An!integrative!study!of!the!effects!of!exposure!to ...UNIVERSIDADE!DE!VIGO! FACULTAD!DE!CIENCIAS!...

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UNIVERSIDADE!DE!VIGO!

FACULTAD!DE!CIENCIAS!

DEPARTAMENTO!DE!QUÍMICA!FÍSICA!

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An!integrative!study!of!the!effects!of!exposure!to!

arsenic!in!Corbicula*fluminea!(Müller,!1744)!

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Ourense!2012!

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UNIVERSIDADE!DE!VIGO!

FACULTAD!DE!CIENCIAS!

DEPARTAMENTO!DE!QUÍMICA!FÍSICA!

D.!JOSÉ!LUIS!CAPELO!MARTÍNEZ,!INVESTIGADOR!IPP!DEL!DEPARTAMENTO!DE!QUÍMICA!

FISICA,! FACULTAD! DE! CIENCIAS,! CAMPUS! DE! OURENSE,! UNIVERSIDAD! DE! VIGO,! Y! D.!

MÁRIO!EMANUEL!CAMPOS!DE! SOUSA!DINIZ,! INVESTIGADOR!AUXILIAR!DE! LA! SECCIÓN!

DE! BIOQÍMICA! Y! BIOFÍSICA,! DEPARTAMENTO! DE! QUÍMICA,! FACULTAD! DE! CIENCIAS! Y!

TECNOLOGÍA,!UNIVERSIDADE!NOVA!DE!LISBOA!(PORTUGAL),!

INFORMAN:!

Que!el! trabajo!descrito!en! la!presente!memória!con!el! título:! “An!Integrative!study!of! the!

effects! of! exposure! to! arsenic! in! Corbicula* fluminea! (Müller,! 1744)”,! presentado! por! el!

Licenciado!en!Química!Aplicada,!Master!en!Biocorganica,!Master!en!Ciencia!y!Tecnologia!de!

los!Alimentos!y!Doctor! en!Bioquímica,!D.!Hugo!Miguel!Baptista!Carreira!dos!Santos!para!

optar!al!grado!de!Doctor!ha!sido!realizado!bajo!nuestra!dirección!en!el!Departamento!de!

Química!Física!de!la!Universidad!de!Vigo,!Campus!de!Ourense.!

Y,!para!que!así!conste,!firman!la!presente!en!Ourense!a!miércoles!17!de!octubre!de!2012.!

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Mário*Emanuel*Campus*de*Sousa*Diniz,*Ph.D*

! José*Luis*Capelo*Martínez,*Ph.D*

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Acknowledgements!

Giving&thanks&because&of&the&successful&ending&of&a&hard&work&is&indeed&something&nice&to&be&

done.&And&this&is&because&with&my&doctoral&thesis&finally&completed,&I&can&see&the&way&I&have&

done&to&get&this&moment.&It&has&been,&it&is&and&I&hope&it&will&be&a&never&ending&way&to&wisdom.&

I&would&like&to&thanks&to&my&supervisors&Prof.&Mário&Diniz&and&Prof.&José&Luis&Capelo&for&their&

help&guidance&through&this&work.&

To&the&University&of&Vigo&for&giving&me&the&possibility&for&work&on&their&facilities.&&

To&the&New&University&of&Lisbon&and&in&particular&to&the&Chemistry&Department&of&the&Faculty&

of&Science&and&Technology,&for&giving&me&the&possibility&to&use&its&facilities.&

To&Dr.&Pedro&Costa,&Prof.&Isabel&Peres&and&Prof.&Maria&Elena&Costa&por&the&advise&during&the&

development&of&the&in&vivo&assays.&

To&my&parents,&Leonor&and&Armindo,&and&to&my&brother&Ruben,&for&being&like&they&are&and&for&

their&constant&support&and&help.&Without&their&constant&presence&and&perseverance&in&pushing&

me&to&give&my&best.&

To&all&my&laboratory&colleagues,&especially,&to&Gonçalo,&Marco&and&Ricardo&for&their&support.&

To& Elisabete,& more& than& a& colleague& a& real& friend,& for& her& always& useful& help,& advice& and&

support.&

To&all&the&people&that&in&a&way&or&another&have&contribute&to&this&work&and&that&I&had&might&

forgotten&of&named&here.&

Finally,&a&special&thanks&to&all&that&truly&friends&that&are&always&with&me…&

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Table!of!Contents!

Acknowledgements.............................................................................................................................vii.

Table.of.Contents..................................................................................................................................ix.

Resumen.................................................................................................................................................13.

Introducción..........................................................................................................................................15.

2.! Los!marcadores!biológicos!de!respuesta!–!Metalotioneinas!(MTs)!...................................!17!

3.! Cuantificación!de!metalotioneínas!por!polarografía!DPPHSDME!........................................!19!

4.! Espectrometría!de!absorción!atómica!de!Cámara!de!Grafito!...............................................!22!

5.! Histología!e!histoquímica!.....................................................................................................................!24!

6.! References!...................................................................................................................................................!25!

Objectivos...............................................................................................................................................29.

Chapter.I..Toxicological.effects.of.arsenic.exposure.in.Corbicula*fluminea.....................31.

I.1.! Abstract!......................................................................................................................................................!31!

I.2.! Materials!and!Methods!........................................................................................................................!32!

I.2.1.! Experimental!Procedure!............................................................................................................!32!

I.2.2.! Histology!and!Histochemistry!..................................................................................................!32!

I.2.3.! Detection!of!Metallothionein!....................................................................................................!33!

I.2.4.! Metallothionein!Determination!...............................................................................................!33!

Table!of!Contents&

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I.2.5.! Arsenic!Determination!.................................................................................................................!34!

I.2.6.! Statistical!Analysis!.........................................................................................................................!35!

I.3.! Results!.........................................................................................................................................................!36!

I.4.! Discussion!..................................................................................................................................................!39!

I.5.! References!.................................................................................................................................................!42!

Chapter. II.. Effects. of. exposure. to. As. in. Corbicula* fluminea:. Evaluation. of. the.

histological,.histochemical.and.biochemical.responses.........................................................49.

II.1.! Abstract!.....................................................................................................................................................!49!

II.2.! Materials!and!Methods!.......................................................................................................................!50!

II.2.1.! Experimental!Procedure!............................................................................................................!50!

II.2.2.! Histology!and!Histochemistry!.................................................................................................!51!

II.2.3.! Quantification!of!Metallothioneins!.......................................................................................!51!

II.2.4.! Arsenic!Determination!...............................................................................................................!52!

II.2.5.! Statistical!Analysis!........................................................................................................................!53!

II.3.! Results!.......................................................................................................................................................!53!

II.4.! Discussion!................................................................................................................................................!56!

II.5.! References!................................................................................................................................................!58!

Chapter.III..Toxicokinetics.of.trivalent.Arsenic.in.Corbicula*fluminea..............................65.

III.1.! Abstract!....................................................................................................................................................!65!

Table!of!Contents&

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III.2.! Materials!and!Methods!.....................................................................................................................!66!

III.2.1.! Experimental!Procedure!.........................................................................................................!66!

III.2.2.! Arsenic!Determination!.............................................................................................................!67!

III.2.3.! Uptake!and!Elimination!Kinetics!and!Bioaccumulation!Factors!............................!67!

III.2.4.! Maetallothionein!Determination!.........................................................................................!69!

III.2.5.! Histochemistry!............................................................................................................................!70!

II.2.5.! Statistical!Analysis!.......................................................................................................................!70!

III.3.! Results!.....................................................................................................................................................!70!

III.3.1.! Arsenic!Concentration!in!Water!...........................................................................................!70!

III.3.2.! Arsenic!Bioacumulation!and!Elimination!........................................................................!71!

III.3.3.! Biodynamic!Accumulation!Model!and!Bioconcentration!Factors!.........................!72!

III.3.4.! Metallothionein!Induction!......................................................................................................!73!

III.3.5.! Histochemistry!............................................................................................................................!74!

III.4.! Discussion!..............................................................................................................................................!76!

II.5.! References!...............................................................................................................................................!81!

Chapter.IV..Conclusions.....................................................................................................................87.

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!Resumen!

Los! bivalvos! han! sido! reconocidos! como! organismos! centinelas! de! ecosistemas!

acuáticos! porque! dan! información! precisa! y! global! sobre! el! impacto! ambiental! y! la!

biodisponibilidad!de!compuestos!químicos.!Corbicula&fluminea!(Muller!1744),&C.&Fluminea,&

es!un!bivalvo!de!agua!dulce!del! sureste!de!Asia!que! se!ha!dispersado!progresivamente!a!

nivel!mundial,!colonizando!varios!países!fuera!de!su!biota!original,!incluyendo!los!Estados!

Unidos!de!Norteamérica!y,!más!recientemente,!algunos!de!Europa.!Presenta!una!dinámica!

invasiva! al! colonizar! ríos,! canales! y! lagos,! donde! vive! semienterrado! en! las! capas!

superficiales! del! sedimento! y! se! alimenta! de! fitoplancton,! siendo! también! capaz! de!

recolectar! material! orgánico! del! sedimento,! lo! cual! contribuye! a! que! C.& fluminea! sea! un!

serio!candidato!para!ser!utilizado!como!molusco!centinela!de!agua!dulce!en!programas!de!

monitorización!ambiental! (Vidal! et! al.! 2002).!Desde!un!punto!de!vista! ecotoxicológico,!C.&

fluminea& es! un! modelo! muy! interesante! ya! que! vive! enterrado! en! los! sedimentos!

superficiales,! filtrando! grandes! volúmenes! de! agua! (cerca! de! 10! L! almeja–1! día–1),! y!

consecuentemente,!puede!bioacumular!grandes!cantidades!de!metales.!!

El! arsénico! (As)! es! un! elemento! que! se! encuentra! presente! en! elevadas!

concentraciones!en!distintas! regiones!geográficas!del!mundo,! contaminando! los! recursos!

hídricos! y,! por! tanto,! representa! un! riesgo! potencial! para! la! salud! humana! y! la! fauna!

silvestre.!En!algunas!regiones!de!Asia,!como!Bangladesh,!el!envenenamiento!crónico!por!As!

constituye! un! serio! problema! de! salud! pública! asociado! con! varios! tipos! de! patologías,!

como!por!ejemplo!el!cáncer.!Además,!las!actividades!antropogénicas!y!descargas!puntuales!

de!contaminantes,!como!las!ocurridas!sobre!la!plataforma!continental!del!Atlántico!NE!por!

el!transporte!del!petróleo!y!sus!derivados,!han!vertido!grandes!cantidades!de!metales!(e.g.,!

Cd,!Cu,!Pb,!Mo,!Ni)!y!metaloides!(As)!al!ambiente.!En!algunos!estudios!se!han!encontrado!

niveles!elevados!de!As!en!(i)!bivalvos!marinos!tales!como!los!ostrones!y!los!mejillones;!(ii)!

Resumen&

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en!los!poliquetos!marinos;!(iii)!en!los!cangrejos!y!(iv)!en!los!peces.!Aunque!este!metaloide!

ha! sido! sujeto! de!muchos! estudios! toxicológicos,! aún! existen! incógnitas! en! cuanto! a! sus!

efectos!en!organismos!vivos,!especialmente!en!los!que!viven!en!ecosistemas!de!agua!dulce.!

La! resistencia! de! los! bivalvos! a! la! toxicidad! causada! por! los! metales! es! atribuida!

normalmente! a! la! acción! de! mecanismos! de! destoxificación.! Así,! una! respuesta! celular!

específica! del! organismos! bivalovos! a! un! ambiente! contaminado! por! metales! es! la!

producción!de!metalotioneínas!(MTs).!Esta!hipótesis!aún!está!en!discusión,!ya!que!existen!

otros!contaminantes!que!pueden!inducir!la!producción!de!MTs!(e.g.,!especies!reactivas!del!

oxígeno,! estrés! físico,! o! una! infección).! No! obstante,! la! producción! de! MTs! causada! por!

otros!agentes!generalmente!es!menor!que! la!causada!por! los!metales! (Kagi!1993),!por! lo!

que!se!ha!propuesto!el!uso!de!las!MTs!como!un!biomarcador!potencial!en!la!monitorización!

ambiental!de!organismos!acuáticos!expuestos!a!metales.!La!producción!de!MTs!fue!descrita!

por! primera! vez! por! Margoshes! y! Vallee! (1957)! en! la! corteza! renal! de! equinos;! desde!

entonces!ésta!ha!sido!identificada!en!otros!organismos!como!peces,!moluscos!y!crustáceos,!

con! las!siguientes!propiedades!comunes:!bajo!peso!molecular,!alto!contenido!de!residuos!

de!cisteína,!ausencia!de!aminoácidos!aromáticos,!y!estabilidad!térmica!y!ácida.!

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Introducción!

La!contaminación!de!los!ecosistemas!acuáticos!por!arsénico!(As)!es!actualmente!un!

tema!de!creciente!interés!para!el!medio!ambiente,!que!afecta!no!sólo!a!la!salud!de!millones!

de!personas!y!otros!organismos!vivos,!sino!también!a!la!sostenibilidad!global.!El!arsénico!

se!encuentra!ampliamente!distribuido!en!diferentes!áreas!geográficas,!como!resultado!de!

procesos! naturales! y! antropogénicos! de! actividad! geoquímica! y! aplicaciones! industriales!

(por!ejemplo,!en!la!fabricación!de!vidrio,!pinturas,!pesticidas,!minería,!y!conservantes!de!la!

madera).!En! cuanto! a! la! contaminación!ambiental! y! los! riesgos!para! la! salud!pública,! los!

casos!más!graves!de!contaminación!por!arsénico!se!registraron!en:!

I. América!del!Sur,!el!caso!de!Chile!y!Argentina;!

II. Europa!del!Este!en!países!como!Hungría!y!Croacia;!

III. Continente!asiático,!en!China,!India!y!Bangladesh!(Cavar!et!al.!2005)!

Del!mismo!modo,! en! Portugal! hay! algunas! regiones! (Alpiarça,! Valbom,!Vila! Flor! y!

Ponte!de!Sor)!donde!los!valores!de!arsénico!en!el!agua!potable!superan!los!niveles!exigidos!

por!la!ley,!poniendo!a!sus!poblaciones!en!riesgo!(Garcia!2006).!

Los!niveles!de!contaminación!asociados!con!el!arsénico!son!particularmente!altos!en!

Banglades! (figura! 1).! De! hecho,! este! país! se! enfrenta! actualmente! a! una! amenaza! grave!

para! la! salud! pública! de! 85! millones! de! personas! expuestas! al! consumo! diario! de! agua!

contaminada!con!arsénico!(Hossain!2006).!

Introducción&

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Figura. 1.! Áreas! contaminadas! por! arsénico! en! Bangladesh.! Presencia! de! arsénico! y!

población! contaminada! (rojo)! .! Presencia! de! Arsénico! (rosa),! Arsénico! no! encontrado!

(azul).!

En!muchas!zonas,!se!observa!un!aumento!significativo!en!el!número!de!patologías!

asociadas! con! la! intoxicación! por! arsénico.! La! exposición! de! largo! plazo! a! este! elemento!

causa!la!aparición!de!enfermedades!cardiovasculares,!hipertensión,!trastornos!del!sistema!

nervioso!central!y!periférico,!alteraciones!del!sistema!reproductivo,!!y!cáncer!del!pulmón!y!

de!piel!(Mouthon!1981).!!

Es!ampliamente!conocida!la!capacidad!de!los!moluscos!bivalvos!para!acumular!altas!

concentraciones!de!metales! en! sus! tejidos.!Debido!a! esta! capacidad,! estos!organismos! se!

puede!utilizar!como!bioHindicadores!de!contaminación!en!el!medio!ambiente!acuático.!

1.. Corbicula*fluminea.Müller,.1744!

La! Corbicula& fluminea! es! una! almeja! de! agua! dulce! originaria! de! Asia! y! que!

progresivamente! ha! colonizado! los! EE.UU.! y! más! recientemente! Europa.! Este! bivalvo!

encontrado!en!el!continente!europeo!en!1980,!fue!observado!por!primera!vez!en!Francia!en!

el!río!Dordogne!y!em!Portugal!en!el! .!En!la!actualidad,! la!Corbicula&fluminea!se!distribuye!

ampliamente!por!los!países!europeos,!presentando!una!invasivos!canales!dinámicos!de!ríos!

y!lagos.!

Introducción&

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Figura. 2.! Corbicula& fluminea& (http://acuatico.foroslatin.com/t61HlaHalmejaHdeHrioH

corbiculaHfluminea)&&

Este! bivalvo! vive! enterrado! en! las! capas! superficiales! del! sedimento! y! realiza!

intercambios!con!la!columna!de!agua!a!través!de!las!corrientes!generadas!por!movimientos!

ciliares! en! la! superficie! de! las! branquias,! los! palpos! labiales! y! el! manto.! Los! sifones!

inhalantes! y! exhalantes! controlan! el! flujo! de! agua! en! la! cavidad! interna! del! manto!

(Mouthon!2001).!

Los! moluscos! bivalvos,! en! general,! tienen! alto! potencial! de! bioacumulación! de!

metales!pesados!y!metaloides!tales!como!el!As.!Desde!el!punto!de!vista!ecoHtoxicológico,!la!

transferencia! del! metaloide! para! el! organismo! acuático! surge! como! resultado! de! una!

compleja! serie! de! interacciones!medioambientales! que! afectan! a! la! biodisponibilidad!del!

arsénico.! Por! otra! parte,! las! vías! de! entrada,! sean! por! ingestión! de! alimentos! o!

directamente!a!través!del!agua!!condicionan!la!distribución!del!elemento!en!los!diferentes!

