ForensicToxicology PrinciplesandConcepts
AMSTERDAM•BOSTON•HEIDELBERG•LONDON
AcademicPressisanimprintofElsevier
NicholasT.Lappas
CourtneyM.Lappas
AcademicPressisanimprintofElsevier
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Preface
Intheprefacetotheir1981book IntroductiontoForensicToxicology,editors RobertH.CraveyandRandallC.Baseltstatedthatitwastheiropinionthatupuntil 1975“. theonlypresentationsofmodernforensictoxicologythatcouldbeusedfor teachingpurposeswerean18-pagechapterbyC.P.StewartandA.Stolmanentitled Thetoxicologistandhiswork intheirbook Toxicology:MechanismsandAnalytical Methods (1960)andthefirsttwochaptersfromA.S.Curry’s PoisonDetectionin HumanOrgans (1963).”Foroneofuswhobeganteachingforensictoxicologyat thegraduatelevelin1975,thislackoftextualmaterialsuitableforbeginning studentsinforensictoxicologywasreadilyapparent.Agreatdealoftheoriginal literatureconsistedofcasereports,which,althoughimportantforpractitioners, didnotprovidestudentswiththeprinciplesandconceptsthattheyrequired.
Inthelastquarterofthetwentiethcenturyandthefirstyearsofthetwenty-first century,therehasbeenadramaticincrease(anexplosion)intheliteratureofforensic toxicology journalsandbookshaveproliferated.Thereareseveralreasonsforthis upsurge,includingrapidadvancesinmethodsofanalyses,animprovedunderstandingoftheinterpretationofpostmortemandantemortemanalyticalresults,andabetterunderstandingofproblemsspecifictoforensictoxicologists,suchaspostmortem redistributionandfactorsinfluencingdrugstability.
Assignificantandimportantastheadvancesintheliteratureofforensictoxicologyhavebeen,therehasbeenrelativelylittleliterature,otherthanreviewarticles andportionsofafewbooks,suitableforstudentsandprofessionalsbeginningtheir studyofforensictoxicology.Manybooksonthesubjectattempttocovertheentire topicinasinglevolume,incorporatingthetheoryofinstrumentalmethodsand immunologicalanalysis,drugdisposition,mechanismsofdrugaction,therapeutic andadversedrugeffects(includingpathologicalfindings),postmortemanalysis, andinterpretationaswellaschaptersonindividualdrugsofabuse.Weareofthe opinionthatatextsuitableforthebeginnershouldintroducethefundamentalprinciplesandconceptsofforensictoxicology,whichintroductorytextsinforensictoxicologyoftendonotcoveradequately.Thedetailsofinstrumentaltheoryandpractice andthetoxicologyofabuseddrugsoftenareincludedattheexpenseofthefoundationalprinciplesoftoxicology.
Thecontentin ForensicToxicology:PrinciplesandConcepts isbasedupontwo graduatecoursesinforensictoxicologythatoneofushastaughtfor40yearsto hundredsofmaster’sdegreecandidatesinforensicsciencesatTheGeorge WashingtonUniversity.Thetextisnotmeanttobeencyclopedicinnature,butrather toprovideanoverviewofthelargelyunchangingcoretenetsofthediscipline: analysis,interpretation,andreporting.
Wehopethat ForensicToxicology:PrinciplesandConcepts willserveasacore resourcenotonlyforupper-levelundergraduatestudentsandbeginninggraduate studentsstudyingforensictoxicologyand/orforensicchemistry,butalsofor scientistswhoarebeginningtheircareersinforensictoxicologylaboratories.
Wehavechosentofocusontopicsthatbeginningtoxicologystudentsgenerally willnothavebeenexposedtopreviously.Assuch,ourtextdoesnotincludetheories ofinstrumentalmethodsofanalysis,theknowledgeofwhich,althoughofparamount importance,iscommontomostbeginningstudentsinforensictoxicologywhoare, orwere,undergraduatechemistrymajors.Thesetopicsareexcludednotonly becauseafamiliaritywiththesetopicshasoftenbeenobtainedpreviouslybystudents,butalsobecausetheyaredealtwithingreatdetailinnumerousotherexcellent sources.However,sincethesestudentsgenerallydonothaveexperiencewithcertain foundationalsubjectsimportanttoforensictoxicologists,includingpharmacokinetics,pharmacodynamics,immunology,andtoxicogenomics,appendicesintroducingthesetopicshavebeenincluded.Inaddition,anappendixcontaininga reviewofselectedcasesinwhichthecoreprinciplesoftoxicologywereapplied isincluded.
Thetextcontainsthefollowingchapters:
Chapter1,TheDevelopmentofForensicToxicology isanintroductiontothe disciplinewithanemphasisonthefoundingscientistsandhistoricallandmarks demonstratingthatroughly200yearsago,thecreatorsofthisdisciplinenotonly identifiedproblemsuniquetothefield,butalsoestablishedmanyoftheprinciples thatcontinuetobeemployedinmodernforensictoxicology.
Chapter2,TheDutiesandResponsibilitiesofForensicToxicologists isasummaryofthecoreprofessionalactivitiesofforensictoxicologists analysis,interpretation,andreporting eachofwhichisthetopicofanentireunitinthebookand willbepresentedingreaterdetailinthechaptersofthoseunits.
Chapter3,ForensicToxicologyResources identifiesanumberofthebooks, journals,onlineresources,andorganizationsfromwhichinformationofdirector peripheralimportancetoforensictoxicologymaybefound.
Chapter4,TheLaboratory examinestheadministrationandfunctionsofa modernforensictoxicologylaboratory.
Chapter5,AnalyticalStrategy describesthevariousprotocolsemployedby forensictoxicologylaboratoriesforthedetectionofdrugsinbiologicalsamples.
Chapter6,SampleHandling focusesontheprinciplesunderlyingtheselection, collection,preservation,andtransmittalofsamplestothelaboratorypriortotheir analysis.
Chapter7,StorageStabilityofAnalytes describesthefactorsthatmayinfluence analytestabilityinstoredsamplesandprovidesanoverviewofthestrategies commonlyutilizedtomaximizeanalytestability.
Chapter8,AnalyticalSamples considersthecommonanduncommonsamples analyzedbyforensictoxicologists,includingthemeritsanddisadvantagesofeach.
Chapter9,SamplePreparation providesanoverviewofthemethodsofsample preparationthataremostcommonlyutilizedinforensictoxicologylaboratories.
Chapter10,MethodsofDetection,Identification,andQuantitation providesan overviewofthecriteriathatshouldbeutilizedforselectingamethodofanalysis, withafocusonthebenefitsanddisadvantages,aswellasthesourcesoferror,of severalofthemethodsthatarewidelyemployedinforensictoxicologylaboratories.
Chapter11,QualityAssuranceandQualityControl describesthecomponentsof aqualityassurance/qualitycontrolprograminaforensictoxicologylaboratory.
Chapter12,TypesofInterpretations assessestheopinionsthatcanandcannotbe madebasedonanalyticalresultsandidentifiesthosefactorsthatmayaffectthe conclusionsdrawnbyforensictoxicologists.
Chapter13,Reports isadescriptionoftheinformationthatshouldbeincludedin officialreportsofanalyticaltoxicologyresultsandanoverviewofthemannerby whichwrittenreportsshouldbeprepared.
Chapter14,Testifying isadescriptionoftheprocessofgivingsworntestimonyat depositionorincourt.Theroleoftheexpertattrial,thepreparationforandmanner ofprovidingexperttestimony,includingapresentationofthe“shoulds”and“should nots”oftestifying,arepresented.
AppendixA,PrinciplesofPharmacokinetics isapresentationofthetheoriesof drugabsorption,distribution,metabolism,andexcretion,emphasizingthosethatare ofparticularimportancetoforensictoxicologists.
AppendixB,PrinciplesofPharmacodynamics considersthemechanismsof drugactionthatareimportanttointerpretationsmadeinforensictoxicology.
AppendixC,Immunoassays explainsthoseaspectsofimmunologythatareof importancetoforensictoxicologists,includinganoverviewoftheimmunesystem andthetheoryofimmunoassays.
AppendixD,Toxicogenomics examinestheeffectsofgeneticdifferenceson pharmacokineticsandpharmacodynamicsanddescribeshowgeneticpolymorphismsmayaffecttheinterpretationofanalyticalresults.
AppendixE,FamousCasesinForensicToxicology isapresentationofspecific casesinwhichforensictoxicologyplayedanimportantrole.
Inreviewingtheliteratureforthepreparationofthisbook,wehavebeen impressedbytheintelligence,insights,andintellectualpowerthatsomanyforensic toxicologists,pastandpresent,havebroughttotheirworkandasaresult,tothe developmentofforensictoxicology.Weareappreciativeoftheireffortsandwe hopethatwehaverepresentedtheirworkaccurately.
