Abstra ct

TIlEEFI'ECrO FCIIANGESINQUANTITYANDQUALITYorFOOD ONrur:t:I:EDINGBEHAVIOUROF ll-IESOFT-SIIELLED CLAM,

MYA ARENARIA,

USING A NEW TECI INIQUETO DETERMINEGUTRETENT IONTI ME.

hy AlisonMargarct Scarratt

A thesissubmittedto the SchoolofGraduateStudies

inpartialfuffilmcmofthc rcquircmcntsIorthcdcgrccof

Mnsrcrof Scicncc

DepartmentofBiology Memor ial UniversityofNewfoundland

March,1994

Newfoundland

. "".

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Dcdicatcdtorny fumily withullmylnve.

Abstra ct

Thi., vtudyinves tigates the effectof changes inthe quantityand quality offood on tnc(cedingbehaviourof ,\{morenoria.tilesoft-shelleddam.M.armaria is a suspension- fe ed ing bivalve.inl:Csling particlessuspen ded inthewatercolumn.The laboratory cumpuncm ofthisstudypresentedM.orenariawith an artificialdiet,consistingofsilicon dioxideISi011pan iclesand the single-celled diatomChaetoceros 1II11t'lIeri,for inorganic antiorguniccomponcmsrespectively.Thesefceding experimentswere performedusing a Ilnw-tluuughapparatus.This includedmeasurementsof clearanceandingestion rates.

abvorprionefficiencies,and gutretentiontimes of bothorganicand inorganicfractions.

In response 10a 3-foldincreaseinfoodconcentration.M.urenoriadecreased ctcurauccrateby almost9-fold,and thereforeingestedlessmaterial, even though more fuml\\:1'<Ivilil'lh!e. Theseclamswithlower ingestionmtcalsohad a shorter¥ut retention time.umlthu' a !>Illallcramountofmaterialin thegut.Itas possible thatclams feedingon the lowquantitydietwere expending.moreenergyinobtainingparticles(higheringestion rate de,pitelower foodconcentrations)butperhapsless energyindigestingthem(clams feedill~'.111thelowquamity diet hadanabsorption efficiencyequal to thosefeedingon the J(Jmg diet, but overalongergut retentiontime.If the net energy gainedinthe digestive proce ss ti.e.lower amountsof enzymes working overalongertime)was greaterthanthat expende din filtering,this wouldbe aneffectivefeeding strategyfor clamstoadoptwhen e.\jlerk-ncillA!low quantitiesof food.

In responseto an increaseinthequalityof the food(proportionof organic material inerclI' es). dams decreased clearance rateand ingestionrate, to maintain a constant il~gc~ti onnueoforganicmaterial.Althoughgutretention time lengthened,absorption efficiency remainedunchanged.Therefore,clams regulatedboththeir intakeoffood and gill reh:ntiolltime\(1keep theiringestionrateoforganic materialconstant.andtomaintain ubsurpnoncrfickmcylcvels.

This~tlld)'abo measuredthe clearancerate,ingestionrate, gut retentiontime and nbsorption efficiencyofM.1Iff'1 1l11";afeeding.on naturalpar ticleassemblages ata clam flatin Planer'sCove,Terra NovaNationalPark,NF. Clearancerates weresignificantlyhigher than.lIlYIlh: .ISl111:Jinthelaboratory study.Field measurements of gutretention time were

compnrabtetothose measured inthe:laboratory study. however.fiddmeasurementsIll' absorp tion efficie ncy werestronglyncgntivc.indicruingpusxiblc mctabnlicf.lc\·all,, :-.,or lXlioui,it~l,fdig csrion intheintertidalpnpulatiun .

Thi, studyntso describesthelk wl opllll'nl,11l11 1lSCof,.ncwtl"'hlliqlll~hlllSSl".'Sthe gutretcruiontimeofsuspension-feeding bivalves.'1111.'greenalgaTetraselmis sII1'cinlwas used asan organicmarker,de tcctublein Iaccnlpelletsbyhighpcrforuuuic cliquid chrununoemphybecauseofitschuructcristicchlorophyllbsi~natllrc.'111eilll'll~allkmarker use dwassiliconcnrbidcpart icle swhich canbede tecte dinFaecalpellets byp;utir k sil c analysis U,illgOJCoulterMuhlvlzcr.Resultsubtuined bythismethodHIel'IlIllj1:1I';lhk III lhos c ofotilerstudies.undthetechniqueis sensitiveenough 10 detectpost-inge.\live selectionIll'panicles withinthe gUI ofindividual clams.

iii

Acknow led gem en ts

The fulluwinl.! p.'llplewere inYoiluah lc intheirsuppon anti assistancelIunughoulthe course of thisproj ect:GregBac on,Be ttyHatfield,!l.brgan:t Mille r,Evan Ward,Anna Redde n,InnathunGardner.Do nnaGa rdn er.Patpa blncu.Ale xand erBochdansky, Ed Downtu n, Ste veSiloley,andRoyBartle tt.Specialthanks goro Dr.D.Innes.my supe rvisorycommit"..-ememhcr.

IthauktheNururnl Sci ences andEngin eeringCounci lofCanada ,Me morial IlniwrsilytlfNewfoun dland. R.J.Thompsonand B.A.M:I~' D\ ' n alt.!for projectfund ing.

Complcrlonofthisworkwould1101havebe enpossiblewithoutthetrem e nd ou s advice andcncoumgc mcntfromIllytwo supervisors,Drs.R.I.Thompsonami B.A.

Mncljonald.

ltinally,Iwouldliketo thank myfriendsandfamilyfor allthei rsup portand kindness.

T;lh ll'of Contents

Abstract..

Acknowledgements _ tv

Lis t of Tables..

List of Figures....

. viii

List of Abbreviations xv

CHAPTE R I. lntr oduc ti nn... . I

1.1ree ding EcologyofSuspension-Feeding Bi\".llvcs I 1.2 The BivalveDigestiveTmct and Measurements of Feedin g 2

1.2.1 The Gills. .. 2

1.2.2. The Labial Palps.; . ..1

1.2 .3 The Gut 5

1.2.4.An EnergyEquationforBivalvc Feeding 7

1.3 Sudy Objc c uv cs.; . H

CHAPTER II:DevelopmentofaNew Techniqueto MeasureGutRcturuion

Timein Suspcnsiun-FcedingBIvalves If)

2.1. Inrrod uc tto n.; . 10

2.2. Tech nique Deve lopment. . 12

2.2.1.Ingestion andDigestionofTetraselmis.fllf 'd mbyM.orenana.

andDetectionof Chlorophyll b llslllgIII'LC. . 12 2.2.2In£cstionandDigestionof SiliconcarbidebyM.((1"('/1(1';(1.• •.••.•• •• •••••If!

2.2.3.Developmentofa ProcedureforQuantifying SiC Mnrkcr

Part iclesin FaecalPellets 20

2.2.4.Acidification of Faecal Pelletsto EnhancetheSiCPan icle Peltk 24 2.2.5.Determinationof theMinimumSonlcuion TimeRequiredto

Disperseall ParticlesinFaecalPullers 26 2.2.6.Enhancementof SiCResponsePeakby SievingPautctcs.; . 211

2.3. Summary.. . 211

CHAPTERIII.Pre- and Post-IngestiveActivityoftheSoft-ShelledCI"m.

Mya arena ria.in ResponsetoDifferentFood Regimes 31

3.1 . Introducti on 31

3.1.1.Importance of Studiesof FeedingBehaviour 31

3.1.2.EvaluatingChanges inFood Supply 31

3.1.3.BivalveFeedingResponses toChangesin Dietary Quaruity.; 33 3. 1.4.BivalveFeedingResponses10ChangesinDietary Quality 33

3.1.5.Particle Selection bySuspension -FeedingBivlaves.... . 34

3.1.6 Dbjcctivcx.,; . 34

3.2. Mutcriul andMet hods 35

3.2.1.C?lIc~t!().nSite,Animals.Transport Conditions.and Holding

Fucili tic s , 35

3.2.2 Pre paratio nofMarkers , 35

.'. 2.3.Experime ntalApparatusandProcedure.. . 35

.l2.4.Experimental Dcxign 37

3.2.5.ClearanceRate and Ingestion RateMcasurcme nts.... . 38

3.2.6. Gut Re ten tion Time Measuremen ts... ...40

3.2.7. AbsorptionEfficiencyMcasuremcnt s.; . , 41

3.2.X. Water Sumpte s.; . 42

3.3. Rc.sults... ... . 42

3,3.1. Actual Diet Loads 42

3.3.2.Use ofTrausfornuuionsand Non-ParamerncStatistics. ....44

3.3.3.Clcnrunce Rate and Relationwith Diet .46

3.3.4. IngestionRateandRelation10Diet .46

3.35.IngestionRateofOrganicMaterialandRelation10 Diet ..46 3.:\.6.AbsorptionEfficiency andRelatio n!ODiet .46 3.3.7.GutRetentio nTime andRel ation toDiet.. 52 3.3.X.Summaryofthe Effects of ChangesinDiet 56 3..l9.Relation BetweenGutRetentionTime and Clearance Rate 56 3.3.1n.Rclathm betweenClearanceIIngestion Rate and Absorption

Brrtctenctcs. .. 56

.':3.Il..SelectionofPart icle s WithintheGUI., ," 60

3.4. DI.Se \ISSlon , , 67

3.4.I.Effec tofvcrladoninNutritional Valueof IndividualDie ts 67 3.4.2.ClearanceRates ofM.arcnariaand Relationto Diet.. 68 3.4.3.AbsortionEfficiency ofM.arenarioand Relatio ntoDiet 71 3.4.4.GutRetentionTimeofM.urenarinand Relation toDie t. 73 3.45.SummaryofthefeedingStrategy ofM.arenarieinResponse

toCh;lllgingQuantity andQualityofSuspendedParticulate

Ma terial. . 75

3.4.6.Hig hIndividualVaraltiun " 78

3.4.7. AvenuesforFurther Research " 79

CIIAI~rERIV,ricldMeasurements od Feeding Behaviour ofMya orenaria in Platter 's Cove,TerraNovaNatio nalPa rk , Newfoundland 80

4. 1. lntroducriun., . 80

4.2 . ~Iethods " " 81

-1.2.1.Study Siteand Animals ". .. " 81

.\.2.2. Exp erimental Apparatus... .. 81

4.2.3. ExperimentalProcedure... ... . .82

-1.2.-1.Clearance RateMeasurements , 82

.\.:!.5.GutRetentionTimeMensuremcnrs.; . 83

-1.2.6.Abso rpt ionEfficienc y Measureme nts . . 83

.\.2.7. Water Sa mples..." 83

4.3. Results ,.... ... .. . ... , " 83

.\..'.1. Scsron Analysis ,,,,, 83

4..\.2 . Cle arance Ra te. . Xl(

4..\..\. Absorp tionEffl:il't ncy XX

4.J.-I.Gut Ret e ntion Ttme..; . Illl

4..t. Discus sion. . lJl

-lA-I. SesronSampling .\l·l·uracy... . 91 4A.2.Clea ran ce R.I.I:... . lJ.:!