órganos!del!organismo!(Baudrimont!et!al.!1997).!

2.. Los.marcadores.biológicos.de.respuesta.–.Metalotioneinas.(MTs).

El! término! biomarcador! se! utiliza! comúnmente! de! modo! general! para! incluir!

diversas! medidas! que! reflejen! la! interacción! entre! los! sistemas! biológicos! y! un! riesgo!

potencial,! que!puede! ser!de!origen!químico,! físico!o!biológico.!Los!biomarcadores! se!han!

Introducción&

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utilizado!en!numerosos!estudios!y!programas!de!vigilancia!del!medio!ambiente!marino.!Sin!

embargo,! el! ecosistema! acuático! a! menudo! ha! quedado! relegado! a! un! segundo! plano!

cuando! se! trata! de! estudios! relacionados! con! la! contaminación! del! medio! acuático! por!

metales! pesados! y! otros! elementos! o! compuestos,! y! por! lo! tanto! surge! la! necesidad! de!

aplicar! el! mismo! tipo! de! enfoque! a! los! ecosistemas! de! agua! dulce.! El! análisis! de!

biomarcadores! en! muestras! de! origen! biológico! permite! obtener! indicaciones! sobre! el!

estado!del!ecosistema!en!que!el!organismo!estudiado!vive.!(Viarengo!et!al.!1997).!

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Figura.3.!Estructura!de!la!metalotioneina.!La!unión!de!los!átomos!de!metal!a!residuos!de!

cisteína.!(http://faculty.virginia.edu/metals/cases/prescott1.html).!

Las! metalotioneínas,! MTs,! son! proteínas! de! bajo! peso! molecular! (6H7kDa)!

(Baudrimont! et! al.! 2003),! con! alto! contenido! de! residuos! de! cisteína! (figura! 3),!

aproximadamente!33%!de!61! aminoácidos,! que!no!presentan! aminoácidos! aromáticos,! y!

que!tienen!una!elevada!tolerancia!a!condiciones!ácidas!u!elevadas!temperaturas.!Presentan,!

además,! una! absorción! característica! a! 254! nm! (Mason! and! Jenkins! 1992).! En! términos!

funcionales,!los!estudios!realizados!sobre!MTs!indican!un!papel!importante!de!las!mismas!

en!el!metabolismo!y!biodisponibilidad!de!metales!esenciales!tales!como!Cu!y!Zn,!mientras!

que!también!participan!en!el!proceso!de!destoxificación!de!metales!pesados!tales!como!Cd,!

Hg,!Ag!etc.! (Baudrimont!et!al.!2003).!Así,! la!utilización!de!MTs!es!particularmente!útil!en!

estudios!relacionados!con!la!contaminación!del!medio!acuático,!y!por!lo!tanto!se!reconocen!

como!posibles!biomarcadores!de!exposición!a!metales!pesados.!

Introducción&

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3.. Cuantificación.de.metalotioneínas.por.polarografía.DPPXSDME.

La! voltametría! ha! sido! ampliamente! utilizada! por! químicos,! físicos! y! biólogos.! en!

diferentes! estudios! sobre! los! procesos! de! oxidación! y! reducción! de!metales! en! distintos!

medios,!en!procesos!de!adsorción!y!en! los!mecanismos!de!transferencia!de!electrones!de!

superficies! de! electrodos! químicamente! modificados! (Skoog! et! al.! 1992).! Esta! técnica!

comprende!un!grupo!de!procedimientos!basados!en!el!comportamiento!electroquímico!del!

potencial! de! flujo! de! un! electrodo! polarizable! utilizando! un! electrolito! de! soporte.!

Teóricamente! cualquier! especie! química! puede! ser! analizada,! siempre! que! sufra! una!

oxidación! o! reducción! en! el! intervalo! de! potencial! aplicada! por! los! electrodos! (Riordan!

1991).! La! reacción! de! oxidaciónHreducción! que! tiene! lugar! en! el! electrodo! se! controla!

mediante!la!aplicación!de!un!potencial!en!el!electrodo!de!trabajo.!La!reacción!de!oxidaciónH

reducción!que!ocurre!en!el!electrodo!es!controlada!por!la!aplicación!de!un!potencial!sobre!

el! electrodo! de! trabajo,! potencial! este! que! varia! en! el! tiempo.! Se! registra! una! relación!

corriente/voltaje! correspondiente! a! los! procesos! de! reducción! que! se! producen! en! el!

electrodo! y! en! la! muestra.! Las! técnicas! electroquímicas,! y! en! particular! los! métodos!

voltamétricos,! constituyen! uno! de! los! métodos! más! directos! para! el! estudio! de! la!

especiación!de!metales! traza! (10H8! a!10H11!mol.LH1),!una!vez!que!no! requieren,! como!paso!

inicial,!extracción!o!preHconcentración!de!las!muestras!(Mota!and!Santos!1995).!

La! identificación! electroquímica! de! metalotioneinas! por! polarografía! de! pulso!

diferencial!(DPP)!es!una!técnica!analítica!que!se!aprovecha!de!la!alto!contenido!de!residuos!

de! cisteína! presentes! en! las,! MTs.! Es! una! técnica! muy! rápida! y! reproducible,! que! es!

utilizada!a!menudo!en!los!estudios!!MTs!en!tejidos!animales,!muestras!ambientales,!etc.!El!

uso!de!la!polarografía!para!la!cuantificación!de!las!proteínas!que!contienen!HSH!fue!descrita!

por!primera!vez!en!1931!por!Brdicka.!Más!tarde,!el!método!fue!modificado!por!Palecek!y!

Pechan,! a! través! del! uso! de! la! polarografía! de! pulso! diferencial! (DPP)! y! que! luego! fue!

adaptada!para!el!análisis!específico!de!metalotioneínas!(Riordan!1991).!

Introducción&

!20!

La!polarografía!de!pulso!diferencial!(DPP)!es!una!técnica!de!voltamétrica!en!el!que!

se!utiliza!un!electrodo!de!gota!de!mercurio!y!donde!la!diferencia!entre!dos!mediciones!de!

corriente! tomadas! en! dos! períodos! de! tiempo! se! realiza! repetidamente! en! una! gota! de!

mercurio! continuamente! renovada! mientras! el! instrumento! explora! una! cierta! gama! de!

valores!de!potencial!de!trabajo.!En!este!método,!el!electrodo!se!polariza!con!una!secuencia!

de!pulsos!cuadrados!y!uniformes!que!se!superponen!en!una!rampa!de!potencial!lineal!!que!

aumenta!lentamente!durante!el!barrido.!La!corriente!se!mide!en!la!segunda!parte!del!pulso!

cuando! la! capacidad! de! corriente! está! disminuyendo! rápidamente.! El! fundamento! del!

método!de!Brdicka!(DPP)!para!proteínas!es!la!generación!de!ondas!dobles!catalíticas!de!las!

proteínas!que!contienen!SS/SH!en!un!electrolito!de!soporte! tampón!y!cloruro!de!amonio!

hexaminocobalto!(III)!(Riordan!1991).!Así,! la!determinación!de!proteínas!que!contienen!H

SH!(incluyendo!metalotioneínas)!tiene!como!base!la!relación!lineal!entre!la!segunda!de!dos!

ondas!H!que!se!siguen!a!la!onda!de!reducción!de!cobalto!y!concentración!de!proteína.!Estas!

ondas!se!denominan!"A"!y!"B"!(figura!4.)!y!se!atribuyen!a!la!reducción!tiólica!con!hidrogeno!

catalizada!por!Co!(III).!!

!

Figura. 4.! Polarogramas! de! Pulso! Diferencial.! 50,! 100,! 150! μL! de! citosol! de! Ruditapes&

decussatus&utilizando!el!electrolito!de!Brdicka,!obtenidos!con!un!electrodo!de!gota!estática!!

de! mercurio.! El! cobalto! (Co),! "A"! y! "B"! se! refieren! a! la! reducción! de! Co! y! proteína,!

respectivamente!(Bebianno!and!Langston!1989).!

Introducción&

! 21!

La!onda!"A"!(H1,35!V!vs!Ag/AgCl)!no!se!puede!determinar!con!precisión,!ya!que!está!

influenciada! por! la! onda! de! reducción! catalítica! del! cobalto,! mientras! que! la! onda! “B”!

presenta!una!mejor!resolución!(!H1,4!V!vs!Ag/AgCl).!Es!de!destacar!que!el!ion!complejo!de!

Co(III)! con!moléculas! de! proteína! juega!un!papel! decisivo! en! el! efecto! generador! de! dos!

ondas!polarograficas!(Bebianno!and!Langston!1989).!

La! altura! de! la! onda! es! una! función! del! número! de! grupos! tiol! adsorbido! a! la!

superficie!del!electrodo!y!es!controlada!por! la!velocidad!de!recombinación!de! los!grupos!

sulfhídricos! ionizados! y! los! protones! (figura! 5).! Las! reacciones! electroquímicas! se!

producen! independientemente! de! si! las! cadenas! laterales! que! contienen! azufre! son! los!

tioles,!los!disulfuros,!o!de!si!forman!complejos!con!los!metales.!

!

Figura. 5.! Polarograma! de! la! línea! de! base! de! electrolito! de! referencia! y! la! onda! B!

resultante! de! 55! pmol! de! MTHI! purificada! de! trucha! arcoHiris.! La! cantidad! de! MT! se!

determinó!mediante!la!medición!de!la!altura!(h)!(Riordan!1991).!

La!técnica!de!DPPHSMDE!aplicada!a! la!determinación!de!MT! ! tiene! la!capacidad!de!

detectar!concentraciones!del!orden!de!pico!moles.!Este!método!es!específico!para!las!TMs.!

Para!ello!primero!se!eliminan!las!proteínas!contaminantes,!a!través!de!su!desnaturalización!

térmica.! Este! enfoque! electroquímico! ha! sido! utilizado! en! la! cuantificación! de! MTs! en!

muchos!estudios!de!inducción!de!metalotioneina!como!resultado!de!la!exposición!a!metales!

y!metaloides.!

Introducción&

!22!

4.. Espectrometría.de.absorción.atómica.de.Cámara.de.Grafito.

La! espectrometría! de! absorción! atómica! (AAS)! es! una! técnica! analítica! bien!

desarrollada!y!lo!suficientemente!robusta!como!para!ser!implementado!en!los!laboratorios!

de! análisis! químicos! a! gran! escala.! Esta! técnica! se! utiliza! para! fines! cualitativos! y!

cuantitativos,!permitiendo!la!determinación!y!cuantificación!de!más!de!70!elementos!y!se!

puede!aplicar!en!diferentes!áreas!tales!como:!análisis!clínicos,!ambientales,!farmacéuticos!y!

en!la!industria.!

El!principio!fundamental!de!la!espectrometría!de!absorción!atómica!es!la!absorción!

de!radiación!electromagmética!en! la!región!del! !ultravioleta!o!visible!por!átomos!neutros!

en!estado!gaseoso.!Para!un!átomo!en!el!estado!fundamental,! los!electrones!de!valencia!se!

encuentran! en! un! estado! E0! de! menor! energía.! Estos! pueden! absorber! energía! sobre! la!

forma!de!fotones!de!energía!hν,!siendo!promovidos!para!un!estado!de!mayor!energía!(E1),!

(figura!6).!

!

Figura.6.!Proceso!de!absorción!de!energía.!

La! diferencia! de! energía! entre! E0! y! E1! corresponde! a! la! absorción! de! un! fotón! de!

energía! igual!a!hν.!Cada!una!de!estas! transiciones!es! característica!para!cada!elemento!y!

por!lo!tanto!requiere!una!fuente!de!energía!que!emita!en!una!longitud!de!onda!discreta!y!

monocromática! ! característica! del! elemento! que! se! va! a! determinar! (Gonçalves! 2001,!

Harris! 2003).! La! formación! de! átomos! libres! en! AAS! es! esencial,! esto! se! obtiene! por!

Introducción&

! 23!

atomización! de! la! muestra.! La! energía! aplicada! durante! la! atomización! de! una! muestra!

determinará! la!población!de!átomos! libres!capaces!de!absorber!energía!de! la! lámpara!de!

cátodo!hueco.!La!energía!necesaria!para!la!atomización!depende!del!elemento!a!analizar!y!

la!matriz!en!la!que!se!encuentra.!

En! la!espectrometría!de!absorción!atómica,!el!analito!puede!ser!atomizado!en!una!

llama,! en! un! horno! calentado! eléctricamente! o! en! un! plasma.! En! la! espectrometría! de!

absorción!atómica!de!horno!de!grafito,!una!pequeña!cantidad!de!muestra,!que!puede!variar!

de! 1! a! 100! μL! se! inyecta! en! el! interior! de! un! tubo! de! grafito.! En! este! caso! la! muestra!

atomizada! permanece! en! el! camino! óptico! durante! varios! segundos,! obteniendoHse! una!

mayor!sensibilidad!(Harris!2003).!

La! espectrometría! de! absorción! atómica! con! cámara! de! grafito! se! basa! en! la!

absorción! de! energía! por! átomos! neutros! en! el! estado! gaseoso,! generados! a! partir! de! la!

deposición! de! una! pequeña! cantidad! de! muestra! en! un! tubo! de! grafito,! que! se! calienta!

mediante! la! aplicación! de! temperatura! elevada! (Harris! 2003).! El! uso! de! la! cámara! de!

grafito! para! el! análisis! de! metales! requiere! un! operador! con! mayor! experiencia!

comparativamente!con!las!técnicas!de!la!llama.!Dependiendo!de!la!naturaleza!de!la!matriz!

en!la!que!se!encuentra!el!analito,!y!el!tipo!de!elemento!que!se!va!a!determinar,!es!necesario!

optimizar! un! conjunto! de! pasos! sin! las! cuales! no! se! obtienen! buenos! resultados.!

Típicamente,!el!horno!de!grafito!se!calienta!en!tres!o!más!etapas:!el!secado,!la!calcinación!y!

la!atomización!(figura!7).!

Después! de! la! atomización,! el! horno! de! grafito! se! calienta,! típicamente! a! 2400ºC!

para! eliminar! cualquier! residuo! restante.! Durante! cada! paso! el! horno! se! purga! con! una!

corriente!de!Ar!o!N2,!excepto!durante!la!atomización,!para!eliminar!el!material!volátil.!Este!

material,! que! no! es! el! analito! es! una! causa! potencial! de! interferencia.! Para! evitar! la!

volatilización!del!analito!de! interés!durante! la!etapa!de!calcinación,! la!muestra!se!mezcla!

con!un!modificador!de!matriz.!Idealmente,!la!matriz!se!descompone!y!se!vaporiza!durante!

Introducción&

!24!

la!etapa!de!calcinación..!El!modificador!de!matriz!puede!actuar!tanto!en!la!matriz!como!el!

analito,! ya! sea! mediante! el! aumento! de! la! volatilidad! de! la! matriz! o! disminución! de! la!

volatilidad!del!analito.!

!

!

Figura.7.!Perfil!de!la!temperatura!en!la!cámara!de!grafito!(Harris!2003).!

5.. Histología.e.histoquímica.

La!histología!(del!griego!histo,!tejido!+!logos,!estudio)!trata!del!estudio!de!los!tejidos!

y!cómo!se!organizan!para!formar!órganos.!Podemos!distinguir!cuatro!tipos!de!tejido:!tejido!

epitelial,!tejido!conectivo,!tejido!muscular!y!tejido!nervioso!(Junqueira!and!Carneiro!2003).!

Los! tejidos! están! formadas! por! dos! componentes! principales,! las! células! y!matriz!

extracelular,! que! formalmente! se! consideran! como! dos! entidades! separadas.! La! matriz!

extracelular! se! compone! de! diferentes! tipos! de! moléculas! altamente! organizadas! en!

estructuras! complejas,! tales! como! las! fibras! de! colágeno.! La!matriz! extracelular! confiere!

soporte! mecánico! a! la! célula! requerido! para! transportar! nutrientes,! metabolitos! y!

productos!de!desecho.!

Cada! uno! de! los! diversos! tejidos! está! compuesto! de! varios! tipos! de! células! y!

generalmente! por! asociaciones! específicas! de! células! y! matriz! extracelular.! Estas!

asociaciones!características! facilitan! la! identificación!de! los! tejidos!por!medio!de!técnicas!

Introducción&

! 25!

histológicas!e!histoquímicas.!

La! aplicación! de! técnicas! histológicas! para! el! análisis! microscópico! tiene!

aplicabilidad! en! campos! tan! variados! como! la! biología! celular,! biología! del! desarrollo,! la!

neurobiología,! la! microbiología! y! patología! experimental,! entre! otros! (Taatjes! and! Roth!

2005).! Los! avances! en! la! química,! la! fisiología,! patología! e! inmunología! así! como! la!

interacción!de!estas!áreas!de!conocimiento!son!esenciales!para!una!mejor!comprensión!de!

la!biología!del!tejido.!

La! preparación! ideal! de! los! tejidos! para! el! análisis! histológico,! debe! preservar! el!

tejido!de!modo!que!mantenga!la!misma!estructura!y!composición!molecular!encontrado!en!

el! organismo! intacto.! Sin!embargo,! el! tratamiento!aplicado!al! tejido,! a!menudo! introduce!

distorsiones! artificiales! y! provoca! la! pérdida! de! algunos! componentes! presentes! en! el!

tejido!original.!