Wearegratefulalsotoourstudents.Asiscommonforteachers,wehavelearned farmorefromourstudentsthantheyhavelearnedfromus.Asitistruethatthedose makesthepoison,itisalsotruethatthestudentsmaketheteacher:forthisweare thankfultoourmanystudents.
NicholasT.Lappas CourtneyM.Lappas
TheDevelopmentof ForensicToxicology 1
OfallofthebranchesofMedicine,thestudyofToxicologyiswithout contradictionthatwhichexcitesthemostgeneralinterest.
1.1 DEFINITIONS
1.1.1 TOXICOLOGY
MathieuJosephBonaventureOrfila
Theword“toxicology”stemsfromtheIndo-Europeanrootword tekw,meaningto fleeorrunfromwhicharederivedtheGreek toxon,bow,and the Latin, toxicum, poison(McKean,2005).
Manydefinitionsoftoxicologyhavebeenproposed,butgenerallyallemphasize thattoxicologyisthestudyofadverseeffectsproducedbydrugsandchemicals.
•“Toxicologyisthestudyoftheharmfulactionsofchemicalsonbiologictissue” (LoomisandHayes,1996).
•“Toxicologyisthestudyoftheadverseeffectsofchemicalorphysicalagentson biologicalsystems:itisthescienceofpoisons”(Hayes,2001).
•“Toxicologyisconcernedwiththedeleteriouseffectsofthesechemicalagentson alllivingsystems”(Plaa,2007).
•“Toxicologyisthestudyoftheadverseeffectsofchemicalsonlivingorganisms” (EatonandKlaassen,2001).
•“Toxicologyisthestudyoftheadverseeffectsofchemical,physicalorbiological agentsonlivingorganismsandtheecosystem,includingthepreventionand ameliorationofsuchadverseeffects”(SocietyofToxicology,2005).
•“Toxicologyisthescienceofpoisonsincludingtheirsources,chemical composition,actions,testsandantidotestheirnatureeffectsandantibodies” (Stedman’smedicaldictionary,2006).
1.1.2 POISON
Theword“poison”isthesameastheOldFrenchwordformagicpotion,which stemsfromtheLatin, potare,todrink(McKean,2005).Theuseoftheword“poison” todescribechemicalsthatcauseadverseeffectsisproblematicsinceitimpliesthat thereexistsubstancesthatproduce only adverseeffectsregardlessoftheconditions CHAPTER ForensicToxicology. http://dx.doi.org/10.1016/B978-0-12-799967-8.00001-3 Copyright © 2016ElsevierInc.Allrightsreserved.
ofexposure aconceptdiscardedbyParacelsusalmost500yearsago(seebelow). Unfortunately,thewordpoisonsisusedinthetitleofthestandardone-volumetoxicologytext, Toxicology:theBasicScienceofPoisons.Wewillattempttorefrain fromtheuseoftheword“poison”inthistextasitisnowknownthatallchemicals canproduceseriousadverseeffectsifadministeredinsufficientlylargedosesby specificroutesofadministration.Inplaceofthewordpoison,wewillusethewords “drug(s)”or“chemical(s).”
1.1.3 DRUG
Theword“drug”derivedfromtheOldFrench drogue bywayoftheMiddleDutch droguevate, whichreferredtothedriedgoodscontainedinvatsgenerally,istakento meanachemicalthatisusedfora beneficialmedicalpurpose. CodeofFederalRegulations(21CFR210.3,2015)makesthefollowingdefinitionsunderRulesfortheFoodandDrugAdministration(withemphasisadded):
•“Drugproductmeansafinisheddosageform,forexample,tablet,capsule, solution,etc.,thatcontainsan activedrugingredient generally,butnot necessarily,inassociationwithinactiveingredients.Thetermalsoincludes afinisheddosageformthatdoesnotcontainanactiveingredientbutisintended tobeusedasaplacebo.”
•Activeingredientmeans anycomponentthatisintended tofurnish pharmacologicalactivityorotherdirecteffectinthediagnosis,cure,mitigation, treatment,orpreventionofdisease,or toaffectthestructureoranyfunctionof thebodyofman orotheranimals.Thetermincludesthosecomponentsthatmay undergochemicalchangeinthemanufactureofthedrugproductandbepresent inthedrugproductinamodifiedformintendedtofurnishthespecifiedactivity oreffect.
•Inactiveingredientmeansanycomponentotherthanan activeingredient.
Basedonthesedefinitions,wewillattempttoadheretotheuseoftheword(s) “drug(s)”torefertosubstancesthatareintendedtofurnishpharmacologicalactivity ortoaffectthestructureoranyfunctionofthebodyofmanorotheranimalsandare usedintentionallyorunintentionallyforappropriateorinappropriatepurposes.We willusetheword(s)“chemical(s)”forthosesubstances,e.g.,volatileorganiccompounds,pesticide,carbonmonoxide,thatarenotintendedeitherformedicalpurposes ortoaffectthestructureoranyfunctionofthebodyofmanorotheranimals,butthat areintentionallyorunintentionallyusedormisusedfortheeffectsthattheyproduce.
1.1.4 FORENSICTOXICOLOGY
Forensictoxicology“ . hasnofutureasitisnoworganizedandwillnothaveuntil anadequatedefinitionofforensictoxicologyisreached”( Kemp,1974 ).Thisstatementdemonstratestheconfusionamongfor ensictoxicologiststhatexistedinthe not-too-distantpastastoadefinitionofth eirprofession.Initially,forensictoxicologywasreferredtoas“postmortemchemistry”andforensictoxicologists
werereferredtoas“coroner’schemists”astherolesandfunctionsthatfellwithin thepurviewofthescienceanditspractitio nerswerethedetectio nand/orquantitationofdrugspresentinpostmortemsamplesandtheinterpretationoftheresults obtained.Underthesecircumstances,fo rensictoxicologycouldbedefinedasthe scienceconcernedwithdeterminingwheth erthedeathofanindividualwascaused by,orrelatedto,theuseofadrug.This“classical”definitionisconsistentwiththe roleofforensictoxicologistsinacoroner’sormedicalexaminer’sofficeinwhich theyarepartoftheteamthatinvestigates thepossibleroleofdrugsinfatalities.As aresultoftheadditionaldemandsplaced onforensictoxicologistsbysociety, forensictoxicologyhasbecomeamuchbroaderdisciplineinthatitpresentlyencompassesadditionalaspectsoftoxicology,principallyastheyrelatetotheliving. Currently,thereareconsideredtobethreedifferenttypesofforensictoxicology: postmortemtoxicology,human-performancetesting,andforensicurinedrugtesting. Thesehavebeendefinedasfollows (SOFT/AAFS),2006)
•“Post-MortemForensicToxicology,whichdeterminestheabsenceorpresenceof drugsandtheirmetabolites,chemicalssuchasethanolandothervolatile substances,carbonmonoxideandothergases,metals,andothertoxicchemicals inhumanfluidsandtissues,andevaluatestheirroleasadeterminantor contributoryfactorinthecauseandmannerofdeath.
•Human-PerformanceForensicToxicology,whichdeterminestheabsenceor presenceofethanolandotherdrugsandchemicalsinblood,breathorother appropriatespecimen(s),andevaluatestheirroleinmodifyinghuman performanceorbehavior.
•ForensicUrineDrugTesting,1 whichdeterminestheabsenceorpresenceof drugsandtheirmetabolitesinurinetodemonstrateprioruseorabuse.”
Theclassicaldefinitionofforensictoxicologydescribesthedisciplineasretrospective,inthatitsaimistodeterminewhetherthereisacorrelationbetweenan eventofinterestandanydrugsdetectedaftertheoccurrenceofsuchanevent. Themorerecentdescriptionofthefieldincludesaprospectiveaspectofforensic toxicology,suchaspreemploymentdrugscreening,inwhichanattemptismade toidentifythepotentialhazardsofdrugusebyapersonbeforethedrugusecauses anyadverseeffects.
1.2 LANDMARKSINFORENSICTOXICOLOGY
1.2.1 EARLYACTIVITYINTOXICOLOGY
Itseemsreasonabletoassumethatthroughouthistoryhumanshavebeenconcernedwiththeadverseeffectsproducedbythenumeroussubstancestheyhave
1Thiscategoryshouldbeexpandedtoincludethedetectionofdrugsinhairandoralfluidasthesesamplesarebeingusedforthesamepurposesasurinedrugtesting.
encounteredintheirenvironment.Thew rittenexpressionofthisconcerndates backatleastasfarasthe EbersPapyrus (Sigerist,1951,p.311),whichisarecord ofmedicalknowledgeandpracticesinEgyptfromapproximately1550BCand whichdescribesnaturallyoccurringtoxicsubstancessuchashemlock,opium, andleadaswellastheirantidotes includingthosethatarenotonlyineffective and/orharmful,butalsorepugnant.Inthefourth-centuryBC,severaldangerous plantsweredescribedinthe DeHistoriaPlantarum writtenbytheGreekbotanist andphilosopherTheophrastus( Gallo,2001).Inthefirst-centuryAD,theGreek physicianPedaniusDioscorides,whoservedwiththeRomanarmyoftheemperor Nero,wrotethe MateriaMedica Dioscoridesiscreditedwiththefirstclassificationofpoisonsintoseparateclassessuchasplants,animals,andminerals( Haas, 1996).