4A.J.GUIRete nti on Time I}J

4AA. Aven uesfor FurtherResearch I) IJ

CHAPTERV.Evaluation of theNewGutMarkerTech nique 95

Append ix A.TIleEffectofFlowRatesandA~nl'l:ofSedimentnn Clt:ar:nlt.·e

Rate sof,\!. urenaria., . .. 1)11

AllpemJixB.EffectofTempera tureonPhysiolul!YofM.arenaria.... ...104 Appt:mlixC.MorpholoEk :l.lIn fOfm :ttilm 00M.arma ria 11)1) Appendix D.Determinationof..Criterion10DeM·rit>.,:GutRctcntiunTime... ....112 Appeodi,E.Effect ofl.ugols and Forma linFix:l!ivcsonSeawaterl'al1kll:

Coun ls ... . II H

LITERAT URE CITED...

vi i

. 110

Tallk.~ . J

T;lhkl7

List ofTables I'rcscoccof chlorophyllbanditscomponent breakdown prOO ucl'>infaecalsamplescclteced fmmM.tlfrntlria exposedtoa45min pulse of

Trmuetmis .ult ci ca 17

Com:cntratiunsofCnoetoceros muelleriandsilicon dioxideused toacbctvcthe4experimental dietleads.

Note thatC.muelleri has approximately72%organic

cem ent by weigh!.... ....39

l\clUaldietloadsdeliveredtothe experimentalapparatus for eachlaboratory experimenl... ....43 MultipkcomparisonofIIlCaIL'\ofpal1iculule drywci~1S andpercentages oforganic content in eachloadsuspension.

Tests weremnoeusinga FisherPLSD andSheffeF·\CSl \ •

•\n aslerhk •indicates !Oignificanceat«:;0.05 45 valuesfromWilcoxonrankI~I(Z)andassociated two-sided p-values (P)ofgutretention limeoforganic

\"sinorg;mic fractions forM.arm ariafeeding on eachdiet...55 Summaryof the effectof changeinquantityandquality of dietonclearancerates,ingestionrate. ingestion rateof (lrg ani~'material.gutretentiontimeof organicand inorganicfractions,andabsorptionefficiencyofM.armaria.

Anasterisk · indicales significanceat(l:::.05.checkprevious

secti ons for more detail. 57

Tbcfollowingtablesummarizestheinstancesof preferentialretentionofparticleswithinthegut (:::gut selectioncatcgory)ofM.armaria,scnedbydiet.N:::6for eachIliet. anddeten11inationofpreferentialretentionisbased on acomparisono(GRTof organicand inorganicmaterial fur eachindiyidual... ..6 1 V"luesrromWilcoxonrank test (Z) andassociatedtwo- sided p-values (P)for gutretentiontimeof organic material(GRTo)and inorganicmaterial(GRTi)

comparisonsamong gutselection categories,forM.arenuria ...62

viii

TallkJ.~

TaMe.1.10

Tahle J.1 TJhk~"2

Table E.I

valuesfromWilco xonrnnk lest(7.):llltla\''l.ll.·i.lIl·dtwo- silk dp-valucslJ' )fnr cil:aT:ulI.'l;'raIl.'{CRlandin~esli l "l T:UCofor~:lni<..materia!OR.,lcc mpari....ins;U\1\lflg gut selectioncategories, 10m~dktsonlyNualilY,,:h;nl~':

datal<l).forAI.orcnor ta _1>5

Valuesfrom wilcoxon rank lesl(Z)and ""'....x·ia led two-sidedp-vntoe stil)fur clcarunccrntclCR)and Ingestion rntc of organicnmrcnal(1R..,)I·tllllJl'lli.\Un.~

;UlluJl~gutSCICI"l iuncategories.10mg 50%nud2m~

50'}dietsonly (1IL13l1litydlilllged:II:1.~t).furAI.o/"l·II" I'ifl (,(1 Results of;l~h"nalys;s ofscstun.1;;1I11ples takenat

