Atlantic of

The Pharmacokinetics of Oxytetracycline in Atlantic Cod (Gadus morhua)

By

to

SusanL.Vatche r,B.Sc., Grad.Dip.Aquaculture

Athesissubmitted to the Schoolof GraduateStudies inpartialfulfillmentof therequirements for

thedegree of Masterof Science

Aquaculture

MemorialUniversity of Newfoundland

November 1999

St.John's Newfoundlan d Canada

Abstract

Under-conditions of intensefishcultureitmay be necessaryto use antibioticsinorder to mitigate disease problems.Inan effort to reduce the impactofantibioticresidual son the environmentand toeliminatethe risk of exposure of these residualstotheconsumer optimal treatmentregimesmustbedeveloped.Theseregimes must accountfor theuseof different antibioti csusedon a widerange of species held under varying culture conditions.Cultureof the Atlanticrod(Gadusmorhua)show sa greatdeal of potential andatpresentlitt leinfonn ationisavailableonthebeh aviou r of Oxytetra cyclin e (OTe) inthisspecies.

Inancffontogeneratebaselinepharmacokineticnumbers and to determinetheclearance timefromorgansfor

aTe

in Atlanticcodtwoexperiments were undertaken.Experiment I examined thepharm aco kineticsoCOTein Atlanticcodfollowinga singledoseby oral gavage {PerOs

(PO»

orintraperitoneal(IP)injection.Eighteenfish wereheldat 1O"C and administeredOTeeither intra peritoneal ly adose leve l of132.8mgOTC!kgfish or orallyatadoselevelof134.5mg OTCJkg fish.Bloodsamplesweretaken at0.5,3,6.

12.36 and 72 hrsand analyzed for OTe levels using High Pressur e Liquid Chromatography(HPLC).Noncompamnental modeling ide ntified a time tomaximum serum concentration(f-JforIPandPOadministrationof18hrs and6 hrsrespectively.

The shorter T-...:in thePO group as compared totheIPgroup isnot as expectedand ma y ha vebeen the resul t ofprecipitati onintheinject abl e solution. Maximum serum concentrations(e,...)for IP and PO were 140.8ppmand 1.04 ppmrespectively.The

higher

em....

inthe IP group is as expected and both numbersare in the same order of magnitude asliteratur e values. The relativebioavailability of OTC was calculated to be 0.8%.This low value compareswell with litera turevalues for orallyadministereddrugs.

T\4 for PO administrationwas calculated to be184hours. The PO T\4valuewasthen usedto prediet a clearancetime forOTC of38.3days from the serum and 55.5days from the muscleofcod. This number compares wellwith literature values and with results from experiment 2.

Experiment 2 examined the absorption,distribution and eliminationofoxyt etr acycline from Atlantic cod held in seawater at temperatures of IO"C and 10"C decreasingto :ZOC.

Fishwere administeredOTCin the feed at a doselevel of 120 mg OTCI kg fish over an IIday period.Onegroup of five fish were held as contro lsand fed nonmedicatedfeed.

The water temperature forhalf the medicated fish was maintainedatlQoe and forcontrol fishand the other half of the medicatedfish was decreased from lOoe to 2°C by daily increments ofO.loC .Samples of blood,muscle, gonad and liver were taken on days I, 5, 10, 20,4 1,6 1 and 101 and analyzed for OTe levelsusing HPLC.OTC in thegonad was detectedin onesampleon day I only therefore no subsequentstatisticalanalysiswas conducted.Data for theremainingthree tissue types was grouped bysampleday and temperature regime.OTe concentration forthe tissue types was plotted versus time.A two factor analysisof variance showedno significan tdifferencesinOTC concentrations between the temperature groups for serum,muscle orliver.Lack ofa temperatureeffect on antibioticclearance isunusualinpoikilothenni c animals and does not compare with resultsfound intheliteratu re.Insufficient differencesin watertemperature between the

iii

two groups,as a resultof smal l dailyincrem entaldecreas es, are believed to accountfor thisresult.

Therewere significantdifferences in OTC conce ntrationbysampleday inthe serum, muscle andlive r. A Scheffes multiplecomparisondetermined thatin serum andmuscle the differe nceswere between O'TCleve ls up to andinc ludingday 10 and OTelevels on and afterday 20.Inliver the differenceslaybetwe en ore levelsup to and includingday 41 and ore levels on and after day 61.It was determinedthat clearanceofOTe inboth temperaturegroups occurredin the serum betweenday 20 and day 41.Thiscompares well with the predictedvalue from experim ent 1 ofJ8.Jdays.Clearancefro mthe muscle in bothtemperature groupsoccurredbetw een day 41 and day 61. This compares well with thepredictedvaluefromexperiment I of 55.5days.Clearance fromthe liverin both temperaturegroupswas still occurring up to day 10 1.

These resultsindicate that residualsofOTC in cod for both temperature groupsfellbelow thelegallyacceptabl elevel of 0.1ppm byday41 in theserum and day61 in the muscle.

These fallwithinthe suggested withdraw al periodof 80 days., at or above10°C.for OTC in salmon.They alsoindicatethat a withdrawalperiodfor OTe of 80days may be acceptable in cod heldbelow lOoe. Clearance of OTefrom theliver wasnot consiste ntlybelow legal limits ineither temperature group withinthesample period.This indicatesthat whenharvesting the liver of culturedcod extendedclearance times shou ld be conside red.

Acknowledgements

Iwould lik etothank my parents for teaching me to work hardandthinkbig,Frank for hisinput during the final stages and myfamilyandfriends forputting up with thisfor the last.. ...years.

Specialthanks toDr.Tor Honberg for reviewingmythesis.Dr. JoeBrown forallowing meto breakmycontractand Dr.lyotiPatelforher well directedinput.

This research wouldnot have been possiblewith out the financia lsupportofSea Forest PlantationInc,the CanadianCentre for Fisheri esInnovation and theNationalResearch CouncilsIndustrial Resear chAssistanceProgram.ThankYou.

AdditionalthankstoJonathan Moir forhis workon developing andimplementing the proposal, Brian Blanc hardfor sharing hisuniquesamplingmethodo logies.Rodney Healeyforlookingafter-samplingat homebase and Charlie O'Driscolland DaveChafe fordeliveringthe fishthrough wind, snow,sleet and hail.ThankYou...

l1Iank:Godits over.

Table of Contents

Abstract••••...•...•....•.•.•..•••••••.•...••••••.•••••••••...•••4 ••• • • •• •• • • • • •• • • • • • • •• •• • • • • •• • ••ii Acknowledgements•••.•••••••..•...•...•••.•••••••••••..•..•..•••••.•••4• • ••••• • •••• •• • •• •• •• • • • ••• • •••• • •V Tab le of Contents•.••••.•••...••...•.•....•.••••••..••••...•... _...•••.•..•..•...vi listofTables....•...•...••••••••.•••••...•.••.••••••••.•..•.•.•••• •.•.•.. •..•...•.••.. ...••••viii Listof Figures ••••••••••••••••••.•..••.••...••.•.••.•..•.•.••.••.••••••••.•.•..•...•..•.•.•••••••••....ix

List of Tenns Used x

1.0 Introduct ion ~..•••.•.••••.••.•...••••1 2.0Materials an d Methods ..•..•...•... .•..._.•..•...•..••...•...•6

2.1Pharmaco kinetics . 2.2Temperatu re..

... ... ... ... ..6 ....11

3.0Results ~ 15

3.1 Pharmacokinetics...

3.2Temp era tu re..

. 15

. ... ...24 4.0 Discussion ...••.•...•.•... ...•...._..••....•.•..•...•.33

4.1Pharmacoki netics . 33

4.1.1Conclusion:Pharmaco kine tics.... . 51

4.2Tempe rature.. . 53

4.2.1 Conclusion:Tempe ratur e 65

5.0GeneralConclusion.•••••••••••.•••.... ...•...•...•.•. ....•... ... . .. ...67 Bibliography••••••.•••••••••.•••.•••.••••.••.•••...•....•.._••...••... .69 Appendix 1:Average Weeldy Feed Consumption of fish(%body weight J day) by IndividualTanks _•...••73 Appendix2:Weightof Fish Usedin PhannacokineticExperiment 74 Appendix 3: Temperature Data: Acclimation and Phannacokinetics Experiment...•••..••..•••..••.••.••.••.••••••.•...•...•••.:._•...•.•..•..•.•...•...•...•75