The HsiYuanLu ,translatedvariouslyasor“Tra nslationstoCoroners”or“The WashingAwayofWrongs”(Kiel,1970;McKnight,1981),amultivolumeseriesof booksoflegalmedicinefromthethirteenth-centuryADChina,isthoughttobethe oldestextantbookonforensicmedicine(Agren,1984).Thisworkincludesalistof thedutiesandresponsibilitiesofthedistrictmagistrate,thechiefgoverningofficial foragovernmentaladministrativearea.Amongtheseveraldutiesofthemagistrate wastheinvestigationofsuspectedhomici des,includingpoisonings.Inthisduty, themagistratewasaidedbyhisassistant, thecoroner,inperformingtheinvestigationandpostmortemexaminationsasdirectedbythe HsiYuanLu.Althoughthe HsiYuanLu predatesbycenturiesthescientificeraoftoxicology,itcontains severalmethodsthatexemplifyearlyattemptsat“scientific”toxicology.One methodcalledfortheinsertionofasilverneedleintothemouthorbodycavity ofthedeceased(McKnight,1981,p.135);blackeningoftheneedlewastaken asasignofapoisoning.Althoughtherei sascientificexplana tionfortheblackeningoftheneedlesincesilvercanreact withsulfur-containingcompoundsto formblackprecipitates,th ismethodisobviouslyinadequateandfallsshortof modernrequirementsofproof,sincemost likelytheblackprecipitatesproduced wouldbeduetothereactionofthesilverwithhydrogensulfide,aproductofputrefactionandnotthedetectionofapoison( Kiel,1970).Asecondprocedurerelied onbiologicalratherthanchemicaldetection( Giles,1924).Boiledricewasplaced inthemouthofthedeceasedw hereitwaskeptforseveralhoursafterwhichitwas fedtoachicken.Theeffect,ifany,onthechickenwasnoted.Althoughthisprocedurehasnotcaughtonwithforensictoxi cologists,theuseofanimalsinforensic toxicologypersistedformanyyears(OfInterest1.1).Asprimitiveastheywere, thedevelopersoftheseearlyattemptsat“scientifictoxicology”shouldbe applaudedfortheiringeniousapplicationofobservationsinanattempttosolve theretoforeinsolubleproblems.
Inthesixteenthcentury,PhilippusTheophrastusAureolusBombastusvonHohenheim,morecommonlyandbetterknownasParacelsus,formulatedhisfamous maxim:“Inallthingsthereisapoison,andthereisnothingwithoutapoison.It dependsonlyuponthedosewhetherapoisonispoisonornot”(Ball,2006, p.229).Paracelsus,analchemist,theologian,physician,and“protoscientist,”rejected
OFINTEREST1.1THEANALYTICALFROG
AlthoughthedevelopmentoftheMarshtestandsubsequentothertestsforthedetectionofarsenic inbiologicalsampleshadbeendevelopedpriortothemiddleofthenineteenthcentury,adequate chemicalmethodswerenotavailableforthedetectionofmanyhomicidalsubstances.Forthis reason,biologicaltests,somewhatmoresophisticatedthanthosedescribedinthe HsiYuanLu, whichwereconductedusinganimalsforthedetectionofthesesubstances,persistedwellintothe latenineteenthcentury.
Reese,aleadingtoxicologistofthetime,suggestedanumberofanimalsthatwouldbesuitable foruseintoxicologicaltesting cats,rabbits,guineapigs,ormicewererecommended,butnot birdswhichweredeemedtobeunsatisfactoryforthispurpose(Reese,1889).Onesuchmethod,for thedetectionofstrychnine,aconvulsivedrug,reportedbyReesereliedontheuseoffrogs,which werereportedtobesensitivetotheeffectsofstrychnine.Thismethodwasrecommendedsince othersubstances,suchasmorphine,wereknowntointerferewithother,nonanimal-basedtestsfor thedetectionofstrychnineinbiologicalsamples.ThemethoddescribedbyReeseconsistedofthe subcutaneousinjectionintoafrogofanextractofstomachandstomachcontentsobtainedfromthe bodyofapersonsuspectedofhavingbeenpoisonedbystrychnine.Apositiveresultforstrychnine bythismethodwastheproductionofspasmsintheanimal.Sincethistestwasalsononspecificfor strychnine,itwassuggestedthatitshouldbeusedinconjunctionwithsmell,taste(theearly forensictoxicologistswerefearless),andcolortestsoftheextractpreparedfromthestomachand stomachcontents.
theworksofGalen2 thathadprevailedforcenturiesandinsteadpromulgated,among severalotherandgenerallylessaccuratetheories,afarfrommodernchemicaltheory ofdiseasesinhis Opusparamirum (Ball,2006,p.260)inwhichheconsideredthe causeofdiseasetobeabodilyimbalanceofthreesubstances salt,mercury,and sulfur.Duringhislife,Paracelsuswhowasattimes“lookeduponasamagician andquackandsometimesasaphysicianofgenius”byhiscontemporaries(Sigerist, 1951,pp.12 14),wasdrunkforagoodportionofhislife,wascastigatedasa discipleofthedevil(Ball,2006),andfailedtocooperatewithhiscontemporaries manyofwhomhetreatedwithoutrightcontemptandscorn(Davis,1993). Nonetheless,regardlessofhispersonalandprofessionalshortcomings,this antisocialpolymathisrememberedtodayasperhapsthefirsttorecognizethe significanceofdoseandoftheharmfulpotentialofallsubstances.Consideringthe scientificallybarrentimesinwhichhelived,wemustexcusehisfailuretorecognize thatotherfactors,suchastherouteofadministration,gender,age,andgeneticsmay accountforthedifferentiationamongbeneficial,innocuous,andharmfuleffects.
Althoughalchemistsandprotoscientistscontinuedtheirattemptsthroughout subsequentcenturiestounderstandtheeffectsofchemicalsonthehumanbody,it wasnotuntilthedevelopmentofthebasicdisciplinesofchemistryandbiology thatmodern,ortrulyscientific,toxicologydeveloped.Intheearlynineteenthcentury, MathieuJosephBonaventureOrfila(Figure1.1),generallyreferredtoas“TheFather
2Galen,wholivedinthesecond-centuryAD,isconsideredtobethegreatestphysicianandmedical researcherofantiquity.Manyofhistheoriesofphysiology,anatomy,andpathology,althoughcontainingseveralerrorsandmistakenconcepts,persistedintothesixteenthandseventeenthcenturies.
FIGURE1.1
MathieuJosephBonaventureOrfila.
ofToxicology,”wasattheforefrontoftheestablishmentofthescientificfoundationof moderntoxicology.3 Hestudiedthebiologicalandchemicalcharacteristicsofseveral toxicsubstancesanddevelopedandappliedmethodsofchemicalanalysisofpostmortemmaterialstodeterminewhetherdeathwascausedbyatoxicsubstance.Oneofhis mostimportantfindingswasthatdrugswereabsorbedintothebloodanddistributedto thetissuesofthebodyandthereforecouldbedetectedintissuesotherthanthoseofthe gastrointestinaltract(Coley,1991).In1813 1814,Orfilapublishedhisclassictwovolumereference, Traite´ deToxicologie:Traite´ despoisonstiresdesregnes mine ´ ral, ve ´ ge ´ talatanimaloutoxicologiege ´ ne ´ raleconsidere ` esouslesrapportsdelaphysiologie,delapathologieet lame`dicinelegale,whichisconsideredtobethefirstbookof moderntoxicology(Borzelleca,2001).Inthiswork,heclassifiedsubstancesintosix categories:corrosives,astringents,acrids,stupefyingandnarcotics,narcotic-acrids, andsepticsorputrefiants.Thispresentationoftoxicologicalprinciplesandconcepts wasanimmediatescientificsensationandtranslationssoonappearedinseveralcountriesincludingan1817abridgedtranslation, AGeneralSystemofToxicology,or,a TreatiseonPoisonsFoundintheMineral,VegetableandAnimalKingdoms,ConsideredintheirRelationswithPhysiology,PathologyandMedicalJurisprudence,inthe UnitedStatesbyJosephNancrede.
3Orfilawasalsoactiveinotherareasofforensicscience.Forexample,hepublishedpapersonthe chemicalidentificationofbloodstainsfollowingtheiraqueousextraction(Gaensslen,1983,p.74).