6.. References.

Bebianno! MJ,! Langston! WJ! (1993)! Turnover! rate! of! metallothionein! and! cadmium! in!

Mytilus!edulis.!Biometals&6:239–244.!

Ćavar!S,!Klapec!T.!Jurišić!R.!Valek!M!(2005)!Science&of&the&Total&Environ.&339:277–282.!

Garcia!R!(2006)!In!Jornal!Público,!12.01.2006–07h49,!PúblicoHPT!onHline!

Hossain!!MF!(2006)&Agriculture,&Ecosys.&and&Environ.&113:1–16!

Smith!AH.!Lingas!EO.!Rahman!M!(2000)!B&World&Health&Organ.&78:1093–1103!!

Regoli!F.!Orlando!M!(1994)!Mar.&Pollut.&Bull.&28:592–&600.&&

Mouthon! J! (1981)! Sur! la! presence! en! France! et! au! Portugal! de! Corbicula& (Bivalvia,!

Corbiculidae)!originaire!d’Asie.!Basteria&45:109–116.!

Introducción&

!26!

http://cars.er.usgs.gov/posters/Nonindigenous/Nonind_Species_Introduced/nonind_speci

es_introduced.html!

J.!Mouthon,!Hydrobiol.452!(2001)!109–119!.!

M.!Baudrimont,!J.!Metivaud,!R.!Maury–!Brachet,!F.!Ribeyer,!A.!Boudu,!Emviron.!Toxicol.!and!

Chem.!16!(1997)!2096–2105!

A.!Viarengo,!E.!Ponzano,!F.!Dondero,!R.!A.!Fabbri,!!Mar&Environ&Res&44!(1997)!69–84.!

M.!Baudrimont,!S.!Andres,!G.!Durrieu,!A.!Boudou,!Aquatic&Toxicology&63!(2003)!89–102!!

A.Z.!Mason,!K.!D.!Jenkins,!Metal!detoxification!in!aquatic!organisms.!In!A.!Tessier!and!D.R.!

Turner,!eds.,!Metal&Speciation&and&Bioavailibity&in&Aquatic&Systems.&John!Wiley!&!Sons,!New!

York,!NY,!USA,!(1992)!pp.!479–608.!

Skoog,!A.D.,!West,!D.M.,!Holler,!F.J.!Voltammetry,!in!Fundamentals!of!Analytical!Chemistry.!

Ed.!6th,!Saunders&College&Publishing,!USA,!(1992)!471!–!507.!

Mota,!A.M.!and!Santos,!M.M.C!Trace!Metal!Speciation!of!Labile!Chemical!Species!in!Natural!

Waters:! Electrochemical! Methods,! in! Metal! Speciation! and! Bioavailability! in! Aquatic!

Systems.!Ed.!Tessier,!A.!and!Turner,!D.R.,!Publ.!Jonh&Wiley&and&Sons,!Ltd.!(1995).!

J.! F.! Riordan,! B.! L.! Vallee,! Methods! in! Enzimology,! Metallobiochemistry& Part& B,&

Metallothioneins&and&Related&Molecules,!Academic!Press!,!INC,!1991!

Bebianno,!M.J.!&!Langston!W.J.!Portugaliae&Electroch.&Acta,!vol.!7,!(1989).!59H64.!

Quím.!Nova,!Vol.!26,!No.!2,!249H252,!2003!

http://www.chemsoc.org/pdf/LearnNet/rsc/AA_txt.pdf!

M.L.S.S! Gonçalves,! Métodos& instrumentais& para& anàlises& de& soluções,! Gulbenkian,! Lisboa,!

2001.!

Introducción&

! 27!

D.C.Harris,!Quantitative&Chemical&Analysis,!!6!th!Ed.!Freeman,!2003.!

L.C.!Jinqueira,!J.Carneiro,!Basic!Histology,!10th!Ed.!(2003)!McGrawHHill!Companies!!

D.!J.!Taatjes,!J.Roth,!Histochem&Cell&Biol!Review,!124!(2005)!547–574!

F.!Regoli,!E.!Orlando,!Mar.!Pollut.!Bull.!28!(1994)!592–!600.!

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Objectivos!

Los!objetivos!de!la!presente!tesis!doctoral!son:!

I. Estudiar!la!respuesta!de!C.&Fluminea!cuando!es!expuesta!a!elevadas!concetraciones!

de!As!durante!!períodos!de!tiempo!prolongado,!de!hasta!4!semanas.!Para!!la!realización!

de! este! estdio! se! determinará! la! concentración! de! As! en! los! tejidos! blandos! de! C.&

Fluminea;&

II. Estudiar!la!distribución!de!As!en!los!tejidos!blandos!y!en!la!fracción!citosólica!de!C.&

Fluminea;&

III. Estudiar!la!producción!de!metalotioneias!como!respuesta!a!la!exposición!a!elevadas!

concetraciones!de!As;!

IV. Estudiar! la!correlación!entre! la!concentración!de!metalotioneias!y!de!As!en!tejidos!

blandos!y!en!la!fracción!citosólica!de!C.&Fluminea;!

V. Estudiar!si!C.&Fluminea!puede!utilizarse!como!bioindicador!de!contaminación!por!As.!

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Chapter!I.!Toxicological!effects!of!arsenic!

exposure!in!Corbicula&fluminea!

I.1.. Abstract.

Contamination!of!aquatic!environments!by!arsenic!is!a!serious!worldwide!problem.!

The! main! objective! of! this! work! was! to! evaluate! the! response! of! a! freshwater! clam!

(Corbicula& fluminea)! to! arsenic! (As! III)! exposure! and! infer! its! potential! as! a! biological!

indicator! of! contamination.!Metallothioneins! (MTs)!were! used! as! indicators! of!metalloid!

toxicity!in!combination!with!a!histological!and!histochemical!evaluation.!After!a!period!of!

acclimatization! in! the! laboratory,! 50!C.&fluminea! (0.4! g! ±! 0.1)!were! exposed! to! different!

nominal! concentrations! of! arsenic! (100,! 300,! 500,! and! 1000! μg.LH1)! for! 7! days.! The!

concentration! of! total! As! III! in! the! water! and! in! the! tissues! of! the! organisms! was!

determined! by! atomic! absorption! spectrometry,! and! MTs! were! quantified! through!

differential! pulse! polarography.! Results! suggest! that! the! organisms! exposed! to! the!

concentrations! of! 300! and!1000!μg!As! LH1!accumulated! the! highest! levels! of! As! III! in! the!

tissues! (17! ±! 9! and! 15! ±! 3! μg.LH1! distilled! water,! respectively),! which! was! confirmed!

through! histochemical! analysis.! An! apparent! induction! of!MTs!was! also! observed! in! the!

organisms! exposed! to! As! III,! suggesting! that! C.& fluminea! possesses! some! capacity! for!

arsenic!regulation.!The!results!suggest!that!the!induction!of!MTs!may!be!of!high!interest!as!

a!biomarker!for!arsenic!contamination!in!aquatic!environments,!and!confirms!the!potential!

of!C.&fluminea!as!a!biological!indicator.!

Chapter!I.!Toxicological!effects!of!As!exposure!in!Corbicula&fluminea&

!32!

I.2.. Materials.and.Methods.

I.2.1. Experimental.Procedure.

The! freshwater!bivalves,!Corbicula&fluminea!Müller,!1744,!were!collected!manually!

from!Rio!Minho’s!hydroHgraphic!basin!(Portugal)!and!maintained!in!a!laboratorial!system!

of!tanks!subjected!to!a!continuous!flow!of!filtered!and!aerated!tap!water.!After!a!period!of!

acclimatization,!50!bivalves!(soft!body!mass!=!0.4!±!0.1!g;!shell!length!=!1.5!±!0.5!cm)!were!

distributed! randomly! into! 10! polyvinyl! tanks! (2! L)! and! exposed! to! different! nominal!

concentrations!of!As!(100,!300,!500,!and!1000!μg.LH1).!The!static!assay!was!carried!out!in!

duplicate,!at!(20!±!1)!ºC,!with!continuous!aeration!and!a!constant!photoperiod!(12:00!light).!

Tap!water!was!used!as!a!control!after!the!removal!of!chlorine.!Temperature!and!pH!were!

monitored!daily.!At!the!beginning!and!end!of!the!assay,!samples!of!water!were!collected!for!

the! determination! of! the! total! As! concentration.! Organisms! were! fed! daily! with! finely!

triturated!fish!food!(Dibaq,!Segovia,!Spain).!Bivalves!were!sampled!after!7!days,!and!whole!

soft!tissues!from!the!replicates!of!each!treatment!were!quickly!excised,!dried!on!absorbent!

paper,!and!weighed!(fresh!weight).!The!length!of!the!shell!was!also!recorded.!Subsamples!

from! the! soft! tissues! (gills,! foot,!mantle,! and!digestive! gland)!were! taken! for! histological!

and!histochemical! evaluation.!The! remaining! tissue! samples!were!preserved!at! H80ºC! for!

the!subsequent!analysis!of!metals!and!MTs.!

I.2.2. Histology.and.Histochemistry.

Subsamples!of!C.&fluminea!tissues!were!fixed!in!a!solution!of!BouinHHollande!for!48!h,!

and!treated!in!accordance!with!the!procedures!used!in!histology!as!described!by!Martoja!

and!Martoja! (1967).! After! immersion! in! paraffin,! sections! of! 5–7! μm!were! stained!with!

hematoxyline! and!eosin! (H!&!E).!The! staining!procedure!adopted! for! the!detection!of!As!

granules!in!the!tissues!(Castel’s!method,!1936)!was!carried!out!according!to!Lillie!(1965).!


! 33!

The! effects! on! the! tissues! were! assessed! through! histological! and! histochemical!

observation!using!an!optical!microscope!(LeicaHATC!2000,!Wetzal,!Germany).!

I.2.3. Detection.of.Metallothionein.

MTs! were! visualized! by! sodium! dodecyl! sulphate! polyacrylamide! gel!

electrophoresis!(SDSHPAGE).!Gels!(0.75!mm!thick)!were!comprised!of!a!15%!acrylamide!in!

a! 0.25!M!TrisHHCl! buffer! (pH!8.8)! running! gel! and!5%!acrylamide! in! a! 0.125!M!TrisHHCl!

buffer!(pH!6.8)!stacking!gel,!and!were!stained!with!Coomassie!Blue!RH250.!Samples!were!

previously!heated!at!1008C!for!2!min!in!SDS!sample!buffer!containing!bHmercaptoethanol!

([99%)! from! Merk! (Darmstadt,! Germany).! BroadHRange! protein! standard! (BioHRad,!

Hercules,!CA,!USA)!was!used!as!molecular!weight!standard!for!electrophoresis,!containing:!

aprotinin! (6.50! kDa),! lysozyme! (14.4! kDa),! trypsin! inibitor! (21.50! kDa),! carbonic!

anhydrase!(31.00!kDa),!ovalbumin!(45.00!kDa),!serum!albumin!(66.20!kDa),!phosporylase!

b! (97.40! kDa),! bHglactosidase! (116.25! kDa),! myosin! (200.00! kDa).! MT!molecular! weight!

was!determined!using!the!software!Quantitity!One!(BioHRad,!Hercules,!CA,!USA).!

I.2.4. Metallothionein.Determination..

Tissue!samples!were!homogenized!individually!in!three!volumes!of!0.02!M!of!TrisH

HCl!(pH!8.6)!in!an!ice!bath!using!a!rotor!(HeidolphHRZR!2100,!Heidolph!Elektro!GmbH!&!Co.,!

KG,!Kelheim,!Germany).! Subsamples!were! taken! for!wet:dry!weight! ratio! determinations!

and!metal!analysis.!An!aliquot!of!the!homogenate!(3!mL)!was!centrifuged!at!30,000!x!g!for!

1!h!at!4ºC.!The!supernatant!(cytosol)!was!separated!from!the!pellet,!heatHtreated!at!80ºC!

for!10!min!to!precipitate!the!high!molecular!weight!proteins,!and!subsequently!centrifuged!

at! 30,000! x! g! for! 1! h! at! 4ºC.! Supernatant! (cytosol)! was! stored! at! H80ºC,! for! subsequent!

measurements.!A!differential!pulse!polarographic!assay!for!thiolic!HSH)!compounds,!based!

on! the!Brdicka! reaction,!was!used! to!determine!MTs! in!heatHtreated! cytosol,!using!a!Met!


!34!

rohm!693!VA!Processor!and!a!694!VA!Stand!(Metrohm,!Herisau,!Switzerland),!with!a!static!

mercury!drop!electrode!according! to! the! techniques!described!by!Thompson!and!Cosson!

(1984)!and!Olafson!and!Olsson!(1991).!The!threeHelectrode!system!consisted!of!a!beveled!

capilary! mercury! electrode,! a! platinum! counter! electrode,! and! an! Ag/AgCl! reference!

electrode.! The! Brdicka! supporting! electrolyte! was! prepared! according! to! the!method! of!

Palecek! and! Pechan! (1971),! and! contained! 1! M! NH4Cl,! 1! M! NH4OH,! and! 2! mM!

(CoCNH3)6)Cl3.!The!electrolyte!was!prepared!weekly!and!stored!at!4ºC!as!recommended!by!

Thompson! and! Cosson! (1984).! Triton! XH100! (SIGMA,! St.! Louis,! MO,! USA)!was! used! at! a!

concentration! of! 2.5! x! 10H2%! (v/v)! to! suppress! secondary! maxima! and! minima,! and! to!

eliminate! baseline! noise.! Supporting! electrolyte! (20!mL)!was! dispensed!directly! into! the!

polarographic!cell,!together!with!250!μL!of!Triton!XH100!and!aliquots!of!standard!solution!

and! sample.! The! cell! was! purged! with! purified! nitrogen! for! 120! s! before! analysis!

(Thompson! and! Cosson,! 1984).! In! the! absence! of! a! standard! for! molluscan! MT,!

determination!of!MTs!in!the!cytosol!of!C.&fluminea!tissues!was!performed!using!rabbit!liver!

MT,!MTHI,!and!II!standard!(SIGMA,!St.!Louis,!MO,!USA).!The!working!standard!solution!was!

10! mg.LH1,! prepared! in! deionized! water.! MT! concentrations! were! determined! using! the!

standard!additions!method!according!to!Bebianno!and!Langston!(1989).!Aliquots!(25–150!

μL)!of! the!heatHtreated! cytosol!were! taken! for!quantification!of! the!heatHstable!MT!using!

differential! pulse! polarography.! MT! concentrations! were! determined! as! milligram! per!

gram!homogenate!dry!weight.!

I.2.5. Arsenic.Determination..

I.2.5.1. Tissue*

Metalloid!analysis!(AsIII)!was!carried!out!on!homogenate!dried!samples!(forced!air!

oven! at! 60ºC! for! 5! days),! and! digested! with! concentrated! nitric! acid! (5! mL! of! HNO3!

Suprapur,!Merck,!Darmstadt,!Germany)!in!a!pressurized!medium!(teflon!cells)!for!24!h!at!


! 35!

room!temperature,!which!was!followed!by!heating!at!100ºC!for!4!h,!and!an!additional!hour!

after! adding!1!mL!H2O2! (Bryan! et! al.,! 1985).!Digested! samples!were!diluted! in! 25!mL!of!

ultrapure! water! (MilliQ! plus)! and! total! As! concentrations! were! measured! by! electroH

thermal! atomic! absorption! spectrometry! (ETHAAS)! with! Zeeman! correction! and! matrix!

modifier! (Pd(NO3)2.2H2O,! Fluka,! Buchs,! Switzerland),! using! a! graphite! tube! atomizer!

(M6Solaar!AAS,!Thermo!Electron!Corporation,!Waltham,!MA,!USA).!The! limit!of!detection!

and!quantification!were!calculated!with!the!criteria!of!3σ!and!10!σ,!and!were!3.1!and!9.3!

μg.LH1,!respectively.!Analysis!of!the!DOLT!3!NRCHCNRC!(National!Research!Council,!Canada)!

reference! material! and! blanks! were! used! to! validate! the! analytical! procedure! analysis,!

where! the! same! analytical! procedure!was! implemented! as! for! the! samples.! The! As! (III)!

measured!(9.4!±!0.3!mg.kgH1;!N!=!2)!showed!good!agreement!with!the!certified!values!(10.2!

±!0.5!mg.kgH1).!The!AsIII!concentration!was!determined!using!a!standard!calibration!curve.!

Standard!solutions!were!prepared! from!a!commercial! inorganic!AsIII! standard! for!Atomic!

absorption!(N!206962,!1!g.LH1,!Aldrich,!Milwaukee,!WI,!USA).!Concentrations!are!expressed!

on!a!dryHtissue!weight!basis!(μg.gH1).!

I.2.5.2. Water*

Two!replicates!were!performed!per!experimental! condition,! and!were!collected!at!

the!beginning!and!at! the!end!of! the!assay,!using!polyethylene!bottles!acidified!with!nitric!

acid! (10! μL.mLH1! of! HNO3! SupraPur,! Merck,! Darmstadt,! Germany)! and! stored! at! 4ºC.!

Samples! containing! As! were! directly! determined! by! ETHAAS! without! filtration,! after!

dilution!with!ultrapure!water.!Blanks!were!analyzed!in!triplicate!using!the!same!procedure.!

I.2.6. Statistical.Analysis..

