromthebodyofthecapsule.Ifthelidhasbecomestucktothebody,lightlytapthecapsuleonitssidesandtryremovingthelidagain.Repeatasmanytimesasittakestoremovethelid.Usingacleanspatula,transferthepulverizedsamplefromthecapsuleandlidbackintothelabeledscintillationvial.Repeatthisprocessforeachsample.

SomesamplesetsmaybelargerthanthenumberofMixerMillcapsuleswecurrentlyhave,sobesuretoproperlycleanthecapsulesbetweenhomogenizationevents.SeeSection7.3.

7.2 Homogenizing Samples without Lipid Extraction

Beginbycarefullytransferringthedriedsampleintoacleanpulverizationcapsuleequippedwithasteelballbearing.Placethelidontothecapsulebody,makingsurethatitisequippedwithanO-ring.ClampthechamberintotheMixerMill,makingsurethatthereisrubbercushioningoneithersideofthecapsuleinsideoftheclamp.Setthetimerfor3minutesandpushthewhitebuttonlocatedinthemiddleofthetimerdialtopulverizethesample.

WhentheMixerMillhasfinished,removethechamberfromtheMixerMillandremovethelidfromthebodyofthecapsule.Ifthelidhasbecomestucktothebody,lightlytapthecapsuleonitssidesandtryremovingthelidagain.Repeatasmanytimesasittakestoremovethelid.Usingacleanspatula,transferthepulverizedsamplefromthecapsuleandlidbackintothelabeledscintillationvial.Repeatthisprocessforeachsample.

SomesamplesetsmaybelargerthanthenumberofMixerMillcapsuleswecurrentlyhave,sobesuretoproperlycleanthecapsulesbetweenhomogenizationevents.SeeSection7.3.

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7.3 Cleaning the Capsules, Spatulas, and Forceps

Takeallofthedirtycapsules,spatulas,forceps,andpaperclipsintheTo Be CleanedcontainertothesinkinRoom253E.FillthecontainerwithwaterandMicro-90soap.Donotleavethemtosoakovernight,asthepaperclipswillstarttocorrode.

Wearinggloves,removethespatulasandwipethemthoroughlywithasoapygloveorbottlebrush,gettinganyleftovermaterialoffbeforeplacingtheminthesolidstainlesssteelsonicatorinsertfilledwithMicro-90soapandwater.

Removeallofthepaperclipsfromthecontainer,thoroughlyrinsethemwithwater,andplacethemontheabsorbentmaterialbythesinktofullydry.

Removethecapsulebodies,rinsethoroughlywithwater,and,usingasmallscrubbrushcoatedinMicro-90soap,scruboutthebodies.Thoroughlyrinsethebodieswithwaterandplacetheminthesamestainlesssteelsonicatorinsert.

Usingapairofforceps,removealloftheO-ringsfrominsidethecapsulelids,rinsethoroughlywithwater,and,usingasmallscrubbrushcoatedinMicro-90soap,thoroughlyscruboutthelids.Rinsethelidswithwaterandplaceeverythinginthestainlesssteelsonicatorinsert.

ThesteelballbearingsandO-ringsleftatthebottomoftheTo Be Cleanedcontainershouldbethoroughlyrinsedwithwateruntiltheyarefreeofanybiologicalmaterial.Addthemtothestainlesssteelsonicatorinsertandmakesurethatthesoapywatercoverseverythinginthetub.Add1,650mLofwaterintothesonicatorbeforeplacingthesolidstainlesssteelinsertwithallofthepartsandspatulasintothesonicator.Sonicatefor30minutes.Whenthesonicatorisfinished,removetheinsertandrinsewellwithwater,makingsurethatnothingislostdownthesink.Layeverythingoutontheabsorbentpapernexttothesinktodryovernight.Donotleavetheutensilstositovernightinthesoapywaterasthesoapiscorrosive.

Afterallofthetoolshavefullydried,placethembackinthesolidstainlesssteelinsertandfillitwithacetoneinafumehood,makingsureeverythingissubmerged(donotsonicatethepaperclipswiththetools).Makesuretowearappropriategloveswhileworkingwithsolvent.Placethetubbackinthesonicatorafteradding1,650mLofwatertotheunit,andrunforanother30minutes.Whenthesonicatorisfinished,wearinggloves,dumpthesolventwasteintoawastecontainerinthefumehoodanddisposeofitintheappropriateflammablewastejug.Layallofthetoolsoutonacleanpieceoffoilunderthefumehoodtoallowthesolventtofullyevaporate(approximately2hours).Wheneverythingisdry,rebuildthecapsules,placingaballbearingineachone,andplacethecleanspatulasbackwiththerestonthetray.

Afterthepaperclipshavefullydried,placetheminacleanbeakerinthefumehood.Usinganacetonesquirtbottle,triplerinsethepaperclips,drainingthembetweeneachrinse.Letthemsitunderthefumehooduntilallofthesolventhasevaporatedfromthem.Oncetheyhavedried,placethembackwiththerestofthepaperclips.

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8.0 Weighing Samples

Throughoutthisfinalprocess,attentiontodetailiskey.Itisimportanttorememberthattheoilsfromyourhandscancontaminatesamples,soitisbesttoalwaysusecleantoolsandweargloves.Itisalsoimperativetodouble-checksampleweightswheninputtingthemintothetemplate,asanerrorwillthrowoffthecalculationofthesample.

8.1 Building a Loading Sheet

Theloadingtemplatesarefoundonthestableisotopeserver.Selecttheappropriatetemplateandsaveitusingthenameoftheset—forexample,ST1111 Loading.Donotoverwritethetemplate.PlacethenewloadingfileintothesetfolderfoundontheserverunderStable Isotope Projects.Inputthedatafoundinthecorrespondingsetup.csvfileintotherightcorneroftheloadingtemplate(startingwithcellI25).TheinformationwillautofillincolumnZandthroughouttheloadingsheet.Typethesetnameintotheboxatthetopofthepage(cellI3),andfillouttheinformationincellsD52–D56;theinformationwillautofillonthenextpage.