TheIndustrialRevolutionandthecontinuingdevelopmentofchemistryand biologyinthenineteenthcenturyandthesubsequentdevelopmentofanalytical chemistry,biochemistry,physiology,pharmacology,anatomy,pathology,andstatisticsfosteredtheinceptionandgrowthofdiversetoxicologicaldisciplines includinganalyticaltoxicology,clinical toxicology,environmentaltoxicology, veterinarytoxicology,genetictoxicolo gy,regulatorytoxicology,andforensic toxicology.Theinterdisciplinarynatureoftoxicologyisdemonstratedbythe numberofscientificdisciplinestowhichithasbeenapplied.Itisunlikelythat toxicologistswillhaveexpertiseinallofthefoundationaldisciplinesoftoxicology,buttheymusthaveatleastaworkingknowledgeofmanyandanextensiveknowledgeofoneormoreofthesedisciplinesdependingupontheirareasof specialization.
Orfilaandmanyofthefirstscientiststorefertothemselvesastoxicologistswere concernedwiththedetectionofhomicidalpoisonings.Theseearlyforensictoxicologists,whogenerallycamefromcareersinmedicine,werecrucialtothedevelopmentandestablishmentofthethreebasicrolesoftheirmaturingscience: analysis,interpretation,andreporting.Theseforbearersofthedisciplinedeveloped chemicalmethodsofanalysisthatcouldbeappliedtopostmortemsamples,applied theirknowledgeofthebasicsciencestotheinterpretationoftheanalyticalresults, andpresentedtheirfindingsinamanneracceptabletoandunderstoodbyjudgesand juries.Inshort,theyidentifiedandestablishedtherolesandfunctionsofpresent-day forensictoxicologists.
Presentedbelowisadiscussionofaselectedgroupofeventsandscientists, whichwhentakentogetherservetoillustratetheearlydevelopmentofforensic toxicology.
1.2.2 ARSENIC
Thelateeighteenthandearlynineteenthcenturiessawthecontinuingdevelopment ofthebiomedicalsciencesincludingthe“new”scienceoftoxicology,whichwas heavilydependentuponadvancesinchemistryandphysiology.Priortothedevelopmentofchemistry,theabsenceofreliablechemicalandtoxicologicalmethodsof analysismadethedetectionofdrugsandchemicals,especiallyinbiologicalsamples,difficultandgenerallyunreliable.Asaresult,suicidal,homicidalaccidental poisonings,bymeansofnaturallyoccurringmaterialssuchasmineralsandplantderivedsubstances,werewidespread.
Arsenicisoneofthenaturallyoccurringchemicalsthathasbeenusedwidely throughouthistoryasafavoredinstrumentofsuicideandhomicide,perhapseven havinghadaninfluenceonhistory.4 Inadditiontoitshomicidaluse,itwasalso
4Livia,thewifeoftheRomanemperorAugustus,wasrumoredtohavebeenoneofthemostnotorious arsenicmurderers.Shewassaidtohavebeenresponsibleforseveralmurderscommittedwitharsenic, includingthatofAugustus,sothathersoncouldascendtothethrone.Herexploitsservedasthefocus inthehistoricalfiction, I,Claudius,byRobertGraves.
widelyavailableduringthenineteenthcenturyasameansofrodentcontrol,asthe activeagentinsheepdipusedtopreventinfestationsoffarmanimals,infoods, householdremedies,andintheformofcopperarsenite(CuHAso3),itwasthe pigmentinScheele’sGreen,popularlyusedforimpartingagreencolortoseveral productsincludinginpaintsandwallpaper.Becauseofitspervasivenessinsociety, arsenicplayedacentralroleinthedevelopmentoflegalmedicineandbecauseof thiswasinstrumentalinthedevelopmentofforensictoxicologyinthenineteenth century.
Thepopularityofarsenic,usuallyintheformofthetrivalentAs2O3 or“white arsenic”asahomicidalagent,isillustratedbyreportsthatitwastheleadingcause ofknownhomicidalpoisoningsintheearlynineteenthcentury(Watson,2006a)and thatitwasthecauseof185ofthe541recordedcasesoffatalpoisoningsinEngland in1837 1838(Coley,1991).TherewereseveralreasonsforthepopularityofAs2O3 asahomicidalagent:itwasinexpensive,readilyavailable,hadasugar-likeappearance,andhadlittlesmellortaste,whichenabledthepoisonertomaskeasilyitspresenceinfoodordrink.Additionally,thesignsandsymptoms(Ellenhorn,1997, p.1540)producedbyarsenicingestion,includingsevereabdominalpain,diarrhea andvomiting,andinflammationofthegastrointestinaltract,weresimilartoother causessuchascholera,theoccurrenceofwhichintothenineteenthcenturywas notrare.Forthesereasons,and,probablymostimportantly,becauseofthelackof areliablemethodforthedetectionofarsenicinhumanremains,theuseofarsenic asahomicidalagentflourishedintheearlynineteenthcentury.
Physiciansrecognizedthatinordertoestablishthatarsenicpoisoningwasthe causeofdeathinsuspectedhomicides,areliablemethodwasrequiredbywhich arseniccouldbedetectedinhumansamples.Thisneedtoidentifyhomicidalpoisoningsbythereliabledetectionofarsenic,andbyextensionofotheragents,wasan importantstimulusto,andparalleledthedevelopmentofforensictoxicology.
Theidentificationofarsenicintheeighteenthandearlynineteenthcenturies commonlyreliedonmethodsthatarenowconsideredprimitive,suchastheproductionofagarlic-like(alliaceous)odorwhenarsenic-containingsubstanceswereheated;reductionbywhicharsenicpresentinsampleswasreducedtoitselementalform byheating;andprominently,“theliquidtests”thatconsistedoftheuseofvarious reagentsthatwouldproducecharacteristicallycoloredprecipitatesconsistentwith thepresenceofarsenic(OfInterest1.2).
Theliquidtestsincludedthereactionofsampleswithreagentssuchasammoniacalsulfateofcopper(coppersulfateinammonia),ammoniacalnitrateofsilver(silvernitrateinammonia),limewater,orsulfurettedhydrogen(hydrogensulfate) (Burney,2002),whichwereexpectedtoreactinthepresenceofarsenictoproduce coloredprecipitates.Thesetestswerenoteasilyadaptabletothedetectionofarsenic inbiologicalsamplessincetheyweredifficulttoperform,hadrelativelyhighdetectionlimits,weresubjecttoerrorsofspecificity,andwerenoteasilyadaptableto coloredbiologicalsamples(Burney,2002).Importantly,theendpointsoftheanalyses,theformationofprecipitatesofspecificcolors,requiredextensivetraining torecognize,werebytheirnaturesubjectiveduetointerpersonalvariationincolor
OFINTEREST1.2 ONTHEROADTOMARSH (CAMPBELL,1965;CAUDILL,
2009;FARRELL,1994;GOLDSMITH,1997)
Theneedforareliablemethodforthedetectionofarsenicproducedanumberofmethods,manyof whichwereincommonusepriortoMarsh’slandmarkdiscovery;allweresupplantedbytheMarsh test.
CarlWilhelmScheele,1775:Developedamethodfortheproductionofarsine(AsH3)in nonbiologicalsamples.
As2O3 þ 6Zn þ 12HNO3 / 2AsH3 þ 6Zn(NO3)2 þ 3H2O
SamuelHahnemann,1785:Developedatestinwhichthepassageofsulfuretedhydrogengas throughanacidifiedarsenicsolutiontoproduceabrightyellowprecipitateofarseniussulfide.
H2S þ HCl / As2S3
JohannDanielMetzger,1787:Determinedthatheatingarsenictrioxidewithcharcoalwould reduceittoitselementalform,amethodknownasthereductiontest.
2As2O3 þ 3C / 3CO2 þ 4As
BenjaminRush,1805:Identifiedthereactionofarsenitesandarsenateswithalkalinecopper sulfatetoproduceagreenprecipitate.
3Cu2þ þ 2(AsO4) 3 / Cu3(AsO4)2 (s)
ValentineRose,1806:AppliedtheMetzger’smethodtothedetectionofarsenicingastrictissue.
JosephHume,1809:Describedthereactionbetweensilvernitratewitharsenitestoformayellow precipitate.
3AgNO3 þ AsO3 3 /Ag3 AsO3
recognition,andweredescribedinspecifictermsthathadunclearmeanings,e.g., “thebloomofanOrleanspeach,”“lively”grassgreen,and“brilliant”lemonyellow (Burney,2006).
Althoughthesemethodsofdetectionwerenonspecific,subjecttoerrorsofinterpretationandgenerallynotapplicabletobiologicalsamples,theywereacceptedas scientificevidenceintrialsofthetime(OfInterest1.3).