The! nonparametric! MannHWhitney! U! test! was! used! to! determine! differences!

between! the! treated!samples!and! the!control! samples.!Statistical!analysis!was!performed!


!36!

with!a!significance!level!of!5%,!using!the!software!Statistica!5.0!(Statsoft,!1995,!Tulsa,!OK,!

USA).!

I.3.. Results.

No!mortality!of!C.&fluminea!was!observed!during!the!experiment.!Measurements!of!

As!in!water!samples!collected!from!the!different!exposure!treatments!of!the!experimental!

assays! are! shown! in! table! I.1.! At! the! beginning! of! each! experiment,! the! average!

concentration!was!measured! to!be!very! close! to! the!nominal! concentration,!but! the!final!

concentrations!decreased!about!4–19%!depending!on!the!As!concentration!added.!

Table.I.1.!Total!As!concentration!(average!±!s.d.!μg.LH1)!

! Control* 100* 300* 500* 1000*

Ci! 1.1±0.6! 108±5! 295±4! 521±8! 993±6!

Cf! 1.4±0.9! 88±6! 276±5! 469±11! 953±4!

Ci:!initial!concentration;!Cf:!final!concentration;!(n=3,!each!replicate)!

The! results! obtained! for! arsenic! stored! in! exposed! wholeHbody! C.& fluminea! are!

presented! in! figure! I.1.! The! highest! level! of! this! metalloid! [29.75! μg.LH1! distilled! water!

(d.w.)]!was!found!in!tissues!of!the!organisms!exposed!to!300!μg!As!LH1!and!the!lowest!level!

was!measured! in!the!control!group!(3.81!μg.LH1!d.w.).!Statistical!analysis!revealed!that!As!

concentrations! determined! in! organisms! exposed! to! 1000! μg! As! LH1! in! water! were!

significantly!different!from!controls!(p!<!0.05).!

!

!


! 37!

!

Figure. I.1.! Total! As! in!whole! soft! tissues! (average! ±! s.d.! μg.LH1! dry!weight);! Significant!

differences!(★!p!<!0.05).!

Electrophoresis!results!of!the!MTs!SDSHPAGE!obtained!from!C.&fluminea!exposed!to!

AsIII!are!shown!in!figure!2.!C.&fluminea!MTs!seemed!to!contain!two!distinct!bands,!18.22!kDa!

and!16.41!kDa,!corresponding!to!MT!forms!I!and!II,!respectively.!In!addition,!lowHmolecular!

weight! contaminants! are! also! visible! in! the! cytosolic! fraction! of! the! exposed! organisms.!

MTs! were! quantified! in! the! tissues! of! C.& fluminea! that! were! exposed! to! different! As!

concentrations,!as!shown!in!figure!I.3.!

!

Figure. I.2.!SDSHPAGE!of!MTs!like!proteins!from!C.fluminea.!Lane!1:!MTs!from!C.&fluminea!

(band!A!and!B);!Lane!2!and!3:!MTs!standard!from!rabbit!liver.!The!main!band!is!identified!

as!both!isoforms!(MT!I!and!II);!Lane!4:!BroadHrange!protein!standard.!


!38!

The!average!MT!concentrations!ranged!from!8.38!mg.gH1!d.w.!in!the!control!group!to!

14.42!mg.gH1!d.w.!quantified!in!organisms!exposed!to!300!μg!!As!LH1.!However,!the!highest!

concentration!of!MT!(28.96!mg.gH1!d.w.)!was!measured!in!organisms!exposed!to!1000!μg!As!

LH1.! An! apparent! induction! of! MT! synthesis! was! observed! along! the! different! exposure!

treatments!(figure!I.3).!Statistical!differences!were!found!between!controls!and!organisms!

exposed!to!300!μg!As!LH1!(p&<!0.05).!

!

Figure. I.3.! Total! As! in!whole! soft! tissues! (average! ±! s.d.! μg.LH1! dry!weight);! Significant!

differences!(★!p!<!0.05)!

The!results!of! the!histochemical!evaluation!showed!the!presence!of!As!granules! in!

the! tissues! of! C.& fluminea,! particularly! in! the! cells! of! the! digestive! gland! (figure! I.4).!

However,! it!was!not!possible!to!establish!a!quantitative!correlation!between!the!granules!

and!the!different!concentrations!of!As!used!in!the!assay.!The!histological!evaluation!shows!

degeneration!of!the!digestive!gland!cells!(figure!I.4!B),!with!the!apparent!loss!of!integrity!of!

the!tissues!in!some!of!the!exposed!organisms!to!500!and!1000!μg!As!LH1.!Regarding!other!

tissues!(foot,!gills,!and!mantle),!the!histological!observation!revealed!few!or!nonexistent!As!

granules.!

!


! 39!

!

Figure.I.4.!(A)!Digestive!gland!cells!of!a!control!organism!and!(B)!histochemical!detection!

of! As! granules! within! digestive! gland! cells! of! an! exposed! organism! to! 1000! μg.LH1! As.!

Arrowheads:!As!granules.!

I.4.. Discussion.

The!main!aim!of! the!experiments!was! to! investigate! the! responses!of!MTs!and!As!

(III)! bioaccumulation! in! C.& fluminea! via! the! waterHcolumn! and! their! potential! use! as!

biomarker!of!As!exposure! in!ecotoxicology!studies.!The! total!As! levels!determined! in! the!

tissues!of! the!control!organisms!are! in!agreement!with! those! found!by! Jonhs!and!Luoma!

(1990)! for!Corbicula& sp.! and! other! freshwater! bivalve! species.! The! bioaccumulation! data!

(figure.! III.1)! suggest! an! increasing! trend! according! to! the! increasing! metalloid!

concentrations! tested,! after! 7!days! of! exposure.!However,! the! levels! of!As!determined! in!

exposed! organisms! showed! some! variability.! Such! variability! commonly! occurs! within!

similar!species,!as!well!as! the!regulation!processes!of!some!metals!(Luoma!and!Rainbow,!

2005).!According!to!Sebesvari!et!al.!(2005),!there!is!an!immediate!and!significant!capacity!

for!C.&fluminea!to!regulate!As.!Therefore,!the!variability!found!can!be!partially!attributed!to!

active!metal!regulation!and!also!to!the!short!period!of!exposure.!Furthermore,!variability!

on!tissue!metal!concentrations!can!also!be!dependent!on!environmental!conditions!and!the!

physiological! status! of! molluscs! (Roesijadi,! 1994).! In! addition,! studies! carried! out! by!

ValetteHSilver!et! al.! (1999)! reported!elevated! levels!of!As! in!oysters! (Crassostrea&sp.)!and!


!40!

mussels! (Mytilus&sp.)! from!southcoasts!of! the!USA,!but!no! link!was! found!between!the!As!

bioaccumulated! by! the! bivalves! and! the! water! column.! MTs! have! been! used! to! assess!

biological!availability!of!heavy!metals,!providing!a!more!sensitive!evaluation!tan!the!direct!

measure! of! metals! in! the! tissues! (Bebianno! and! Langston,! 1993).! The! increase! in! MT!

concentrations!has!usually!been!associated!with!elevations!in!tissue!metal!conH!centration!

(Olsson! and! Haux,! 1986),! suggesting! a! correspondence! between! bioaccumulated! metals!

and! MT! induction! (Roesijadi,! 1994).! Detection! of! MTs! in! invertebrate! tissues! by!

electrophoresis!is!a!common!procedure!to!assess!the!presence!of!this!protein!in!cytosolic!

fractions! (Duquesne! et! al.,! 1995).! SDSHPAGE! electrophoresis! showed! proteins! of! similar!

molecular!weight!to!rabbit!MTs!and!two!main!isoforms!seem!to!coexist!(figure!III.2).!Thus,!

SDSHPAGE!results!support!the!detection!of!MTHlike!proteins!in!C.&fluminea!and!show!protein!

fractions! of! 18.22! and! 16.41! kDa! (approximate! molecular! weight),! consistent! with! the!

typical! size! of! dimeric! MTs! (10–20! kDa)! found! in!many! aquatic! invertebrate! organisms!

(Engel,! 1999;! Demuynck! et! al.,! 2006).! Additionally,! the! polarographic! response! in! the!

cytosolic! fractions! indicates! the! presence! of! containing! HSH! groups! in! MTHlike! proteins!

(Bebianno!et!al.,!1992),!confirming!the!electrophoresis!data.!The!MTs!induction!in!exposed!

organisms! showed! statistical! differences! between! the! control! group! and! organisms!

exposed!to!300!μg!As!LH1!(p!<!0.05),!but!we!could!not!find!significant!statistical!differences!

between! any!other! experiment!with! the! control! organisms.!Nevertheless,! results! suggest!

that! there! is! a! tendency! of! MTs! to! increase,! at! least! until! 300! μg! As! LH1,! which! can! be!

indicative! of! active! As! detoxification! mechanisms.! On! the! basis! of! histological! data,! the!

capacity! to! regulate! As! might! be! affected.! Some! authors! reported! MTs! induction! in! C.&

fluminea! after! exposure! to! Cd,! (Doherty! et! al.,! 1988;! Inza! et! al.,! 1997;!Baudrimont! et! al.,!

1997)! and! Marie! et! al.! (2006),! transplanted! two! freshwater! bivalves,! C.& fluminea! and!

Dreissena&polymorpha,!along!a!gradient!of!polymetallic!pollution!and!found!that,!in!spite!of!

stronger! metal! accumulation! factors! in! D.& polymorpha,! C.& fluminea! showed! a! higher!


! 41!

sensitivity!in!MT!response!along!the!pollution!gradient,!most!likely!due!to!more!efficient!Cd!

and! Zn! detoxification! mechanisms! being! activated! in! C.& fluminea.! Thus,! under! higher!

contamiH!nation!pressures,!C.&fluminea!developed!a!more!efficient!adaptative!response!than!

did!D.&polymorpha!with!respect!to!Cd!and!Zn!accumulation.!Nevertheless,! the!tendency!of!

MTs! to! increase! after! metal! exposure! is! not! systematically! observed,! especially! when!

exposure!conditions!are!too!severe!and!MTs’!metal!sequestration!capacity!is!overwhelmed!

(Baudrimont! et! al.,! 2003).! In! a! few! organisms,! the! histological! evaluation! showed!

degeneration! of! the! cells! of! the! digestive! gland! (figure! III.4! B),!with! an! apparent! loss! of!

tissue! integrity! in! organisms! exposed! to! 500! and! 1000! μg.LH1! of! As.! These! organisms!

exhibited!lower!levels!of!MTs!in!comparison!with!organisms!from!the!same!treatment!that!

revealed! a! normal! digestive! gland! cell! condition.! This! suggests! that! the! homeostatic!

capacity,! and! consequently! the! regulation! and!neutralization! of!As,! can!be!diminished! in!

organisms! where! cells! are! severely! affected! by! exposure! to! high! concentrations! of! As.!

According!to!Cajaraville!et!al.!(1995),!the!digestive!cells!are!involved!in!MT!induction!and!

intracellular! digestion! via! a! lysosomal! system.! This! is! important! for!metal! sequestration!

and!detoxification,!but!excessive!amounts!of!metals!overload!the!storage!capacity!of!these!

organelles!and!cause!alterations!of!the!lysosomal!membrane!stability.!Thus,!MT!induction!

and!metal! detoxification!may! be! affected.! The! intracellular! storage! and! sequestration! of!

metals! by! vesicleH! bound! granules! and! cytosolic! proteins! have! been! shown! to! play! an!

important!role!in!controlling!both!the!kinetics!of!bioaccumulation!and!the!manifestation!of!

toxic! effects! via! the! reduction! of! the! metal’s! accessibility! to! functional! binding! sites!

(Roesijadi! and!Robinson,! 1994).! It! is! possible! that! an! analogous!process!may!occur!with!

respect! to! arsenic! and! this! mollusc! species.! The! histochemical! results! show! some! As!

granules! within! tissues,! particularly! in! the! digestive! gland! cells,! demonstrating! As!

bioaccumulation!and!supporting!the!hypothesis!that!C.&fluminea!digestive!gland!cells!play!a!

major!role!in!As!detoxification!mechanisms.!The!evaluation!performed!in!the!gills,!mantle,!


!42!

and! foot! revealed! an! absence! of! (or! few)! granules,! suggesting! that! they! are! less! able! to!

bioaccumulate! As,! and! may! be! less! involved! in! detoxification! processes.! The! results!

obtained!in!this!study!suggest!that!C.&fluminea!have!a!high!potential!to!be!used!as!indicative!

organisms! for! As! contamination.! Further! exposure! studies,! using! organisms! of! different!

sizes,!as!well!as!longer!exposure!periods!is!highly!recommended.!Other!biomarkers!should!

also! be! analyzed! (e.g.! lipid! peroxidation,! gluthationeHSHtransferase,! genotoxicicity)! to!

complement!the!available!information!on!toxicological!effects!of!As!in!these!molluscs.!

I.5.. References.

Banerjee! K,! Helwick! RP,! Gupta! S! (1999)! A! treatment! process! for! removal! of! mixed!

inorganic!and!organic!arsenic!species!from!groundwater.!Environ&Prog&18:280–284.!

Basack!SB,!Oneto!ML,!Fuchs!JS,!Wood!EJ,!Kesten!EM!(1998)!Esterases!of!Corbicula&fluminea!

as! biomarkers! of! exposure! to! organophosphorus! pesticides.!Bull&Environ&Contam&Toxicol&

61:569–576.!

Baudrimont!M,!Metivaud!J,!MauryHBrachet!R,!Ribeyre!F,!Boudu!A!(1997).Bioaccumulation!

and! metallothionein! response! in! the! asiatic! clam! Corbicula& fluminea! after! experimental!

exposure!to!cadmium!and!inorganic!mercury.!Environ&Toxicol&Chem&16:2096–2105.!

Baudrimont!M,!Andres!S,!Durrieu!G,!Boudou!A!(2003)!The!key!role!of!metallothioneins!in!

the!bivalve!Corbicula&fluminea!during!the!depuration!phase,!after!in!situ!exposure!to!Cd!and!

Zn.!Aquat&Toxicol&63:89–102.!

Bebianno! MJ,! Langston! WJ! (1989)! Quantification! of! Metallothioneins! in! marine!

invertebrates!using!different!pulse!polarography.!Port&Electrochim&Acta&7:59–64.!

Bebianno!MJ,!Langston!WJ!(1991)!Metallothionein!induction!in!Mytilus!edulis!exposed!to!

cadmium.!Mar&Biol&108:91–96.!


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Bebianno! MJ,! Langston! WJ! (1993)! Turnover! rate! of! metallothionein! and! cadmium! in!

Mytilus!edulis.!Biometals&6:239–244.!

Bebianno!MJ,!Langston!WJ,!Simkiss!K!(1992)!Metallothionein!induction!in!Littorina!littorea!

(mollusca:!prosobranchia)!on!exposure!to!cadmium.!J&Mar&Biol&Ass&UK&72:329–342.!

Bilos!C,!Colombo!JC,!Presa!MJR!(1998)!Trace!metals!in!suspended!particles,!sediments!and!

Asiatic!clams!(Corbicula&fluminea)!of!the!Rio!de!la!Plata!Estuary,!Argentina.!Environ&Pollut&

99:1–11.!

Bissen!M,!Frimmel!FH!(2003)!Arsenic—A!Review.!II.!Oxidation!of!arsenic!and!its!removal!

in!water!treatment.!Acta&hydroch&hydrob&31:97–107.!

Britton! JC,! Morton! B! (1982)! A! dissection! guide,! field! and! laboratory! manual! for! the!

introduced!bivalve!Corbicula&fluminea.!Malacol&Rev&3:1–82.!

Bryan!GW,!Langston!WJ,!Hummerstone!LG,!Burt!GR!(1985)!A!Guide!to!the!assessment!of!

heavyHmetal! contamination! in! estuaries! using! biological! indicators.! Mar& Biol& Ass& UK&

(Occasional&Publication)&4:74.!

Cajaraville!MP,!Robledo!Y,!Etxeberria!M,!Marigomez!I!(1995)!Cellular!biomarkers!as!useful!

tools! in! the! biological! monitoring! of! environmental! pollution:! Molluscan! digestive!

lysossomes.!In:!Cajaraville!MP,!editor.!Cell&Biology&in&Environmental&Toxicology.&University&of&

the&Basque&Country&Press&Service,&Bilbo,&Spain.&pp&89–134.!

Carpene!E!(1993)!Metallothionein!in!marine!molluscs.!In:!Dallinger!R,!editor.!Ecotoxicology&

of&Metals&in&Invertebrates.&FL&USA:&CRC,&Boca&Rato.&pp&55–72.!

Croteau!MN,! Luoma! SN,! Stewart!AR! (2005)! Trophic! transfer! of!metals! along! freshwater!

food! webs:! Evidence! of! cadmium! biomagnification! in! nature.! Limnol& Oceanogr& 50:1511–

1519.!


!44!

Demuynck! S,! Grumiaux! F,! Mottier! V,! Schikorski! D,! Lemiere! S,! Lepretre! A! (2006)!

Metallothionein! response! following! cadmium! exposure! in! the! oligochaete! Eisenia& fetida.!

Comp&Biochem&Physiol&C&144:34–46.!

Doherty!FG,!Failla!ML,!Cherry!DS!(1988)!MetallothioneinHlike!heavy!metal!binding!protein!

levels! in!Asiatic!clams!are!dependent!on!the!duration!and!mode!of!exposure!to!cadmium.!