Platter's Cow.TerraNO"aN,llil1llotlI'ark... . K7 Tbe relative;1Il10l1n1s IIf~ank:chlumphyll h1II.In.e r

~~~~"i~~~~~;;~~:~: ~nn~,~~h~~~f~:~~I:~~:~rl~~I:J "

fromSludicsufM. orcnortaall'lancrs('u\"c. TerraNIIva N:ttion alParle I\na\tcrisk(.)inllil"lless;gn;lk:ml dirrl"fCl1l'e fro mfacl'l:s.\OImplcstakenImmconuolindividuals

un

Ihe~1l 11 e dayOIln=0.05(one-tailedr-rcsr. t=2 .lU2 .d.f.=4 1 9(J Mea nsandsla nd anJdeviationsof purticlc

concentrations(pmticles1111-1)ofseawatersamples beforeandafter treatment withI%eachl.ugols ami formalinfixatives.The re are no sij;.nifk:tnt diffe rcnl'es betweeninitialandlaterconcem rauonsuf ca-·'1unple Ill)

ix

Lls t of Figures

Fig.1.1 Schematic diagram ofthe componentsof feedingand digestioninasuspension.feeding bivalve.Leiters in bold type rercrto thcEquationl.L'Circtcdarcasindicarc poinlSa1ungthe digestive tractwhere protideselection

mayoccur .. . 3

Fig.2.1

Fig. 2.'2

Fig.2.3

Fig.2.4

Exumplc(IfIIchnnnatngram showingpigment cOlllpooiliulin f fllcccsfmmadmll fcedingnnc lllixturellfC./Il11l1eriands ilicondioxidepimicks,as wellasaJtlminpulsc of T. .l'lIw ·co and silicon clirhidcpaniclcs.Peaksca n beidelltiliediLs: l.chlorophyllc.2·ph;lcophorhidea.3·chlorophyll b, a-chtorcphyl!a.5·phncophytin h, 6·phncophytina 16

Il1\cmalandcxlernalfe alurcso f thcexperimenlal apparatus.AlFeaturesoftheIlow-rhmughheader tank.HT=hcudcrtank.J=Inflow,0 «overflow,D

=dietinnow, C=experimentalcontainers(n:..:6 maximum).B)Featuresofthccxpcrir ucntal comalncrs.1T

=

tygon tubing,FR

=

flowrcsutcror.I

=inflow,"B=Plexiglasbaffle,H=holderfor clam.

SP=standpipe,0

=

^outnow.Arrowsindicatethe

ofrccuonorscawacr now.; ...19

SizedistributionofsuspendedSiCpaniclesas determined by Coulter Multisizcr.Thc SiCpeak

r.mgcs fromapproximalc1y4.71o J7.9).1nl... ....23

Particle sizedistributionsof4 faecalsunplcs. two s,ullpleSnom clamsnotexposed10 SiC(A),andtwo

samplesfromclamsexposedloSiC {B)... . .. 27

Fig. 25 Pan iclesizedistributions ofcnc faecalsamplefroma ctamnotexposed10 SiC(A,B,C).andonefaecalsample fromadiUIIexposedto SiC (D,E.F),measuredal20,40 an d60 minuteintervalsinthe sonicationbath.... ... 29

Fig. 3.1

Fig 3.2

Fig.3.3

Clearance rateuf M.11rt'l lII ria!"l'~d inl111n.1difk renl fuodsuspensions overa 10 11ll1lTperiod:---0-- 10 lIlg/l.75'l-mganks;- - - o -IOmg/1.50'}

organics;- - f t - - 10111£11.:!5 '}(l1'l1illlk'S:_..__

2lIlg/l.50'1organics.verticalburs arc~1;Uldanl crrorcrmcmcaosaodnelLfur cuchpulm...

Clcarallccratco fM.u/"t'111/I·j" inrcliltillllhlt hel[Ualil)'{Al andquantity(B) of food insuspension.Euur barslifeIhe standarderrorofthemean.n :::(iOfill'each.Different lettersaboveeach bariudicnrcsignilkanldifferenceat alpha=0.05,STDtest...

JllgeMionrale llfM.flrt'lWI";1Iinrchuiontu thel[uality (Alandqurmthy(B)uffood inslispensiun.Errnrhars representthestandardCrTOfufthc mean,andn",(ll) fIlT eat'h. Diffcrcnt lcllcffi;lhuvClhc hou's illllic:lte slgnifirumdiffcrcnccatalpha:::0.ll5,STDleSI...

...·17

...·IX

...•.••]lJ

Fig3.4 RateofingestionoforganicmaterialufM,urenarinin relation10thequalityCAlandqanmhy(BJIll'Iucnlin suspension.Error barsarcthcstnndardcrrurofthc mean, andn=60 foreach diet Differentlcuc rsilhllY\:

thebarsIndica esignificant differenceatalpha:::0.05.

STOiCS!.... . 50

Fig.3.5

Fig.3.6

AbsorptionefficiencyofM.orenariainIt:lalinnIIIIlie quality(Alandquantity (B) orroodinsuspension.EITnrhaffi represent thestandarderrorofthe mean,nndn:::6 fureach diet.There was nosignificantdifferencebetweenmeansuf

all)'dietsalalphae0.05,STOles1... . 51

Gutretentiontime'ISsonbodymarssIorindividualclams feedingonIOmg 50%(A),10mg25'.t{B ),2mg50',;;

(C)and10Ins75%(D)diets.Thereatenoabvtous allometrictrends.0 =GRToforganicfraclion,. :::

GRTofinorganic fraction... .. .. 53

xi

Fig.:U Gutretention time of organit;(solid barsj und inorganic (slriped hars)d ielary fraetionsin M. affllllrirli nrc spllnsc 10differingquality(A)andquantity(B lof foodin suspension. ErrorbarsIndicatethestandarderror ofthe means, andn=6foreach diet.Differenticucrsabove ban;indicate significantdifferenceatalpha=0.05, WilcoxonRank test. Note.there isno signiflcam diffcrcnccbc tweenorga nic lLlld inorSanicgulretr nlion timeswithindietsatalpha=0.05,Wilcoxon Ranktest. 54

Fig .3.R GutrcrcnnontimeofM.(lfelU/ fir,vsclearancerates for changein food quality(A)andquantity(B)datasets.

Openulrclcsrcprcscntthegutrctcnuonttmcofthc orgaulc

r racuon. sond

clrclcs rcprcscnnhcgut retention lime of theinorganicfraction.Spear man correlation cocfflcicntsbetween gut retentionlimesandclearance rales, lmd significanec lcvcls arc s ivcn inlhe lexl... . 58

Fig.3.9 Clearance rate (A)andingestionrate (B) vs nbsorptlon cffide ncy of M.rlrellllria.Thercarcnosignifieanl

corrctauons.... . 59

Fig.3.10 Meangutretentiontimeoforganic(solidbars)and inorganic(stripedbars) particles inM.orenariaadopting onco flhrcediffen"nt gulsclcctionstrategics:prefcrcntial retention of inorganic particles, organic particles, or no selection. Errorbarsare standarderrorclthcmean.• lindUabove twobars indicatessignificantdifferenceat alpha= 0.05and0.1 respectively,WilcoxonRanksrest, 63

Fig.4.1 Results of5eSI0l1analysisatPlaner'sCove,NF,on August5 (A)andAugust12(B), 1992.Indicatedarcthe totnldrywcighrofthescstonperlitreorscawacr (- - 0 - - )andpercentorganiccontentofthescston

(_ ) 85

Fig.4.2 Resultsofscston analysis atPlatter's Cove,NFon Augu~t5(A) and August 12 (B),1992.lndicatcdarcthe amounts of organic( ~dinorganic (_ _)materialpresentinltarcsamplesorseawater.

xii

... 86

Fig.4.3

Fill. A.I

Fig.A.2

Fig. 8.1

Fig.8.2

Fig.C.I

Clearancerues ofclamsfl'cdillilon a naturalpanicle assemblageat PI suer's Cove.NI:.onAu ~u st5 (----0----)and August12(_ ), 1992.Conccntnui onofsus pended p;utkul;ltemuucr W;Lo;;

approx.4XgrcnrcronAugust12'han(\1\AuguSI5.

verticalhal'Sarc standarddeviationsofthemean,n'"12

foreachpoint. 1\1)

Clearancemtc ofM.f!H'I/(l/'il/ held in holders(A)andin sand(Bjfnrctattouronewrntcufscawntcrthrongh experimentalc ontainers.At 0111pointsmcusunxl, clear-mel' mIl'isindcpcndaut ofnowrntc.Errorhal'san:

~.land;lrd de\'iations,antin'"Sforeach point 1111

ClcaranCe faleS of M//I'{'/lllfil, hcld inpl :lstich uh1cr.o;; (o) andinsandre). Absenceorscdurcmdocsnorancct clear.lIlCenlles n f M.lIIl '/ItI /·it,... . 10:\

HffcctortcmpcranncunclearancenucufM./lff'I1I/1';1l feedingonCII/It'lI/am,\'//lul/a i.Error bursarcthe

standarderrorof thelIIean". . I06

Effect oftempermurconabsorptkm efficiencyIffM.

IIr eUl/flafeedingonCtmc tocerosmulleri,Errorbarsare thcstandard crrcrofthcmc nn.i. . J0 7 Relation betweendry tissue weightandshelllength inM.

arenariafromRiverhead(0landPlaner's Cove(.) populations. Equruiuns urc:

Riverhead W=2.167X10,4Luo~

Plaucr's Covc W",1.208 X10.4L².2K7

110

Fig.0.1 Exampleofmarkerprofilegraphsshowi~ggond(A)and pnor (B)fil 10the non-llnenrmodcl describedin Appendix 8.Circlesarcobservedvalues;crossesarc finedvalues...

xiii

...113

Fig.D.2

Fig.D.3

Fig.D.4

Examplesofpooled profilegraphsshowinggood (Aand B)andpoor(CandOJlittothe non-linearmodel describedinAppendix D.n;::6for each,errorbarsarc

standarddeviations 114

Gutretentiontimeof organic(solidbars) andinorganic (stripedhars)materialdeter mined bythetimeinterval containing themedian(A)andmaximum(8)values.

Verticalbars=standarderrors,n=6.Similar!cUeTS ahoveeaehh.vindicillcllosignilicMtdiffercnecalaJpha

=0.05,Wilcoxon RankTest. Note:There wereno signilicantlHtTercnccsinthe gutretentionumcsor organicandinorganicImcuons within each diet at alpha=

0.05, WilcuxonRank Tcst.i. . 116

Exaruplcsofmar kcrprofllcgraphsofthe amount of urganlcmarkcrO.M.(solidbars)and inorganicmarker I.M.(stripcd hars) infaecalsamplescollectedfromtwo M.orenano(AandB)overan18h

urnc

periodaftcra30 minexposuretomarkerpanicles.Annsterisk(·) indiclltcs the har containing lhclllcdiim villuc andh cncc thegutretentiontimcdcsignillion.Note that both clams iI1ustr-dlcdhcrcrctuincdtheinorg,micfraclionlongcr than

thcnrganicfmction 117

xiv

ListofAbb r e vla t ic us

AE-Ab~orll tionefficiency CR- Clearance rate GRT·Gutretentiontime

HPLC -High performanceliquidchromatography IR -Ingestion rate

IR.:,-[ngcstion ratcof orgi,nic malclial SiC-Siliconcarbide SiD! -Silicondioxide

CHAPTE R I Int roduction

~....fu:W!WYfirSuspension.FeedingBiya!.n£

Suspension-feed ingbivalvesform a significantcomponentof thebent hic populations ofmarine ecosystems,dominatingmnnyestuarineand coastalhabitats world- wide,andarc thereforethesubjects

o r

many physiologicalandecologicalresearchprojects. Becauseoftheir dominanceinmanymarine ecosystems,itis necessaryto investigatethe ecologicalrole of bivalvesin ordertounderstand productivityandenergy dynamicsof benthicpopulations.Many bivalvespeciesarealso importanteconomicallyforboththe harvesting of naturalpopulations andin aquaculture.somuchresearchisbeingdirectedOIl maximizing growth efficienciesinthese species(GriffithsaridGriffiths1987).