Theproblemsintheapplicationofthe“liquidtests”tocomplexsamplesserved tospurinterestinthedevelopmentofanalyticalandforensictoxicology.In1813, OrfilaattemptedtodemonstratetohisstudentsinParisthattheliquidtestscould beusedtodetectarsenicincomplexsamples(Nieto-GalanandBertomeuSanchez,2006).Tohisdismay,theprecipitatesthatformedwhenthereagents wereaddedtoasampleofcoffeetowhichhehadaddedarsenicwerenotofthe anticipatedcolors.Asaresultoftheseunexpectedresults,Orfilaissaidtohave exclaimed “Toxicologydoesnotexist.”Hisextensiveground-breakingscientific effortsfollowingthisepisodewereinstrumentalinthewritingofhisclassicwork, Traite´ deToxicologie.Publicationof Traite´ deToxicologie.ThisbookandOrfila’s research,whichincludedthedevelopmentofanalyticalmethodsforthedetection ofpoisonsandthedemonstrationthatchemicalswereabsorbedintothegeneralcirculation,weremomentouseventsinthedevelopmentoftoxicologyasascientific disciplineandledtoOrfilabeingcelebrateddeservedlytodayasthe“Fatherof Toxicology.”
OFINTEREST1.3WHATA“GRUEL”DEED(ANONYMOUS,1752;EMSLEY, 2005,PP.145–147)
IforgivetheemyDearandIhopeGodwillforgivethee;buttheeshouldsthaveconsidered better,beforetheeattemptistanyThingagainstthyFather;theeshouldsthaveconsideredI wasthyownFather.
ThisstatementwasmadeshortlybeforehisdeathbyFrancisBlandy,whowasconvincedthathis sicknesshadbeencausedbyhisdaughterMary.MaryBlandy,a26-year-old“spinster”livingin Henley-on-ThamesfellinlovewithLieutenantWilliamHenryCranstoun,amarriedmanwhohid hismaritalstatusfromMary.However,Cranstoundidnothidehisdesiretomarryher,inspiteofthe objectionsofherfather.Cranstoun’sardornodoubtwasspurredonbythe10,000pounddowrythat Mary’sfuturehusbandwouldacquire.CranstounconvincedMarythatthe“powderstocleanScotch pebbles”thathegaveher,ifadministeredtoherfatherwouldchangeherfather’sresistancetotheir marriage.Mary,apparentlyextremelygullible,believedhimandperiodicallyaddedthepowderto herfather’sfoodoveraperiodofmonths,untilafinaldoseofthepowderaddedtohisgruelin Augustof1751provedfatal.MarywasbroughttotrialinFebruaryof1752forthefatalpoisoning ofherfatherwitharsenictrioxide.
DrAnthonyAldington,whohadcaredforMrBlandy,providedmedicalandscientifictestimony fortheprosecution.Hismedicalopinionswerebasedbothontheclassicsignsandsymptomsof arsenicpoisoning severepainofthegastrointestinaltractaccompaniedwithseverevomitingand diarrhea thatMrBlandyexhibitedaftereatingthegruelaswellasonpostmortemfindingsthat wereconsistentwitharsenicpoisoning.Aldington’sidentificationofarsenicwasbasedonthe detectionof“. theStenchofGarlick”uponheatingofsamplesandtheresultsofseveralofthe chemicalcolortestscommonlyusedfortheidentificationofarsenic.Hesummarizedhisresultsof thesetestsbytestifyingthataknownsampleofarsenicandthepowderfoundinMrBlandy’sgruel. correspondedsonicelyineachTrialthatIdeclareIneversawanytwoThingsinNature morealikethantheDecoctionmadewiththePowderfoundinMr.Blandy’sGruelandthat madewithwhiteArsenic.”
MaryBlandywasconvictedandsubsequentlyhangedonApril6,1752.
AdditionalcriticismsoftheliquidtestwereleviedbySirRobertChristison (Figure1.2),thepreeminentforensictoxicologistofthenineteenthcenturyinGreat Britain:
Ifwhathasbeensaidofthemodificationswhichtheliquidtestsforarsenicundergointheiractionwhentheyareappliedtovegetableandanimalfluidsbe reconsidereditwillatoncebeseenthattheyarequiteuselessinrelationto suchfluids.Ifthesolutionindeedcontainsalargeproportionofarsenicandis notdeeplycolouredallthethreewillactintheusualmanner.Butinactualpracticethesolutionsarealwaysdilutedandinthemtheliquidtestswiththeexception ofsulphurettedhydrogengaseitherdonotactatallorthrowdownprecipitatesso materiallyalteredintintfromthosewhichalonearecharacteristicoftheiraction thattheiremploymentwouldleadtofrequentmistakes.
Christison(1829)
FIGURE1.2
Christison’scharacterizationoftheproblemsoftheliquidtestswasaccurateand carriedgreatweightsinceRobertChristisonwasthepreeminenttoxicologistin GreatBritaininthefirsthalfofthenineteenthcentury.Histext, ATreatiseon PoisonsinRelationtoMedicalJurisprudence,PhysiologyandthePracticeof Physic,whichwaspublishedin1829whenhewasprofessorofmedicaljurisprudenceandpoliceattheUniversityofEdinburghinScotland,wasthefirstwork devotedtoforensictoxicologyinGreatBritain(Anonymous,1830)andthefirst bookontoxicologywritteninEnglishandpublishedinthe19thcentury(Christison, 1829,p.i).Thispublication,hisdevelopmentofanalyticalmethods,hissuccessas anexpertwitnessinforensictoxicology,andhispositionasmedicaladvisertothe CrowninScotlandfor37years(Coley,1991),broughthimsuchacceptanceand famethathefelt“. hisreputationinScottishcourtsbecamesooverpowering thathisevidencewasrarelyquestioned”(Crowther,2006).
TheproblemsofarsenicdetectioninhumanremainsraisedbyOrfila,Christison, andotherswassuccessfullyaddressedfirstbyJamesMarsh,alow-salariedchemist employedbytheEnglishgovernment,whoseworkinthisfieldwasstimulatedbythe 1832trialofJohnBodlewhohadbeenchargedwiththemurderofhistyrannical grandfather(Thorwald,1964).Marshhadparticipatedinthiscaseasanexpertfor theprosecutionandhadconductedtheprevailingstandardcolortestsforthedetectionofarsenic.Hereportedthepresenceofarsenicinthecoffeepreparedbythe defendantforhisgrandfatherandhewasconfidentofthedefendant’sguilt.
RobertChristison.
However,Bodlewasfoundinnocent.Marshwasconvincedthathisinabilitytopresentdemonstrableevidencetothejurywasinstrumentalintheacquittal.5 Asaresult ofhisfailuretoconvincethejuryofhisanalyticalfindingsinthiscase,Marsh workedtodevelopamethodofanalysisforthedetectionofarsenicinhumantissues thatwouldsolvethecourtroomandscientificproblemsassociatedwiththeexisting methods.BasedonthepriorworkofCarlWilhelmScheele6 in1775andothers (Watson,2006a),Marshdevelopedamethod,whichnowbearshisname,thatcould beemployedforthedetectionofarsenicinbiologicalsamplesandwouldproduce demonstrablepositiveresultsthatajurycouldsee(Marsh,1836).Thebasisofthe Marshtestisthereactionofarsenic-containingsamplesincludingbiologicalfluids ortissueswithhydrogengasgeneratedbythereactionofzincwithanacid,suchas sulfuricacid.Whenheated,arsinegas(As2H3) theproductofthisreaction is reducedtometallicarsenicthatmaybecollectedonasolidsurfacesuchasaglass orporcelainplate.Thepresenceoftheshinydeposit,knownasanarsenicmirror, isapositiveresult.Inthepaperreportingthedevelopmentofhismethod,Marsh statedthat
Notwithstandingtheimprovedmethodsthathaveoflatebeeninventedofdetectingthepresenceofsmallquantitiesofarsenicinthefood,inthecontentsofthe stomach,andmixedwithvariousotheranimalandvegetablematters7 aprocess wasstillwantingforseparatingitexpeditiouslyandcommodiously,andpresentingitinapureunequivocalformforexaminationbytheappropriatetests.
TheMarshtestwasananalyticalsensationbecauseitpresentedforensictoxicologistswithamethodforthedetectionofarsenicinbiologicalsamples.Althoughthe testwasnotspecificforarsenic,itcouldbeusedforthedetectionofverysmall amountsofarsenic,wasreliableinthehandsofanexperiencedchemist,andproduceddemonstrableresultsthatcouldbeshowneasilyandexplainedtoalayjury comprisedofindividualsunfamiliarwithanalyticalassays.However,inspiteof itsanalyticalmerits,theMarshtestinitiallywasmetwithmixedreviews.Alfred SwaineTaylor(Figure1.3)(Coley,1991;Rosenfeld,1985),whohadbeenappointed lecturerinmedicaljurisprudenceatGuy’sHospitalinLondonin1831andsubsequentlydevelopedawidespreadreputationandfameasaforensictoxicologist duetohistextbooksinmedicaljurisprudenceaswellashiseffectivenessasan expertwitness,wasanearlyadvocateoftheMarshtest,althoughincertaincases hedeemedittobeunnecessaryandreliedonmoretraditionalmethodsofdetection.