Water&Res&22:927–932.!

Domouhtsidou!GP,!Dailianis!S,!Kaloyianni!M,!Dimitriadis!VK!(2004)!Lysosomal!membrane!

stability! and! metallothionein! content! in! Mytilus& galloprovincialis! (L.),! as! biomarkers:!

Combination!with!trace!metal!concentrations.!Mar&Pollut&Bull&48:572–586.!

Duquesne! S,! Flowers! AE,! Coil! JC! (1995)! Preliminary! evidence! for! a!metallothioneinHlike!

heavy!metalHbinding!protein!in!the!tropical!marine!bivalve!Tridacna!crocea.!Comp&Biochem&

Physiol&C&112:69–78.&

Engel!DW!(1999)!Accumulation!and!cytosolic!partitioning!of!metals!in!the!American!oyster!

Crassostrea!virginica.!Mar&Environ&Res&47:89–102.!

Fournier! E,! Adam! C,! Massabuau! JC,! GarnierHLaplace! J! (2005)! Bioaccumulation! of!

waterborne!selenium!in! the!Asiatic!clam!Corbicula&fluminea:! Influence!of! feedingHinduced!

ventilatory!activity!and!selenium!species.!Aquat&Toxicol&72:251–260.!

Hassan! MM,! Atkins! PJ,! Dunn! CE! (2005)! Social! implications! of! arsenic! poisoning! in!

Bangladesh.!Soc&Sci&Med&6:2201–2211.!

Inza! B,! Ribeyre! F,! MauryHBrachet! R,! Boudou! A! (1997). Tissue! distribution! of! inorganic!

mercury,!methylmercury!and!cadmium!in! the!asiatic!clam!(Corbiculafluminea)! in!relation!

to!the!contamination!levels!of!the!water!column!and!sediment.!Chemosphere&35:2817–2836.!

IRAR!(2005)!Instituto!Regulador!das!Aguas!e!dos!Resıduos:!Relatorio!Anual!do!Sector!das!


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Aguas! e! Resıduos! em!Portugal! (RASARP! –! 2004),! Vol.! 4,! Controlo! da!Qualidade! da! Agua!

para!Consumo!Humano.238!p.!

Jonhs! C,! Luoma! SN! (1990)! Arsenic! in! benthic! bivalves! of! San! Francisco! bay! and! the!

Sacramento/San!Joaquin!River.!Sci&Total&Environ&97/98:673–684.!

Kojima!Y!(1991)!Definitions!and!nomenclature!of!metallothioneins.!In:!Abelson!JN,!Simon!

MI,!editors.!Methods&in&Enzymology.&San&Diego&CA,&USA:&Academic.&pp&8–10.&

Labrot! F,! Ribera! D,! SaintHDenis! M,! Narbonne! JF! (1996)! In! vitro! and! in! vivo! studies! of!

potential! biomarkers! of! lead! and! uranium! contamination:! Lipid! peroxidation,! acetylH

cholinesterase,! catalase! and! glutathione! peroxidase! activities! in! three! nonHmammalian!

species.!Biomarkers&1:23–30.!

Liao! CM,! Chen! BC,! Singh! S,! Lin! MC,! Liu! CW,! Han! BC! (2003)! Acute! toxicity! and!

bioaccumulation!of!arsenic!in!tilapia!(Oreochromis!mossambicus)!from!a!Blackfoot!Disease!

area!in!Taiwan.!Environ&Toxicol&18:252–259.!

Lillie!RD!(1965)!Histopathologic!Technic!and!Practical!Histohemistry.!New!York:!McGraw!

Hill.445!p.!

Luoma!S,!Rainbow!P!(2005)!Why!is!metal!bioaccumulation!so!variable?!Biodynamics!as!a!

unifying!concept.!Environ&Sci&Technol&39:1921–1931.!

Marie! V,! Baudrimont! M,! Boudou! A! (2006)! Cadmium! and! zinc! bioaccumulation! and!

metallothionein! response! in! two! freshwater! bivalves! (Corbicula& fluminea! and! Dreissena&

polymorpha)!transplanted!along!a!polymetallic!gradient.!Chemosphere&65:609–617.!

Martoja!R,!Martoja!M!(1967)!Initiation!aux!tecniques!de!l’histologie!animal.!Paris:!Masson!

and.!Cie.345!p.!

Mouthon! J! (1981)! Sur! la! presence! en! France! et! au! Portugal! de! Corbicula! (Bivalvia,&


!46!

Corbiculidae)!originaire!d’Asie.!Basteria&45:109–116.!

Mouthon!J!(2001).Life!cycle!and!population!dynamics!of!the!Asian!clam!Corbicula&fluminea!

(Bivalvia:!Corbiculidae)!in!the!Saone!River!at!Lyon!(France).!Hydrobiol&452:109–119.!

Ngu! TT,! Stillman! MJ! (2006)! Arsenic! binding! to! human! metallothionein.! J& Am& Chem& Soc&

128:12473–12483.!

Olafson! RW,! Olsson! PE! (1991)! Electrochemical! detection! of! metallothionein.! Methods&

Enzymol&205:205–213.!

Oliver!LM,!Fisher!WS!(1999)!Appraisal!of!prospective!bivalve!immunomarkers.!Biomarkers&

4:510–530.!

Olsson! PE,! Haux! C! (1986)! Increased! hepatic! metallothionein! content! correlates! to!

cadmium!accumulation!in!environmentally!exposed!perch!(Perca&fluviatilis).!Aquat&Toxicol&

9:231–242.!

Palecek! E,! Pechan! Z! (1971)! Estimation! of! nanogram! quantities! of! proteins! by! pulse!

polarographic!techniques.!Anal&Biochem&42:59–71.&

Roesijadi!G!(1994)!Behavior!of!metallothioneinHbound!metals!in!a!natural!population!of!an!

Estuarine!mollusc.!Mar&Environ&Res&38:147–168.!

Roesijadi! G! (1999)! The! basis! for! increased! metallothionein! in! a! natural! population! of!

Crassostrea!virginica.!Biomarkers&4:467–472.!

Roesijadi! G,! Robinson! WE! (1994)! Metal! regulation! in! aquatic! animals:! Mechanisms! of!

uptake,!accumulation,!and!release.!In:!Malins!DC,!Ostrander!GK,!editors.!Aquatic&Toxicology:&

Moleck&ular,&Biochemical,&and&Cellular&Perspectives.&FL&USA:&CRC&Boca&Raton.&pp&387–420.!

Sebesvari! Z,! Ettwigb! F,! Emonsc! H! (2005)! Biomonitoring! of! tin! and! arsenic! in! different!


! 47!

compartments! of! a! limnic! ecosystem! with! emphasis! on! Corbicula& fluminea! and!

Dikerogammarus!villosus.!J&Environ&Monit&7:203–207.!

Thompson! JAJ,! Cosson! RP! (1984). An! Improved! electrochemical! method! for! the!

quantification!of!metallothioneins!in!marine!organisms.!Mar&Environ&Res&11:137–152.!

ValetteHSilver!NJ,!Riedel!GF,!Crecelius!EA,!Windom!H,!Smith!RG,!Dolvin!SS!(1999)!Elevated!

arsenic!concentrations! in!bivalves! from!the!southeast! coasts!of! the!USA.!Mar&Environ&Res&

48:311–333.!

Viarengo!A!(1989)!Heavy!metals! in!marine! invertebrates:!Mechanisms!of! regulation!and!

toxicity!at!the!cellular!level.!Aquat&Sci&1:295–317.!

Viarengo!A,!Ponzano!E,!Dondero!F,!Fabbri!RA!(1997)!A!simple!spectrophotometric!method!

for!metallothionein!evaluation!in!marine!organisms:!An!application!to!Mediterranean!and!

Antarctic!molluscs.!Mar&Environ&Res&44:69–84.!

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! 49!

Chapter!II.!Effects!of!exposure!to!As!in!Corbicula&

fluminea:!Evaluation!of!the!histological,!

histochemical!and!biochemical!responses!

II.1.. Abstract.

Arsenic!(As)!is!a!common!element!in!aquatic!environments!and!it!is!originated!from!

both!anthropogenic!and!natural!processes.!It!is!a!toxic!metalloid,!especially!in!the!trivalent!

form! (arsenite),! to! humans! and! wildlife,! and! consequently! its! contamination! of! aquatic!

environments! (especially! groundwater! and! drinking! water)! is! a! serious! public! health!

problem!in!several!regions!around!the!globe,!particularly!in!Asia,!since!it!is!known!to!cause!

cancer!and!other!types!of!pathologies.!The!main!goal!of!the!present!study!was!to!assess!the!

toxicological! effects,! bioaccumulation! and! ability! to! regulate! As! in! Corbicula& fluminea!

(Müller!1744),!exposed!to!different!concentrations!of!As! in!the!water.!One!hundred!fresh!

water!bivalves!(1.6!±!0.3!g)!from!the!Minho!River!(Portugal)!were!randomly!distributed!in!

ten!20L!tanks!and!exposed!to!different!nominal!concentrations!of!As!(100,!300,!500,!and!

1000!μg.L–1)! for!28!days.!A!tank!with!tap!water! free!of!chlorine!was!used!as!control.!The!

assay! was! performed! in! duplicate,! at! a! constant! temperature! of! 20! ±! 1ºC.! The! results!

showed! a! significant! increase! (p! <! 0.01)! of! As! in! the! total! concentration! and! cytosolic!

fraction! after! 28! days! of! exposure! in! all! treatments.! A! significant! increase! (p! <! 0.01)! in!

metallothionein! concentration! was! also! detected! in! bivalves! exposed! to! the! different!

concentrations!of!As,! in!comparison!with!the!controls.!The!histological!and!histochemical!

evaluation! provided! clear! evidence! that! As! accumulated! in! tissues,! especially! in! the!

digestive!gland,!and!caused!tissue!alterations!in!50%!of!the!organisms.!

Chapter!II.!Histological!histochemical!and!biochemical!responses&

!50!

II.2.. Materials.and.Methods.

II.2.1.. Experimental.Procedure.

The! biological! material! used! to! carry! out! the! experiments! was! the! freshHwater!

mollusc!C.&fluminea.!Animals!were!collected! from!the!Minho!River! (northern!Portugal)! in!

December!2005!and!then!maintained!under!laboratory!conditions!in!a!system!of!tanks!with!

recirculated,!filtered,!and!continuously!aerated!tap!water,!at!a!constant!temperature!of!20!±!

1ºC.!After!a!twoHweek!period!of!acclimatization,!100!individuals!(soft!body!mass!=!1.6!±!0.3!

g;! shell! length!=!1.9!±!0.2!cm)!were!randomly!distributed! in! ten!20L!polyvinyl! tanks!and!

exposed! to! different! nominal! concentrations! of! trivalent! As! (0,! 100,! 300,! 500,! and! 1000!

μg.L–1)!added!to!the!water!in!each!tank!at!the!beginning!of!the!experiment.!A!solution!was!

prepared!from!standard!inorganic!trivalent!As!purchased!from!Aldrich!(St.!Louis,!MO,!USA),!

to!which!distilled!water!was!added!to!obtain!a!final!concentration!of!100!mg.L–1.!Aliquots!of!

this!solution!were!added! to! test! tanks!according! to! the!nominal!concentrations! intended.!

The! assay! was! static! and! carried! out! over! 28! days,! in! duplicate,! at! 20! ±! 1ºC,! using!

dechlorinated!tap!water,!with!continuous!aeration!and!constant!photoperiod!(12!h!light;!12!

h! dark).! Two! tanks! with! tap! water! (chlorine! free)! were! used! as! control.! Some!

physicochemical! parameters! were!monitored! daily:! temperature! and! pH! using! an! Orion!

potentiometer!(model!290,!Orion!Research!Inc.,!USA),!and!electrical!conductivity!using!an!

Orion!conductivityHsalinity!meter!(model!140,!Orion!Research!Inc.,!Germany).!The!bivalves!

were!fed!minced!fish!pellets!daily!(Dibaq,!Spain).!Organisms!were!sampled!after!28!days!of!

exposure,!and!soft! tissues!were!removed!using!a!scalpel! to!cut! the!adductor!muscles!and!

force!open!the!valves.!Fresh!soft!tissues!were!weighed!(fresh!weight)!and!dried!in!a!forced!

air!oven!at!60ºC!for!five!days!(dry!weight),!and!then!stored!at!–80ºC!for!further!analysis.!

The!length!of!the!shell!was!also!recorded.!Samples!of!water!from!each!tank!were!taken!at!

the!beginning!and!end!of!the!assay!for!the!determination!of!the!total!As!concentration.!


! 51!

II.2.2..Histology.and.Histochemistry.

Subsamples! of! C.& fluminea! ! soft! tissues! were! taken! and! fixed! in! BouinHHollande’s!

solution!for!48!h.!The!samples!were!processed!using!the!standard!histological!procedures!

described! by!Martoja! and!Martoja! (1967).! After! paraffin! inclusion,! sections! 5–7! μm! thin!

were!stained!with!haematoxylin!and!eosin,!and!mounted!for!microscope!observation.!The!

histochemistry!was!performed!according!to!Castel’s!method!as!described!by!Lillie!(1965)!

to! reveal! As! granules! in! the! tissues.! The! effects! on! the! tissues! were! assessed! through!

histological!and!histochemical!observation,!using!an!optical!microscope!(LeicaHATC!2000,!

Wetzlar,!Germany).!

II.2.3.. Quantification.of.Metallothioneins.

The! procedures! adopted! to! determine!MTs!were! previously! described! by!Olafson!

(1981),!and!Thompson!and!Cosson!(1984).!Briefly,!MTs!were!extracted!by!homogenizing!

the! soft! tissues! from!C.& fluminea! in! TrisHHCl! (0.02!M,! pH! =! 8.6)! in! a! volume! ratio! of! 1:4!

(tissue:buffer),! in! an! iceHbath,! using! a! rotor! equipped! with! a! TFE! pestle! (HeidolphHRZR!

2100,!Heidolph!Elektro!GmbH!&!Co.!KG,!Kelheim,!Germany).!Subsamples!were!then!taken!

for!wet:dry!weight!ratio!determinations!and!metal!analysis.!The!remaining!homogenate!(3!

mL)!of!each!sample!was!centrifuged!(30,000!×!g!at!4ºC)!for!1!h.!The!cytosol!(supernatant)!

was!separated!from!the!pellet,!placed!in!a!80ºC!water!bath!for!10!min!to!denaturate!high!

molecular!weight!and!nonHheat!stable!proteins,! followed!by!centrifugation!(30,000!×!g!at!

4ºC)!for!1!h.!The!cytosolic!fractions!obtained!were!stored!at!–80ºC!for!MT!determination.!

The! quantification! of! MTs! in! the! heatHtreated! cytosolic! fraction! was! carried! out! by!

differential! pulse! polarography! for! thiolic! (–SH)! compounds,! using! the! Brdička! reaction.!

The! determination! was! carried! out! using! a! Metrohm! 694! stand! and! 693! processor!

(Metrohm!Ltd.,!Herisau,! Switzerland),!with! a! static!mercury!drop!electrode,! according! to!

the!methods!described!by!Thompson!and!Cosson!(1984)!and!Olafson!and!Olsson!(1991).!


!52!

The!threeHelectrode!system!consisted!of!a!bevelled!capillary!mercury!electrode,!a!platinum!

counter!electrode,!and!an!Ag/AgCl!reference!electrode.!The!Brdička!supporting!electrolyte!

was!prepared!weekly!according!to!the!method!proposed!by!Palecek!and!Pechan!(1971)!and!

Thompson! and! Cosson! (1984),! and! contained! 1! M! NH4Cl,! 1! M! NH4OH,! and! 2! mM!

[Co(NH3)6]Cl3,! being! stored! at! 4ºC.! Triton! xH100! (Sigma,! St.! Louis,! MO,! USA)! at! an!

appropriate!concentration!of!2.5!×!10–2!%!(v/v)!was!used!to!suppress!secondary!maxima!

and! minima! and! eliminate! baseline! noise! (Bebianno! and! Langston! 1989).! Given! the!

absence!of!a!molluscan!MT,!a!rabbit!liver!MT!(forms!I!and!II,!Sigma,!St!Louis,!MO,!USA)!was!

used! as! a! standard,! prepared! to! a! working! standard! solution! of! 10! mg.L–1! in! deionized!

water.! The! measurements! were! performed! by! adding! 20! mL! of! supporting! electrolyte!

directly! into! the!polarographic!cell,! together!with!250!μL!of!Triton!XH100!and!aliquots!of!

standard!solution!of!rabbit!MT!(250!μL)!and!sample!(25–150!μL).!The!cell!was!purged!with!

purified! nitrogen! for! 120! sec! before! analysis! (Thompson! and! Cosson! 1984).! The! MT!

concentrations! were! determined! using! the! standard! additions! method! according! to!

Bebianno!and!Langston!(1989),!and!the!results!are!expressed!as!mg.g–1!dry!weight!whole!

body!homogenate.!

II.2.4.. Arsenic.Determination..

Soft! tissue! samples! were! dried! at! 60ºC! for! five! days,! followed! by! digestion! with!

concentrated! nitric! acid! (5! mL! of! HNO3! –! Suprapur,! Merck,! Darmstadt,! Germany)! in!

pressurized!Teflon!cells!over!24!h!at!room!temperature.!The!samples!were!then!heated!at!