Despitethisgreat volumeof research (seeBayne1976,Bayne and Newell1983, Gri fliths and Griffiths 19K7,Ja rgensen1990, Gosling1992for reviews),there remain manyaspects of bivalvephysiologywhich arenot well understood.Recently,attentionhas turned towards bivalvefeeding ecology, particularlyin response10changes inthe available food source(e.g.Bayneet al.1984,Hawkinsand Bayne 1984, see reviewsby Bayne 1976,Bayneand Newell1983,Griffithsand Griffiths1987, Bayneetal.1988, 19 89).

Althoughbaste modelsoffeeding behaviourhave beendeveloped (tobediscussed more fullyina later sectionof this chapter),bivalveresponsesdo not alwaysconform10 expectedres ults.Thisisfurt her complicatedby thefact that responsesto environmen tal stimuli,suchasachangein the composuionor concentrationof afood source,fall into essentially twocategories:there are responsesforsurvivalatenvironmentalextremes, and therearc responsesforthe maintenanceof rate functions at optimallevels overa normal environmenta lrange.Furthermore, theadaptationalrespo nsescanbeexpressedaseither decreased energyexpenditure(energy-conse rving)or increasedenergy input(energy- supplementing)tGlllmcr 1982,Green et al.1985).

Thereisusually a hierarchical responseto environmentalstimuli(Slobodkin 1968).

Theinitial response levelto environmentalchange is usuallya changeinthe behaviouror physiologyof theanimal.Thistype of compensation"y many individualscan, intum,

affectsuch popular.onparameters nxmortalitynucs.fecundityanddlspcrsnlofIlCl:lg il' la rvae . which couldintum'Illerthegeneticcomposition of futuregcnc nuiuus.Fmnlly, changesin the structureof a populationcan alterthestructureofrhccommunityUS:lwhole.

Bivalve shavebeen identifiedasa group whichelmshow adaptationIII cnviruruucutnl st im uli at all hierarchicallevelsof rcspon .se •agaln making them good suhjl'elsfu r experime ntalresearch(Greencr al.1985).Theresearchof thb lhesisden!...withrcSi1UIl~S of bivalvesatbehavio ural andphysiological levels .Infe re nceswillbe made into the re sulting physio logicalenergetics oftheanimals,intermsof!!a ins andlossesof energyand energy transformation(Hibbert1977.Rayne andNewell19&]).

In orderto understand the runge ofadapuuious available toabivalveintermsIll'ils feedingbehaviour,it isfirstnecessary10understandthennatomicnlSITllI'l lJ l"C.~(andtheir functio ns ) involved.TIlefeeding anddigestive structuresof a bivalvehasbeenilhl.~tralcd schematically inFig.1.1 (modifiedfromwiddows ctHI.1971), Bayne and Newell1910. Bricel]and Malouf 1984 ).

! 2!TheQj1ls

Suspension- feeding(=filter-feeding)bivalvesing estmicro-and macroscopic particulatessuspendedin the watercolumnabove thebivalvepopulation.Ciliarycurrents pump water throughthe bivalvemantlecavityandacrossthe animal'sgills.Particles arc capturedfromsuspensio nbytheIa tero fro mal ciliaof thegills(Jfl"rgenscn 196 6.Dral 1967).Pum pin...I.cSprobablycorrelatewiththe beat frequencyofthecilia,althoughthe relation betweenthe actio nofthe cilia andthe transport of waterisMilldisputed (Jflrgensen and Riisgiird1988,Jsr ge nsen 1990, J,l:1rgensen etat 1990. Wardctal.199 ] a). Rates of watertra nsportare alsocontrolled byregulat ion of thediameter orthe exhalantsiphon (Foster-Smi th1976b). and inthe degree of gape of thetwo valves(Bayneand Newell 198 3).

The rateoffecding by abivalve is dependentnotonly ontherareofwate ruanspurt (=pum p ingrate)butalso onthefuncti onal slateof thegill. Jfl"rgenscn(1976 )describes threefunctional statesofthe gillsofsuspension-feeders:I)non-retentive,indicativeof

I

Su!>pc ndedP;uticuhlles

I

~ ~ ---

c

I

^Ingc5lcdP ;uticlcs

I

Absorbed Ration

,It""

P R U

Fig.1.1:Schematicdiagramofthe componentsof feedinGanddigestion ina sus(lI:nsion-fccding.bwalve.Lcncrsin boldtyperefer totheEquation1.1.Circled areasindicarcpointsa!ClnGlhedigestivetractwhereplutklcselectionmayoccur.

stressedor disturbedanimals,2)acleaning state. dlaracterill'd hycopious!l1l11'IIS

production,whichmaybefavourable incoudirions ofhigh particleccnccruratlonund increasedfrequency ofclogging ofthegill,and3)a"nor mal"state, withhigh!luillping rates andhigh particleretentionefficiency,Researchis stillneededtounderstandthe morphological basis andthe physiological roles of thesethre e functionalstaresO."rgl'nsen 1990).However.itis knownthatsomebivalvesCallchangetheircffictcncyofparticle retention inresponseto changes in thescstonbychangiugtheporosity ofthe gillstDml 1967,Hummel1985).

Because of the combined effectsorpumptngrateand the flilKtiunalstaleofthe gill on rates offeeding, muststudiesof bivejveshave used the"clearance nne"as allimlkatin ll of feedingbehaviour.Clearance rare{CR)can bedefinedas the volumeofwutcrdC:lredof particlesabove a certain minimumsize ina givenperiodoftime.

The gillsareknown to be thefirstciteof particle selectionwitbtuthcbivalves.

Selection bythe gills isbased primarilyon particlesize. whichin rumdependson the gill ostiasize:paniclessmallerthanthe ostia generally arenot retained.Mostbivalvesdonor retain particlesbelowabout1.5~111indiameter(Griffiths andGriffuhs 19K7).

!22TheIabja!Palos

Once panicles have been retainedby thegills,theyare boundinJ1l11Ctl.S(Wnrdetnl.

1993a, butsee commentsby J.'l'l'gcnsen 1993andWard etal.199JbJ andnavel"Iong :l food groove on the gillsto structuresin front ofthe mouth calledthe labialpulps(Fig. 1.1).

The palps are thesecondsiteofparticleselection,where rejectedparticlesareexpelledfrum the mantle cavityby muscularcontractionof thevalves,reversingthedirectionofwater now andforcing panicles back throughmeinhalent siphon.Theseparticlesrejectedbythe labialpalps, as well as particles arisingfromthe mantle surfaceafterdroppingoff the gills.

are collectivelytermed"pscudofueces". Unlike the gills,thepalpsareknowntoselector rejec t pan icles onthe basis of their chemicalor nutritional properncs(Ki(lrboeand Mohlenberg1981).Many studieshavedemonstratedthat certainbivalve speciesarc capable ofpreferentially retainingtheorganicfraction ofa food suspension,withinorganicpan icles beingrejected aspseudotaeccs (Kier boeand Mdhlcnberg 198I, NewellandJordan19K3, Shumwayctel.1985).The abilitytoretain selectively morenutritious particles i.s of great

ecologicnl ndvantage, since energy wouldotherwisebewastedontrying todigest poor qualityfoodparticles.

Althoughparticleselection by thepalps has been demonstrated (e.g.Kierbceand Muhlenberg1981),themechanism behind itis still underdispute(Jjlrgensen1990).

Selectionlikelyoccurs inresponse to surfaceproperties ofthepan icle (such ascharge, biochcrniculcnerectcrtsucs.shape, etc.).butthis is complicatedbythe factthatpanicles reachingthepalpsnrc boundin mucus.whichprobably confounds detectionofchemical prope rties(Kierboc andMUhlenberg 1981)andsize(widdo wset al.1979).Itispossible thatdifferent particleshaveadifferentlikelihoodofbeing caught in mucus(basedOntheir surfacechemicalproperties)andhencehavedifferent probabilitiesof beingexpelledas IlselH.lufaeccs(Ki~rbocand Muhlenberg 198\).Regardless of thecompositionofthe scstou. pscudoracccsproduction occurs abovea certain threshold concentration which variesconside rablybetweenspecies (Foster-Smith1975b, Bayne and Newell 1983, Griffithsand GriffithsIIJ1l7).Whatdeterminesthat thresholdconcentration is not known.

Pseudofaecesproductionis importa ntinthe re gulation offoodintakeby suspension-feeding bivalves(Widdowsct a!. 1979,Iglesias etal. 1992). For thisreason manystudies refer10"ingestion rate"(lR),which is theamountofmalerial ingestedbyan animal ofa certainstzein a given periodoftime.When no pseudofaecesareproduced.the amount ofmaterialcleared(=CR) is equal10theamount of materialingested.However, whenpseudofaccesare produced,IR islessthanCR.Ingestionrate cantherefore be controlledI)by changing thepumpingrateandIortheCR(Winter 1978.Bayneeral.

1989).and2)through theproductionof pseudofaeces (Widdowsetal.1979,Iglesiasetat.

1992).

Aftermaterial hasbeenacce piecbythelabialpalps.it passes throughthemouthand entersthedigestivetract,whichconsistsof an esophagusleadingto the stomach andstyle soil'withassociateddigestive gland. followedby alongintestine,and endingatthe anus whichislocatedat thehaltomof theexhalentsiphon.

Thestomachisthe thirdlur aliunOIlwhichselection\If fl.1rtirlcsmilYoccur.The mechanismsforselectio n in thegUIan: IIlII clearly undCl1ihll.)d.:Lhhuugh ibcy an: pn.Ili;Il1ly basedonbiochemicalsensing{Bri«l j !!131.19lWl.SelectioncantakeIWOdiffcrem form s.

First. the retentionume of food withi,1jhegUIcanbemanipulal!!dinn.'SJ'IlllIO<10 11l1:

chemical prcpenlesof thefoodIre-us.Fooditems of lowdietaryqualitymay1'Cpa....'\CJ through lhe gutmore rapidlymantbcseofhigbcrdietaryllu,lIity(Selfand Jumars197K, Briceljeral. 1984).This,coupledwiththefiIClth:11itemsrct:liucdlongerinuegUIare expectedto bedigested ,1IIdlorabsorbedwithgreaterdficicm'Y(faghlMI19RI),resull'l in an efficientstrategy10minimizeenergywastagefmmdi~'l:.'ling.nUllitiunallypoor IImleri:l!.

Bivalvescapableofthis type of digestiveselectionllI:Lybeatan ecologicaladvumagcwhen comparedwith those which arenot. Secondly,bivalvescan usctwoIunus ofdigestion (VanWeeI1961,Widdows et OIl.1979, BayneandNewell,19KJ ):I) extracellular

"intestinal"digestionoccurringinthestomachandresultingin luw digcsticuand absorptionefficiencies,and2)moreprolongediruracullular "gl:ll1 J ul:lr^Mdigestionlaking placeinthe digestiveglandwithmuch higherdigestionandabsorptionefficiencies.Itis possiblethaisome bivalvesare capableofshuntingpeorcrquulhy1l<1l1it:lcsdirecuyintuthe intestine tobeeliminatedquickly,while higher qualitypan ides aredirected imuthe digestive gland formorethorough digestion,Bivalvescapableufdirectingpanidesinlllthe differentdigestivepathsmay be31anecologicaladvantage.ThcJ;C twolypc... ofsekcuonin the gut (manipulatinggut passage limes and pathsofdig~slion)are likely tobe clcsety linked,and insome cases theformermaybea direct resulto(thc latter.

It is imponanlto note thatthe sequence of dige'llionisas yet unknown.IIwas traditionally believedthatimCSlinal ::mdglandulardigcMionoccurredsimultaneou.sly(Owcn 1966, Purchon 1968).However,this view ha.s beenchallenged,anda newthl.:ory ha.sbeen proposed that feeding andimestinaldigestionarecompletedbeforegtandular digestion is inltianJ(i.e.thereisa temporala!'>wellas a spatialseparationbetweentheIWO Icrms of digestion;McQuiston 1969,Purchon 1971,Morton1973,Langton 1975.1977).

However.a studybyRobinson etOIL (1981)docsnot support this lattertheory:these researchersfound highintra-and inter-animalvarianceinthedigcsrivcstagesof tubules withinthedigestiveglandofimcrtidal bivalves,meaningthai differentpartsofthe digestive gland wereat different Magesoftbedigestlvecycle,Theseresearchershypothesizethat despite cyclesof foodavailabilityimposedbyenvironmen talfactors(e.g. tidal rhythms),

bivalves arcessentiallyopportunisticreeders. and utilize digcst h'c strategies to exploitan erratlcfoodsupply.

Toevaluatethe treatment of foodwithin the gut,researchersge nerallymeasurethe lengthoftimethatfoodisretainedwithin the gut(=gUIretentiontime(GRT),gutpassage lime,-rcsidencctime, -throughputtime or-clcamnccrate)andthe absorption efficiency (AE)orIigesnveefficiencyofthefood.The methodsformeasuringthesevariables willbe describedin detailinlate r portionsof thisthesis.Previousstudieshave alsolooked at factorssuch1.1.\compartmentalization(=gutarchitec ture)and the totalamountofmaterial withinthegut,whenstudyingthefeedingecology ofdifferent bivalvespecies.

J.J~.crg~Eql!'I1iotJforBiyalyeEcedinJ:;

MaterialwithinthegUIcan eitherbeabsorbedby theanimalfor useingrowth.

metabolismorexcretion (substancesother thanfaeces suchasmucusandurine) orit can be eliminatedasfaeces(Fig.1.1).This completesthedigestive pathway ofsuspension- feedingbivalves.

This entirepathwaycanbesummarizedbytheenergyequation

C= P +R +U+F Equation1.1

where Cistheamount ofenergyconsumed(CRx foodconcenuatic n.foodrejectedas pscudoraeccs),P1.\ theenergyincorporatedas production(growthand reproduction),R is theencrgyallocated to basic metabolic functions,Uisthe energyconte nt ofexcreta, and F istheenergy voided asfaeces (BayneandNewell 1983). Thesesymbolshavebeen included in theappropriateplacesinFig.1.1.

By altering their feeding behaviour,bivalvescan effecti vely change energy nllocntionswithinthisequation, therebyalteringtheir physiological andmetabolic state.An animalmustbe ableto balanceitsmetaboliclossesagr.mstene rgygainsfromthe environmentin ordertomaintainpo sitivesomatic andreproductivegrowth.This

"balancing" oftheenergy equation iscarriedout by anumberofphysiologicaladaptations, lind is broughtabout,in part,by beha viouraladaptations.In general, bivalves can

compensate forincreased metabolictkm:mdsby inl'rc"sint:cousurnpnontC',EquJtitln 1.1), usua llybyincreasingCR,Howe ver,anincrease in ('Ralsomeanstha tflltld mol}'he processed throughIlledit:Ooliveuact morequickly (lk.'\'rcascd GRl)andl·uuhl lhen:f~lrcbe digestedwithlowerefficiency(Tat:hun 1981),The interactions~1W\.'\:nC'R, AI:, nndGirl' arc furthercompoundedbythingssuch as changes in gutvolume.enzyme,,·a ('ldl y, selectionoffood paniclesatvariouspointsinthe digcsth'c tract.:lndgenelicerrccts(e,~, pre-dispo si tiontowardscertainfoodcompositions)amongothers.Therdnrc.fl'\'Jing responsetoenvironmenlal changcs can beclltTelllclyeull1ple!(,

Theobjectiveof thisuresis was10investigatepre-andpos t-ingestivefl'\'lling activity(measured as CR.IR,GRTof organicandinurgunicIracnons,midAE)inthesoft·

shelledclam.My"arenarlu L.. particularly inresponsetochangcsinfondconccmrarion andcompos ition.Sincemoststudies offcedingphysiolugyhaveused epifaull;llanimnls, this study specificallychose aninfaunnl species.since!l's...is geucrully knownabouttheir feedingphysiology,

Thesoft-shelled clam.MYQarmaria(K,Anlmatla,P,Mollu sca. C.lJivah'ia.O.

Eulamellibranchia (Sub-orderHete rodcma),F.Myidae.was selected forthis study because.althoughmuchisknownaboutitsnaturalhtsrory ,il~physiologyand f« ding behaviourhavenot beenextensjvefystudied,In general,mcasuremcr usoffe~di ng behaviourorphysiologyofM.arma riahavebeen madea...poinL.. ofcomparisonwilt!mhcr species(e.g.MOhlenberg andRilsgird1919.Ki~rbocand Muhlenberg 1981.Shum wayer al.1985.Kruegeretal.199 2),Studies ce nteri ngonM.arenariahaveincluded morphological work (MacDonaldandThomas1980,zwnns and Wanink19119).biod:pnsil analyses(Allen1962,Brown 1986), andfeedingmechanics (Foster-Smith197611.

JergensenandRiisg!rd1988.Ward et al.1991,1993).Otherstudic...haveinvestigated variousaspectsof thebehaviourand physiology ofM,armaria,butwith respecttofuclms otherthanfeedingstrategies, includinggeneralculture andecology(HiduandXcwcll 1989). metabolic stale (Lowe and Trueman1972). sub~I"'ol tCtype (Swan1952).roleinthe benthic co mmunity(Emersonetnl.1988),sedimentdisturbance(Emerson1990),benthic- pelagiccoupling(LooandRosenberg1989).ratesofrecruitment (Andre and Rmc nhcrg 1991), andoilspillage(Gilfillan eral.1976,MacDonald and Thomas1982),

Mya urmoriaW3.\alsoselected sinceitis an importantspeciesinmany benthic marinecornnumitiesthroughout thenorthernhemisphere{HiduandNewell1989).There h...sbcenincreasedinterestinstudyingM.armariaasanaquaculturespecies throughoutthe Auamiccoast of NewEnglandand theMaritimes.Myaarmariaalso occurs abundantlyin manyNewfoundland tidal flats,making it an important speciesecologicallyif not economically,

Measurcmentsof CR,JR,GRT,andAE weremadetoassessthefeeding physiology ofM.unmariainthis study.Methodsfor measuringCR,IR andAE are well established.However.before these behaviouralstudiescouldbeundertaken.itwas necessaryto developanappropriatemethod for determining the ORTof suspension- feedingbivalves.Chapter11explains the methodscurrentlyavailable,why theywere consideredinappropriate for thisstudy, and the technique developed inresponseto this problem.

Chapter IIIusesthis new ORT technique inalaboratorystullytoinvestigatethe effectofchanges intheconcentration andcompositionof a foodsuspensionon the feeding behaviourofMyanrcnana.Theresults fromthis studyarc thencompared inChapter IV10 field measurementsof feedingbehaviourofanaturalpopulationofM.arenana.Thefinal chapterofthis thesisis a criticalevaluationofthe newORT technique.

CHAPTER II

Development of a New Techniqu e to Measure Gut Retention Time in Susp ension -feeding Bivalves

21 Illlr Qdn rllon

Thereis substantialevidencethatbivalvesrcguercfeedingbehaviourunder differentenvironmental conditionsin order to optimize net energyyiclll,~(secreviewsby Winter 1978.BayneandNewell 1983.Buynectat 1988).ThlsmayIncludethercgutution of clearanceandingestionrates,absorptiondfki.: ndcs. and theprcfcrcnti'll.~kclj(Jnof particles bothbeforeandafteringestion.Considerable workhasinvestlgatcdhuwthese physiologicalvariables are interrelated.particularlyinresponsetoquantitativeand qualitativechangesin the food ration available to the animals.Inresearch ofthistype.it is ofte nuseful todeterminethelengthof limeIhal food isretained withinthegut,or thegut retentiontime, since it often bears3closerelation10clearancerate (Baynectal.19H4, 1989)and absorptionefficiency (Bayne et al1984, Siblyand Calow1986 ,Bayne etal.

1987), andifGRT is compared fordifferenttypesof particles,canhi:usedWide ntifypost- ingestiveselectionof particleswithinthe gut (Briceljernl.1984).