Appendix4:Dose levels of aTC Administered Per as for IndividualFish With Sample Calculation••...••...•..•...•.•••...•...••••.••••••••••..••76 Appendix5:Doselevels of aTC Administeredby IP Injec tionforIndividual Fish•....•...•... .•••..•.•...••...•••....•.•••...•.•.••.•.••.••....••..••••...•....•••....•. 77 Appendix 6: Administration and Sample Times for Pharmacokinetic Experianent••.••...•...•.•...••...•.••••••••.••••••••••••••.••.••••••••.•...•...••••..••.••••.78 Appendix 7:HPlC Method for Detection of aTCin Cod Fish Serum...•••••19 Appendix8: Recoveriesof aTC from Spiked Serum Samples..•...•••80 Appendix9: Water Temperatures for Experimental Fish 81 Appendix 10:Weights ofFish Usedin EliminationExperiment. 82 Appendix 11:Sub Weights of Fish for Elimination Experiment.•...•••.•... 83 Appendix 12:Water Temperature Profile:Bay Bulls. NF Farm Sit e,July to

November, 1994 84

Appendix 13: Water Temperatures, and Differences Between the Temperature Groups,for the 10"C,10"CDecreasing to 2"Cand Control Tanks.••...••••••.•••••••..•••.•.•.•••••...•...•••.•••••...•••••.••.•...•.•••••...85 Appendix14: Number of Fish Sampled Per Sample Day forth e Elimination Experiment ...•.•••.••.•.•...•...•...•••••.•...••...86

vii

List of Tables

TABLE 1:OXYTE'IllAC'YCLI."a LEVELS(PPM)BY SAMPLE Pf.RJOD(fIRS) INTHE SERUM OrINDIVIDUALFIsHFOR INTRAPERIToNEAL ANDPI:Ros ADMINISI1tATION...17 TABLE2:AVERACEOXYT'ETRACYCLINE CONC£N'1'RATIONS(PPM)IN THE SDUJM OF

FIsH..GROUPED BY SAMPu:Pl:RJ:oo(RRS). FORIP ANDPOADMINISTRATION...18 TABLE3: PltARMAcoKlNLllCPAIlAMETERSFOR.PEROsANDlNIRAPERCTONEAL

AoMlNlSTRATI ONOr O T C IN COD... ••...•...19 TABLE4:SUMMARY OF PuBUSHEI) PliARMACOKINETlCPARAMETERS FOR

OXYr£TRACYCUNE FORDIFFDlENT FrSHSPECIES BI:LD UNDER VARVINC ENvIRONMENTAL COl"IDrTION5.... •••••••••••••... .•....•...••...••..•••.20 TABLE5:CONCENTRATIONS OF OXYTETRA CYCL INE (PPM). GROllPEDBY SAMPLEDAY

AND TEMPERATUREREGIME" INTHE SERtlM,Museu:.UVE:RAND GoNAD OF INDIVIDUALFISH

wrra

MUN.STANDARD[RRO RANDSTANJ)ARD DEVlATION.26 TABLE 6:AVERA GE SERUM CONCENlRATIONS OF OXYrETRACY CL INE(PPM) .

GROUP ED BYTEMPERA TUREREGIME AND SAMP LE DAv,IN THESERUM. MUSCL E.,

LIvERANDGoNAD OF FISH . 28

TABLE 7:REsuLTSOFTHETWOFACTOR ANO V ATESI1NGTHEEFFE Cf OFDAY, TEMPDlA 11JR£, ANDlNrERAcnON ON THE CONCl:NTRATION OF

OlOTElllACYCLINE (PPM)IN THE SERUM OF FISH.... ...28 TABLE II:Rl:suLTS OFTHE TWO FACTORANOVATESllNCTHEEFFECf OFDAY,

TEMPtRA ltIRL. AND L"'ITERAcrION ON THE CONCl:NTRATION OF

OXYrElRACYCUNI:(PPM)INTIlEMUSCL E OFFIsH.... . . 29 TABLE':REsuLTSOFTHETWOFACfOR ANOVA~GTIlEEFlLCTOFDAY,

Tl:MPERATUJU:. AND INTERACTION ON nil: CQlIiCI:NTRATION OF

OXYTEI'RA CYCLINE(PPM) INTIlEUVI:ROf'FISH...•...•...•.29 TABLE10:Sou:Fn:'SPAIR·WISECOMPARISONSFOR EFFECT OFDAY (p<o .OS)ONTlIE CONCENTllATlON OF OXYI'ETRACYCUN'E (PPM)1NlHI: SmUM OFFIsH...•...29 TABLE11:ScHEFn::'SPAIR-WiSECOMPARISONSFOREFn:CTOFDAY(P<O.OS)ONna:

CONCENTllAnON OF OXYI'ETRACYCLINl:(PP M)1NlHI:M1..lSC1.EOFFIsH.•••...30 TABLE 12:SCHEfTE'SPAIR- WiSECOMPARISONSFOREFn:CTOFDAY (P<O.OS)ON"llIE CONCENTRATION" OF OXYTETRACYCUNI: (PPM) INTlIE LIVEROFFISH. 30

"iii

List of Figures

FIGURE 1:AVERAGEOXYTETRACYCLlNECONCENTRATIONS (PPM) lNTHE SERUMOF FISH (N=3),GROUPEDBY SAMPLE PERIOD(fIRS).FOR PER Os ADMINISTRATION

OVER TIME... ....•... ...18

FIGURE 2:AVERAGEOXYTETRACYCLlNE CONCENTRATIONS(PPM)INTHE SERUM OF FISH(N =3)"GROUPEDBY SAMPLE PERIOD(fIRS).FOR 1NrR.APERlTONEAL ADMINlSTRATIONOVERTThfE••••••••••••••... ...••••••.••••••• ••••••••.•.••••19 FICURE 3:AVERAGE OXYTETRACYCLINE CONCENTRATION(PPM),wrmSTANDARD

ERROR BARS" IN THE SERUMOF FISHOVD.TIME(DAy S) ]1 FIGURE 4:AVERAGE OXYTETRACYCLINE CONCENTRATIONS (PPM) .wrmSTANDARD

ERRORBARS, IN THE MUSCL E OF F'ISH OVER 1)ME(DAYS)... . 31 FIGURE 5:AVERAGE OxYTETRACYCLINECONCENTRATIONS(PPM), WITH STANDARD

ERRO R BARS, IN THELiVEROF FISH OVER TIME (DAYS)•... ...32

List of Terms Used

Ave- area under the plasmadrug co ncent rationversustime curve(zerotoinfinity: zero mom ent ).

ADeM- areaunder theconcen tra tio n- timeversus time curve (firstmom ent).

f3- beta.Eliminat ion rateconstant. Obtained fromterminal slopeof a semilogarithmic plotof plasma drugconcentra tionversustime curve.

Cmax- maximum observed concentra tionofadrug.

Cl- Volume of referencefluid(such as plasma)clearedof a drug by various eliminationprocessesperunit time.

Clb- Total (orsystemic)clearance.Sumof individ ual cleara nces thatcontribute tothe ove ra ll elimination of a drug.

IP- intraperitoneal.

~T- meanresiden cetime.

PerOs (PO)- Oraladminist ration.

T..,.,.- timeof maximumobserved conce ntrationof a drug from atissueor organ.

Tl/2 - half-lifeof eliminatio n.Timerequired for a givenconcentrat ionofadrug in a tissueororgantodecline by500/0 duringtheexponentialterminal phaseof the drug concentration-timeprofile.

1.0 Introduction

The Atlantic cod(Gadus morhua )isabottom dwelling gadoidwhich inhabitsthe cool- temperatewatersof the continentalshelf(Scott andScott, 1988).Codhasbeen the corne rsto neoftheAtlantic Can adian fishery for 500 yearsduringwhich time there have been marked fluctua tionsin annual landings.In1988,inaneffortto allevi ate marketi ng prob le msassociatedwiththes e fluctuations, Sea ForestPlantationLtd. ofNewfo undland inve stigat ed the potentialofcodfarmin g. Cod werecapturedlive during thehighvolume trapseaso n in the spri ng and heldand fedoverthe summer. Highqualityfillet sthenwent tomarke tduring thetraditionall y lowvolumeseason oflate fall.Althoughcodprovedto beasuitable species tofarmVibriosisbecameaproblemunderconditionsofintense culture.

Vibrios isisa diseasecausedbybacteriabelongi ng to theVibri ospp..Itis commo nin manyculturedspeciesandcauses the deathof infect edfishifnottreated.The antibiotic of cho icefortreatingVibrios is is oxytetr acycl ine(a Te).O'I'C is abroad spectrum antibiotic thatis effective agai nst arange ofGra m negat ive bacteria(Combeset al, 1972).It isused extensivelyin aquacultur e as itisrelativel yeffective, inex pensive, has lo w toxicityandiseasil yacce ssi ble (Alderm an, 1988) .