Dependingonhowmanysampleshavebeenassignedtotheset,rowswillhavetoberemoved.Highlighttheemptyrowsthatarenotneededanddeletethem,shiftingthecellsup.Ifthesetissmall,itisnotnecessarytorunallofthestandards.AllsamplesetsmusthavefiveSRMs(tworunatthebeginningoftherun,oneruninthemiddle,andtworunattheendofthesequence),andaminimumof5eachofasparticacidandhistidinestandards(twoeachatthebeginningoftherun,oneeachevery15orfewersamples,andtwoeachattheendofeveryrun).

Oncetheloadingsheetisfullyfilledout,printoffahardcopytofillouttheweightsofyoursamples.TheSample & Standard Weight Guide,foundonthethirdtabintheloadingtemplate,canbereferencedifyouareunsureoftheweightsforthestandardsandsamplesintheset.

8.2 Pilling of Samples for Analysis

Forallpillingoperations,beextremelycarefulnottotouchthetincups,theworkingendsofthetools,orpartsofthemicrobalancewithyourfingers,oryoucouldcontaminatethemwithforeignmaterials.Makesurethatallofthetoolsyouwilluseforpillinghavebeenpreviouslyrinsedwithacetoneandarecleanbeforebeginning.Usingthebalancebrush,makesurethatthebalanceisfreefromanydebris.Labelthelidofa96-wellplatewiththesamplesetnumber.Alwaysbeginfromweighingoutthesamplesofasetbeforemovingontothestandards.Itisbestpracticetoweighthestandardsthedaythatyou

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willrunthemontheIRMS.ThewellplateshouldalwaysbestoredinadesiccatorbetweenpillingsandpriortorunningthesamplesetontheIRMS.

Usingforceps,beginbytakingone4×6tincupfromthecontainerandplaceitonthemicrobalance.Allowthebalancetostabilizewiththedoorclosedbeforetarring.Next,openupthedoorand,usingthesmallsamplespoon,scoopasmallamountofsamplefromthevial,makingsurethatyoudon’tgetanyskin,bones,largechunks,oranythingthatdoesn’tlookhomogenized.Weighouttheappropriateamountofsampleforthematrixyouareworkingwith.Closethebalancedoorandlettheweightstabilizebeforerecordingtheweightofthesampleontheloadingsheet.

Afteropeningthebalancedoorup,useforcepstocrimpthelipofthetincupshutsothatnosampleislostduringfolding.Then,usingtheflat-tippedforceps,foldthetincupintoasmalltightcubeontopofthestonesurface.Makesuretherearenosharppiecesstickingoutofthecubeandthatthetincapsuleisflatsothatthecubedoesnotgetcaughtupontheautosampler.Usingforceps,placethesampleintheappropriatecellinthewellplateandmoveontothenextsample.Makesuretowipedownallofthetoolsalongwiththestonesurfacewithapapertowelbetweeneverysample,soasnottocross-contaminatesamples.Repeatthisprocessforallofthesamplesandstandardsintheset.StorethewellplatesinthedesiccatoruntiltheyarereadytobeloadedontheIRMS.

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Whenyouaredoneusingthetools,thoroughlyrinsetheminacetoneandstorethemawayuntilthenexttimetheyareused.Alwaysplacethelidonthetinthatthecapsulesarestoredinbetweenuses.Thebalanceshouldalwaysbeleftwiththedoorshutbetweenuses.Turnoffthebalanceattheendoftheday.

8.3 Finalizing the Loading Sheet

Beginbyinputtingalloftheweightsofthesamplesandstandardsintotheset’sloadingsheet.Doublecheckthatallweightsweretypedincorrectlybeforemovingon.

Clickonthesecondtabofthetemplate,labeledData,andmakesurethatalloftheinformationfromthefirsttabhastransferredoveraccurately.Ifyouhaveremovedrows,youmighthavetostartatthefirstrowanddragitdownandthenmanuallyremovetherowsthatarenotneeded.Youshouldbeleftwiththesamesequencenumberattheendofthedatatabasonthesetsheet.

SavethisloadingsheettoaUSBsothatyoucanaccessalloftherequiredinformationupstairswhenitistimetoloadthesampleset.Makesurethatyoukeepthehand-writtenloadingsheetintheset’sfolderincasethereisadiscrepancywithsampleweights.

9.0 Loading the Samples on the Elemental Analyzer (EA)

Thedaybeforeyouplanonrunningtheset,checktheinstrumentlogbookformaintenanceneedsandactaccordingly.Ifthesystemrequiresanewreactor,allowthesystemtoequilibrateandflushovernightbeforerunninganyair/watertestsorcheckingthebackgroundoftheinstrument.

9.1 Instrument Maintenance

ThedaybeforeyouplantorunasetontheIRMS,checkthestatusoftheinstrument.Checkhowmanysampleshavegonethroughthewatertrap,reactor,andashtube.Replacethewatertrapafterevery500samples.Replacethereactorafter300samples.Replacetheashtubeafter124samplesand/orbetweensamplesets.

Checkthelevelsonallofthegastanksalongthewall,recordingthemontheclipboardhangingnearthetanks.Ifanytankisbelow500psi,changeitoutwithanewtank,andiftheheliumtankislow,usetheswitchingvalvetoswitchtotheothertankthatshouldbehookedupandopen.Thismaintenanceshouldbedonethedaybeforearuntoallowtheinstrumenttostabilize.YoushouldalsomakesurethatthecorrectinstrumentconfigurationhasbeenselectedinIsodat Acquisitioninthebottom-leftcorner.

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9.2 Running Linearity and On/Offs

Onthemorningofthedayyouareplanningtorunthesampleset,on/offsandlinearitychecksneedtobeperformedforbothcarbonandnitrogen.MakesurethattheConFlo and EAsystemsetuphasbeenselectedinthebottom-leftcornerofIsodat Acquisition.