In previo usstudiesof feeding behav ior insuxpension-Iecdingbivalves,a variety of techniquesto assess GRT have been used. All makeuse of marker particleswhich arc either traced throughthedigestivesystem.or quantified infaecal material. Gut retention time isestimate dby determiningthe amountof markerparticlespresentinsamples collected atknownlime intervalsfrcm bivalve sfed a "p ulse"of markermateria l.Inmoxl experiments,completeeliminatio nofthe markerrequire sa long sam pling period ,so previousstudieshave arbitrarilydefined GRT accordingto a specifiedcriteri on,themost comm onbeing thetime at which90%ofthereco veredmarkerhas accum u latedinthe faeces(e.g .Bayne et a1.1987,Hawkins et al.1990).

Severaltypes of markerparticles ha ve been usedinpre viousstudiesof ORT.

including radiola belle d particles,bovinered blood cells,andfluorescentorstrongly 10

coloured paniclesorbeads.TIle most commongutmarkerisperhaps mdiolabelled panicles (e.g. Bricclj et al.1984,HawkinsandBayne1984,Bayne et31.1987,Hawkinset al.

1990.Pechaand Luoma1991)whichcanbe detected in faecal pelle tsby standar d sci ntillation countingtech niques.Thismetnodhastheadvantagethat thelabelled panicles canbea naturalcomponent ofthebivalve'sdiet,suchasalgaleel' s. andthe labelled particlesareeasilyquantified. Radiolabelling can also verysensitive: the highspecific activityofcertainlabels, suchas14C. bicarbonatc.enables use ofaverysbo n exposure time to the markers,therebynarro wing the responsepeakand givingbetter tem poral resolution.However,therearepracticallimitations to the use ofradioisotopes,especially whendealingwithnon-recirculating,flow-throughsystems. or field-andship-based rcsearcf projects.

Bovineredbloodcells, whichare readilyingestedby suspension-feeding bivalves.

havealsobeenemployedasmarkers(Bayne etal.1984) by usinghistochemicaltechniques todetect peroxidase activitywithinthe.se cellsasthey passthrough the bivalve digestive gland.Allhough bovineredbloodcensareingested bybivalves,it is notknown whether theirsubsequenttreatmentinthe gutis similar tothatof typicaldietcomponents.Bivalves are capableof sortingdifferent algalspeciesinthe gut, eliminatingthemore indigestible speciesmorerapidly(Brieeljetat.1984, Shumway etal.1985). Thus,itislikelythat foreign panicles suchas bovinered bloodcells arealsosubjected tosorting withinthe bivalvegut,giving inaccurate estimates ofthe GRT of morestandarddietarycomponents.

Colouredpaniclessuchaslatexpanicles orbeads are a third type ofmarkerin use (e.g.Bricel]er al.1984,Hummel1985),These canbe detectedvisually.but aregenerally not quantifiedinfaecalpellets. therebygiving onlyapproximateestimatesofGRT.

Furthermore,Ihesepaniclesmayalso be subjected to post-ingestivesorting withinthegut.

Itwouldbepreferabletousemorenaturalpanicles.suchas algalcells,whichmay alsobe subjectedto sorting, butareanatural dietarycomponent.

Thcre is someevidencethattheinorganic fractionof thediet is voidedfrom the gut more rapidlythanrhe organic fraction (Briceljeral.1984).Therefore.it maybe desirable in somestudies of GRTtoutilize two markers, oneorganic and oneinorganic, to detect any differentialselectionwithinthegut. Theuse of twodifferentmarkerpanicles,(organic and inorganic),administeredsimultaneously(0bivalvesinIInow-throughexperimentalsystem,

II

is the basisforthetechniquedescribed inthis~·hajller.Both marker particlescan be detectedineachfaecalpellet. The gl...ccuflagellateTetmsclmis.~IU·C;CIIwas II."Cdasthe organicmarker,because its characteristicchlorophyllf,signatureis notfoundinnHlstother types of algaeincludingdiatoms. dtnonagctlarcs. ,IOU cryprornonnds. The amount of chlorophyllbin thefaecesGInbe determined byhigh performanceliquidehrmnutogr uphy (HPLC), and used as anindicatorof thenumber ofT..medcu cells present. Silicon carbide part icles(SiC.#600grit, used for polishin ggeologlrulsections) werechosenas the inorganic marker.Thesecan beobtainedinthesame sizerange asT.SI//'cil'llcells(npprux. 8· 12pmdiameter) which eliminatestheproblem ofpanicleselection or retention based solelyonsize. In addition,theywill pas!ithrough the gutundigestedamitheirIlre!icncein the faeces canbedetected usingstandanlpaniclesize analysis.

This section describesthemajor stepstakenin thedcveloprue ntofthedouhlc- marker technique. Additionalinfcrmruicnisincludedin AppendixD.

For allexperimentsinthis chapter, clams of similarstzc(approx.45-55nunshell length) werecollectedfromthe mid-andlowerlittoral zonesatRiverhead. 51. Mary's Bay, Newfoundland.Collections weremade approximately1-2weeksprior 10experimentation.

Clams weretransported immediately onice(trip duration=apprcx.211)toholding facilities at theOcean SciencesCentre.MemorialUniversityofNewfoundland.Therewerefew transport-relatedmortalities.Clams were heldin a30 1 lanksuppliedwithunflucred.

flowingseawaterat ambienttemperature.Allseawaterused in thelaboratoryexperiments was obtained fromtheOceanSciencesCentre main seawater line.The waterisnot filtered, heatedorrecirculated, and ispumped directlyfrom LogyBay.Naturalsestc nwas supplementeddailywithCnaetoceros muelleriand/ortsachrysisI;ulhunllal anapproximate concentration of66x103 cellsmr-!for9 0 min(=approximately9.5mgdry weight foud-I clanrlday-I),duringwhichtimethe seawater flowwasshutoff.

22IIngestion3ndDigest ioll ofTflrmelmjtwerimby Mqmwriu"lOciDelection of ChlorophyllbusjngHpIC

12

The purpose of this study was10 ascertainwhetherM.arenariawould ingest T.

.Hlf'cim.and whetherthechlorophyllbpresentinT.suecicacouldbe detected from piglllcnl~extractedfrom faecalpellets.

Methods

rive damswereplacedin individualfingerbowlswithsiphonsoriented in the same direction.andlabelled AlOE.The fingerbowlsweresubmergedin a plastic tray.witha constantIIminot now ofunfiltered seawaterat ambient temperature.Clams wereleft undisturbedfor 2-1 h 10 adjustto theseconditions.Afterthis adjustment period,faecal pclle txIsamplesIl

h) were collected fromallbilltwo clams,whichhad not produced anyfaeces. and stored for ptgmcnranalysisasdesc ribed below.

After theinitialfaeces sampleshad beencollected,the bonom ofeachfingerbowl was siphoned clean of settled debris,andthe seawater inflowline was shutoff, Approximatelyone-halfthevolumeof seawaterwas siphonedfrom eachfingerbowl and replacedwith 70 mlofT.suecica culture,to afinalconcentrationof 50,000cellsml-t.

Clams wereallowedto feedon theT.suecicafor 45 min,before theseawaterinflowline was reopened, therebyflushing theT.suecicamarker from thefinger bowls. Faeces samples were collectedatI,2 and 4 h afterintroductionoftheT.suecicacells,and again from the fifthclam (E) after 24 h. One clamproduced abright greenpiece ofpseudoteeces one hour afterdeliveryof the marker,whichwas also collectedforanalysis(sample PF).

Allsampleswere preparedandanalyzedon theHPLC asdescribedbelow,

Ascstonsamplewas taken24 hafterdeliveryof the markercells byfiltering 700 011ofseawateronto a WhatmanGF/Cfiller, fromwhich pigmentswereextractedovernight al ·20OCin90% acetoneand analyzedby HPLC as describedbelow.

Pigment analysisof faecalpellets:

Pigmentsampleswerestored in 1.5mlEppendorftubes (withallseawater removed)in darknessat-200Cuntilanalysis,Sampleswerethen thawed, suspendedin1.5

13

01190% acetore.ecnicmed in aniccbath for 10·15min.and hddin d:ulncsS31·2()'C fur 24hfor extractionof pigments.Sample...wen:the n cemrifugcdatIb,OOOref for3min.

The supernatant wascollectedtransferredintoanother1.5 ml EP1"'=Ollorf dispusahlc:rcbe.

andsto re d in darkncSli at·200cfor subsequentpie-men! ana lys is by HPLC.Pigmem extractionis95·I()()'t. complete:b)'thismethod.

Before analysisbyHPle.pigmentsamples wen: dilutedwith90~acciore to a total pigmen t concen trationofapproximately 0.7 5•I.UOmt:1-1^3.\deicrmtne d with^II TurnerDesigns (mode11U IO)Fluorometer.Five-hundredpiofthediluted samplewas mixed with150 pi ionpairing solution (7.7gemmc mumeccurcin100IIII mi1li·Qwater) and250 pI ofthis mixture wasinjected intothe100~II IUllPufaBeckman reverse-phase IIPLC.Sampleswererun through a10min.gradient of HIl%methanol,15%Milli-Q wmcr and 5%ion pairingsolutionto7Wkmethanoland30%acetone.Pluorcsccnremissionwas detectedbya GilsonModel121detectorand displayed asastnndnnlchromatogram(Fig.

2.0 .Pigments were identified by comparisonswith known standardsobtaine dfrom Sigma ChemicalCompanyorTLCextraction,

Results andDiscussion

Thepresence or absenceof chlorophyllband iL'icornpcoenrbreakdown prodUl.·l'i in each sample islisted inTable 2.1.In most samples,chlorophyllb wa.'ipresentin either trace:amounts.or couldnotbedetected at all.This include...the seston sample.andinitial (Oh)faecal samples,whichindicates that background levels ofchlorophyllb were very low.Clams C andD did not pump duringtheperiod of exposure toT.suecica.and therefore didnotshow any chlorophyll b in subsequent faecalsamples.uowever,two clamsdidshow presence of b-pigments in theirfaeces:clam Aat 4haneresposure toT.