100ºC! for! 4! h,! before! adding! 1!mL!H2O2! (Bryan! et! al.! 1985).! After!waiting! 1! h,! digested!

samples!were!diluted!in!25!mL!of!ultrapure!water!(MilliHQ!plus).!Water!samples!(n!=!3!per!

each!experimental!condition)!were!collected! in!polyethylene!bottles!at! the!beginning!and!

the! end! of! the! assay! and! acidified! with! 10! μL.mL–1! HNO3! (Suprapur,! Merck,! Darmstadt,!

Germany).!Samples!were!stored!at!4ºC.!Arsenic!was!determined!by!electrothermal!atomic!


! 53!

absorption! spectrometry! (GFAAS)! with! Zeeman! correction! and! matrix! modifier!

(Pd(NO3)2.2H2O,! Fluka),! using! a! graphite! tuve! atomizer! (AAS,! Thermoptec! M6Solaar),!

without! filtration,! after! dilution!with! ultrapure!water.! Standard! solutions!were! prepared!

from!a!commercial!inorganic!As!standard!for!atomic!absorption!(N!206962,!1!g.L–1,!Aldrich,!

USA).!The!total!As!concentration!was!determined!using!a!standard!calibration!curve.!The!

detection!and!quantification!limits!were!3.1!and!9.3!μg.L–1,!calculated!according!to!the!3σ!

and! 10σ! criteria,! respectively.! Quantification! of! total! As! in! digested! tissue! samples! was!

achieved! by! electrothermal! atomic! absorption! using! the! same! procedure! aforesaid.! The!

reference! material! DOLT! 3! (NRCCNRC,! Canada)! and! blanks! were! analyzed! in! order! to!

validate! the!analytical!procedure!analysis,!using! the!same!analytical!procedure!as! for! the!

samples.!The!total!As!measured!from!the!digested!samples!of!reference!material!(9.4!±!0.3!

mg! kg–1;! n! =! 2)! showed! good! agreement! with! the! certified! values! (10.2! ±! 0.5! mg.kgH1).!

Concentrations!are!expressed!on!a!dry!tissue!weight!basis!(μg.g–1).!

II.2.5.. Statistical.Analysis..

Statistical!analysis!was!performed!using!the!nonparametric!MannHWhitney!U!test!to!

determine! differences! between! treatment! and! control! samples.! Correlations! were!

performed!using!the!nonparamatric!Spearman!rankHorder!correlation!coefficient!(R).!The!

Statistica!5.0!software!(StatSoft!Inc.,!USA,!1995)!was!used!in!all!statistics!using!a!0.01!level!

of!significance.!

II.3.. Results.

Throughout!the!28Hday!experimental!period!no!mortality!was!registered!regardless!

of!treatment.!The!physicochemical!parameters!were!stable!and!showed!good!values!during!

theexperiments:!temperature,!20!±!2ºC;!pH,!7.0!±!0.2;!and!conductivity,!545!±!10!μS.cm–1.!

The!measurements! of! total! As! in! water! samples! at! the! beginning! and! at! the! end! of! the!


!54!

experiment!are!shown!in!table!II.1.!

Table.II.1.!Total!As!concentrations!(average!±!s.d.!μg.LH1)!measured!in!water!samples!

at!the!beginning!and!end!of!the!experiment.!

! Control* 100* 300* 500* 1000*

Ci! <3!μg.LH1! 98±6! 297±5! 495±8! 993±5!

Cf! <3!μg.LH1! 94±3! 289±3! 475±5! 971±4!

Ci:! initial! concentration;! Cf:! final! concentration;! (n=3,! each! replicate).! Limit! of!

detection:!3.1!μg.LH1!

The! results! obtained! for! total! As! accumulated! in! soft! tissues! from!C.& fluminea! are!

shown!in!figure!II.1.!The!highest!average!level!(145!±!26!μg.g–1!d.w.)!was!determined!in!soft!

tissues!from!organisms!exposed!to!1000!μg!As!L–1.!The!lowest!average!level!(0.7!±!0.1!μg.g–

1!d.w.)!was!measured!in!the!cytosolic!fraction!of!the!control!group.!

!

Figure. II.1.! Concentration! of! metallothioneins! (average! ±! s.d.! μg.LH1! dry! weight),! and!

concentration!od!As!in!the!soft!tissue!and!in!the!citosolic!fraction!(average!±!s.d.!μg.LH1!dry!

weight):!Significant!differences!from!controls!(★!p&<!0.05).!

!

!


! 55!

The! statistical! analysis! of! the! results! showed! that! total! As! concentrations!

determined!in!exposed!organisms!were!significantly!different!(p!<!0.01)!from!those!of!the!

controls! in! all! treatments! and! in! whole! tissues,! as! well! as! the! cytosolic! fraction.! The!

quantified!levels!of!MTs!for!the!different!exposure!concentrations!are!also!shown!in!figure!

1.! The! average! MT! concentrations! per! test! ranged! from! 6.8! ±! 1.2! mg.g–1! (d.w.),! in! the!

control! group,! to! 19.1! ±! 3.7! mg.g–1! (d.w.),! in! organisms! exposed! to! 100! μg! As! L–1.! The!

statistical!analyses!showed!that!MTHlike!protein! induction!was!significantly!different!(p!<!

0.01)!from!controls!in!all!treatments.!Statistics!showed!a!significant!correlation!(R!=!0.6,!p!<!

0.01)! between! cytosolic! As! and!MTHlike! proteins;! however,! only! a!marginally! significant!

correlation! (R!=!0.3,!p! =!0.09)!was! found!between!whole! tissue!As!and!MTHlike!proteins.!

Nonetheless,!a!better!significant!correlation!(R!=!0.7,!p!<!0.01)!was!observed!between!As!in!

tissues!and!cytosolic!As.!The!percentages!of! total!As!determined! in! the! cytosolic! fraction!

ranged!from!5.5%!in!controls!to!17.3%!in!organisms!exposed!to!100!μg!As!L–1!relative!to!As!

in!soft!tissues!(figure!II.2.).!

!

Figure.II.2.!Distribution!of!As!in!soft!tissues!and!in!the!heatHtreated!cytosolic!fraction.!

The!histochemical!evaluation!results!support!the!bioaccumulation!results,!revealing!

that! As! granules! are! present! in! the! tissues! of! C.& fluminea! exposed! to! the! different! As!

!


!56!

concentrations,! mainly! in! the! cells! of! the! digestive! gland! (figure! II.3).! The! histological!

observations!also!showed!that!organisms,!especially!those!exposed!to!1000!μg!As!L–1,!have!

degeneration!of!the!cells!from!the!digestive!gland.!

!

Figure. II.. 3.! (A)! Digestive! gland! cells! of! a! control! organism! and! (B)! histochemical!

detection!of!As!granules!within!digestive!gland!cell!of!an!organism!exposed!to!1000!μg.LH1.!

Arrowheads:!As!granules!

II.4.. Discussion.

The! total! As! concentrations! found! in! the! tissues! of! the! control! organisms! are! in!

agreement! with! the! levels! reported! by! other! authors! for! the! same! species! (Johns! and!

Luoma! 1990),! as! well! as! for! other! bivalve! species! such! as! oysters,! clams,! and! mussels!

(Suñer! et! al.! 1999,! ValetteHSilver! et! al.! 1999,! Argese! et! al.! 2005,! Orescanin! et! al.! 2006,!

Santos! et! al.! 2007).! The! results! of! total! As! accumulated! in! whole! soft! tissues! showed! a!

significant! increase! (P! <! 0.01)! after! 28! days! of! exposure! according! to! the! different!

treatments.!Similarly,!total!As!levels!found!in!the!cytosolic!fractions!registered!a!significant!

increase!(p!<!0.01)!and!showed!a!good!correlation!with!As!in!whole!soft!tissues!(R!=!0.7,!p!<!

0.01).!The!levels!of!total!As!found!in!the!water!samples!showed!a!decrease!at!the!end!of!the!

experimental! period! for! all! tested! concentrations.! This! difference! can! be! due! to! As!

bioaccumulation!and!eventually! to!As!adsorption!on!the!tank!walls.!The!MTHlike!proteins!


! 57!

have!been!used!as!biomarkers!of!exposure!to!high!levels!of!metals,!where!an!induction!is!

usually! associated! with! elevations! in! tissue! metal! concentration,! suggesting! a!

correspondence!between!bioaccumulated!metals!and!MTHlike!protein!induction!(Bebianno!

and!Langston!1993,!Roesijadi!1994).!The!detection!and! identification!of!MTHlike!proteins!

from!C.& fluminea! tissues! is! a! common! procedure! and! has! been! done! in! previous! studies!

(Baudrimont! et! al.! 1997,!Marie! et! al.! 2006,! Diniz! et! al.! 2007),! showing! protein! fractions!

consistent! with! the! typical! size! of! dimeric! MTs! (10–20! kDa)! found! in! many! aquatic!

invertebrate!organisms.! In!addition,! the!same!authors!showed!that! this!bivalve!species! is!

able! to! induce!MTHlike! proteins!when! exposed! to! high!metal! concentrations.! Our! results!

show!a!significant!increase!in!MTHlike!proteins!for!all!tests!when!compared!to!controls!but!

no! significant! differences! were! found! between! treatments,! which! may! suggest! that! the!

capacity!to!sequester!the!metalloid! is!diminished!as!the!result!of!an!excess!of! total!As,!as!

also! suggested! by! Baudrimont! et! al.! (2003).! The! increase! in! total! As! concentrations!

observed! in! whole! soft! tissues! was! accompanied! by! a! significant! induction! of! MTHlike!

proteins!in!comparison!to!the!controls;!however,!the!best!correlation!was!found!between!

the! cytosolic! fraction! As! and! MTHlike! proteins! (R! =! 0.6,! p! <! 0.01),! indicating! a! close!

relationship!between!the!metalloid!present!in!the!cytosol!and!MT!induction.!These!results!

suggest! that,! most! probably,! other! detoxification! mechanisms! are! involved! in! the!

neutralization! of! the! toxic! effects! of! As,! by! reducing! the! intracellular! metal! availability.!

Some! of! those!mechanisms!may! be! related! to! intracellular! storage! and! sequestration! of!

metals! by! vesicleHbound! granules! and! other! nonHMT! cytosolic! proteins! (Roesijadi! and!

Robinson!1994,!Baudrimont!et!al.!1999,!Cecílio!et!al.!2006).!Sebesvari!et!al.!(2005)!showed!

that!this!species!is!capable!of!actively!regulating!As,!while!Marie!et!al.!(2006)!reported!that!

C.&fluminea!has!more!efficient!detoxification!mechanisms!than!another!freshHwater!bivalve,!

Dreissena!polymorpha,! even!under! exposure! to!higher! concentrations! of!metals! (namely!

Cd! and! Zn),! probably! due! to! a! more! efficient! adaptive! response.! Moreover,! the! low!


!58!

percentages!of! total!As! found! in! the! cytosol! supports! this!hypothesis,! suggesting! that! for!

the! concentrations! tested,!C.& fluminea! is! capable! of! regulating! the! excess! of!metalloid,! at!

least!to!some!extent,!using!different!detoxification!mechanisms!that!may!also!act!together!

for! this! purpose.! In! addition,! it!must! be! noted! that! biotic! parameters! such! as! body! size!

and/or! weight,! phenotypic! differences,! sex,! physiological! conditions,! and! reproductive!

state!may! influence!metal! bioaccumulation! (MartínHDíaz! et! al.! 2006).!The!histochemistry!

revealed!the!presence!of!As!granules!accumulated!in!the!bivalve!soft!tissues,!mostly!in!cells!

from! the! digestive! gland,! supporting! the! higher! levels! of! total! As! determined! in! tissues!

according! to! the! different! exposure! concentrations.! These! results! emphasize! the!

hypothesis!of!the!participation!of!other!detoxifying!mechanisms!other!than!As!chelating!by!

MTs.! The! present! study! shows! that!C.& fluminea! bioaccumulates!As! according! to! different!

concentrations!of!this!metalloid!in!water.!The!induction!of!MTHlike!proteins!can!be!inferred!

as!a!detoxifying!response!to!contamination!pressure;!however,!the!total!As!concentrations!

found! in! the! cytosolic! fractions! were!much! lower! than! the! total! As! found! in! whole! soft!

tissues,!supporting!the!hypothesis!that!other!detoxifying!mechanisms,!besides!MTs,!play!an!

important!role!in!detoxification,!since!MTs!are!cytosolic!proteins.!This!study!also!reinforces!

the!suitability!of!using!C.&fluminea! !as!a!potential!biological!indicator!of!As!contamination,!

but! highlights! the! need! to! develop! more! studies! using,! for! example,! other! types! of!

biomarkers!(e.g.,!reactive!oxygen!species,!glutathioneHSHtransferase,! lipid!peroxidation)!to!

complement!the!As!ecotoxicologic!traits!found!to!this!date!in!this!bivalve.!

II.5.. References.

Argese!E,!Bettiol!C,!Rigo!C,!Bertini!S,!Colomban!S,!Ghetti!PF!(2005)!Distribution!of!arsenic!

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267–277.!

Baudrimont!M,!Metivaud!J,!MauryHBrachet!R,!Ribeyre!F,!Boudu!A!(1997)!Bioaccumulation!


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and! metallothionein! response! in! the! Asiatic! clam! Corbicula& fluminea! after! experimental!

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Bebianno! MJ,! Langston! WJ! (1993). Turnover! rate! of! metallothionein! and! cadmium! in!

Mytilus!edulis.!Biometals&6:&239–244.!

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(2004)!Biomarkers!in!Ruditapes!decussatus:!A!potential!bioindicator!species.!Biomarkers&

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Mar.&32:&439–445.!

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99:&1–11.!

Bryan!GW,!Langston!WJ,!Hummerstone!LG,!Burt!GR!(1985)!A!guide!to! the!assessment!of!


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heavyHmetal! contamination! in! estuaries!using!biological! indicators.!Occas.&Publ.&Mar.&Biol.&

Assoc.&UK&4:&1–74.!

Carpéne!E!(1993)!Metallothionein!in!marine!molluscs.!In:!Dallinger!R!(ed.),!Ecotoxicology&

of&Metals&in&Invertebrates.!CRC,!Boca!Raton,!pp.!55–72.!

Cecílio!P,!Raimundo!J,!Canário!J,!Vale!C,!Sequeira!M!(2006)!Relationships!between!total!and!

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Domouhtsidou!GP,!Dailianis!S,!Kaloyianni!M,!Dimitriadis!VK!(2004)!Lysosomal!membrane!

stability! and! metallothionein! content! in! Mytilus! galloprovincialis! (L.),! as! biomarkers:!

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Fernández!N,!Cesar!A,!González!M,!DelValls!TA!(2006)!Level!of!contamination!in!sediments!

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Sacramento/San!Joaquin!River.!Sci.&Tot.&Environ.&97:&673–684.!

Kagi!JHR!(1993)!Evolution,!structure!and!chemical!activity!of!class!I!metallothioneins:!An!

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Margoshes!M,!Valee!BL!(1957)!A!cadmium!protein!from!equine!idney!cortex.!J.&Am.&Chem.&

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!64!

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! 65!

Chapter!III.!Toxicokinetics!of!trivalent!Arsenic!in!

Corbicula&fluminea!

III.1.. Abstract.

Arsenite! (AsIII)! uptake! and! elimination! kinetics! were! studied! in! a! freshwater!

bivalve,!Corbicula&fluminea,!exposed!to!several!nominal!concentrations!of!AsIII!(0,!100,!300,!

500,!and!1000!μg.LH1)!in!a!static!28Hday!assay,!folH!lowed!by!a!depuration!stage!of!14!days.!

At! the! end! of! each! sampling! time! (days! 0,! 7,! 28,! and! 42)! wholeHbody! portions! were!

surveyed! for! total! As! concentrations! and,! compliH! mentarily,! surveyed! for! wholeHbody!

metallothionein!(MT)!induction!to!assess!its!role!as!a!defense!mechanism!against!exposure!

to! AsIII.! Histochemical! evaluation! of! the! digestive! gland! was! performed! to! verify! As!

deposition! and! eliminaH! tion! in! the! tissue.! Results! show! a! significant! increase! in!wholeH

body!total!As!after!28!days!of!exposure!for!all!treatments,!followed!by!a!decrease!at!the!end!

of! the! depuration! phase.! Biodynamic! kinetic!models! for!As! uptake! and! elimination!were!

obtained! from!bioaccumulation!data!during! the!exposure!phase,! for!all!As! treatments,!by!

estimating! uptake! and! elimination! rate! constants.! Bioconcentration! factors! (BCFs)! were!

estimated! by! the! ratio! of! these! constants.! Results! revealed! that! exposure! to! higher!

concentrations! of! AsIII! causes! a! decrease! in! BCFs,! suggesting! that! C.! fluminea! triggers!

effective!regulatory!mechanisms!when!exposed!to!higher!concentrations!of!the!metalloid.!

Significant! induction! of! MT! was! detected! during! the! exposure! phase,! followed! by! a!

decrease! in! MT! concentration! to! control! levels! after! depuration! for! all! treatments.! No!

significant! differences! in! MT! concentrations! were! observed! between! treatments.! This!

finding! may! confirm! the! role! of! MT! as! part! of! the! As! regulation! process,! but! its!

independence!relative!to!concentrations!of!AsIII!in!water!suggests!that!MT!induction!is!not!

Chapter!III.!!Toxicokinetics!of!trivalent!As!in!C.&fluminea!