5Theabilitytoconvincejurorsofthevalidityofscientificevidenceisperhapsthemostimportantrole oftheexpertattrial,butitisalsooneofthemostdifficult.
6ScheelehasbeencreditedwiththediscoveryofoxygenyearspriortotheclaimsofPriestley,whois generallycreditedwiththediscovery,orLavoisier,whoclaimedthepriorityofdiscovery (Severinghaus,2003).
7Unfortunately,neithertheworkofSheelenoranyoftheotherswhodevelopedthemethodstowhich hereferredandwholaidthefoundationforhisbreakthroughwasmentionedbyMarshinthepaper describinghismethod.
FIGURE1.3
AlfredSwaineTaylor.
LessenthusiasmfortheMarshtestwasexpressedbyFresenius,therenowned Germanchemistwhocreatedthefirstjournaldedicatedexclusivelytoanalytical chemistry.FreseniusopinedthattheMarshtestwasnotsuitableforthedetection ofarsenicinorganicmatterandthattherewasapossibilitythatzincandsulfuric acidusedinthetestcouldbecontaminatedwitharsenic(Coley,1991).However, becauseMarshhadbeenawareofthe“ambiguity”(false-positiveresults)thatmight resultifhisreagentsorapparatuswerecontaminatedwitharsenic,hehadrecommendedthattheprocedureshouldbeperformedintheabsenceofasampletoensure thatanyarsenicthatwasdetecteddidnotoriginatefromeitherofthosesources.He describedtheanalysisofablank(althoughhedidnotusethatterm)consistingofthe zincandsulfuricacidreagentsintheabsenceofasampleasfollows:
Itis,therefore,necessaryfortheoperatortobecertainofthepurityofthezinc whichheemploys,andthisiseasilydonebyputtingabitofitintotheapparatus, withonlysomedilutesulfuricacid;thegasthusobtainedistobesetfireasitissuesforthejet;andifnometallicfilmisdepositedonthebitofthatglass,andno whitesublimatewithintheopentube,thezincmayberegardedasinafitstate foruse.
Marsh(1836)
Marsh’smethodnotonlygreatlyimprovedexistingmethods,butitalsostimulatedthedevelopmentofothermethodsforarsenicdetectionbyBerzeliusand Reinsch,whodevelopedamethodbywhicharsenicandothermetalsweredetected
bytheirplatingontoacoppercoilinaboilingHCLsolution(Reinsch,1842).Additionally,Gutzeitdevelopedasemiquantitativemethodforarsenicdetectioninwhich arsinegasisreactedwithnitricacidtoproduceaprecipitate,which,withnumerous modifications,wasusedintothetwentiethcentury.
TheMarshtesthadusheredintheeraofscientificanalyticaltoxicologyandwith itthemodernageofforensictoxicology.
1.2.3 THELAFARGEAFFAIR(SAUNDERS,1952;THORWALD,1964)
TheMarshtestplayedaprominentroleinacaseofhomicidalpoisoningthatcameto beknownastheLaFargeaffair.Thiscaseprovokedthesametypeofwidespread publicattentioninthenineteenthcenturyastheO.J.Simpsoncasedidinthetwentiethcentury.
TheprincipalcharactersintheLaFargeaffairwereMarieCappellandherhusband,CharlesLaFarge.Beforetheyweremarried,CharlesLaFargehadrepresentedhimselftoMarieastheownerofathrivingfoundryandafinecountry estate,neitherofwhichwastrue,andwhichcausedagreatdistresstoMarie whenshefirstsawthe“estate”afterhermarriagetothismanwhoshehardly knew.InDecember1839,shortlyaftertheirmarriage,whileMonsieurLaFarge wasinParisonabusinesstrip,hereceivedacakepreparedforhimbyhiswife. Charlesbecameillaftereatingthecakeandreturnedhomewherehewascared forbyMarie.Inspiteofor,aslaterwascharged,becauseofMarie’scare,Charles diedonJanuary13,1840.SomeoftheservantsontheLaFargeestateweresuspiciousofMadameLaFarge’sbehavior(shewouldnotallowanyoneotherthanherselftocareforherhusband)andsuspectedfoulplay.Asaresultoftheir investigation,which,amongotherfindings,revealedthatMadameLaFargehad purchasedarsenicinDecember1839 priortoMonsieurLaFarge’striptoParis, theauthoritiesconcludedthatMadame LaFargehadpoisonedherhusbandand shewaschargedwithhomicide.
Inadditiontothenonscientificevidencethattheyuncovered,theauthorities madeseveralattemptstodeterminewhethertheremainsofCharlesLaFargecontainedarsenic.Apanelof“experts”comprisedofphysiciansfromBriveswascalled upontoconductanalysesoftheexhumedremainsofCharles.Theyreportedthat theyhaddetectedarsenicinLaFarge’sstomachandstomachcontents.However, Orfila,whowasconsultedbythedefense,concludedthatthesephysicians,who wereunawareoftheMarshtest,hadusedanoutdatedandnonspecificmethodof detectionandtheirresultswerethereforenotreliable.Thecourtthenappointeda secondpanelof“experts”consistingoftwoapothecariesandachemistfrom Limoges.Respondingtothecriticismoftheresultsproducedbythephysicians fromBrives,theyappliedtheMarshtest,amethodtheyhadneverusedbefore; theyreportedthattheydidnotdetectarsenicinLaFarge’sstomachorstomachcontents.Inordertoresolvetheseveraldiscrepanciesamongtheanalyticalresults,the courtthenordereda“tie-breaker”inwhichthe“experts”formBrivesandLimoges wouldworktogethertoanalyzesamplesfromLaFarge’sexhumedbodytodetermine
whetherarsenicwasdetectableinanyoftheorgans.Thecombinedexpertsreported thatarsenicwasnotdetectedintheorgansobtainedfromtheexhumedbody.However,arsenicwasdetectedineggnogpreparedforCharlesbyMarieandalsoin Marie’smalachitebox,whichcontainedawhitepowdershehadbeenseenputting intheeggnog.
Inthemidstofthisscientificchaos,OrfilawascalledupontoexamineLaFarge’s remains.Employingwhatwasthenthestate-of-the-artMarshmethodforhis determinationofarsenic,OrfilaanalyzedthesamplesobtainedfromLaFarge’s bodyandtestifiedthathehaddetectedarsenicinthem.8 BasedonOrfila’sscientific testimonyandtheinvestigativefindings,MadameLaFargewasconvictedand sentencedtolifeinprison,althoughhersentencewascommutedaftershehadserved afewyears.MarieLaFarge’scasewasa causece´le`bre andgeneratedextensivescientificandpopulartumultsinceshehadmanysupporterswhodefendedherinnocence.Sheevenwroteamemoirthatwasapopularsuccess.
Orfila’sworkintheLaFargecasewasre ceivedenthusias ticallybymanywho welcomeditasthedawnofmoderntoxicology,whichheldthepromiseofdetectingpoisonsaswidelyusedasarsenicinthetissuesofavictimbymeansof state-of-the-artchemicalmethods.Orfila’sroleinthiscasecontributedtohis eminenceas“. oneofthefirstinternationalstarsofscience”(Crowther,2006). However,theanalyticalresultsandtheverdictwerealsogreetedwithcontroversy bythosewhoarguedthattheMarshtestwassubjecttonumerouserrorsofprocedureandinterpretation(OfInterest1.4).AmongthecriticismsoftheresultsobtainedbyOrfilabymeansoftheMarsh testwere(1)thattheresultsdidnot agreewiththoseproducedbyoriginalexpertsand(2)themethodwassosensitive thatcontaminationofthepostmortemsamplesbyarsenicinthereagentsorinthe cemeterysoilcouldhaveproducedfalse-positiveresults.However,Orfilahadconductedanalysesandobtaineddatathatanticipatedandbluntedthese,aswellas othercriticisms.Heexplainedthattheinconsistencybetweenhisresultsandthose ofthelocal“experts”wasduetotheirlackofexpertiseintheperformanceofthe test,e.g.,theyusedsamplesthatweretoosmall,theyusedaflamethatwastoo large,andtheydidnotwaitlongenoughfortheformationofthearsenicdeposit. Thesecondcriticismwasdiscountedsinc ehehaddemonstratedthatneitherthereagentsheusednorthesoilfromthecemeterycontainedarsenic,asdeterminedby theMarshtest(OfInterest1.5).Inaddition,Orfilaexplainedthatthearsenichehad detectedinthesamplestakenfromLaFarge’sbodywaspresentinaquantitythat wasmuchgreaterthantheamountofarsenicfoundnaturallyinthehumanbody.