BeginbyopeningupIsodat Acquisition.SelecttheON_OFFs.seqsequencefromthesequenceslistinthebottom-leftcorner.Everythingisallsettogoonceithasbeenproperlyloaded.ClickthegreenStartbuttonandselecttheOKbuttononthenextscreen.Thesequencewillcontinuethroughalloftheon/offsandlinearitychecks.Ittakesabout2hourstorunthewholesequence.

9.3 Checking the On/Offs and Linearity Checks

Whenthesequencehasfinished,openupIsodat WorkspaceandselecttheResultstab.Openuptheon/offsequencethatwasrun—itwillhavethedateattachedtothefilename.Openeachfilebydouble-clicking.Thestandarddeviations/slopeshouldbecheckedandshouldbelessthanorequalto0.06.Iftheyaregreaterthan0.06,theon/offsshouldberunagain;itmayindicatethatthesystemneedsmaintenance.

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TocalculatetheCO2on/offs,doubleclickonthefiletitledCO2 on_off 4.Selectthelastonetoruninsequence(ittakessometimeforthesystemtostabilize).Whenitopenstothescreen,clickonthecolumntitledd13C/12Ctowardsthebottomofthepage—thisshouldselecttheentirecolumn.Right-clickonthecolumnandselectCalculate.Thestandarddeviationshouldbe≤0.06.

TocalculatetheN2on/offs,double-clickonthefiletitledN2 on_off 4.Thisisthelastonetoruninasequenceofthree.Whenitopenstothescreen,clickonthecolumntitledd15N/14Ntowardsthebottomofthepage—thisshouldselecttheentirecolumn.Right-clickonthecolumnandselectCalculate.Thestandarddeviationshouldbe≤0.06.

TocalculatetheCO2linearity,double-clickonthefiletitledCO2_linearity_(2).MinimizeIsodat WorkspaceandopenLinearity Calculator(anExcelfilelocatedonthedesktop).OpenIsodat WorkspacebackupandselecttheentireAmpl. 44column.CopyandpasteitintotheAmplitude 44columninLinearity Calculator.Next,selecttheentired13C/12CcolumninIsodat Workspace.Copy–pasteitintothed13C/12CcolumninLinearity Calculator.Excelwillcalculatetheslope.Theslopeshouldbe≤0.06.

TocalculatetheN2linearity,doubleclickonthefiletitledN2_linearity_(2).MinimizeIsodat WorkspaceandopenLinearity Calculator(anExcelfilelocatedonthedesktop).OpenIsodat WorkspacebackupandselecttheentireAmpl. 28column.CopyandpasteitintotheAmplitude 28columninLinearity Calculator.Next,selecttheentired15N/14NcolumninIsodat Workspace.Copy–pasteitintothed15N/14NcolumninLinearity Calculator.Excelwillcalculatetheslope.Theslopeshouldbe≤0.06.

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9.4 Checking the Background in the IRMS

Afterthediagnostictestshavebeencalculated,theIRMSbackgroundshouldbechecked.BeginbyopeninguptheBackgroundtemplateonthedesktop.Logthedateandfollowtheinstructionsinthetemplatetorecordallofthenecessarybackgroundmeasurementsintheworksheetforthatdate.Makesurethatyousavewhenyouarefinished.YoushouldalsomakesurethatyouhaveCO2selectedasthereferencegasand44incup2,45incup3,and46incup4(ifyouright-clickoncup2andenter44itwillautofilltherestifCO2hasbeenselectedasthereferencegas).

9.5 Building a Sequence in Isodat

Tobuildasequence,selectFileandthenNewandselectSequence.TypeinthetotalnumberofsamplesandstandardsinthesetandclickOK.Thiswillgenerateablanksequencetemplate.PlugtheUSBcontainingthesamplesetloadingfileintothecomputer.OpenupthefileandclickontheDatatab.BegincopyingtheappropriatedatafromtheDatatabandpastingitintothecolumnsintheblanksequencetemplateinIsodat Acquisition.TheSample NamecolumninformationispastedintotheIdentifier 1columninIsodat Acquisition.TheSample TypecolumninformationispastedintotheIdentifier 2columninIsodat Acquisition.SelecttheentireEA Methodcolumn,right-click,andselectFill grid with Data.SelectthemethodFlash Default\HTN_C_4sec_O2.eam.SelecttheentireMethodcolumn,right-click,andselectFill grid with Data.SelectthemethodMethods\C_N\N_C_karl_jonelle_new.met.

Whenallofthecolumnshavebeenfilledin,makesurethatthePeak Centercolumnhaschecksinalloftheboxesforalloftheruns.Forthefirstblank,selectStart Blank MeanfortheTypecolumn.Forthesecondandthirdblanks,selectAdd Blank MeanfortheTypecolumn.AlloftheothersamplesandstandardsshouldhaveSampleastheType.Right-clickanywhereinthesequenceandselectFit Cells to Grid.

Whenthesequenceiscompletelyfilledout,gouptoFileandselectSave As;namethesequencethenameofthesampleset.Next,gouptoFileandselectPrint.Placethisprintedcopyofthesequenceinthesamplesetfolder.

SelectthegreenStartbuttonandbegintofillouttheset’sinformation.FortheFolder NameselectPreandselectDate.FortheFile NameselectPreandselectIdentifier 1.DeselecttheAutoEnumoption.SelectExcelfortheexportandselectModify Template,add08Dec2016testEAN_CO2.wke,andselectOk.FortheExport File Name,typeinthesetname.Finally,selectInterface Standby After Sequencesothattheinstrumentwillautomaticallygointostandbyafterthelastsamplehasrun.BeforehittingOK,loadtheautosamplerwiththesamplesandmanuallyadvancethetraytodropthefirstpillintothechamber;seeSection9.6.