!66!

dose! dependent.! The! histochemical! evaluation! provided! clear! evidence! that! As! was!

effectively! accumulated! in! the! digestive! gland! during! exposure! and! eliminated! during!

depuration.!The!present!work!demonstrated! that!C.!fluminea! is! capable!of! regulating!As,!

even!at!exposures!as!high!as!1000!lg!LH1!of!waterborne!AsIII.!

III.2.. Materials.and.Methods.

III.2.1..Experimental.Procedure.

The! freshwater! bivalves,! Corbicula& fluminea,! collected! from! the! river! Minho’s!

hydrographic!basin!(northwestern!Portugal)!were!acclimated!in!a!closed!continuousHflow!

system!with!filtered!and!aerated! tap!water.!Animals!were! fed!daily!with!a! suspension!of!

minced!fish!pellets!(DibaQ,!Segovia,!Spain).!The!assay!was!carried!out!in!static!mode!and!in!

duplicate,!at!20!±!1°C!and!pH!7.06!±!0.07,!with!a!12:12Hh!light:dark!photoperiod.!Bivalves!

(soft!body!mass,!1.6!±!0.3!g;!shell!length,!2.0!±!0.3!cm)!were!randomly!distributed!by!20LH

capacity!polyvinyl!tanks!(15!individuals!per!replica),!with!constant!aeration,!and!exposed!

to! different! nominal! concentrations! of! AsIII:! 0! (control),! 100,! 300,! 500,! and! 1000! μg.LH1.!

AsIII! stock! solutions!were! prepared! from! standard! inorganic! trivalent! arsenic! (Sigma,! St.!

Louis,!MO,!USA).!The!control!test!was!performed!with!clean,!dechlorinated,!tap!water.!The!

assay!was!divided!into!exposure!and!depuration!phases.!The!exposure!phase!lasted!for!28!

days,!with! three!sampling! times,!at!days!0! (T0),!7! (T7),! and!28! (T28).!After!T28!animals!

were! allowed! to!deH! purate! in! clean!water! for! a! further!14!days! (depuration!phase),! the!

assay! was! terminated! at! day! 42! (T42).! At! T0! 10! animals! from! acclimation! tanks! were!

sacrificed!for!analysis,!and!at!subsequent!sampling!times!five!individuals!per!replica!were!

processed!for!wholeHbody!As!and!MT!concentrations!and!histochemical!evaluation.!At!each!

sampling!time!water!samples!were!collected!to!determine!the!real!As!concentrations.!

!


! 67!

III.2.2..Arsenic.Determination.

Total!As!in!tissue!was!determined!in!homogenized!whole!soft!body!samples,!dried,!

and!digested! in!pressurized!TFE!cells!with!HNO3!and!H2O2!(Bryan!et!al.!1985).!Total!As!

concentrations! were! measured! by! electrothermal! atomic! absorption! spectrometry! (ETH

AAS),!with! Zeeman! correction! and! a!matrix!modifier! (Pd[NO3]2.2H2O! from! Fluka,! Buchs,!

Switzerland),! using! a! graphite! tube! atomizer! (M6Solaar! AAS;! Thermo! Electron! Corp.,!

Waltham,! MA,! USA).! Analysis! of! DOLT! 3! reference! material! (National! Research! Council,!

Ottawa,!Canada)!and!blanks!followed!the!same!analytical!procedure!used!for!the!samples!

to!validate!the!procedure!and!the!measured!values!were!found!within!the!certified!range.!

The! calibration! curve! was! obtained! from! standard! inorganic! As! for! atomic! absorption!

spectrometry!(N206962!standard;!Aldrich,!Milwaukee,!WI,!USA).!Concentrations!are!given!

as! micrograms! total! As! per! gram! tissue! dry! weight! (d.w.).! Total! As! in! water! was!

determined! using! the! same! procedure,! from! water! samples! acidified! with! 1! μg.mLH

1Suprapur!grade!65%!HNO3!(Merck,!Darmstadt,!Germany).!

III.2.3..Uptake.and.Elimination.Kinetics.and.Bioaccumulation.Factors.

FirstHorder! twoHcompartment! kinetic! models! were! obtained! to! estimate! the! As!

uptake!and!elimination!(excretion)!rate!constants!k! .!According!to!the!biodynamic!models!

detailed! by! De! Kock! and! Kramer! (1994! )! and! Luoma! and! Rainbow! (2005! ),! the!

bioaccumulation!of!a!contaminant!results!from!the!balance!between!uptake!from!water!and!

elimination! from! tissues,! which! depends! on! the! concentration! of! the! bioavailable!

contaminant!in!the!surrounding!medium.!!

CAs =k1CAs(water )

k2(1− e−k2t )+CAs(init.)e

−k2t! Equation!III.1!

Arsenic! bioaccumulation! may! therefore! be! given! by! the! model! where! CAs. is! the!


!68!

concentration!of!total!As!in!tissue!at!time!t,!k1!!is!the!uptake!constant,!k2!!is!the!elimination!

constant,! CAs(init.). is! the! baseline! As! concentration! in! tissue! (i.e.,! at! the! beginning! of! the!

exposure! period),! and! CAs(water)& is! the! As! concentration! in! the! water,! assuming! that! it!

remains! constant! and! no! decay! parameter! is! necessary! in! the! model.! Since! tissue! As!

concentrations!are!given!as!micrograms!per!gram!tissue!d.w.,!water!As!as!micrograms!per!

liter,! and! time! as! days,! k1! ! is! given! as!micrograms! As! per! gram! tissue! d.w.! per! day! per!

microgram!As!per!liter!water,!which,!simplified,!becomes!liters!per!gram!per!day!(L.gX1.dayX

1).! The! constant!k2! is! expressed! as! daysX1! ,! since! the! elimination! rate! over! time!may! be!

obtained! by! multiplying! this! constant! by! the! As! concentration! in! tissue! (Luoma! and!

Rainbow! 2005).! The! constants! k1! ! and! k2! are! thus! random! coefficients! related! to! the!

removal! of! the! element! from! the! water,! as! is! its! incorporated! in! the! tissues! and! its!

elimination! from! the! organism,! respectively.! Under! steadyHstate! conditions,! the!

bioconcentration!factor!(BCF)!can!be!estimated!as!the!ratio!of!k.constants!(e.g.,!Bailer!et!al.!

2000):!

BCF = k1k2

! Equation!III.2!

Bioconcentration! factors!are! thus!given!as! liters!per!gram!(L.gX1! ).!Standard!errors!

(SE)!for!BCFs!were!estimated!as!described!by!Bailer!et!al.!(2000!),!according!to!the!formula!

SE(BCF) ≈ σ12

k22 −

2k1σ1,2k23 +

k12σ 2

2

k24 ! Equation!III.3!

where! r2! ! is! the! variance! of! each! k! ! estimation! and! r1,2!is! the! covariance! between!

both!estimations!since!they!were!obtained!by!the!same!model.!Estimation!was!done!using!

bioaccumulation!data! from!the!exposure!phase.!Since!wáter!As!concentrations!were!kept!

constant!during!the!exposure!phase!in!each!experimental!treatment,!four!different!models!


! 69!

(corresponding!to!the!nominal!exposures!to!100,!300,!500,!and!100!μg.LX1!)!were!obtained.!

Rate!constants!k1!!and!k2!!were!estimated!by!iterative!least!squares.!Model!goodness!of!fit!

was!assessed!by!the!r2!statistic.!

III.2.4..Maetallothionein.Determination.

MT! was! determined! by! quantification! of! thiolic! (–SH)! groups! in! heatHtreated!

cytosols!from!wholeHbody!samples!according!to!the!Brdicˇka!reaction,!as!described!by!Diniz!

et!al.!(2007).!WholeHbody!tissue!samples!were!homogenized!in!the!cold!(4!ºC),! in!3!ml!of!

0.02!M!TrisHHCl!buffer!(pH!8.6)!and!centrifuged!at!30,000!g!!for!1!h!at!4!ºC.!The!supernatant!

(cytosol)! was! then! separated! from! the! pellet,! heatHtreated! at! 80! ºC! for! 10! min,! and!

subsequently!centrifuged!at!30,000!g!for!1!h!at!4!ºC!to!precipitate!residual!high!molecular!

weight! and! nonHheatHstable! proteins.! Quantification! of! MT! equivalents! in! heatHtreated!

cytosols!was!performed!by!means!of!differentialHpulse!polarography!with!a!static!mercury!

drop! electrode! (DPPHSMDE)! using! a! 693! VA! processor! and! a! 694! VA! stand! (Metrohm,!

Herisau,! Switzerland).! The! threeHelectrode! system! consisted! of! a! mercury! capillary!

working!electrode,!a!platinum!auxiliary!electrode,!and!an!Ag/AgCl!reference!electrode.!The!

Brdicka!supporting!electrolyte!was!prepared!according!to!Palecek!and!Pechan!(1971!)!and!

contained!1!M!NH4Cl,! 1!M!NH4OH,! and!2!mM!(CoCNH3)6Cl3! (hexaminecobalt! III! chloride;!

Sigma).!Triton!XH100!(Sigma),!2.5!9!10H2!%!(v/v),!was!added!to!the!electrolyte!prior!to!use!

to!suppress!baseline!noise.!The!quantification!procedure!by!DPPSMDE!was!adapted! from!

Thompson! and! Cosson! (1984).! MT! concentrations! were! determined! using! the! standard!

addition!method.!In!the!absence!of!a!standard!mollusk!MT,!determination!of!C.&fluminea!MT!

was!performed!using!a!rabbit! liver!MT!(forms!I!and!II)!standard!(Sigma),!as!validated!by!

Diniz!et!al.!(2007).!Results!are!expressed!as!milligrams!MT!per!gram!tissue!d.w.!

!


!70!

III.2.5..Histochemistry..

Fresh!samples!of!wholeHbody!C.&fluminea!!were!placed!in!Castel’s!fixative,!containing!

10%!(v/v)!formaldehyde!and!2.5%!(m/v)!copper!sulfate,!for!5!days.!After!washing!for!24!h!

with! distilled! water,! samples! were! embedded! in! paraffin! for! histological! sectioning! as!

described!by!Martoja!and!Martoja!(1967).!After!inclusion!in!paraffin,!5H7!μm!sections!were!

counterstained!with!Nuclear!Fast!Red!(Sigma).!Castel’s!method!for!arsenic!histochemistry!

was! employed! as! described! by! Lillie! (1965! ).! Histochemical! evaluation! was! performed!

using!a!DMLB!optical!microscope!(Leica!Microsystems,!Wetzal,!Germany).!

II.2.5.. Statistical.Analysis..

The!nonparametric!statistics!Mann–Whitney!U!and!Kruskall–Wallis!H!were!used!to!

identify! differences! between! exposure! and! control! treatments! and! within! groups,!

respectively,! after! failure! to! demonstrate! homogeneity! of! variances! by! Levene’s! test.!

Correlation!analysis!was!obtained!with!Spearman!rankHorder!correlations!R!!or!Kendall’s!s!!

statistics.! Inference!on!BCF!estimates!was!obtained!with! the!zHtest.!The!significance! level!

was! set! at! 5%.! Statistical! analysis! and!model! parameter! estimation!were!done!using! the!

Statistica!6.0!software!(StatSoft!Inc.,!Tulsa,!OK,!USA),!after!Sheskin!(2000!).!

III.3.. Results.

III.3.1..Arsenic.Concentration.in.Water.

Arsenic!Concentrations! in!water!during! the!experimental!period!no!mortality!was!

observed!in!any!treatment.!Total!As!concentrations!determined!in!water!samples!collected!

from!the!different!treatments!were!found!to!be!very!close!to!the!nominal!concentrations!of!

AsIII!exposure!(table!III.!1).!No!significant!differences!were!detected!between!the!beginning!

and! the! end!of! the! experimental! period! (Mann–Whitney!U,! p>0.05),!which! confirms! that!

CAs(water)!(Equation!III.1)!can!be!regarded!as!constant.!!


! 71!

Table. III.1.! Average! ±! standard! deviation! values! of! effective! total! As! concentration!

(μg.LH1)!measured!in!water!samples!at!the!exposed!assay.!

! Control* 100* 300* 500* 1000*

T0! <3!μg.LH1! 96±2! 306±5! 540±8! 1010±3!

T28! <3!μg.LH1! 92±3! 299±3! 530±5! 991±4!

Limit!of!detection:!3.1!μg.LH1!

III.3.2..Arsenic.Bioacumulation.and.Elimination.

The!data!obtained!from!clam!tissue!samples!collected!at!T7!and!T28!!regarding!the!

different!exposures!to!AsIII!!(100,!300,!500,!and!1000!μg!As!LX1!)!are!shown!in!figure!III.1!,!

which!also!reports!total!As!concentrations!for!depurated!organisms.!!

!

Figure. III.1.! Average! total! As! accumulation! in! exposed! organisms.! Error! bars! represent!

standard! deviation.! Significant! differences! (★)! from! control! organisms! and! (†)! from! the!

other!exposed!groups,!respectively!(MannHWhitney!U,!p&<!0.05).!


!72!

Total!As! concentration! in! control! clams!was!10.8!±!6.4! μg!As! gH1! d.w.! and!did!not!

vary! significantly! along! the! experiment! time! (p& >! 0.05).! The! levels! of! As! in! organisms!

sampled!at!T0!!(11.7!±!4.4!μg!As!gX1!d.w.)!were!not!significantly!different!from!controls!(p&>!

0.05).! During! the! exposure! phase,! a! very! significant! increase! in! accumulated! As! was!

observed!for!all!treatments!at!T28!(p!<!0.05)!but!not!at!T7!.!At!the!end!of!the!exposure!term!

a!strong!correlation!between!As!in!water!and!As!in!mollusks’!whole!soft!body!(Spearman!R!

=! 0.75,! p! <! 0.05)! was! observed.! After! 14! days! of! depuration! a! significant! decrease! was!

observed! (p! <! 0.05)! in! the! contents! of! total! As! in! the!whole! soft! tissues! of! the! clams! in!

comparison!with! levels!measured!at!T28,! for!all! treatments.!The!decrease! in!wholeHbody!

total!As!ranged!between!79%!and!91%.!

III.3.3..Biodynamic.Accumulation.Model.and.Bioconcentration.Factors.

Bioaccumulation! data! from! the! exposure! phase! were! successfully! fitted! to!model!

(equation! III.1),! yielding! four! different! models! that! explain! 95.5%–98.4%! of! the! total!

variance!(figure!III.2).!

!

Figure.III.2.!Estimated!models!for!all!AsIII!treatments!during!the!exposure!phase!


! 73!

Estimated! k! constants! and! respective! BCFs! are! presented! in! table! III.2.! The! BCF!

estimated!for!the!100!μg.LX1!exposure!was!found!to!be!significantly!higher!than!the!other!

treatments!(p!<.0.01).!Although!no!other!significant!differences!were!found!between!BCFs,!

these!were!found!to!be!negatively!correlated!with!As!exposures!(Kendall’s!τ!=.X.1,!p!<!0.05),!

decreasing!with!increasing!exposures!to!waterborne!AsIII.!

Table.III.2.!Results!of!the!kinetics!study!of!the!exposure!phase!

Treatment!

(μg!AsIII!.LH1)!

k1*

(L!gH1!dayH1)*

K2*

(L!gH1!dayH1)*

BCF.±.SE.

(L!gH1)*

100! H0.019! H0.176! 0.110±0.018!

300! H0.003! H0.119! 0.024±0.015!

500! H0.004! H0.173! 0.021±0.004!

1000! H0.002! H0.162! 0.010±0.004!

Note:!BCF!bioconcentration!factor;!k1*uptake!constant;!k2*elimination!constant;!SE!standard!

error!

III.3.4..Metallothionein.Induction.

The!MT!induction!in!exposed!organisms!was!found!to!be!significantly!different!from!

controls!only!at!T28!(figure!III.3).!Induction!was!higher!in!organisms!exposed!to!100!μg.LX1.!

The!concentrations!of!MTs!in!the!organisms!used!as!controls!(7.8!±!0.8!mg.gH1!d.w.)!did!not!

vary! significantly! during! the! experimental! period! (Kruskall–Wallis! H,! p! >! 0.05).! The!

increase! in! MT! concentrations! ranged! approximately! between! twoH! and! threefold,!

corresponding! to! induction! rates! ranging! between! 0.52! and! 0.79! mg! MT! gH1! dayH1,!

respectively,! from!T0!to!T28.!After!the!depuration!phase,!a!decrease!was!observed!in!MT!

concentrations!in!all!exposed!groups,!reaching!levels!similar!to!those!of!controls!(p!<!0.05).!

A!moderate! but! significant! correlation! (Spearman!R! =! 0.5,!p&<!0.05)!was! found! between!


!74!

bioaccumulated!As!and!MT!concentrations!after!28!days!of!exposure!but!not!at!any!other!

sampling!points.!

!

Figure. III.3.! Average! woleHbody! MT! concentration! in! tested! organisms.! Error! bars!

represent! standard!deviation.! Significant!differences! (★)! from!control! organisms! and! (†)!

from!the!other!exposed!groups,!respectively!(MannHWhitney!U,!p&<!0.05).!

III.3.5..Histochemistry.