Theefficacy ofnot only O'I'C,but anyantibioticregim e,is depe nde nton thedrug reaching thetargetorgan.This, in turn,isdependenton fourfactors:

• absorption -transport of adrugfromsite ofapplication tosyste mic bloodsupply;

distribution- transport ofdrug from blood to tissues and organs.Efficacy is further dependent on the drug accumulating in tissues and organs at concentrationsadequate to killOTinhibit bacterial growth.This concentrationis referredtoas the minimum inhibitio n concentration (MIC).

metabolism - enzyme mediated anabolismor catabolismof a compound; and

elimination- excretion of a compoundfrom the body (Baggott, 1988).

Thesefourfactors represent thepharmacokinetics of a drug.Phannacokineticsis affected by the properties of the particular drug, the biologyof the host and the environmental conditionsin which itlives.Drugproperties includesuch factors as molecular weight, solubility,degree ofionization(pH and pICa) and chemical structure.These affect a drugs abilitytobepassivelydiffused, actively transported or metabolized (Guarino,1987, 1991;Ingebrigtsen, 1991).Hostfactors includethepresence of metabolic or active transpo rtsystems,plasmaprotein binding,fat or protein contentof tissues and organs.

vascularization, gastric emptying timeand nutritional status (Jacobsen,1984;Grandelet ai, 1989;Bjorklundand Bylund,1990,1991;Black, 1991;Uno,1992). Specieswhich undergo profound changesin physiology(suchasmetamorp ho sis or smoltification)or seasonalvariations inmetabolism may show variationsintheir ability to utilize a drug {Bruno,1989; McSwain, 1992;O'Haraet ai,1997).Of the many envi ronmentalfactors whichcan potentiallyaffect pharmacokinetics. the main one for poikilotherms is temperatu re.Highertemperaturesincreasetherateatwhich kinetic processes occur, lowertemperatures decreasethe rate {Bjorklund andBylund. 1990;Burka etai,1997) .

Salinity also affectspharmacokin eticsthro ughitsinfluenceonbioavailability(EJemaet at.1996;Burkaet al,1997).

The widespread use ofOTCin aquacu ltureimpliesthe existence ofan extensivedatabase onits therapeuticandchem ical pro pertie s.This isnotthe case.The main body of work conducted has been on wannl tempera te,freshwaterspecies such as carp (Grendelet al 1987,1989;Nouwsetal1992) orfreshwaterphase salmon and trout (Grond elet al, 1989;BjorklundandBylun d,199 0,19 91;BlacketaI,1991;Rogstader aI,199 1;Nouws etaI,1992;Uno,1996;Uno etaI,1992 , 1997).This impactson maricultureactivitiesin that regimes forcold!temperate, salt water species are often based on pharmacological assumptions fromwarm! temperate,fresh water species.Differencesin drug disposition indicatethat extrapolatio nof data among species maybe invalid(Ingebrigtsen,1991;

Unoet aJ,1992).Inappropriateadministrationofa drug may alsolead to problems with environmentalbuildup(BjorklundetaI,1991;Rogstad etaI,1991;Hektoen,199 5)and bacterial resistance (Lunestad etaI,1990 ;Smithetai,1994).

Forcod aquaculture it is inte nded thatthe liverandgonad,inadditio n to the fillets,will bemarketed.Because there is variability inclearancetimes for OTC,not only among speciesbut between organ systemsaswell,the industrymustworktoensurethattheyare in compliancewiththelegallimit of0.1 ppm for OTCresidu als whensettingwithd rawal times.This study,preci pitated byalack of publisheddata, representsapreliminaryeffort to establisha pharm acoki neticdata baseforcod.This datawasgeneratedbycond uct ing twoexperiments.

Ellperiment1:Pharmaco kin etics of oxytetracyclineinAtlanticcod(Gadus morhua) following asingle doseby oral gavag e or intra pe rito neal injection.

Thisexperimentwasdesign ed tomap serumconce ntrationsofOTCovertime in orderto determinemaximu mserumconcentration(C-x).timeto maximumconce ntration(T_ .) . bioavaitabi lity(F)and elimi nation half-life (T'4).This would allow for thegenerationof preliminaryestimatesofclearancetime for OTC from the bloodandmuscleof cod.

Thesenumbers were subsequently compared totheresults ofexperiment 2 andwith literaturevalues forcompara tivepurposes.

Ellperiment 2:The absorption. distribution andeliminatio nof oxytetracyclin efrom Atlant iccod(Gadusmo,hua)heldin seawater at temperatures ofconstant10°C and IOoe decreasi ngtoZOC.

This experiment wasdesign ed with three aims.To : 1) determinewhet her theabsorption.

distributionand eliminatio nof oxytetracyclineoccurs differen tiallyattwotemperatur e regim es.One regimemim ick ed thatofa cod farmbetween August (hig h riskof diseaseat 10°C) and December (harvest at2°C)andthesecondservedas a controlled comparison (constant seawater temp eratureoflO°C), 2)ide ntify the affinity ofOTefordifferent organs, and3)determi nethe eliminationtime,for regulatorypurposes,of OTefromthe serum. muscle. liver andgonadofcod.These goalswereachievedbyadministeringO'I'C tocod in thefeed.Bloodandorgansamples wereanalyzedandtheconce ntr ationofOTe mapped overa 101 dayperiod.Thesenumbers werecomp ared to resultsfrom Experiment I andtoliteraturevalues for compara tive purposes.

This projectmarks thefirsttime such pharmaco kinericexperi ments were conductedin a 100% recirculation,saltwater system.Ahigh degree of effortwas made to establishand maintai nthefacility toexperim entalprotocols.Inaddition,theseexperimentswere conductedto GoodLaboratory Practice (GLP) standards.GLP is a set of regulations, enforced bylaw in theUnitedStates.whichare designedto ensurethe qualityand validity of non-clinical laboratory studiesof materials intendedfor market.GLP guidelinesdeal withthe test facility,itspersonnel.theStandardOperatingProcedures (SOPs),the performanceof thestudy,thereporting of study resultsandthe archivingof records and materials.Itis anticipatedthat GLP (oritsequivalent) will beimplementedin Canada.When that happens.experimentsnot conductedto GLP specifications willbe invalid insofarasregulatory proceduresareconcerned.This experiment wasconducted to GLP standardsin order to familiarizeall parties concernedwiththe proceduresrequired and to commence building adatabase forpharm acoki neticsin cold,saltwaterspecies whichwouldhaveahighdegree of validityfrom a regulatory standpoint.

2.0 Materials and Methods

Atotal of189Atlanticcod(Gadusmorhua),targeted weightof 2 kg, wereobtained from SeaForestPlantation's grow out siteat Bay Bulls, Newfo undlandon January4,1995and landtransportedtothe AtlanticVet eri naryCollege facilities inPrinceEdward Island wheretheyarrived onJanuary S.199 5. Upon arrival thefishwer e rando mlyassignedto nineL9m^lfiberglasstanks in either of two 1000/0recirculati onunits(Modul es 7and 8).

Temperatureduringtransport was 2.1°C and thearrivaltemperature was 3.O"C in Module 7and4AoCin Modul e 8.The temperaturesafthemoduleswere raised over the cours e of the holdingperioduntil the experimentaltemperatures oflODewere reached.

Allfishweretagged threeweeksafter arrivalin thefacility.The fish werefed moist pellets orcho pped herringadlibitumover thecourseaf the acclim ationperiod.Dissol ved oxygen levels in all tanks weremaintained above 8ppm.

2.1Pharmacokine tics

Eighteenfishwere used in this experiment and had been heldfor74daysprior tothestart ofthe experiment.Spawningwasin progre ssand fishwerefeed ing at 1.0"/0body weight moistpellet s!day onconunencementof experiment(AppendixI).Foodwasdeniedfor twodayspriortothestartofthe experiment.

The fish were dividedequally intotwotankson March3(thre eweeks priorto experiment start) at which timethe averageweightswere2755g in Tank 2 and 3443 g in Tank3(Appendix2) with stockingdensitiesof 13.lkgfm³and 16.3/m³respectively.

Watertemperaturesin both tanks were 6.9"<;fourteen daysprior to commencemen t, 7.2"Cseven days prior to commencementand10.O"Catcommencement of experiment (Appendi x 3).

aTC for IF administration at a dosagelevel of 132.8mgt kg fish was preparedby mixing 10.79gramsofOTC·HC Iwithdoubledistilled~Oto atotalvolumeof 50mL.A period of approximately1.5 hourselapsedfrom time ofsolution preparation toinjectionby which timesome precipitati on ofthe solutionhad occurred.

Fororally administered(PO)OTC, a modified Luer Monovettesyringewithan outside diameter of15.3 mm was used for delivery.The feed! OTC mixture was made up using herring which was thawedand processedinto a paste.Thepastewaspremeasured into labeledsyringes tothe3/4 mark. The weight of each fishwas determined (Appendix2) and the requisiteamountof OTCinjected into thesyringe. Theremainingvolume in the syringewaspackedwithherring paste.Fish were orallygavagedat a dosagelevelof 134.5mgOTCIkgcod (Appendix 4).