suecica,andclamE at 24h.(IIshouldbenotedthat the 24hsamplecould havebeen voidedfrom the gutatanytime between4and24hafterdeliveryofthemarker).

Furthennore, bothof these samplesshowed bre akdown of chlorophyllbintheform of phaeophytinb,indicating that theT.suedeacellswerepartiallydegradedin the gut.Some T.suecicawas rejectedaspseudoreeces.butthisisprobablydue tothehigh conceniration ofT.slIecicQadministeredto theclams.

14

Fro m this experimentitW1l5conclude d(hal!of.armariawillingestanddigestT.

mecicocells,makingthisalgaappropriatefor use asaGRT marker.

15

.[- - -=

I'

Table2.1:Presence of chlorophyllbandiLScomponent breakdownproductsin faecal samples collectedfrumM.armariaexposed(0a 45minpulseofTttrasrlmis sutcica.

h·Oh T""" ^Absent liltl.:backgrOl:::d b-pigmcnt

B - Oh Trace Absent littlcback8roundb-ptgmcm

E-011 T""" Ahscnt linkbackgroundb-plgmcnt

E-Ih Absent Absent noTetraselnus

3-2h Ahsent Absent noTetrosrlmi s

C· 2h Ahscnt Absent noTetrasdnds

1)·2h Absent Absent noTetmsrlnds

A-4h Trace High degradedTetmselnus

B·4h T""" ^Absent noTetraselmn

E-4h Trace Absent noTnrasetmis

E-24h High High Trtrwt lmiscells present

PF lIigh Absent pscedoracces isTetraselmu

Sesron T""" Absent liltkbackgroundb-pigmcnt 17

222Ingest;nn andDigestionQ(Sj licon Cam hI..'brAtlIm",riq

Tbepurpose ofthefouo wfngexperimentW:L"10detcrmircwletbcrM.arrnaria~·an ingestsilicon carbide (SiC)panicles.Detectionof panicles was done (rum a\;s\l:ll examinationofthefaecal pellets:SiC is grey in colourand easilydislin~uWll:tlfrHIIl otherwisenaturallybrown pellets.

Methods

Allthefollowing experiments wereconducted intheflow-through systemshownin Fig.2.2. A20 I plastic bucketservedas a headertank. ScuwntcrW,ISIihcrcdIII 100um and flow10theheader Ulnkwas maintained:l\..\Imin-Ithrnughmuthecourseufeach experiment. Seawaterwasdistributedsimultaneously10the experimentalconmincrs throughIem internaldiametertygon tubing.Thefreeendsof theselinesweresecured inside theheaderlankthrougha15 emdiameterPlc ltiglasdisc,raisedaOllul9. 5em above thebottomofIhebucket.Flow throughthesenncs into theexperimentalcontainerswas controlledby plasticplugsdrilledwitha0.5mmbit.Thisresultedin aMandanJi1.et.Inowof 100 - 120mlmin-Ito eachcontainer.predeterminedtobean appropriaterJte forM.

orenaria: see AppendixA.A constantwater headpre...UfCwas muintaircd byensuring thai water continuouslyexitedvia theoverflowoutlet.

Experimental conntners were madefrom1.0 I plastic containers(170Jt80Jt110 mm,Fig.2.2).A I em internaldiameterinflo... tube was pa....sed througha hole drilled throughthewallof thecontainer.3 em above the base.A 0.5 eminternal diameter standpipe waspassed through the base with its upperendIembelowtherimoflhe contai ner.Jointswere sealed with siliconewherenece...sary.A four-prongedplastic

"holder"forpositioningthe clamsInthenaturaluprightpositionW3 .Ssecuredby aplastic screw throughthe baseorthecomalner.4.5 emforwardorthestandpipe.Finally, a 5.5em tallPlexiglas bafflewas affixedwith silicone 4.3embackfromlheinrlow.10ensureanon- recirculatingflowthroughthecontainer.

Preliminaryexperimentswiththisflow-throughsystemshowedthattherewa.s nil significant differenceinpanicle concentration betweenallexperimentalcontainers.Itis

18

"i g.2.2:Intenlal

and

externalfeaturesof the experimentalapparatus.

A) FeaturesoftheOo...-throughheadertank.HT

=

headertank.I

=

inflow,0=overflow,D

=

dietinflow,C=experimentalcontainers (n=16maximum).B)Futuresoftheexperimentalcontainers.IT=

~~O~~~b~;f~r~a~~S~:~~~~~~~Jn~~:n~~~A~l~I~:d~:~?ee.

thedirectionofseawaternow.

19

therefore reasonable to assumethatinthese experiments,mixin,ginthebende rrank was sufficienttoensuretha tidentical suspcnxionxwen: deliveredtoall hnlividuulronmincrx,

In thisexpenmem.uvedams wereheldin individualI.lli plasticpatsin theIl ow- throughapparatusat ambienttemperature.Clamswen:allowedIIIadjustto these('tlndi l i(ln.~

for24 h.beforea fewdrops of SiCin suspension(al1.6 x 1()6 particles ml-l)were introducedintothe wetcrabovethe incurrentsiphons of4 clams.The firth servedOISa control (no SiCadded). Clamswer e allowe d10feed undisturbedfor 24 h beforefact'lll pelletswerevisuallyexaminedforthe presenceofSiC.

Results

After 24 h.all clam s exposedto theSiC particlesproducedgrey faCC:11relict', indicating thaiSiCwas ingestedand voidedfromtin: gut.There werealso grey pseudoteecesinsome containers.which were moretoo....elyboundthan faecalpelletsand floatedatthe surface.The containersalsohad a lightfilmofgrey particles acrossthe bottom.presumablyfrom SiCsettlingoutof the watercolumn.Thisfilm waseasily distinguishedfrom the greyfaecalpcljets.

Fromthis experiment. itwas concludedthat SiC isingestedbyM. lm -fw rill.and could thereforebeusedas a markerforGRT.

223 pevelopmcntofa ProcedureforOlliJDlifyin? SiCM'!rkcrPilrtic!csjnF·ttTilll'l'llc!s

Althoughthe presenceof SiCin faecalpelletscouldbedetectedvisuallyby their greycolour. it wasnecessaryto developamethod forquantifyingtheamountof SiC particlespresentin each sample.Three possiblemethods wereexplored.I)microscopy.in whichSiCpanicles wouldbeindividuallycounted. 2) a.shweightanalysis,in whichfaecal samplescontainingSiC wouldhaveahigherinorganiccontent.and3)particlecount analysis. in whichtheSiC panicleswouldbequantifiedfromaxizcdistributionof partides in each sample.

20

Methods

Fived"Il1~,labelledA·E. wereplacedinindividual 1I plasticcontainersin theflow throughapparatu s describedin section2.2.2.Clamswereallowed to adjust tothe experimental conditions(or 24h, beforeSiC markerpaniclesweredeliveredtothe .Jamsat aconce ntr ation of approx imatel y5000panicles ml-'for30min.Thiswasachievedby pouring a pre-determinedamountofSiCparticles in suspensioninto thetopoftheheader lank.The inf'luwline 10ClamAwaspluggedduringthis time. toprevent exposure toSiC andthus actas acontrol.After 30 minfeeding,the flowrateofthe incurrent seawater line W3.~increasedtoFlushmarker particles OUIofthesystem quickly through the overflow line (M:CFig.2.2),andthe linetodamAwas unplugged. Faeces sampleswerecollected atfi, 12,and24h after deliveryof themarkerparticles. This experimentwasrepeated3 times.

withexaminationofthefaecalpellet sforSiC bymicroscopy,ashweight analysisand particleslzcanalysis respectively.

Faeces samplescollected formicroscopeanalysis werepipetted into1.5ml Eppcndorf tubesfilled with 1.0I.Imfilteredseawater,sonicated forJ5mintobreak up mucus-boundclumps,filtered ontownarmanGF/Cfilters , and examinedatlOXon a dissectingmicroscope.

Samples collectedfor ashweightanalysis were filteredonto pre-ashedand weighed WhatmnnGFICfilters,rinsed with3%ammoniumformate,placedinanovenat800cand dried10constantweight. Filters werethenheated at 4500Cfor6 htocombust organic compounds.cooledinadesiccator10roomtemperature,reweighed. and the proportionof organic compounds ineachsample determined.Organic content wasalsodeterminedfor samplesof pureSiC.

Samples collected for analysisof the panicle size distributionwerepipenedinto 1.5 ml Eppendorf lubes filledwith1.0 11mfiltered seawater, andsonicated for30 minto dispersetheparticles.Eachsamplewasthen addedto250ml of 1.011m filteredseawater and analyzedfor 2minon :1CoulterMuhixizerequippedwith a 100urn diameterorifice tube (whichrepresentsavolumeof4.5mI).Thisproducedafreque ncy histogramof particlesbetween approximately 2and62 urn diameterfor each sample. TheSiC particles werefound byMultisizer analysistohave a particlesizedistribution of about 4.7 to17.9

2 I

11m(Fig. 2.3). Therefore.sampleswit ho ut SiC won',1have:1"bM.c1inc"numberIl f particles between·.1.7and17.9 1J0ldiameter.Fael'alS<lmplcSCUIl13ininA:SiCweuhlhavea larger"peak"of paniclesin this size range.Funhatnorc.theamplitulkofthe4.7.17.9 umpeakisanindication ofhowmanySiCpaniclesan:presentinthesample. Results

Mic roscopy: SiCparticleswerevisibleonIhefillersasshiny greychips.

However,countingIhrnumberofchipsoneachfilter provedtobedifficuhundlaborious.

IIwasdec ided thatthis^W3Snotnnefficientway totluantifyI~SiCmarkerpanu-lcs ineach sample.

Organiccontent :'111('underlyingtheory Inthisaunlysisistluumurc SiCpresent inasample should lowerits organic weight cumcnt).0,1111A (cwl1ruljdidnetproduce sufficientfaecesfor thisanalysis.sofourfreshfaecalpelletsweretakenfromduminthe holdinglank(wherelheyhadbeenexposedtothesame watertempenuurcsand.scMtlll lu:ld asmoseintheflew-through apparatus)as samples offaCt:eswhhoutSiC.Th esehada meanof 23.2%organiccementbyweight(SO

=

8.3).Three greypclhnxprodun::d by clamsexposed10SiCwere analyzed. andfound tohavea^meanof18.1'-"organic^content (SO=1.5).Twosamplesof pureSiCwereaboanalyzed.andfoundtohaveamean organicecrueruof9.75%(SO=2.5).Theorgankcomentuf faeceshumdam...expu..alIU SiCandcontrolsamples didnotdiffer signirlCantly(indcpernJcmt-Iest.t

=