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9.6 Loading the EA Autosampler

Removeallofthetraysfromthetopoftheautosampler,makingsurethatthegearissettozero(0).Removetheclearlidoffofthefirsttrayandbeginloadingthesamplesintheslotsonebyoneuntilallofthesampleshavebeenloadedinthetrays.Takecarenottoskipaslot,ortoplacetwosamplesinoneslot.Afterallofthesampleshavebeenloaded,stackthetrays,startingwiththe0–31trayatthebottomandsequentiallyaddingallofthetraysontopofeachother.Makesuretoplacetheclearlidontopofthestackoftraysbeforeplacingitontopoftheautosampler.Ensurethatthestackoftraysissittingevenlyandlevelontopoftheautosamplerandmanuallyadvancethefirsttray,turningitclockwiseoneclicktodropthefirstpillintothechamber.Makesurethereisaclickandthatthetrayhasfullyadvancedto1.

9.7 Starting the Run

Afterthesampleshavebeenloadedandthetrayhasbeenadvanced,presstheOKbuttoninIsodat Acquisition(whereyouleftitinSection9.5).Checktomakesurethatyouhaveselectedtoputtheinstrumentinstandbyafterthesetisdonerunning.Alsochecktomakesurethereisn’taflagwavingtoindicatethatthesystemisonlygoingtorunaselectednumberofsamplesoutofthesequence.Itisbesttowatchthefirstsampleruntoensurethateverythingisrunningproperly.Besuretoupdatethewhiteboardbythecomputer,alongwithboththecorrespondingbookandbinder,withthesampleinformationandnumberofsamplesthroughthereactor,watertrap,andashtube.

9.8 Post-Run

Afterthesequenceisfinishedrunning,makesurethatallofthesampleshavedroppedfromtheautosamplerbycheckingthetraysandchamberforanythatmighthavegottencaughtorleftbehind.Resetthetraybymanuallyrotatingthegearbacktozero(0)alongwithallofthetrays.Checkthegastanklevelsandchangeoutanytanksthatmightbelow.Makesurethatthesystemwentintostandbymodeuponcompletionofthesequencebycheckingthattheflowsareturneddowninthemethod.

Ifeverythinglooksgood,usingtheshortcutonthecomputerdesktopcalledEA Results,findtheExcelfilegeneratedforthesetyoujustranandsaveitontheUSByoubroughtupwiththeloadingtemplate.TaketheUSBbackdowntoyourcomputertoprocessusingR.

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10.0 Excess Sample Archival

Allunanalyzedfieldsamplespreparedforbulkstableisotopeanalysiscanbestoredatroomtemperatureindefinitely.SamplesarestoredwithtightcapsinthestableisotopecabinetinRoom253E.Samplesetsshouldstaytogetherinavialflatwiththeappropriatevisiblelabelingforeasylocation.Samplesetinformationshouldbewrittenontheoutsideofeachvialflat.

Sampleswillbearchivedforuptooneyearoraspecifiedtimeperiodafterthedatahavebeenvalidated,asagreeduponbytheprojectleaderandtheprincipalinvestigator(s).

11.0 Processing the Data

StartbyplacingtheExcelfilegeneratedbyIsodatintotheset’sfolderonthestableisotopeserverintheStable Isotope Projectsfolder.Theloadingtemplateandsetupfilesshouldalreadybeinthefolder.MakeacopyofallofthefilesinthetabR Code,andpastethemintoyourfolder.

11.1 Manipulating the Raw Data

BeginbyopeninguptheManipulate raw data.Rfileintheset’sfolder;itdoesnotneedtobesaved.Fillouttheset’snameonline35,replacingtheXXX,andplacethecursoranywhereaboveline35.HitthebuttonRunatthetopuntilawindowopensuptoselecttherawExcelfilethatwasgeneratedbyIsodat;double-clicktoselect.ContinuetohittheRunbuttonuntilitgoesthroughallofthedataandyoureadalinethatsaysSaved.Double-checkthatthereare4peaksforeachrow.Ifthereisanerrormessageortherearelessthan4peaksforeachrow,troubleshootaccordingly.Wheneverythinglooksgood,youcanexitoutoftheprogramwithoutsaving.

11.2 Processing the Data in R

OpenuptheC/N ProcessingfolderandSave Asthenameoftheset.Insertthesetnameonlines8,59,and118.Answerthequestionsonlines61,62,and63.Inserttherundateonline120.Inserttheprojectnameonline128.SavethechangesatthetopofthepageandhitKnit.ReadthroughthePDFfilethatisgeneratedtomakesurethatallQApass.Copy–pastethepast_histidine28_plus_new_run.csv,past_SRM1946_plus_new_run.csv,andpast_aspartic_plus_new_run.csvfilesintotheR Codefolder,replacingtheolderversions.PlacethefolderinJennie’sinboxtobeinputtedintoFileMaker.

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12.0 Data

ThelaboratorywillprovidedatatablesandQAdocumentationsuitableforQAassessment.Alloriginaldataanddatadocumentationdevelopedbythelaboratoryforagivendatapackagewillbekeptbythelaboratoryforatleastoneyearafterthedatahavebeenvalidatedandreported;and,ifrequested,thedatawillbestoredinthecollectionformatforuptofiveyears.

12.1 Data Review

Alldataarereviewedandevaluatedbylaboratorypersonnel.Thisreviewisundertakenbyanalystswhoareresponsibleforensuringthattheanalyticaldataarecorrectandcomplete,theappropriateSOPshavebeenfollowed,andthattheQAresultsmeettheacceptancecriteria.Itistheprojectmanager’sresponsibilitytoensurethatallanalysesperformedbythelaboratoryarecorrect,complete,andmeetprojectDQOs.Theprojectleaderhasfinalreviewauthority.