Doselevels were to have been120mgOTCIkg fish.A calculationerrorresultedinthe administrationofhigherdoses.Asthesedoses wereat clinical lyacceptablelevelsandof the sameorderofmagnitudefor bothgroupsthenumberswereused insubsequent pharmacokineticcalculations.Theyare not consideredtohaveimpacted onthe integrit y of theexperiment.

For intraperitoneally(IP)administratedOTC,Hamilton Micrchtre Syringes(cal ibrated to 0.1milligramprecision) wereused.On administration,fish weight was determined

(Appendix2) andtherequisite amount ofOTCwasaspiratedintothe syringe.FISh wer e injectedata dosage level of132.8mg OTCIKgcod(Appendi:x5).

To collectand holdfishforadministrati on oftheOTCandblood sampling,a1- mesh vexerbarrier wasplacedintheholdingtank.dividing it into three compartmentsof adjustable size.Thefish wereheld in one compartme nt. Fishwere dip-nett ed and anaesthetized using MS222.Anaesthetized fish wereweighed and theweightsusedto calculatethe appropriate dose for thePO andIPgroups.

ForPO administration, the syringewasprepared withtheappropriateamount ofOTC sandwichedbetweentwoLayers ofherring paste.Thesyringe was placed in the oesophagusoft.'lefish,theplungerdepressedand dosedelivered,Fish were then placed inan oxygenatedrecovery bathandmonitored forsigns ofregurgitation and stress for IS minutes.After recovery,thefish werereturnedto compart ment2 of the holdingtank.

Ninefishwere orally gavaged.

For injectedfish,therequisiteamount ofOTCwas aspiratedintothe syringeand fish injectedinuap erltonea1ly ontheirleft. ventral side.Thesefish were alsoplaced in an oxygenated recovery bathandmonitoredforsigns of stress for 15minutes.After recovery the fishWCf"ereturnedtocompartment3oCth e holding tank... Nine fishwereIF injected.

Blood sampling startedathr0.5forthe IFgroup and at hr3 forthe PO group.Sam ples forboth groupswere takenthereafter at 6,12,18, 36and 72hrs(Appe ndix6).Eachfish was sampledfor blood twiceinorder to obtaintherequisite data.Atthe scheduled times

fishwere removed from theappropriatetankcompanrnentand anaesthetizedinMS222.

lbeywereplaced,ventralside up,onadampspo nge and5mLofblood extracted by venipuncture ofthecaudalvein.After the0.5,3,6and12hrbloodcollections.fishwere revivedin anoxyg enated recovery bath and returned to the housingtank.Oncompletion ofthe 18,36and

n

hrbloodcollections.fish were euthanized bysubmersion in the MS222anaestheticbathuntilISminutesafterlast signs ofopercular beat. Necrop sies were performed on allfish.

Bloodwas collectedfromthe fish using 24G1IY.l •needleswithSecbarrels.The blood washeldin5mllithiumheparin vacutainer tubesuntil centrifuged.Lithiumhep ari n is an anticoagulantwhichnormall y doesnot interfere withHPLCanalysis.Blood samples were transferred from thesyringeinto alithium heparinvacutai ner andstoredon ice.On complet ionof eachblood collection, sampleswerecentrifu ged in aBeckm an TJ-6 Centrifugeat 2,700RPMfor2Sminutesat ambienttemperature.Plasmawasthen pipert edfromthe vaeutainertubes intolabe ledayovialtubesandstored at-200<:until analysis.

All plasmasampleswereanalyzedusing High PressureLiquid Chromatograph y (HPLC) witha method refined forcod serum to a LimitofDetection(LoD)of0.05 ppm (Appendix 7).Theinstrumentation used was aPerkin ElmerISS· IOOHPLC syste m with a Series410LC pump.anLC 90 UV Spectrophoto metric Detectorand anLCI-loo LaboratoryComputing Integrator.Spikedserumsamp leswererunto determinethe recoverylevelsof theHPLCprocedure(Appendix 8).

Results were recordedandgraphedusingQuattroProPlus.The data were modeled using a least squares,nonlinear regression package. PCNONLINversion4, SCI. This package was made availablecourtesy of Dr.Tor Horsberg, Norwegian Schoo!of Veterinary Medicine,Oslo.Modeling was noncompanmentalwith extravascular administration.

10

2.2Temperature

Atlantic codWU"eheldfor 133daysina100-10recirculatedsaltwater systemprior to commencementoftbeecpe:rimern.. During this timewater"tempe:ratures were raised from 3.00c inModule 7and4.4°CinModule 8to10.1"Cinbothmodul es(Appendix9).

Waterquality was monitoredcontinuously andoncommencementof experimentOz leve ls wereat9.4 ppmand 11.4 ppminModules7and8, respectively,andun-ionized ammo nia levelswere at 0.01mgll.

Thefishhad completed a spawning eventduringthisperiod.Organic loadinginthe recir culationsystem resultedin poorwater quality witha subseque nttotal mortality of 106 fish outofl 7l overtheacclimati on period.Mortalitieshadstabilized tozerooverthe mo nth precedi ngthe experiment.

Sixtyfivefish wereusedinthis experiment,They wereweighedonMarc h29(Appe ndix 10) anda sub-sample of9fishwasagainweighed onMay2(Appendix Il).Anaverage weigtudecrease of2_6%wascalculated andthis numberwasusedtodetermi ne theOTe dosagefOTthe experiment.Tenfisheachwereallocated to six1.9m)tanks.These were the experimenta.lfish.Fivecontrolfishwereallocated to a separate 1.9m) tank.The stocking densitiesfOT the fishrangedfrom9.6 kg.'m)to14.9kg.'m).

Ofthe experimentalfish,thirtyweremaintain ed atIO"Cthroughout theexperiment (constan t temperature group).Watertempera tures forthirtyexperime ntalfish and5 co ntrolfish weredecreased from IO"C,byO.IOC daily, to 2"Con the finalsample day (decreasingtemperaturegroup).The water temperature regime of the decreasin g

11

temperature groupwascalculatedfrom theavera gedailytemperaturesatacodfarmsite betweenAugustandDecember, 1992(Appendix12).

Theconstant temperature group,decreasing temperaturegroupandcontro lfishwere held atIO"C for the IIdayperiodduring wbich theOTC wasadministered(Appendix13).

Fordelivery ofthe OTC toall experimental fish,OTC-HCLwasinjectedintothe body cavity ofwholesmelt inquantities calculatedto deliver120mgOTC/kgcod.Thesmelt were stored at -20"<:inplast icbagsina freezerandwereremo vedfourhoursprior to feeding timeand thawed.The orderof deliverytotanks was rando m.Thewaterwas disturbed byhand andsmeltoffered slowly to thecod.Thefishwere closelymonit ored and non-feeders weregavage d.Due to excessive noise in the tank roomon Administrationday S,feedin g was cancelled for thedaytoallow fishto reacclim ate.Asa result.the 10dayregimeorOTCwasadministeredover ant1dayperiod.

Variations intemperatur econtro l resulted in aO.SOCdifferencebetween the two temperaturegroups atsample day10when thereshouldhave been aIOCdifference.As thiswasnotconsideredsigni ficant. fishfrom thetwotemperature groupsweresampled as acommontemperaturegroup(to"C)tosampledaylO.Thereaftertheyweresampled astwotempera ture groups:IO"C(co nstant temperature)and1O"C decreasi ngto2"'C (decreasingtemperature) (Append ix14).Thefishwere lethally sampledathour12and ondaysS,la,20,41,61 and 101.Fish tobesampledwere selected randomlyfrom within thesamplegroupsusingMinitab generated numbers.On thefinal sampledaythe

12

weightswen:recordedforthe1O"Cdecreasingto2"Cgroup.1O"Cgroupandthecontrol (Appendix10).

Sampledfishwereplaced inananaestheticbathof MS222andleftuntil moderate anesthes iawasachieved.Bloodwastaken viacaudalvenipunctureusing5mLsyringes with 24G1 1'h.-needles.Thebloodwashelduntil centrifugedin5mLlithium hepari n Vaanainer®.CentrifugationwasdoneinaBeckman TJ-6 centrifuge at2,700RPMfor 25minutesatambienttemperatu re.Plasmawasthenpipett ed fromthe vacutainer tubes into labeledcryovial tubesandstoredat-20"'Cuntil analysis.

Fish were then euthanized by subme rsion in theanaesth eticbathuntil15minutesafter thelast signsof opercu lar beat, transferredtoa laboratoryanddissectedusin gaseptic technique.Samples of liver,gonadand muscleweretakenwith double setsof tissue placed indivi duallyinbags marked with the date,sample type,fish number and destination,All sampleswere quickfrozenandstoredlU-2O"Cinacontrolledaccess freezer.One tissue set was shipped toRPC Laboratorylnc.,Fredericton, NewBrunswick.