TheLaFargeaffairdemonstratedthatnewermethodsofchemicalanalysis employedinthedetectionofchemicalsfrompostmortemsampleswerereliable onlyifprecautionsweretakentoavoidcontaminationofsamplesandifthey 8Orfilawaseminentlyqualifiedfortheseanalysessincehewasthefirsttoextractarsenicfromnongastrointestinalorgans(Eckert,1980).
OFINTEREST1.4HESHOULDHAVETAKENTHETRAIN(WEINER,1959)
OneofOrfila’sleadingcriticswasFranc¸oisVincentRaspail,adistinguishedscientistinhisown rightwhohasbeencalledthe“founderofmicrochemistry”andwhoformulatedanearlyversionof thecelltheory.
OrfilaandRaspaildisagreednotonlyaboutscientificmatters,buttheyalsohelddifferingpolitical views,whichmayhaveexacerbatedtheirscientificdisagreements Raspailwasanantimonarchist republicanwhowasjailedandexiledforhispoliticalviews,whereasOrfilasupportedthe monarchy.OneofthelongestboulevardsinParisisnamedforRaspail.
RaspailwastotestifyforthedefenseintheLaFargecase,butdidnotarriveatthecourtintimeto dosobecausehefellfromhishorseinhishastetoreachthecourtinTulle(Thomas,1974).
OFINTEREST1.5THESOILDIDIT
AlthoughthecriticismintheLaFargeaffairthatarsenicinthesoilhadcontaminatedtheremainsof CharlesLaFargewasansweredbyOrfila,the“soildidit”defensepersistedintothetwentieth centuryinthecaseofMarieBesnard(Thorwald,1964)whowasaccusedofthefatalarsenic poisoningofherhusbandandseveralrelativesandneighbors.Theexhumedbodiesofseveralofher allegedvictimswerefoundtocontainelevatedconcentrationsofarsenic.After3trials,overa periodof9years,Mariewasacquittedofallcharges,inpartasaresultofthedefensepositionthat thepresenceofarsenicintheexhumedbodiesoftheallegedvictimsmayhaveresultedfromthe actionofsoilmicrobesthatcausedthediffusionofarsenicfromthesoilintotheburiedbodies.
wereperformedbyscientistswhowerewelltrained,experienced,andexpertintheir use.Thesecaveatsremaintothisday.
1.2.4 THEBOCARMECASE(ANONYMOUS,1882;THORWALD,
1964;WENNING,2009;WHARTONANDSTILLE ´ ,1855)
Asecond“crimeofthecentury,”theBocarmecase,wassignificantnotonlyfor itssensationalism,butalsoforitsimpactonthedevelopmentofanalyticaland forensictoxicology.In1843,AlfredJulietGabrielHippolyteVisart,theCount deBocarme ´ ,marriedLydiaFougnies,thedaughterofaprosperousgrocer,in theanticipationthatfinancialgiftsfromherfatherwouldenablehimtomaintain hislifestyle onethatincludedalargemansionstaffedwithmanyservants,elaborateparties,andhuntingexpeditions.TheCountsoonrealizedthathiswife’s yearlyincomefromherfather’sestatecoupledwithhisownincomewasinsufficientforthemaintenanceofhispreferredlifestyleandhegeneratedhugedebts ofseveralthousandfrancs.GustavFougnies,theCountess’brother,whohad inheritedthemajorportionoftheirfather’sestatewasunmarriedandhadbeen inpoorhealthsincethelossofhisleg.Bocarme ´ becameimpatientwaitingfor GustavtodieanaturaldeathandthereforeplannedtomurderhimsinceLydia, herbrother’sonlyheir,wouldinherithissizableestate.Hisplanshadtobe
acceleratedwhenGustavsurprisinglyannouncedhisplanstomarry.Ofcourse, Bocarme ´ ,whowishedtospendhisanticipatedlargess,desiredtocommitthe murderinamannerthatcouldnotbeidentifiedasahomicide.Hedetermined thatpoisoningwouldbethebestwayofachievinghisgoal.
Usinganassumedname,Bocarme ´ approachedProfessorLoppens,achemist,in Ghentforinformationconcerningthepreparationofnicotinefromtobaccoleaves.9 Loppensdescribedhimthemethodtobeused,andBocarme ´ hadtheequipment necessaryfortheproceduremanufactured.Hisfirstattemptswerenotsuccessful, butultimately,afteralmostayearofeffort,Bocarme ´ obtainedasampleofnicotine thatwaslethaltotheanimalsonwhichhehadtestedit.AfterBocarme ´ hadprepared twovialsofnicotine,anamounthejudgedtobesufficientforhispurpose,heandhis wifeinvitedhisbrother-in-lawtodinneratwhichtimetheyattackedhimand attemptedtopourthenicotinedownhisthroat.Thebrother-in-lawresisted(some peoplejustwillnotcooperate)andintheensuingstruggle,nicotinewassplashed onhisclothingandbodyaswellasthefloor.However,asufficientamountwas forcedintotheGustav’smouthandhedied.TheCountesstoldtheservantsthat herbrotherhaddiedofapoplexy.AfterGustav’sdeath,Lydiadirectedtheservants towashorburnherbrother’sclothingandcrutchesandtowashthefloorwithvinegar.VinegarwasforcedintoGustav’smouthandhisbodywaswashedwithvinegar.Theservantsthoughtthattheeventsofthateveningandthebehaviorofthe Bocarme ´ swereunusualandsuspiciousand,therefore,reportedtheirconcernsto theauthoritieswhoinitiatedaninvestigation.Duetothesuspiciousbehaviorof theCountandCountess,thepresenceofchemicalburnsonthesideofGustav’s mouthandahumanbitemarkonhishand,theauthoritiessuspectedthatthecause ofdeathwasnotapoplexy.Therefore,theyhadthebodyexaminedbyphysicians whoconcludedthattherewasnosignofnaturaldeathandthatpoisoningwas indicated.10
JeanServaisStas,a37-yearold,brilliantchemistattheE ´ coleRoyaleMilitairewasaskedtodeterminewhetherany poisonscouldbedetectedinthetissuesofGustavFougnies.Itwaswidelyacceptedatthistimethat“vegetable alkaloids,”i.e.,nitrogenousbasesfoundinplants,couldnotbedetectedin humantissuebecauseofthecomplexityofthetissuematrixwithitsmany potentiallyinterferingsubstancesthatmadeitdifficulttopurifythealkaloids sufficientlytoapplyavailablemethodsofdetection.EventhegreatOrfila, whomStashadassistedinParis,was ofthisopinionandhadstatedonlya fewyearsearlierthattherewasnoacceptedmethodfortheextractionofvegetablealkaloids,suchasnic otine,fromhumanremains, andthatthedetectionof thesematerialsfromhumanremainsmightneverbepossible(Wenning,2009 )!
9Bocarme ´ developedhismethodsundertherusethathewaspreparingaunique eau-de-cologne or pesticide(Wenning,2009)!
10Thephysicianserroneouslysurmisedthatthechemicalburnswereduetosulfuricacid;itwaslater concludedbyStasthattheywereduetothevinegarusedbytheBocarme ´ s.
However,Stasdevelopedamethod,nowknownasliquid liquidextraction,by whichthenicotinewasextractablefromsamplesintoorganicsolvents.Themethod involvedtheseparationofthenicotinef rom“animalmatter”throughaseriesof extractionsofanalkalinizedaqueousportionofthesamplewithether.Theresidue thatremainedaftertheevaporationofetherwastestednotonlywiththestandard testsofthedayfortheidentificationofpurenicotine,butalsobytheodorof nicotineandthatofmouseurine,anodorassociatedwithnicotine aunique eau-de-cologne indeed(Wenning,2009)!Onthebasisofhisanalysis,Stas concludedthatthebodyofthebro ther-in-lawcontainednicotine.
BasedontheevidencepresentedbyStasaswellasadditionalevidencedevelopedbytheinvestigators,theCountdeBocarmewasconvictedandguillotined. However,theCountessdeBocarmewhosaidthatsheknewofherhusband’sactivitiesandgoals,butdidnothingtostophimbecauseherhusbandhadthreatenedher andshefearedforherlife,wasacquitted.Lydiaindeedledacharmedlife;shortly afterheracquittalshereceivedabequestofseveralhundredthousandfrancsfrom theestateofanEnglishmanwhosepriorproposalofmarriageshehadrefused (Anonymous,1885).
ThemethodofStaswasmodifiedin1851byOttofortheremovaloffats.ThesocalledStas Ottoliquid liquidextraction,althoughmodifiedseveraltimesinthe ensuingyears,remainsthebasisfortheliquid liquidandsolid-phaseexactions usedinforensictoxicologylaboratoriestothisday.