12.2 Data Storage

ProcesseddatageneratedbyIsodataremaintainedinelectronicfileswithfrequentbackupandstorageontoanexternalharddrive.AllfinalanalyticalresultscalculatedinRaremaintainedinelectronicdatabasefilesonthestableisotopeservermaintainedbyIT,withfrequentfilebackupsandweeklybackupsontotapeforoffsitestorage.BulkstableisotoperesultsarealsostoredintheEnvironmentalChemistryProgram’sFileMakerdatabase.

13.0 Quality Assurance

TheEnvironmentalChemistry’sbulkstableisotopeestablishedqualityassuranceprovidesguidelinesformonitoringanddocumentingthequalityofanalysessothatadesiredlevelofperformancecanbedemonstratedandmaintained.TheseguidelinesarebasedonprotocolsestablishedpreviouslyforspecificprojectsundertheNationalOceanicandAtmosphericAdministration(NOAA)andtheEnvironmentalProtectionAgency(EPA),andhavebeenadaptedtonewtypesofanalysesandcurrenttechnologies(Sloanetal.2019).

13.1 Quantitation Range

FortheEA/IRMSmethod,theδ13Candδ15NvaluescanbeaffectedifthemassspectrometerresponsesfortheCO2andN2peaksaretoosmallortoolarge.Forfieldsamples,resultsarereportedifpeakamplitudesforN2(mass28and29)andCO2(mass44and46)arebetween500and12,000mV.Samplesthatdonotmeettheabovecriteriaarereanalyzed.Ifpeakamplitudesareneartheirlimits,theaccuracyoftheresultislesscertain.SampleresultsarefootnotedtobeusedwithcautionifthepeakamplitudesforN2(mass28or29)orCO2(mass44or46)arebetween9,000and12,000mVorbetween500and750mV.Thecorrespondingdeltaandweightpercentage(Wt%)measurementswillnotbereportedifthesample’speakamplitudeformass28ormass29is>12,000mVorifitspeakamplitudeformass44ormass46is<500mV.

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13.2 Instrument Calibration

TheEA/IRMSmethodrequiresatleasttwodeltalevelsofcalibrationstandards(histidineandasparticacid,preparedin-house)foranalyteratiomeasurements.Theδ15Nandδ13CvaluesforthecalibrationstandardsareassignedusingprimarystandardmaterialsfromIAEA(IAEACH-7)andtheUnitedStatesGeologicalSurvey(USGS40andUSGS41a).δ15Nandδ13Cmustbecalculatedusinglinearcalibrationandatleastfivereplicateanalysesofeachcalibrationstandard,includingatleastoneofeachatthebeginningandendofthebatchafterextremepoints(ifany)areidentifiedandexcludedduringthecontinuingcalibrationverification(Section13.3).Theδ15Nresultsandδ13Cresultsfortheincludedreplicateanalysesofthecalibrationstandardsversustherespectiveassignedδvaluesmusthaveacorrelationofr>0.9900;ifthiscriterionisnotmet,thenthesamplesinthebatchmustbereanalyzed.

MinimumFrequency:Atleasttwoofeachcalibrationstandardatthebeginningandendofeverybatch,andbetweenevery15orfewerfieldsamples.

13.3 ContinuingCalibrationVerification(CCV)

FortheEA/IRMSmethod,theCCVstandards’δ15Nandδ13Cvaluesareevaluatedusingtheapplicablefourstepsasfollows:

1. TheamplitudesofN2(mass28and29)peaksmustbebetween500and12,000mVandtheamplitudesofCO2(mass44and46)peaksmustbebetween500and12,000mV;otherwise,thatanalysisofthestandardisexcludedfromthedatasetandnotfurtherevaluated.Atleastoneanalysisofeachstandardmustremainatthebeginningandendofthebatch.

2. Thestandarddeviationofδvaluesinthereplicateanalysesofeachstandardmustbe≤0.25permil(‰)forδ15Nand≤0.35‰forδ13C;otherwise,extremepointswillbeidentifiedandremoved(seeStep3).

3. AnextremepointisdefinedastheCCVstandardwiththegreatestdifferenceinδ15Nandδ13CfromthemedianofallreplicateCCVstandards.ExtremepointsareidentifiedandexcludedinastepwiseprocessuntilthestandarddeviationsmeetthecriteriainStep2.

4. Nomorethan20%ofδ15Norδ13CvaluesinthereplicatesofeachCCVstandardcanbeexcludedduetoextremevalues.Atleastoneanalysisofeachstandardmustremainatthebeginningandendofthebatch.

5. MinimumFrequency:Atleasttwoofeachcalibrationstandardatthebeginningandendofeverybatchandbetweenevery15orfewerfieldsamples(samestandardsasthoseusedforinstrumentcalibration).

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13.4 Reference Materials

ThereisnotissueSRMcertifiedforratiosofstableisotopesofcarbonandnitrogen.However,NationalInstituteofStandardsandTechnology(NIST)SRM1946(fishmuscletissue)isusedasanIRM.Theassignedreferencevaluesforδ15N,δ13C,Wt%N,andWt%CforSRM1946arethemeanofrepeatedin-houseanalysesofthisIRMforstableisotopesofcarbonandnitrogen.Thelaboratory’sperformancefortheEA/IRMSmethodisconsideredacceptableifaminimumofthreesamplesofIRMperbatchmeetfourcriteriaasfollows:

1. ForeachIRMsample,theamplitudesofN2(mass28and29)andCO2(mass44and46)peaksmustbebetween500and12,000mV;otherwise,thatsampleoftheIRMisexcludedfromthedatasetandnotfurtherevaluated.

2. Themeanoftheδ15Nvaluesandthemeanoftheδ13Cvaluesmustbewithintheirrespectivecontrollimits(Equations7–10).

3. Thestandarddeviationoftheδ15Nvaluesmustbe≤0.3‰andthestandarddeviationoftheδ13Cvaluesmustbe≤0.2‰.

4. ThemeanoftheWt%NvaluesandthemeanoftheWt%Cvaluesmustbewithintheircontrollimits(Equations11and12).

Ifbothδ15Nandδ13CmeettheacceptancecriteriabutaWt%doesnot,thentheδ15Nandδ13CarereportedforallsampletypesinthebatchbutWt%andC/Nratioarenot.