Onshipmentday,ocgan sampleswere packedininsulated,heavy duty styrofoam contai ners with temperatures maintainedbelowfreezin g usingmedigrad eicepacks. Shipment was via air courier withtransittimemaintai ned belowfour hours.Thesecond tissuesetwasstoredattheAtlanticVeterinary College.

HPLC analysis wasconducted byRPC Laboratory onserum(LimitofDetection (LoD) 0.5 ppm], muscle(LoD0.05ppm),liver(LoD 3 ppm) and gonad(LoD3 ppm).Results from theanalyseswere storedon QuattroProPlus.Data for thefourtissuetypeswere

13

groupedbysampledayandtemperature regime.Groupmeans,standard errors and standarddeviationswere celcelated. OTC concentra tions were plottedversus time forthe differenttissuetypes.

TheBoxCox procedure wasom to examine the shape of the observeddatadistribution andtoidentifyanydepartu res from normality.A twofactor analysis of variancewasom with sample day and temperatureasthe main effects.Significance wasset atp=O.05.If significant differen ce swere found a Scheffesmultiple compariso n(posthoc, two way.

pair wisep=O.05) wasruntodetermine wherethesediffere nces lay.

•4

3.0 Results

3.1Pharm a cokinetic s

OnMarch 22 (initiation of experiment) the average weight of PO fishwas 2445gand IP fish was30 12g.Thisrepresentsan averageweight lo ss0£31 0 g for PO fish and 431 g for lP fish from the March 3weights of2755g and3443grespectively (Appendix2).

ThePO fish received, on average, 134.5 mgOTCJkg fis h(Appendix 4)whilethe IPfish received anaverag e close

or

132.8mgOTCJkgfish (Appendix 5)

Exami nati onof the data revealed an outlier inthe PO treatmentgroupathours 3and 18 (17 ppm and6.3ppm respectively(Table 1).As thesenumbers were excessiv el yand in exp lica blyhighrelativetotheothers they were not used in thesta tist ical analysi s.It shouldbe noted that theseoutliers were from the same fishsampledattwotime periods and thiswasthe first fish gavaged.One fish also diedin thePOgroup after thefirst sam ple period.The averageserum concentrations fOTthis groupshow an initialriseto 0.76ppm athour 3withaslight rise to 1.04 ppm at hour 6.After thistimethe num bers le vel out with 0.67,0.61,0.49and 0.68ppm OTe at hours12, 18,36 and 72respectivel y (Table 2,Figure1).

One fish died inthe IP group after the firstsampleperiod.The averageserum concentrationsriseinitia llyto 67 ppm at hour 0.5 and declineto 44.8ppm then 51.2ppm at hours 6 and 12 respectively(Table2,Figure 2).Amaximum concentration of140.8 ppm is reached at hour18.This falls to 64.9ppm at hour 36andrisesagain to88.6ppm at hour 72.

15

Necropsieson all sampled fishrevealed highlevelsof mucousin thegastrointestinal

The OTCrecoveriesonspiked serum samples usingtheHPLCmethod wereconsistently high,ranging from 83 %to 108%(Appendix 8).

Modelingusing PCNONLIN gave the following results:T-... for IP and PO administrationwasat18hrs and 6 hrsrespectively.

c....x

^forIP and PO was140.8ppm and 1.04 ppmrespectively.AUC for the72 hour timeperiod forIP and PO was5802.5hr g1mland 43.8hr glm!.MRT(0-72hrs) was 36.8hr and35.9hr respectively(Table3).

The relativebioavailabilityof OTC was calculatedto be0.8%.T'I.zfor IP administration was calculatedtobe154.2hours.T'I.zforPO administrationwascalculatedto be 184 hours.

Thesenumberswere compared to literaturevalues generatedfromoth er pharmacokinet ic experiments.Asyno psis of pharmacokinet icnumbersfound in theliteratureisgiven in Table4.

16

Table 1:OI)'tetracyciineLevels (ppm) by SamplePeriod (hrs) in the SerumorInd ivid ual Fisb forIntraperitonealand PerOs Administration

March22,1995

March23, 1995

March24. 1995

Man:h25,1995

Sample Period(bn) 0.' 0.' 0.' J J J 6 6 6 6 6 6 12 12 12 12 12 12 18 18 18 18 18 18 J6 J6 J6 J6 J6 J6 72 72 72 72 72 72

17

SerumOTeLevels

".3 71.4 65.4 0.67 0.85 17 0.79 1.46 0.87 46.1 18.3 70 0.49 0.78 0.73 4.21 15.3 87 0.62 0.59 6.34 88.5 170

'"

0.61 0.37 18.6 53.1 l2J 0.47 0.87 0.70 lOS 72.1

Table 1: Anrace (hytetracydine CODteDtrations (ppm) ill theSerumor Fish, Grouped by Sample Period (hn),rorIP and PO AdministratioD.

SampleInterval (hrs) AveraeSerum Coecentrettcas m

IP PO

0.' 67.0

3 0.76

6 ".8 ^1.64

12 51.2 0.67

18 140.8 0.61

36 64.' 0.49

72 88.6 0.68

1.2

~ o

~ _ 0.8 o E

~! ^0.6

:il

0.4

u

S 0.2 o

' /\ \-.

---

----..-

o

o ^{20 40}

Tim e(h rs )

60 80

Figure1:Aver ageO.q-tdutydine COllte nt r atiolls (ppm) ill the Ser um of fish(n=3 ), Gro upe d bySamplePeriod(hn),for PerOsAdmini~rationovertime

.8

150 -r-- - -- - - - -- - -, u b

'0 c _ 100

-t--+-~---j

:8[ ~.E!: 50 +''''''...

- j

..

u

c

8

^o+---.---.----~----!

o 20 40 60

Time (hrs)

80

FiCllre2:Ave r a c cOIytetrac ydineConcen trations (ppm)in the Seru m of Fish (n:3), Gr o uped bySamplePeriod (hrs),fo rIntrape ri toneal Administr ation over tim e Table3: Pbarmacokine ticParamete rsforPe rOs and Intra pe r itone al Adm inis t ra t ion of OTCinCod

Aver a eV alues Ma:l:imumValues Minim um Values

0.79

• mI 34.035 hr-umI

1158 .84 hrs-uml

Intra PerOs

ritoneal

I '"

325 \. 9 hr-u mI

40.7hr 132466.2

~

'"

53.37 hr.mI

1.46

• mI

1957.59 ... ml

lotra PerOs

rhon eal

I' "

170 u mI

35.9br 8389.7 lu-.mI 301142.1 ... ml 1.04 umI

3S.9 hr 43.77 ta-u mI 1572.48 lu'~

la in Pu Os

ritoDcal

I '"

5802.5 hr.mI 140.8 u mI

213653.7 ... ml

~ t;~I~84~±==:i===t===±==~

^0.8%

Tmax concentration.

Cmax- Maximwnserum concentration(ppm) AUC- Areaunder thecurve

AUMC - Areaunder theconcentration-time curve (first moment) MRT- Mean retentiontime

T",-Haltlifeofctimination

19

Table 4: SummaryorPublishedPharmarokinetkParametersror O.ytetracydineferDirrerent Fish SpeciesHeldunder Varyinjl EnvironmentalConditions.

Species Researcner Year SalinityTemp. Roule Do" ^TmaxcmexT" Ellmlnaton F (,~) (celsius) (mglkg) _(h~ (ppm)(h~ (days) Atlantic BjOfldund&Bylund1990 Fresh 13-17 Per os 100110 0 127

"

salmon water

Rainbow Bjorklund&Bylund1990 Fresh 17-20 Per Cs 100/100 40.8 9

Iro", water

RaInbow Bjorklund&Bylund 1990 Fresh 5 Per Cs 75 24 3.2 213 72

trout water

Rainbow BJOOJund&Bylund1990 Fresh io Peros 75 12 5.3 "6 41

trout water

Rainbow BjOOJund &Bylund 1990 Fresh 16 PerCs 75

,

2.' 115.227

Iro", water

Rainbow_Iro"' BJorldund&Bylund 1991Freswatehr '6 Intravenous 2D

•

^{113 60.3}

Rainbow BJOOJund&Bylund 1991Fresh '6 Per Cs 75 '2 2 74.9 5.60%

trout water

Rainbow Black. 1991 to Intravenous 5 o.5 18.8 81.5

trout etal

AtlanUc Elema

'990

^{Salt 7} Intravenous 20 50.4

salmon

.ta'

^water

Atlantic Elema 1996 Salt 7 Per os 50 12 0..42 1.94%

salmon

.'01

^water

cere Grondel 1987 Fresh 2. Intravenous 60 1 247.5 139.8

"01

^waler

e""

^Grondel

.'01

^{19a7 Freshwater 2. Intramuscular 60}

"

^{58.8 78.6 80%}

e.",

Grondel 1987 Fresh 2. Per Os

eo

2. 0.11 0.60%

.'01

^water

Aflican Grondel 1989 Fresh 25 Intra 60 7 43.4 74.4 65%

catfish eta/ weter muscular

29

SpeCIes Researctlflr Vear SallnRy Temp. ROlJle

D,,,.

rmexcmexT111 Ellmlnaton F (PI'I) (celsius) (lTllVkg) II«) (ppm) Ih~ (da)'t)

Rainbow Grondel 1989

F,,"'

^{12 Intra}

^eo •

^{56.9 94.7 80%}

lto<rt

.10'

^{waler muscular}

African G...,." 1989fresh 25 Intravenous 60 0 ,O? 80.3

calfish etsl water

Rainbow Grondel 1989Fresh 12 Intravenous 60 0 7531 89.5

'ro~

""

^water

Atlantic MCSwain (MSc) 1992 Sail 15

' '''"

¹⁰⁰

,

31.5

56.'