1.024.df=5.p '" 0.353).Thismayhavebeenbecause thesestonhad¹¹lowurgan!c-content.orbecausethe clamshadahi!hAEfororganiccompounds.Itwasl"tlllclutlcdthatash-weight determinationisnota reliablemethodforquantifying theamoum of SiCpresentinIaecal pellets.fortworeasons.First,alargeamount ofIaecalnuterialis neededfuranan'ur;llc analysisby weight. andnotall clamsproduced surriciemfaecalmaterialinthecourseof this experiment. Second. foranalysis bythismethod.itiscsxentialthata constantAEhe maintainedbyeachclamthrougho uttheexperiment.Otherwise.observedfluctuationsin theproportion oforganicsduetochangingAEcoulderroneouslybe interpretedas fluctuatingamountsof SiC.Clamsmaywelladju.'ilAEnvcrshuntimeperiods.

Par ticlesizeanalysis:lillie Iaceal materialwa...producedduring(he 24h period.sofaecalpelletsfromclamsexposed10theSiCparticleswere pooledinlOUIlC

22

4.0 3.5 3.0

~

^2.5_2.0

.I~

g -'I

ee1.5

1~~

1.0

0.5

~~

0.0

10 50 60

ParticleDiomel er(llm)

Fig ure 2.3:Size distribu tion ofsuspended SiC part icles as determine dbyCoulterMult isize r.TheSiC peak ranges fromappro ximat ely 4.7 to 17.9

~IT'l.

2.\

sample. Likewise, Iaccalpelletsfromtheone clam notexposed totheSiC panicleswere pooledinto asecondsample.

Seventy-fourpercentof theSiC panicles weredetenuincdto be between4.Mant!

17.86urn india.oeter(Fig.2.3),sothe proportionof particlesin thi.-; size rungecompared tothe2to62urnsize rungescanned was calculated for eachsample.F:lCCCSfromclams exposed toSiCshowed a greaterproportionofpanlclexin this sizerangeth,m eitherfaeces fromthe clam notexposedto SiC or natural seuwarcr (40.6% compared to 3J.5",f- and 33.1%respectively).Thiswas interpretedas evidence of the presenceof SiCin thefaeces of clams exposedto the pulseof SiCparticles.Itwas concludedihutSiCwas mcsteasily andaccuratelydetectedandquantifiedby useof the CoulterMultisizcrto .maly/e thesize distributionsof particles in the faccalpcl1ets.

Itwas necessarytomodify theproceduredescribedabovein orderto acquire a stronger. sharperSiCparticlepeak inthehistograms.Thefollowingthreesectionsofthis chapter dealwith theproceduresdevelopedtoobtainast ronger SiCsigrwl.

224 AcidificatjonofFaccalPe!!elstoEnhancetheSiC PjlrticlePeak

Thefirst stepinenhancingtheSiC paniclepeak intheparticlesin.::distributionswas to remove as many other paniclesfromthe pelletsaspossible.Themethod employedwas to treatfaecal pellets withacid to dissolveanyorganic particles.leaving behindonlythose whichcouldwithstand this treatment,including SiC.This wouldfacilitate detection of the SiCparticlepeak.

Methods

The flow-throughapparatus describedinsection2.2.2wassetup with6 clams.

each in individualcontainers: 3 wereexposed to SiCpaniclesand 3 (thecontrols)were nor.

Forthis experiment. theinflowingseawate r wassupplementedwitha mixtureof microalgae(c.mlulleri),andsilicon dioxide(Si02) panicles(not tobeconfusedwiththe silicon carbide markerpanicles)to stimulate theclams to produce more faecalmaterial.

Ehaetocerosmuelleri wasgrownin 200Icylindersat 22"Cusingconstantilluminationand F2+medium.

Suspensionsof algaeandsilicon dioxideweremixed separately in60 1buckets to predeterminedconcentrations.Alargestirbarwasplacedinthe bouomof thebucket containing silicon dioxide.In addition,the silicon dioxidebucketwas plungedmanually every1-2hto limit settling.Anairstoncwas placedin the algaebucket toensure proper aeration andmixing.Particles fromboth bucketsweredeliveredto the header tankbya peristalticpump. The inflow linesfrom theperistalticpump wereattached 10 thelarger seawaterinflow linetofacilitatemixing.A large stir-bar wasalsoplacedinthe bottom centreoftheheaderlank.Thenowrarerequired by theperistalticpump to producethe desiredparticleconcentrationwas determinedbytheformula:

Equation2.1

whereC,

=

theconcentrationof thestock culture, ChI

=

theconcentration required in the header tank,P,=thenowrateinthemainseawaterinflowline (into theheadertank),and PI'!'=thenow ratethroughthe peristalticpumpdelivering stock panicles tothe header lank.

The artificialdiet wasdeliveredat afinal concentrationof10,000particlesrnl-!of eachparticletype.Clamswere allowedtoadjust to these conditions for 24 h.SiC marker panicleswerethenintroducedata concentrattonofapproximately10.000particles ml-!for 30 min.Linestothe 3 controlclamswereplugged duringthis time to preventthem from ingestingthe SiC particles.

Two faecalsamples fromclams exposedto SiCpanicles.and two samples from clamsnot exposedto SiCpanicles.werepipeued separatelyinto 1.5 nilEppendorf tubes, coveredwith a few dropsof concentratednitric acid.and allowed to stand overnight.The tubeswere centrifuged at16.000ref for3 min.thesupernatantdiscarded, and the pellets washedoncein distilledwater,Pellets werethencovered with 1.5 mlof 1.0urn filtered seawater.sonicatedfor 10minto de-aggregatethe panicles, added to 250 ml of 1.0 11m filteredseawater,and analyzedbyCoulterMultisizcrasdescribedinsection2.2.3.

25

Results

Treatme nt withacid did nOI alterthe size distnbmion ufrbe SiC particles.Pa" id e:

size distributionsforthefour samplesare givenin FiS'2A.Doc sample (Treamu:nl2) showeda highe rproportionofparticlesbetweenapproximately"and8 11mthan the Treatment1 and ControlI samples.Indicaungthe presence of SiC.ThisWbfurther supportedby visual observations oftlussam ple.which was grey incolour unlikethe others.It shouldbe:notedIhalthe SiCparticlepeakis present only in thesmallerhalf of ils sizefraction, i.e.thepeakdocs not continue from8 tol.pproximately18J.111las expected, This isprobably duetosettlingoftheSiCmarker particles intheheade rtank,wilhlhe largerparticles settling outfirst.In allsubsequent experiments.thehe aderWI!.;W:L~

manually stirredduringtheperiodof markerparticleintnxluctiunIu keep asIllan~partid es as possibleinsuspension,The Control Isa mple also hat!n high prop o rti onofpanicles between3 and8umdiameter,butwasnetgreyin colour.IIwaspossiblelh,lllll", 10min sonication timewasnotsufficicm10fully disperseallp'lrtkks inII..:samples(fI:\ullingin twoextremelydifferentparticledistributionsforIhe controlsamples.Fig. 2..5AJ.solhe foUowingexperiment was designed toinvestigatethis.

22 .5PcJefmjnalionof!beMjnimumSonicp!iQnTImeRcuu;n;d toDisQt·fSCal!Panicle:;in

Methods

Twofaecalsamplesfrom theaboveexperimentwen:selectedforIhisanalys is:one from a controlclam. and the oneknownto con tainSiCpanicles. Sampleswen:sonicated for 60 minand analyzedbytheCoulterMuh isilcraftcr20 min,andthereafterat10 min intervals.Theparticlecountanalysis wasperf ormed as describedinsection 2.2.3.

26

27

1.4

(A)

[JOnl'O:]

1.2 • Control_~

1.0

~

^0.8

<;

> 0. 6

"

_0.4

yd~~

0.2 0.0

12 16 20

1.4 (B)

1.2

. ~

1.00.8

g

_0.6

" ""'1'1/

0.4

U '

0.2

.!~ tii?;

0.0

12 16 20

Particle Diametertu.m)

Figu r e2.4:Particlesize distri butionsof4 foecolsamples, two sample sfrom cla m s not exposedto SiC(A), andtwosamples from clams expos ed to SiC(B) .

Results

Theparticlesize distriburlonsur1~20.~oand 60 min.interval"are given inFig.

2.5.Inboth samples.tbereislillie changeinI~panicle size distributions lifterabout':0 min.Basedon these: graphs.a conservativedecisionwa..madeto sonicate alls..uuplesfur 60min before analysisbytheCoulter Multisizer.

22 6 Enhans:tmcn! o[SjC ResnonsePrakbySir r jngparticles

To define theSiC particlepeakfurther, SiC paniclesweresievedusingNucx screens toretainonly those particles between 10 and15urndiameter.This servedtwo purposes:I) bynarrowingthesin~distributionfrom itspreviousrange of approximately 4.7·17.9urn,detection oftheSiCparticlesis caster andmore:ICCUnt IC.and2) torender the size range of SiCmore equivalent to thai of the Tetraselmis.I"III' I'iCltmarkerparticle, with asize distributionofapproximately 10·12 urn.Usint:the sieved particlesdidnot affect any ofthe conclusionsmadefrom the precedtng expertmemsustngunsieved particles.Furthermore,sievingtheparticles. ratherman justnarrowingtheparticle size rangeon !he Multisiz.c:r.increased theamplifudc oltbe particlepeakonihc Multisil.cr.l/lus givinga strongerandmore accuratesignal.

Theexperimentsincluded in thischapterhave demonstratedthedevelopmentofa newtechniqueto assesstheGRTof suspension-feedingbivalves.Thetechniquemakes usc of2 markerparticles:Tetrasetmis slIrcica and silicuncarbide (SiC)10 estimatetheGRT of organicpaniclesandinorganicparticlessrespectively,Trtrusrlmis suecica is uaced in faecal samples by idemifying andquantifyingitscharacteristic chlorophyllhcomponentsu.o;ing highperformanceliquidchromatography. SiC panicleswere identilicd andquantified throughpanicle sizeanalysisonthe CoulterMuhisizer.SiCpanicleshave adiscrete size distribution. which isenhancedby sievingtoretain only thosebetween10andISurn.

Thus.faecalsampleswitha peakofparticlesbetween10and15 ~mnOIpresentincontrol sampleswillindicatethepresenceof SiC.Purthermcre.the amplitude oflhatpeak isused

28