Equations:

• δ15Nuppercontrollimit=referencevalue+0.3‰ (7)• δ15Nlowercontrollimit=referencevalue−0.3‰ (8)• δ13Cuppercontrollimit=referencevalue+0.2‰ (9)• δ13Clowercontrollimit=referencevalue−0.2‰ (10)• Wt%uppercontrollimit=(1.05×Wt%referencevalue) (11)• Wt%lowercontrollimit=(0.95×Wt%referencevalue) (12)

MinimumFrequency:Tworunatthebeginningandendofeverybatchandoneruninthemiddle,withaminimumofthreesamplesofIRMperbatchmeetingtheacceptancecriteria.

13.5 Method Blanks

FortheEA/IRMSanalyses,themethodblanksaretincupswithnosampleadded,whichareanalyzedinthesamemannerastheenvironmentalsamples.Theseblanksareusedtocorrecttheδ15Nandδ13Csamplevaluesfortracesofnitrogenorcarbonmaterialsinthetincups,andarenotameasureofcontaminationthatoccurredduringsampleprocessing.TheN2mass28andCO2mass44peakamplitudesforallofthemethodblanksmustbe<50mV,orthesourceofcontaminationmustbedeterminedandcorrectiveactiontaken.

MinimumFrequency:Threeatthebeginningofeverybatch.

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13.6 Sample Replicates

ForEA/IRMSanalyses,replicatesamplesoftheIRMareanalyzedbetweenevery15orfewerfieldsamples,withaminimumofthreeperbatch,toshowtheperformanceoftheEA/IRMSsystem.Duplicateortriplicatefieldsamplesaresuggestedforapproximatelyevery10fieldsamples,butarenotusedforQA.Therearenoacceptancecriteriaforreplicatefieldsamplesbecausemanyexplanationsexistforwidelyvaryingvalues(e.g.,problemswiththesampleprocessingorthehomogeneityofthestartingsample)thatareoftenoutsidethecontroloftheanalyticallaboratory.However,within-samplevariabilityoftheresultsforreplicatefieldsamplesmaybeusefultotheresearcher.

13.7 Reported Results

Forstableisotoperatios,δ15Nandδ13Cvaluesarereportedas‰,whereδ13isthedifferenceinratioofcarbonisotope13Ctocarbonisotope12CinasamplerelativetothatinthePeeDeeBelemnitestandard,andδ15Nisthedifferenceinratioofnitrogenisotope15Ntonitrogenisotope14Ninasamplerelativetoatmosphericnitrogenusedasthestandard(Equations13and14).

Equations:• δ13C={[(13Csample/12Csample)/(13Cstandard/12Cstandard)]–1} (13)• δ15N={[(15Nsample/14Nsample)/(15Nstandard/14Nstandard)]–1} (14)

Forstableisotoperatios,Wt%N,Wt%C,andC/Nratiosarealsoreported.

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ReferencesHerman,D.P.,D.G.Burrows,P.R.Wade,J.W.Durban,C.O.Matkin,R.G.LeDuc,L.G.Barrett-Lennard,

andM.M.Krahn.2005.FeedingecologyofeasternNorthPacifickillerwhalesOrcinus orcafromfattyacid,stableisotope,andorganochlorineanalysesofblubberbiopsies.MarineEcologyProgressSeries302:275–291.

Sloan,C.A.,B.Anulacion,K.A.Baugh,J.L.Bolton,D.Boyd,P.M.Chittaro,D.A.M.daSilva,J.B.Gates,B.L.Sanderson,K.Veggerby,andG.M.Ylitalo.2019.QualityAssurancePlanforAnalysesofEnvironmentalSamplesforPolycyclicAromaticHydrocarbons,PersistentOrganicPollutants,DioctylSulfosuccinate,EstrogenicCompounds,Steroids,HydroxylatedPolycyclicAromaticHydrocarbons,StableIsotopeRatios,andLipidClasses.U.S.DepartmentofCommerce,NOAATechnicalMemorandumNMFS-NWFSC-147.https://doi.org/10.25923/kf28-n618

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https://doi.org/10.25923/kf28-n618

Appendix A: Instructions for Submission of Samples

for Stable Isotopes Analyses

NWFSC Environmental Chemistry Program

1. Priortofillingoutanypaperwork,pleasediscussyourprojectwithPaulChittaro,(206)861-7617,and/orJonelleGates,(206)302-2445.

2. Foranideaofsampleweightsrequiredforstableisotopeanalysis,refertothistable:

TypeMinimum Sample Weight for Bulk C/N

Minimum Sample Weight for CSIA

WholeBodyFish 1g 3gFishMuscle 1g 3gMussel/Shellfish 2g 3gMarineMammalskin/Blubber 0.5g 1gVegetation 2g 3gInvertebrates 1g 3g

3. ObtaintheSubmissionFormforStableIsotopesAnalyses1(see Appendix B)fromthelinkprovided,orviae-mailfromJonelleGates([email protected])orJennieBolton([email protected]).Thisinformationwillbeusedtoscheduletheanalyses,trackthedataandresults,andanticipateproblems.Note:Non-NOAApersonnelmayneedtorequestpermissiontoaccesstheform.