^85.8m~hr

.. -

^{water artel1a1}

AlIantlc McSwIin(MSe) '''2 S., 10 Intra 100

,

39.9 62.2 8O.5mlfhr

salmon waler artel1a1

Atlantic MCSwain (MSc) 1992S. h 10 P" ", 100

,

0.22 12.5 5522 mt/ 1.5Mt

salmon waler hr

All. ntlc MCSwain (MSc) 1992 S. h 15 eeros 100

,

0.77 171.6933.4mlf 7.40%

salmon water

'"

Allantlc MCSwain 1992S. , _{15 tntra 100}

,

31.5 '2

salmon

""

^{water alterial}

AtlantIc MCSwain

""

^{S., 15 PerOs 100}

"'mon ""

^waler

C.", Nouws 1992Fresh 21l intra 80

"

⁵⁷

"

""

^{water muscular}

C.", NOUW5 1992Fresh

,

Intra

eo ,

"

¹⁵⁷

.,,,

waler muscular

E .,

No.... 1992 FreSh 22 Intra

"

^{18 100}

^'96

""

^{waler mllSCUlar}

RaInbow Nouws 1992 Fresh 10 Int..

eo

^{10 35 150}

'''''' . ^,,,

^{waler muscular}

eo", N"""

.,,,

1992freshwaler loontonealIntra

eo •

^{es 36}

C.", Nouws

.,,,

1992walerFresh 20 Intravenous eo >100 52 11

-

^{Researcher Year 5alinMy Temp.}

^R "", oese

'rmex crnexT,n EllminBlon F (ppt) (celsius) (mgl kg) (h~ (ppm) (hr) (days) C,,,, etalNouws 11192waterFresh 8 Intravenous 60 >'DO

'89

Rainbow No.... 11192 Fresll

"

Intrallenous 60 >100 76

"

^Rainbow

"" ""

^Nouws

""

^{waterFmh 10} Intravenous 60 >' DO 130

,rout

""

^Wiler

Rainbow

... ^, .. ^,

^{Fresh 10 PerOs 80}

..

^{0.35 50 1.25%}

lrout

, .. ^,

^wllef

C. ", N_

, .. ^,

^{Fmh 20 PerOs 80 24 0.46 58 0.36%}

""

^water

Rainbow Rogslad 1991 Fresh 1 Per OsI '50 12 '.1 278.4>1.. 2.60%

lro~

, .. ^,

^{water casulll}

~A=~lveliS ^Uno

"00

^Fresh_water ^{18 Per Os/}_cansule ^{100 34.2 53.1 9.60%}

P.altiveUs uno

"00

^{Fresh 18 Per Os! 100 2.17 83.2 3.80%}

IA" waler

'u••

~A:IiS ^Uno

''''

^{Fresh 18} Intravenous 2S 0 122 52.1 waler

Rainbow Unoet 81 1897F" 'h 13 Intt8Venous 50 0 331

"

,rout waler

Am... Uno etal

, .. ,

Fro'" 13 Per OS

'00

^{24 2.05 18}

salmon water

Rainbowtrout Uno9tal 1'192waterFresh 13 PerC s

'00 •

^{1.14 23,2 0.80%}

Vel owlail Uno ef aJ 1992

S.,

22 Per Os 'DO 3 0.89

"

water Gadus VatcherfRalnnle 1995

s.,

10

':;:oneal

^{132.8 18 140.8 154.2 0.80%}

mortlua water

Gadus Vatcher/Ralnnle

,..s S "

^{10 Per Os 134.5 8 1.04}

," _"

momua water

22

g

~ '"

-.:

;l.

s

^~

'" i ^'"

~ e.

iii

;;

!!!

:.1

~

e-

_II

_§

;l :;;

8

~

.. ..

^{ci ci}

~ ~

11

13-

s :<:

~ :;;

~

~

I e

:il

§

~

§

~ H H

~ ^~e

~ ~ ~ ~

Ii- a 0..<;0.. <lf~~5

! i ^:;; .. .. _- ..

i

Ii

~ '" ..

! ~ ! ! ~ !

^.

"

II

_{8 8 ·}

H

I ^{I I}

^!!l

^{s £!}

^!l'~

^:!

^!l'~

_~ Hu ^nt

~ HU U~i ^{s ,}

^{I I}

^J!~

~

t!l<i~Git!lca-t!liii

li~S ~I

3.2Temperature

Gonadal concentrationsof aTeintheexperimental fishwereat1.0ppmatHour12 (Table5).Therewereno detectab lelevels ofOTeatdays 5.10,20,41,61or 101.

Serumeoeceetrau cn s of OTein the experimentalgroups rose from0.8ppm (standard error0.2ppm) at hour12toamaxi mum concemntion of1.3ppm(standarderror0.2 ppm) atday 5(Table 5).Atday10OTelevelswereat1.2ppm(standarderror0.3ppm).

Afterthispointthe groupswere sampledas two temperatureeffects.Levelsinthe constant temperature group were at0ppmonday20,41, 61and10 1.Thedecreas ing temperature group had0.1ppm(standarderror0.1 ppm)on day 20and0ppmon day41, 61and 101. TheaverageserumconcentrationsofOTe in theexperimentalgroups by sampleday show aTmucatday5witha

e-..

^{of1.3ppm(Table 6;Figure 3).}

MuscleconcentrationsofOTe rosesteadilyfrom0.43ppm(stand ard error0.16 ppm) at hour12, 0.51ppm(standar derror0.13ppm)onday5 and peaked at0.76ppm (standard error0_23 ppm) on sampleday 10(Table 5).After this poilUthe groups were sampledas two temperatureeffects.The constant temperaturegroup was at 0.14ppm(standarderror 0.05ppm),0.02ppm (standard error0.01ppm), 0ppmand0ppm on sampledays20, 41,6l and10 1respectively.The decr easingtemperature group wasat 0.18ppm(standard error0.07ppm),0.08ppm (standard error0.05ppm), a ppm andappmonsampledays 20,41,61 and 101 respectively.Theaverag emuscle concentr ations ofO'FCinthe experimental groupsby sampleday show aT...atday10withae.-,.of0.76ppm (Table 6,Figure4).

Liver OTC concentrationsrose steadilyfrom 7 ppm (standarderro r 2 ppm) at hour12to 15 ppm (standard error0.7ppm)atday5to a maximum conc entrationof42 ppm (standard error 6 ppm)at day 10 (Table5). After thispointthe groupswere sampledas twotemperature effects. The constanttemperaturegroupwasat28 ppm (standarderror 5 ppm),27ppm (standarderror 8 ppm), 2 ppm (standard error I ppm) and0ppm on sample day20,41,61 and10 1respectively.The decreasingtemperature group was at17 ppm (standard error 8 ppm),17ppm(standard error3ppm),0 ppmand1ppm (standard erro r 1 ppm)on sampledays 20, 41,61and 101respectively.Theaverage liver concentrationsof OTCin theexperimental groupsbydayshow a Tnwo:at day10witha Cmaxof 42 ppm (Table6;Figure5).

One contro l fishdied during theexperiment.Liversamples in onecontro l fishatday5 and onecontrol fish at day 10 had residualsofOTC(Table5).

Analysisof theobserveddata distribution patternsfromthe BoxCox procedureindicated that a GompensTran sformation (11SQRT(LogX+1.5)wasrequiredtonormalizethe data.Thetwo factor (sample day and temperature) analysis of varianceshowed no sig nificant differencesin GTC concentrationsbetweenthe temperaturegroups for serum (Table 7),muscle(Table8) or liver(Table 9). There were significa nt differences in OTC concentration bysample day in theserum (Table7), muscle(Table8) and liver(Table9).

ASc heffesmultiple comparison detennined that in serum (Table10)and muscle(Table II) significant differenceslay were between OTClevelsup toandinclud ingday 10 and OTClevelson and after day 20.Inliver (Table12) significant differences were between OTClevels up to and includi ngday41andOTC levels on and after day61.