Apartfromitssignificanceinthedevelopmentofanalyticaltoxicology,itisalso ofinteresttonotethatthemethoddevelopedbyStas,whichwaslargelyresponsible fortheconvictionofCountdeBocarme,hadbeendevelopedspecificallyforthis caseandhadnotbeenevaluatedpreviouslybyotherforensictoxicologistsprior tothetimeatwhichtheresultsobtainedfromitsusewerepresentedandaccepted astrialevidence.Theuseandacceptanceofanovel,untestedanalyticalmethod inacriminalinvestigationwasalsosignificantinacase themurdertrialofCarl Coppolino thatwouldoccuralmost100yearslater.
Inthenineteenthcentury,theworkofOrfila,Christison,andMarshspearheaded thedevelopmentofforensictoxicology.Theauthorsofseveraltextsofmedicaljurisprudenceattemptedtoincorporateforensictoxicologyasanintegralcomponentof medicaleducationandpractice.However,bythelatenineteenthandearlytwentieth centuries,thecomplexityofforensictoxicologyhadbecome“ toodelicateforthe medicalprofession”(Crowther,2006),anditwasentrustedtothosescientistswhose trainingandeducationhadpreparedthemforthisspecializedprofession.Forensic toxicologyhadbecomeascienceuntoitself.
1.3 FORENSICTOXICOLOGYINTHEUNITEDSTATES
Thedevelopmentofforensictoxicology whichwastakingplaceinEuropeinthe nineteenthcenturywasslowincrossing theAtlantic.Thepublicationofbooksin theUnitedStatesinthemid-eighte enthcenturyonthetopicofmedical
jurisprudence(Niyogi,1980)suchasDean’s AManualofMedicalJurisprudence in1845and ATreatiseonMedicalJurisprudence in1855coauthoredbyWharton, anattorney,andStille ´ ,aphysician,devotedsignificantspacetoforensictoxicology issues,includinggeneralconceptsofforensictoxicologyandthediagnosisof poisoningbyelements,organicandmineralacids,andvariousnaturalproducts. However,thebookconsideredtobethefirstAmericanbookdevotedtotoxicology, MicrochemistryofPoisons ,byT.G.Wormley,wasnotpublisheduntil1867 more than50yearsafterOrfila’sclassicwork( Borzelleca,2001 ).Wormley’stext includedathoroughpresentationofthe chemistryandtoxicologyofanumber ofpoisons,aswellasanoverviewofdetectionmethodsincluding,asappropriate, drawingsofthecrystalsproducedbythe reactionofvarioussubstanceswithspecificreagents.Thesuccessofthefirsted itionledtothepublica tionofasecondeditionin1885thatwaspraisedasmeriting“ aseparateplaceinmedicalliterature occupyingthemiddlegroundbetweenlegalmedicineandmedicalchemistry.To eachofthesebranchesitisaninvaluable,and,wemaysayindispensableadjunct” (Anonymous,1885).Subsequently,intheearlytwentiethcentury,severaltexts andresearchpapersdealingwiththesymptomsanddetectionofpoisonswere published.
ThelandmarkeventinthedevelopmentofforensictoxicologyintheUnited StateswastheestablishmentofaforensictoxicologylaboratoryintheNew YorkCityMedicalExaminer’sOfficein1918,whichfollowedtheestablishment ofamedicalexaminer’ssysteminthatcity(Freimuth,1983).AlexanderGettler, thefirstdirectorofthislaboratory,tookonthisdutyinadditiontohisdutiesas apathologicalchemistatBellevueHospita landaninstructorattheBellevueMedicalSchool(Freireich,1969).Gettlerandhislaborator ystaffdevelopedoradapted methodsforthedetectionforanumberofsub stancesincludingethanol,methanol, carbonmonoxide,cyanide,andchloroformandprovidedinterpretationsofanalyticalresults.ThethoroughnessofGettler’sworkisexemplifiedbyhisreportthathe evaluated58methodsfortheidentificatio nofmethanolinapproximately250liquorsandmorethan700samplesofhumanorgans(Gettler,1920)!Inlaterlife, herecountedthecircumstancesofseveralcasesinwhichthepresenceorabsence ofsubstancessuchasfluoride,chloroform,andcarbonmonoxideledtotheresolutionofthecases(Gallo,2001;Gettler,1956).NotonlywasGettler’sdirectinfluenceonanalyticalandforensictoxicolog yextensive,buthisinfluenceultimately spreadfarbeyondNewYorkasseveraloftheforensictoxicologistswhomhehad traineddisseminatedtheirknowledgean dskillsthroughouttheUnitedStates. ThesescientistsandthelocationsoftheirownlaboratoriesincludeHenryFreimuth inMaryland;LeoGoldbaumatTheArmed ForcesInstituteofPathology;C.J. UmbergerinNewYork;IrvingSunshineinOhio;andSidneyKayeinPuerto Rico( Eckert,1980).ThesepioneerswhohadbeentrainedbyGettlerinturn trainedanewgenerationofscientists, manyofwhomareactivepractitioners, whofurtherdisseminatedtheknowledgean dspecialskillsofforensictoxicology. Forensictoxicologytrulyisayoungandcontinuallydevelopingscientific disciplineintheUnitedStates.
1.4 FORENSICTOXICOLOGYGROWINGPAINS
Eventhoughthepioneersofforensictoxicologywerebrilliant,perceptivemenwho laidthefoundationsofmanyofthepresentprinciplesandconceptsofforensictoxicologyoftenfoundthemselvesenmeshedincontroversyresultingfromeitherthe growingpainsoftheprofessionortheirlessthanstellarbehavior.
AlfredSwaineTaylor,1806 1880,arenownedtoxicologistofhistime,hasbeen called“thefatherofBritishforensicmedicine”(Rosenfeld,1985)andwas“recognizedastheleadingmedicaljuristinEngland”(Coley,1991).Hewroteseveraltexts onmedicaljurisprudenceincludingthe ManualofMedicalJurisprudence in1846, morethanone-thirdofwhichdealtwithissuesoftoxicology.Tayloralsowasan earlyadvocateoftheMarshtestandgavethefirstcourseonmedicaljurisprudence inEngland(Rosenfeld,1985).However,Taylor’sreputationwassulliedbyhis participationintwonotablecases(Coley,1991).In1856,DrWilliamPalmerwas accusedofmurderingJ.P.Cook,agamblerandhorseowner.His1856trialwas oneofthefirstofseveral“trialsofthecentury”thatweretooccurthroughoutsubsequentdecades.Thecasehingedontwopiecesofevidence:Cookhaddiedwhile sufferingsevereconvulsionsandTaylorhadfailedtodetectstrychnineinCook’s stomachcontents(Watson,2006b).Taylortestifiedfortheprosecutionthatinspite ofhisfailuretodetectstrychnine,theconvulsivesymptomswereconsistentwith strychninepoisoning.Thistestimonywasgiveneventhoughhehadwritten“Inrelationtoexternalappearances,therearenoneindicativeofpoisoninguponwhichwe canrely”(Burney,2006).Theseveraldefenseexpertscounteredthattherewere severaldiseasesthatcouldcausethetypeofconvulsionsthatCookhaddisplayed (Watson,2010).However,despitethetestimonyofthedefenseexperts,Palmer wasconvictedandsentencedtodeath.Interestingly,onthegallowshisfinal wordswere“. Iaminnocent,ofpoisoningCookbystrychnine”not“Ididn’t killCook”(Anonymous,1856).ThePalmercasedemonstratedtheproblemsthat mayoccurwhenthereisaninconsistencybetweenanalyticalresultsandsigns andsymptoms suchproblemspersisttothisday.
Tayloragainwasinthecenterofcontroversyinthe1859trialofthephysician, ThomasSmethurst,whowasaccusedofthemurderofIsabellaBankes,thewoman withwhomhewaslivinginanapparentlybigamousrelationship.Taylorwasasked toanalyzeasampleobtainedpriortoMissBankes’deathandhereportedthathehad detectedarsenicbymeansoftheReinschtest.ThisfindingledtothearrestofDr Smethurstandhewassubsequentlyconvictedofmurder.BecauseofdefensetestimonyrefutingTaylor’sevidenceandhisadmissionthathisresultwasafalsepositive,duetotheuseofarsenic-contaminatedcopperfoilintheanalysis,Smethurst wasgrantedapardononappeal;thispardonwasapparentlywarrantedsinceamodernopinionisthatMissBankeswassufferingfromCrohn’sdisease(Fielding,1985).
EventhegreatOrfilawasnotimmunetoignominy.In1818,hepublisheda bookonpoisonsintendedforanontechnicalreadershipandwasaccusedofplagiarismbyHectorChaussierwhoclaimedthatOrfilahadtakenideas,“eveninthe errors,”fromChaussier’searlierpublishedbookandincludedtheminhisown