1https://drive.google.com/file/d/1Zan5tFQUOtoMvsyRkdfhtAcpkV2SRNUl/view

4. Filloutinformationaboutyourprojectandthegeneralnatureofyoursamplesonthefirstpageoftheform(theProject Informationtab).• Investigator:ThepersonatNWFSCsubmittingthesamplesforanalysis,orthe

contactpersonforanexternalagency.• Project Name:Aconcise(4–8word)namefortheproject,includingthe

samplingyear,especiallyiftheprojectismulti-year.• Project Description:Abriefdescriptionofwhattheprojectisabout(nomore

thanaparagraphisneeded).• Charge Code:Thebillingcode,contractnumber,etc.,fortheworktobedone.• Referring Agency:EithertheProgram/DivisionatNWFSC,orthenameofthe

externalagency/office.• Date of Request:Dateonwhichtherequestforanalysisismade,afterthesamples

areallavailabletoNWFSCpersonnelandanyfinancialagreementsarefinalized.• Date Data are Needed:Thisdateshouldbesetbyagreementoftheinvestigator

andNWFSCpersonnel,dependingonprioritiesandworkload.Ifyouhaveameetingwheredatawillbepresented,pleaseincludethisinformationtohelpsetthelaboratorypriorities.Wewilltrytoaccommodatetheserequestsasbestwecan.

• Analyses Requested:Checktheboxesforeachmeasurementrequired.

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https://drive.google.com/file/d/1Zan5tFQUOtoMvsyRkdfhtAcpkV2SRNUl/view

https://drive.google.com/file/d/1Zan5tFQUOtoMvsyRkdfhtAcpkV2SRNUl/view

• Sample Prep. Required:Checktheseboxesforanysamplepreparationthatwillnotbedonebyyourgroup.Normally,thegroupsubmittingsampleswillfreeze-dryandpulverizesolidsamplesaccordingtoourrecommendations(NOAAProcessedReportNMFS-NWFSC-PR-2020-04,Standard Operating Procedures for Measuring Bulk Stable Isotope Values of Nitrogen and Carbon in Marine Biota by Isotope Ratio Mass Spectrometry (IRMS),willbeprovided),andweighthesamplepillsalongwithanappropriatenumberofstandardpills.AdditionalchargeswillbeincurredtotheinvestigatorifNWFSCpersonnelarerequiredtodothisprepwork.Weadvisegroupssubmittingsamplestofreeze-dryandpulverizesolidsamplesaccordingtotheprovidedSOP.Ifyourgrouphasnoexperienceinpillingsamplecapsulesforstableisotopeanalysis,EnvironmentalChemistrypersonnelcanweighthefinalsampleandpreparethecapsulesforanalysis(subjecttoagreement).

• General Description and Number of Samples:Providesampledescription(matrix)andnumberofsamplestobeanalyzed.

• What Precision is Required (in per mil) for the Analyses:Ifknown,pleaseindicatetheprecisionfortheanalysis.Atrophiclevelrepresentsadifferenceof~3‰ford15N.Differencesdownto0.5‰canbemeasured,butthisrequiresalargernumberofreplicatestobeanalyzed.

• Expected %N Range:Ifknown,pleaseprovidetherangeofvaluesexpected.Sampleswithveryhighorverylowvaluesmayneedtobepreparedoranalyzeddifferentlyfromothersamples,sothisinformationneedstobeknowninadvanceanddiscussedwithNWFSCpersonnel.

• Expected %C Range–ifknown,pleaseprovidetherangeofvaluesexpected.Sampleswithveryhighorverylowvaluesmayneedtobepreparedoranalyzeddifferentlyfromothersamples,sothisinformationneedstobeknowninadvanceanddiscussedwithNWFSCpersonnel.

• Anything Unusual About Samples:Listanypropertiesofanyofthesamplesthatmightbeexpectedtocauseproblemswiththeanalyses.Forexample,homogeneitybecomesaproblemwithsamplesthathaveahighpercentageoflipid.Weadviselipidextractionortheuseofliquidnitrogeninthesecasesbeforehomogenization.Anysamplesexpectedtobelessthan5%nitrogenshouldbenoted.ProblemsamplesshouldbediscussedwithPaulChittaroorJonelleGatesattheoutset(priortosamplepreparation).

• In What Format Would You Like the Final Data: Checkwhetheryouwouldlikehardcopieswithtables,anExcelworksheet,orboth.

5. FilloutinformationaboutyoursamplesontheSample Informationtabofthetemplate.Notethatofthefollowinginformation,thosemarked*areabsolutelyrequired;theotherswillappearonthedatareportyoureceiveandwillaidusintrackingandidentifyingyoursamples,andinprovidingtheresultsinamorecompleteformat.Onehundredsamplerowsaregiveninthistab,butyoumaysubmitmoresamplesifyouhavethem.Makethesheetaslongasyouneed.• Sample Category:*Selectoneofthefollowing(pleasedon’tabbreviate):

vegetation,fish,invertebrate,marine mammal,filtrate.• Sample Type:*Describeeachsampleasfollows:Tissue – skin,Tissue – muscle,

Tissue – whole body,Tissue – fat,Tissue – egg,Tissue – blubber,wood,leaf,berry,bark,periphyton,algae.Forsamplesfromtheanimalkingdom,pleaseusetheformatTissue (space) (dash) (space) typeasshownintheexamplesabove.ContactJennieifyouhaveanyquestions.

27

• Site:Wherethesampleisfrom.Examples:Snohomish Station 3,Prince William Sound.Ifknown,pleaseincludethelatitudeandlongitudecoordinatesofwherethesampleswerecollected.

• Species:PleaseprovidetheLatingenusandspeciesnamesifpossible,ortolowestlevelofclassification(genus,family,order).Examples:Tsuga heterophylla,Thuja plicata,Oncorhynchus tshawytscha,Tricoptera.

• Common Name:Thecommonnameofthespecies.Examples(fromLatinnamesabove):western hemlock,western red cedar,Chinook salmon,caddis fly.

• Field/Animal ID:*Yourfieldidentifier/specimennumberforthissample.Examples:SN3-0095-02,006,145-SNOmmmp.

• Jar/Vial #:Ifthesamplecontainerhassomeadditionalidentifyinginformation,enterthatinformationhere.Thisisatextfield,soanyshortcombinationoflettersandnumbers(excludingspecialcharacters)isallowed.Youcouldalsousethisfieldtodesignateatreatmentgroup.Examples:1 of 2,4cc WX.