25

Tab le 5:Concentrations of Osytetracycline (ppm),Grouped bySampleDay and Tem peratu reRqime,in theSerum,Muscle, Liver andGonadofIn divid ual Fis bWith MeanStandardErTorand Standard Deviation

Sam p le Tempel'2tufe F.... Oc r CODttlltratiOD ro

Do, G~ Num ber S<ru ro MU5(:le Livcr Gonad

1 Common 1 0 0.17 5 0

1 Ccmmcn z 0.5 0.21 0 0

1 Cemmcn 3 I.' ^1.24

,

3

1

""- - .

^{1 0.18 n 0}

1

""-- ^,

^{0.9 026 13 0}

1

""- -

^{6 1.> 0.S2}

.

⁰

~ _, ,

⁵

""--

^Common<:0=Common^Colltrolca 11⁸10^{79 0.80.'0.1LI}ULS^{01 0.430.380.160.52}0.890.s5^{00 11}

. ,

⁷⁵¹⁸8 ⁰⁰⁰⁰00¹¹

~

1 ^CommonCemmcn^{Control 1113}14 ^0.70.'0.10.51.3^{L3 0.6O.Sl02 90.13}0.25^{01 4S18}38^{isis7 00}0

10 Common rs 0.' 1.28 63 0

10 Common 16 LS 0." 34 0

10 Common 17 1 0.21 51 0

10 Common 18 0.8 1.41 15 0

A 1.1 0.76

"

St. 0.6 05 13

e, 0.3 0_23 6

1O"C

"

^{0 0.06 13 0}

10 1000C 10 0 0.32 19 0

10 1000C 11 0 O.ll 0

10 IO"C 11 0 0.19 0

10 1000C 13 0 0 41 0

~

^1"C1"C1"C 16^{1415 0.50}0 ^{0.14O.ll0.050.490.08}0.14 ¹⁸¹¹¹⁴

^,

^{3 00}0

10 1"C 17 0 0.12

,

0

10 1"C 18 0 0.08 46 0

Mao 0.1 0.18 17

SLDcviation 0.1 0.16 17

SLE rror- 0.1 0.07 8

"

^{ic-c 19 0 0 31 0}

16

5&nJplc Tn-pcrabIrt

" .. ^ocr

Coacealntioll DO'₄₁ G~._IO"C N..._JO

... _•

^Mud<_e.cs

^u.... .... ..

"

⁰

41 1O"C JI

•

^{O.O~ 26 0}

..

^{IO"C J2}

· ^{• " •}

..

^{IO"C ] ]}

·

^{0 7 0}

..

^IO"C

_" ·

⁰

^"

⁰

M= 0.02 27

5t.Devial:ioD 0.03

"

Sc _

.. .. .. _..

^'_'^'OC'OCOC_·C

_"

^{26J7 000 0.33}0.0 1^{0.130 za}

_" • ^,

⁰⁰⁰

"

^{0 0 26 0}

..

^'OC

_"

^{0 0 17 0}

41 'OC eo 0 0 is 0

M~ 0.0 8 17

St.DeviatioQ 0.12 7

st.ueor O.O~ 7

61 ro-c

..

^{0 0 s 0}

61 ro-c 42 0 0

,

0

61 ro-c .o 0 0 0 0

61 ro-c

"

^{0 0 0 0}

§i

^{sc ro-c}_,OC

^" _..

⁰₀ ⁰₀ ⁰

^{, ,}

₀^{1 0}₀

,OC 47 0 0 0 0

a-c

..

^{0 0 0 0}

z-c

.,

0 0 0 0

a-c

so 0 0 0 0

h

^eoa...

"

^{0 0 0 0}

~

^'O"C

_"

^{0 0 0 0}

1O"C

"

^{0 0 0 0}

IO"C

"

^{0 0 0}

.

IO"C

"

^{0 0 0 0}

IO"C 56 0 0 0 0

IO"C

"

^{0 0 0 0}

~

101 ^2"C2"C ^{ss 0 0 0 0}

"

^{0 0 3 0}

101

'·C

^{60 0 0 3 0}

101 ,OC 61 0 0 0 0

10. '·C

"

^{0 0 0 0}

17

...

_D••

10 1 10 1

F;"

NVlIl w

"

..

^se~oo OCTCoocarratioa ..

Mli sde Lner

o 0

o 0

Goa. . o

...

o

SL DeviaDoo S<Emr

Tab le 6:Avera&<, Senna Coo ceutratiou of Oxytetncycliae(ppm),Grouped by TempeuturelUVme andSampleDay.illtheSenua,Musde, LiveraDd GooadofFlSb

" 'pl< Tem peratu re 0 Coattatntioll m

D'~ ^G~

...

^Muscle

...

^{Goa. ,}

I

c. _ _

0.' 0.43 7 I

5

c.__

I.J O.SI 15 0

10

c. __

1.2 0.16 42 0

20 Tcn DeereasUtIl10 0 0.14 28 0

T~ 01 0.11

"

⁰

41 TenDea'easingto 0 0.02 27 0

T~ 0 0.08

"

⁰

61 TenD«:reas ing10 0 0 2 0

T~ 0 0 0 0

101 TenDcacasing lO 0 0 0 0

T~ 0 0 I 0

"'D

^{0.5 O.OS 3 3}

Table 7:ResultsofthetworactorANOVAtestiaCtbefired. ofDay,Tempcnl vre, and (olen moa00theCODcet:ltr at iooofO¥)'tcrr aeydioc(ppm) iatheSeru mofrlSh.

T1ItoFactorAIW .rlVarialM:e <0.

I:fl'tct DF*

I

FV....

0.161

51if"1Ca.ItCe*

NS 5 NS 0.0001 0.ssec 60."