• Life Stage:Examples:adult,larvae,fry,juvenile,lactating,pregnant,spawning.• Collection Date:Datethesampleswerecollectedinthefield,ifyouwouldlike

thisinformationtobedisplayedonyourreportofresults.• Sample Weight:*Weightofthesamplepilledforanalysis,ifyouarepillingyour

owncapsules.• Notes:Noteanythingaboutthesamplesthatmaycauseproblemswiththe

analyses(forexample,individualsampleswithnitrogencontentlessthan5%couldbenotedaslow N).

• Sample Location/Contact:Letusknowwhomtocontactwhenwearereadytoanalyzeyoursamples,sotheycanshowuswherethesamplesarelocated.

• Cassette Identification:*Identifyeachcassetteincludedinyoursubmission.Aresearcher,group,and/orprojectnameisagoodidea,alongwithanumber.Example:Elwha River Nutrients 2010.

• Cassette position:*Providethewellnumber(A1,C3,etc.)foreachsampleinitscassette.Putthepositioninasinglespreadsheetcolumn(e.g.,don’trecordthepositionwithanAinonecolumnanda5inanother;recordthepositionasA5).Youmayaddcolumnsasneeded;forexample,forReachorTransect,andwewillaccommodatetheseinreportingtotheextentpossible.Important:Aftercompletingthelistofsamples,checkoverthelisttoensurethat:1) Emptypillsorpillsthathavetoolittlesampletoeffectivelyanalyze(check

withJonelleorPaulifyouhavequestions)areremovedfromthelist.2) ThesamplelistissortedfirstbyCassette IDandthenbyCassette position.3) Therearenospecialcharacters(i.e.,/,*,?,‘and,).Youcanuseanunderscore

(_)toseparatedescriptions,ifneeded.Specialcharactersaredifficulttocheckforwhenloadingsamples,andtheywillcausearuntimeerrorintheinstrumentduetoOSlimitations.

WerecommendthatsamplesbesubmittedinordersothattheloadingoftheSIautosamplergoessmoothly.Thiswillgreatlyreducethechanceoferrorsfromsamplesbeingloadedinthewrongorder,assamplesareusuallyloadedexactlyintheorderpresented.Theautosamplercanhold124samples/standardsinonerun.Thesubmittedlistofsamplesmaybebrokenupintomultiplesetsrunoverseveraldays;youwillneedtoensurethatsufficientstandardandSRMpillsarealsosubmitted.

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6. Ifyouhaveadditionalinformationthatyouwouldliketohavedisplayedaspartofyourreportofthestableisotoperesults,orifyouneedthedatapresentedorprovidedinaparticularformat,pleasediscussyourneedswiththedatamanager,JennieBolton([email protected],(206)860-3359,Room248E).

7. OncetheExcelsubmissionformhasbeenfilledout,emailacopytoJennieBolton([email protected])sothatthesamplescanbescheduledforanalysis,accordingtotheprioritiessetinStep4.

8. Asequencewillbegeneratedbasedontheinformationprovidedalongwithsomebasicpillinginstructionsforthesample/standardpreparer,printedoff,andhandedtothepreparerpriortopilling.

9. IfthesamplesarefromamaterialthathasnotbeenanalyzedbeforebytheEnvironmentalChemistryProgram,youmaybeaskedtoprepareseveraltestsamplessothatanoptimalsampleweightrangeforthematerialcanbeestablished.

10. If collecting your samples requires filtration:• Makesuretoonlyuseglass-fiberfilters.Do not use paper filters.• Trytogetaslittleoftheglass-fiberfilterintothesampleaspossible.Piecesof

glassfibercancauseproblemswiththeinstrumentduringtheanalysis.• Saltfromseawatercancompromisetheanalysis.Whencollectingsamplesin

seawater,trytogetaslittleofthewateraspossibleinwiththesamplebeforefreeze-drying.

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Appendix B: Submission Forms for Stable Isotopes Analyses

Project InformationProject Information

Fill out the following information regarding your request. See the Instructions for more information.

Investigator:

Project Name:

Project Description:Charge Code:

Referring Agency:Date of Request:

Date Data are Needed †: Data Manager use only

Analyses Requested: %C %N C/N Ratio ∂13C ∂15N Date in projects db:

Extract Lipid Freeze Dry PulverizeSample Prep Required: Date in whitesheet db:

General Description and Number of Samples: Edit: 9/13/2004 JLBWhat Precision is Required (in per

mil) for the Analyses?*Expected %N Range:Expected %C Range:

Is %N Content Less Than 5% for Any Samples?

Anything Unusual About the Samples:

In What Format Would You Like the Hard copy data tables Excel worksheet BothFinal Data?

* A trophic level is a difference of ~ 3 per mil, but measurements down to 0.5 per mil can be made using a suitable numberof replicate analyses.† "ASAP" is not a date - please see the submission instructions.

Submission Form for Stable Isotopes Analyses

30

Sample Information

* Indicates information that is required - please see submission instructions for how to format the entries.

51

Sample Information

* * * * * *Sample Category Sample Type Site (include Lat./Long. if possible) Species Common Name Field/Animal ID Cassette ID Vial/Well # Life Stage Collection Date Sample Wt (mg) Notes Sample Location/Contact

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950

525354

Submission Form for Stable Isotopes Analyses

31

U.S. Secretary of Commerce

Wilbur L. Ross, Jr.

Acting Under Secretary of Commerce for Oceans and Atmosphere

Dr. Neil Jacobs

Assistant Administrator for Fisheries

Chris Oliver

February 2020

fisheries.noaa.gov

OFFICIAL BUSINESS

National Marine Fisheries ServiceNorthwest Fisheries Science Center2725 Montlake Boulevard East Seattle, Washington 98112

https://www.fisheries.noaa.gov