0.lS2

.. _. ^, _,

RcsiduaI 62

~~~~r:-d .su.tislia.ll"siomific:ant resu.ll

Tab le8: ResultsDC tbe two Cactol;'ANOVA tesueg the effect ofna y,Tem pera tu l:'e,and Inter acti oDon tbeConce ntr ati o Dof Oxytetracycli ne(ppm)io tbeMuscl eofFisb.

TwoFactol;'An'"lisofVari lUll:e

Etrcd DF~

T= I

6 6 62

FV"'ue 0.135 18.735 0.060

PV...e 0.7150 0.0001 0.9991 ofFrecdom.

Si cance·-"SMdenotesaSlatistit:al.lsi cantresult,

Table9:Resultsof tbetworect orANOVAtestin g the effect ofDay,Tempera ture,and Int era ction011theConce ntra t io nof Oxytetracycli ne (pp m ) in theLiverof Fish .

FVlllue 0.0004 19.372 l.012

PValue 0.9838 0.0001 0.4261

Table 10:Scheffe'spair-w ise com pa l:'isons for Effect of nay(P<O.OS)on the Concen t ra tio n of Oxytetracycl in e(ppm) in theSerumofFish

D. 1 s 10

"

⁴¹

"

,

N5

10 N5

zo

5 S

41 5 5 NS

61 5 5 N5 N5

101 5 5 N5 NS N5

51

= '

-S"'dcnotes a

•

c:antresult.

29

Table 11:Scheffe'spair-wisecomparisonsfor Effectof Oay(P<O.05 )on the Concentrati on ofOxytetracydin e(ppm)in theMuscle of Fish.

D. I s 10 10

"

⁶¹

s NS

10 NS

10 NS 5

"

^{5 5 NS}

61 5 5 NS NS

101 S S NS NS NS

Si =~" "S't denctesa =We

Tablet:z: Scheffe'spair-wise comp arisons(or Effectof Oay(P<O.05)on the Concentratio n of Oxytetracyclin e(ppm)in theLiverof Fish.

D. 1 s 10 10

"

⁶¹

s NS

10 NS NS

20 NS NS NS

"

^{NS NS NS NS}

61 S 5 S S S

101 S 5 5 S S NS

Si

= "

~S"dcnotcsastalislicallvsi earnresult,

30

_ O. c....llino T p lu••

_ ConIlUlntT I",••

150 10 0

;j ^{! I -\-- \ ---'}

0.4 _ .\

o~ h .

o

50

FiE;ure 3: AvengeO~etraeydineConcentra ti on (ppm ),witbSta nd a r d ErrorBars,inthe Seru m nfFi!;hoverTime(Da~

•

^1.2

.-

~

~

0.8

~

^0.6

•

j 0.4

~

0.2

•

s

onsan em p... ",r.

I

T "- W\

\. ~

50 100 150

Doy

Figure4:AverageO~etraeydineConcent r ati ons (ppm),with StandardErrorBars,in the MusdeofFish OverTime (Days;

31

I_ De crea sin GTemp ...

tu" j

___Constant Tempenlture 1

I

⁶⁰

I

! ~ 1~~ ~ +- I --=---4 ---'

i ~

50

'"

100 150

Fiiure5:AveraieO.,-tl:1racydin eConcentrations(ppm).with Standard ErrorBars.in the LiverofFishoverTime(Days).

32

4.0 Discussion

4. 1Pharmacokinetics

The graphed datafrom POadministration of

aT e

reveala curvewitha risein concentration to1.04ppmaTeatbour6anda steady decline to 0.49 ppm athour 36.

Theslightrise intheterminalportion oflhecurveoccurred aftertheanticipated pointof absorpti on of OTC in theserum.PO serumcurvesoften manifest as a smallpeakwith a slowbutsteady declinetonegligibleresidues (Grondeletai,1987;Nouws etat1992;

Uno,1996). Dissection s of the codfollowi ngthe finalsamplings revealedthatthe herri ngpastewaspartiallydigested atthe 36hou rsampleperiodand was fullydige sted by the 72 hour.sampleperiod. This indicatesthatdigest ionwas occurringwithin the norm alrange of 24-48hrs at lOoe(Ellisetai, 1989) .Therewere, however,large quanti tie s of gastrointestinal (GI)mucous.GImucoushasbeen noted in non-feed ingand newlyfeed ingfarmedcod (Brian Blanchard, Pers.Corron.).Feeding in wild and farmed cod is greatlyreducedduri ng the winter and throughoutthe springspawningevent.The codused inthis experimentwere transported inJanuary at2.1"Cand acclimated toIDee over a 74 day holdingperi od.Atthestanof the experimentthecod hadjust completed spawni ng and werestill atlowfeedinglevels(1.0% body weigh t! daymoist feedas opposedtothenonnal dailyintakeof5%body weight! clay).The presen ce ofGImucous was, therefore,not considered unusualhoweverthemucousmayhaveinterferedwith the efficien cy of absorptionofOTeacross the gutwall.Ingebrigtse net at(1985) fed radiolabele d tetracyclinetofishand thenfolloweditsflow through theorgansystems.

Theyfound thater day2 therehad beennegligibleabsorptionoftheOTC;most of it

33

residedinthe mucosallining ofthe gastrointestinaltract.This mayresult in a prolonged absorptionphase of OTC to systemiccirculationandexplainwhythe OTC in this experiment was still beingabsorbed past the final sampleperiod.

The grapheddata from[Padministrationreveal a curve with a doublepeak: at hours 0.5 and 18(at levels of67ppmand140.8 ppm respectively).Althoughthe concentrat ionof OTCdeclined rapidlyto64.9 ppm athour 36, there was a slightrise in thetennin al portionof the curveto the finalsample point.This differs from publisheddata inthat most absorption curves (intravenous,intra peritonealor intramuscular)are manifested as a singlepeak immediatelyafter injection witha steadydecline to negligible residuals (Grendeletal,1987. McCrackenet aJ,1975.Uno, 1996).Inthis experiment,the first peak occurs at30minutesafter injection, which is inlinewith publishedfigures.The second peak at 18 hoursis an anomalyand maybe the resultof precipitationof OTC in theinj ected solution.OTC which remainedin solutionwas absorbedimmediately;

precipitated OTC wasnot absorbed untilitdissolved.The presence of precipitous material in the viscera ofdissect edfish at 72hourssupports thistheory.This may also have contributedto the rise in the curve to the finalsampleperiod.

A physiologicalcontributionto the pattern of absorptio n(andtherefore theshapeof the curve) for bothtypes of administrationmayhavebeen enterohc:paticcirculation.This describes the process wherebya compoun d,such as OTC.isrecirculatedbetweenthe gastrointestinaltract andtheliver.OTCis transportedin blood via thehepaticportal system from the intesti ne(for orallyadministereddrugs) orfrom systemiccirculationvia thecoeliacartery (for IV,1M or IP administered drugs )to theliver.Intheliver it is

34

metabolized or biotransfbrmed,actively transported to the bileand then excretedviathe biliary routeinto theintesti nal lumen. It can thenbe excreted from the body via the faeces or reabsorbedback into the livervia the hepatic portal system.This createsa loop which allowsfor a portion of the compound tobe reabsorbedand a portion to be eliminatedfrom the body with eachcycle.The rate of reductionof the compoundwould be dependenton hepatobiliary function(lngebrigtsen, 1985;Bruno,1989;Ellis, 1989;

McSwain,1992 ; Buricaet ai, 1997).Secondary peaks for POdataatIScC support the presence of an enteroh epaticcycle(McSwain,1992). The cod used in this experiment had just completedspawning andwere in transitionfrom a nonfeeding phase where energy was beingderived fromthe liver,to a feeding phase where a portion ofthe energy was being directed to production of the liver.Longperi ods of food deprivation may induce general changes inhepato biliary function(McSwain, 1992). Strasdine and McBride (1979)found evidenceof differentialabsorption between fasting (sexually mature) and non-fasting (immature) salmon.The effect ofhepatic metabolismin species, such as cod,which undergo marked seasonal variations in feedingand use the liverasan organ of storage,might impactsigni fican tly on the pharmacokinetics of a drug andbear furtherinvestiga tio n

Thereare two methodsavailable for model ingphann aco ki net ic data:compartmental and non-compartmental.Inthe classiccompartmentalapproachthe body is viewedasone, twoorthreecompartmentswith bloodandhighlyperfused organsbeing the central compartment.Drug transport betw een theseabstractcompartments occurs with concentration differencesacting as the driving force.The most frequently usedmodelis

3S

thetwocompartment model whichdictates that theplasma concentratio ncurvesatisfy a hi-exponentia lequation.The mod elparameter sareclearance, volumeand flow.Rate constants govern transport betweencompart ments.The majori ty ofpharmacokinetic work in fishismodele dthiswayhow everthetheoretical limitationsof amodel which reduce sthe body[0twocompart ments mustberecogn ized.As aresult of these limitations,classicalpharmacokinetic modelsarc designe dto render conservative estimatesfor the elimination ofdrugs.Thisprovide s awidemargin ofsafetyforthe consumer butresultsin extendedholdingperiod sfor farmers with recently treated market fish. Bjork lundand Bylund(1990)predicted eliminationtimesof n,41 and 27 days for OTC from Atlanticsalmon heldatSoC,lOoC and 16°Crespect ivelyusing theclassical approac h.Actual elimi natio ntimeswere 40,30and20days.Thisrepresent s an overestimationof27"/oto 45% in elimination time.

Innon-co mpartmentalpharmacokineticsthebodyis see n asan arrangement of organs andsubsystemsthroughwhich blood flows andbehavesas a system controlledby positive feedback. The importantparametersarethe meanbody transit time,cardiac output,extraction ratio,clearance,number of recirculatio ns,meanresidence timeand volumeofdistribution.Assumptions madeabout the static natu re of thebody systemare greatly reducedin non-compartmentalanalysis.This mathemat ica l acknowledgement of thecomplexnature of the bodysystemrequires an increa sein themathematical complexityof the computer packagewith fewerassociatedassumption s.Italsorepresents a movetowardsincreasedprecision inpredicting elimination timesunderchanging conditionsofadministra tion,dose,temperature,age,diseasestatus andspecies offish.

36

Thismust be donewhile maintaining an acceptablemarginof safety for the consumer.

Brocklebanketal(1997) comparedthepredictedeliminationtimes of classical and noncompartmental analysis for OTe in salmon.Fora givendosage regime;theclassic pharmacokinetic approachpredicted a180 day withdrawal period, and thenon- compartmentala 108 day withdrawal period. Actualanalysis ofthe tissuesshowed that there wereno detectable residuesat day U8.Therewereno samplestaken on day108.

Assuming that the OTehad not cleared byday 108,the noncompartmental analysis renderedan under-estimation of clearance timeof9"lo.The classical approach renderedan overestimationof 34%.It is important tomovetowardsthe increasedprecisionof non- compartmental modeling however given the variationsin physiology within and among speciesthe collection of a more extensive databaseis required.Data in thisexperiment was mode lednon-co mpanmentally.

Inthefollowingdiscussionitshould be noted that there is litt lestandardization between pharmacokineticexperiments. Experimentaldifferencesindesign, data modeling,water temperatur e,salinity.therapeuticdose; route of administration,species, individualsize and seasonal variatio nsinphysiology make compariso nsbetweenpublished numbers a benchmarkingprocessratherthana precisecompariso n.Table 4 providesasummaryof pharmacckineric experiments. Only onereferenceto OTein nonsalmonid, marine fish was found(Unoet ai,1992).

Cmaxisthemaximum concentrationof a drugfound in the tissue andismeasured in parts permillion (ppm).T-.; is thetime (hi) tomaximumplasma concentration and provides an estimateof absorptio nratefora givendosage form (Barron etai,1990) _Inthe present

37