The!histochemical!analysis!revealed!the!presence!of!As!granules!in!the!tissues!of!C.&

fluminea,! particularly! in! the! cells! of! the! digestive! gland! (figure! III.! 4)! from! organisms!

exposed! to! higher! concentrations! (500! and! 1000! μg! As! LX1).! Histochemical! analysis! of!

depurated!individuals!depicts!a!distinctive!loss!of!accumulated!As,!shown!by!the!reduction!

to!total!or!nearHtotal!absence!of!copper!arsenite!granules,!which!is!in!accordance!with!the!

reduction!of!tissue!As!to!control!levels!measured!by!AAS!(figure!III.!5).!


! 75!

!

Figure. III.4.!Histochemical!analysis!of!exposed!individuals!showing!an!increase!in!copper!

arsenite!granules!(arrows)!after!28!days!of!exposure.!a.Digestive!grand!tubules!of!a!control!

individual;!b!digestive!gland!tubules!of!a!clam!exposed!to!500!μg.!As!LH1.!

.

Figure.III.5..Histochemical!analysis!of!digestive!glands!of!depurated!individuals!showing!a!

decrease!in!copper!arsenite!granules!(arrows)!in!digestive!gland!tubules!of!clams!exposed!

to!100!μg.!As!LH1.!a!After!28!days!of! exposure,! exhibiting! clusters!of! granules;!b! after!14!

days!of!depuration!..

!

!


!76!

III.4.. Discussion.

The!present!work!demonstrated!that!C.&fluminea! is!capable!of!bioaccumulating!and!

eliminating!As!as!a!result!of!exposure! to!high! levels!of!arsenite! in!water,!which! indicates!

that! the! species! is! capable! of! regulating! the! metalloid! even! following! contamination! by!

what! is! recognized! as! the! element’s! most! toxic! form.! According! to! Baudrimont! et! al.!

(1997a)!C.&fluminea! is!capable!of!accumulating!high! levels!of!waterborne!elements!and! is!

an! interesting!model!organism! for! toxicological! studies! in! freshwater!ecosystems!since! it!

lives!buried!in!the!upper!layer!of!sediments!and!filters!large!quantities!of!water!(10!L!clamH

1!dayH1).!Nevertheless,!only! limited! information! is!yet!available!regarding! the!exposure!of!

this! freshwater! bivalve! to! As,! although! Sebesvari! et! al.! (2005)! have! demonstrated! that!

Corbicula&sp.! is!capable!of!accumulating!As!and!stated!that! the!asiatic!clam!is!suitable! for!

monitoring!environmental!heavy!metals!and!metalloids.!A!field!study!carried!out!by!Jonhs!

and! Luoma! (1990),! who! collected! Corbicula& sp.! from! the! San! Francisco! Bay! and! the!

Sacramento/San! Joaquin! river! delta! (USA),! showed! that!mean! As! concentrations! ranged!

from!5.4!to!11.5!μg.gX1g!whole!tissue!d.w.!at!all!stations!(with!small!temporal!fluctuations!

being!detected),!generally!being!consistent!with!concentrations! found! in!uncontaminated!

systems.!The!control!organisms!in!the!present!work!show!concentrations!within!the!same!

range.! The! results! show,! however,! that! As! bioaccumulation! is! linked! to! As! in! water,!

contradicting!previous! findings!when!bivalves!were!employed! to!monitor!waterborne!As!

(e.g.,! ValetteHSilver! et! al.! 1999;! Orescanin! et! al.! 2006).! Most! of! these! field! studies! with!

bivalves! failed! or!were! unable! to! relate! As! concentrations! in!water!with! the!metalloid’s!

levels! in! typical! contaminant! storage! compartments! such! as! sediments.! It! is! likely! that!

waterborne! As! represents! a! much! more! bioavailable! form! of! the! metalloid,! especially!

concerning!filterHfeeding!animals!such!as!bivalves,!hence!the!exponentialHtype!curve!of!the!

bioaccumulation! models! estimated! in! the! present! work.! Bioaccumulation! of! toxic!

substances!by!aquatic!organisms!is!a!complex!phenomenon!that!results!from!the!relation!


! 77!

between!uptake!and!elimination!rates!and!therefore!from!the!intrinsic!biological!variables!

from!which! they! derive.! From! the! uptake! constants! low!BCFs! (<1)!were! obtained.! Since!

accumulation!depends!on!uptake!constant!x!As!concentration!in!water!(1),!low!BCFs!do!not!

necessarily!mean!that!the!organisms!are!not!bioaccumulating!but!that!the!proportion!of!the!

metalloid!actually!being!removed!from!the!water!and!incorporated!into!the!tissues!is!low,!

at!least!at!the!tested!concentrations!of!AsIII.!It!is!noteworthy!that!the!tested!concentrations!

are!realistic! in!terms!of! freshwater!highly!contaminated!with!this!metalloid!(reviewed!by!

Mandal! and! Suzuki! 2002).! The! results! show! that! As! concentrations! in! soft! tissues!

decreased!in!all!treatments!during!the!depuration!phase.!Furthermore,!the!BCF!estimates,!

which! were! found! to! decrease! with! increasing! exposures! to! As,! may! indicate! that! the!

organisms! underwent! a! regulation! process! by!modulating! the! uptake:elimination! ratios.!

Even! empirically,! it! can!be! observed! that! exposure! to! 1000!μg.LX1,! for! instance,! does!not!

correspond!to!a!10Hfold!increase!in!bioacumulated!As!relative!to!100!μg.LX1,!at!any!sampling!

time.!According!to!our!results,!this!can!mean!that!the!animals!were!responding!to!exposure!

especially!by!restricting!uptake,!since!the!estimated!elimination!constants!failed!to!show!a!

clear! trend!with! increasing!exposure!concentrations.!On! the!other!hand,! the!results! from!

the!depuration!phase!(confirmed!by!histochemistry)!indicate!that!these!bivalves!have!good!

mechanisms! to! eliminate! the! metalloid! at! the! tested! concentrations! of! exposure.! These!

results!confirm!the!strong!metabolic!capacity!of!the!species!regarding!As!detoxification,!as!

already! suggested! by! Sebesvari! et! al.! (2005).! The! phenomenon! of! decreasing! BCFs!with!

increasing! metal! exposures! has! also! been! observed! for! other! elements! (essential! and!

nonessential)! and! described! as! a! factor! that! may! compromise! the! employment! of! this!

guideline!as!a!biomonitoring!tool!of!aquatic!biota!(McGeer!et!al.!2003).!Still,!much!research!

is!lacking!regarding!the!actual!biological!factors!that!are!responsible!for!this.!Nevertheless,!

even! if! BCFs! alone! are! not! capable! of! providing! a! direct! measure! of! environmental!

contamination,!our!work!showed!that!BCFs,!when!associated!with!uptake!and!elimination!


!78!

kinetics,! may! provide! solid! insight! into! the! species’! sensitivity! and! regulatory! capacity!

toward! contamination.! The! release! of! contaminants! from! cells! depends! on! the! different!

turnover!times!of! the!various!biochemical!compartments!to!which!the!pollutant! is!bound!

(reviewed! by! Ravera! 2001):! the! pollutant! fraction! bound! to! substances! with! a! short!

turnover!time!is!the!one!initially!released!(such!as!phosphorous!in!membranes),!and!only!

subsequently! are! the! fractions! bound! to! compounds! with! a! longer! turnover! time! (like!

metals! precipitated! in! granules).! Regarding! As,! these! compounds!may! consist! of! several!

detoxifying! mechanisms! that! are! activated! to! neutralize! the! toxic! effects! and! rapidly!

eliminate! the!metalloid! from! tissues! (e.g.,! formation!of!metal! thiolates!by!MTs!and!other!

chelating! processes! like! As! methylation! [Aposhian! 1997]).! The! kinetic! differences! in! As!

elimination! in! organisms! exposed! to! different! concentrations! of! contaminants! can! be!

attributed!to!differences!in!uptake!and!depuration!rates!(Luoma!and!Rainbow!2005).!Our!

results! show! that!C.& fluminea! can! efficiently! regulate! As! by!modulating! bioconcentration!

factors!according!to!exposure!concentrations,!possibly!by!enabling!a!series!of!responses!of!

which! MT! induction! and! intracellular! storage! of! arsenic! granules! may! be! important!

components.!The!kinetics!analysis!performed!in!this!work!was!effectively!based!on!a!twoH

compartment! (water/! organism)! partitioning! model! which! is! a! simplified! equation! that!

reflects! the!overall!processes!within! the!organism.! It! is!not!possible!at! this! stage! to! infer!

whether! uptake! is! reduced! by! specific! responses! to! exposure! or! if! physiological! effects,!

such!as!a!decrease! in! filtration!rates,!are! involved,!or!how!elimination! is!modulated.!The!

low! mortality! and! relative! constancy! of! elimination! during! uptake! (and! the! effective!

depuration! that! followed)! should! indicate,! however,! that! exposure! to!waterborne!As! did!

not! considerably! impair! the! animal’s! metabolism,! suggesting! that! direct! responses! to!

exposure!have!occurred.!The!BCFs,!on!the!other!hand,!reflect!the!combination!of!all!effects!

and! responses! that! affect! uptake! and! elimination,! a! clear! negative! correlation! with!

increasing! exposures! having! been! observed.! Even! though! some! inconsistencies! and!


! 79!

divergences!remain!in!the!scientific!community!regarding!the!physiological!role!of!MTs!and!

their!use!as!biomarkers!in!environmental!monitoring!programs!(Amiard!et!al.!2006),! it! is!

generally! accepted! that! these!proteins! play! a! role! in! the!homeostasis! of! essential!metals!

(e.g.,! Cu! and! Zn),! as! they! can! act! as! essential!metal! stores! ready! to! fulfill! enzymatic! and!

other!metabolic!demands!(Viarengo!and!Nott!1993;!Roesijadi!1996).!It!is!also!believed!that!

the!binding!of!MTs!to!metals!has!a!detoxifying!effect,!due!to!their!ability!to!sequester!these!

elements! and! consequently! decrease! their! bioavailability! to! other! cellular! ligands!

(Baudrimont!et!al.!1999;!Ravera!2001).!It!is!noteworthy!that,!at!least!concerning!mollusks,!

there! are! several! agents! that! are! also! able! to! induce! MT! biosynthesis! such! as! physical!

stress,! tumor! necrosis! factor,! and! reactive! oxygen! species! (Baudrimont! et! al.! 1997a;!

Viarengo!et!al.!1999).!The!present!study!reveals!that!a!significant!MT!induction!over!time!of!

exposure! occurs,! followed! by! a! decrease! to! baseline! levels! when! the! organisms! are! no!

longer!subjected!to!waterborne!As.!These!results!clearly!indicate!that!C.&fluminea!MTs!play!

a!significant!role!in!As!contamination,!confirming!the!results!of!previous!studies!(Santos!et!

al.!2007;!Diniz!et!al.!2007).!The!induction!of!these!proteins!was,!however,!higher!in!clams!

exposed!to! lower!concentrations!of!AsIII! in!water!(100!and!300!lg!LH1),!showing!that!MT!

overexpression! in!this!species! following!exposure!to!As! is!unlikely!to!be!dose!dependent,!

which!may!result!from!other!detoxification!mechanisms!that!may!have!a!more!prominent!

role!in!As!detoxification,!a!subject!already!debated!by!other!authors!(e.g.,!Apeti!et!al.!2005).!

Although!many!field!and!laboratory!studies!were!performed!with!C.&fluminea,!only!a!limited!

number! of! studies! have! been! devoted! to!MT! induction! in! this! species! and,! in! particular,!

regarding!AsIII!exposure!(Baudrimont!et!al.!1997b;!Santos!et!al.!2007;!Diniz!et!al.!2007).!In!

fact,! studies! focusing! on!MT! induction! in! freshwater! bivalves! following! As! exposure! are!

scarce.! In! accordance!with! the! present! findings,! Baudrimont! et! al.! (2003)! observed! that!

fieldHcontaminated& C.& fluminea&MT! concentrations! rapidly! decreased! by! 37%! following!

transfer! to! unpolluted! conditions.! According! to! Viarengo! et! al.! (1987)! the! elimination! of!


!80!

metals! by! bivalves! seems! to! be! mediated! by! an! active! mechanism! involving! their!

accumulation! in! lysossomes! (with! the! subsequent! excretion! of! the! metalHrich! residual!

bodies).! Since! our! results! show! that! C.& fluminea! is! able! to! regulate! this! metalloid! by!

accumulating! and! eliminating! the! excess! of! As! in! tissues,! it! is! thus! possible! that! MT!

induction! may! partially! function! as! part! of! a! primary! defense! mechanism! in! favor! of!

bioaccumulation! and! other! biochemical! mechanisms.! The! presence! of! intracellular! As!

deposits,! as! detected! by! the! histochemical! evaluation,! may,! in! fact,! be! another! primary!

defense! mechanism! against! contamination,! since! precipitated! forms! of! the! metalloid!

should! not! be! made! easily! available! to! affect! cellular! functions.! Arsenic! concretions,!

observed!to!form!large!clusters!in!tissues!of!the!digestive!gland!in!the!individuals!exposed!

to!higher!concentrations,!appear!to!be!linked!to!the!metalloid’s!uptake!and!elimination!and!

might!explain!the!good!potential!of!this!species!to!bioaccumulate!As;!in!addition,!it!may!be!

an! important! factor! regarding! its! tolerance! to! contamination.!From! the!present! results! it!

can!be! inferred!that!C.&fluminea!may!be!suitable!as!a!monitoring!organism!of!exposure!to!

AsIII!in!water,!being!able!to!accumulate!and!effectively!eliminate!the!metalloid!even!when!

exposed! to! relatively! high! concentrations.! The! present!work! also! shows! that!C.& fluminea!

may!have!a!good!resistance!to!this!metalloid,!mostly!by!being!able!to!modulate!uptake!and!

elimination!relative!to!exposure!concentrations,!from!which!result!low!BCFs.!Some!caution!

may!thus!be!required!when!interpreting!As!kinetics!data!on!this!species.!Finally,!although!it!

has! been! demonstrated! that! MT! induction! occurs! as! a! result! of! exposure! to! AsIII,! the!

unclear! relationship! between!MTs! and! exposure! concentrations!may! signify! that! caution!

should!be!implied!when!considering!it!as!a!standHalone!biomarker!of!exposure!to!arsenite!

in!C.&fluminea.!


! 81!

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.

!

! 87!

Chapter!IV.!Conclusions!

Results! obtained! for! the! organisms! exposed! during! 7! days! to! different!

concentrations!of!As!suggest!that!the!organisms!exposed!to!the!concentrations!of!300!and!

1000!μg!As!LH1!accumulated!the!highest!levels!of!AsIII!in!the!tissues!(17!±.9!and!15!±.3!μg.gX1!

distilled!water,!respectively),!which!was!confirmed!through!histochemical!analysis.!

Fort! he! 7! days! exposure! an! apparent! induction! of! MTs!was! also! observed! in! the!

organisms!exposed!to!AsIII,!suggesting!that!C.&fluminea!possesses!some!capacity!for!arsenic!

regulation.!!

Results! obtained! for! the! organisms! exposed! during! 28! days! to! different!

concentrations!of!As!shows!a!significant!increase!(p!<!0.01)!of!As!in!the!total!concentration!

and!cytosolic!fraction!after!28!days!of!exposure!in!all!treatments.!A!significant!increase!(p!<!

0.01)! in! metallothionein! concentration! was! also! detected! in! bivalves! exposed! to! the!

different! concentrations! of! As,! in! comparison! with! the! controls.! The! histological! and!

histochemical!evaluation!provided!clear!evidence!that!As!accumulated!in!tissues,!especially!

in!the!digestive!gland,!and!caused!tissue!alterations!in!50%!of!the!organisms.!

Results!show!a!significant!increase!in!wholeHbody!total!As!after!28!days!of!exposure!

for!all!treatments,!followed!by!a!decrease!at!the!end!of!the!depuration!phase.!Biodynamic!

kinetic!models! for! As! uptake! and! elimination!were! obtained! from! bioaccumulation! data!

during!the!exposure!phase,!for!all!As!treatments,!by!estimating!uptake!and!elimination!rate!

constants.!Bioconcentration!factors!(BCFs)!were!estimated!by!the!ratio!of!these!constants.!

Results!revealed!that!exposure!to!higher!concentrations!of!AsIII!!causes!a!decrease!in!BCFs,!

suggesting! that! C.& fluminea! triggers! effective! regulatory! mechanisms! when! exposed! to!

higher!concentrations!of!the!metalloid.!

Significant!induction!of!MT!was!detected!during!the!exposure!phase,!followed!by!a!

decrease! in! MT! concentration! to! control! levels! after! depuration! for! all! treatments.! No!

significant! differences! in! MT! concentrations! were! observed! between! treatments.! This!

finding! may! confirm! the! role! of! MT! as! part! of! the! As! regulation! process,! but! its!

Independence!relative!to!concentrations!of!AsIII!in!water!suggests!that!MT!induction!is!not!

dose! dependent.! The! histochemical! evaluation! provided! clear! evidence! that! As! was!

Chapter!IV.!Conclusions!

!88!

effectively! accumulated! in! the! digestive! gland! during! exposure! and! eliminated! during!

depuration.! The! present!work! demonstrated! that!C.& fluminea& is! capable! of! regulating!As,!

even!at!exposures!as!high!as!1000!μg.LX1!of!waterborne!AsIII!.!

The!results!suggest!that!the!induction!of!MTs!may!be!of!high!interest!as!a!biomarker!

for! arsenic! contamination! in! aquatic! environments,! and! confirms! the! potential! of! C.&

fluminea!as!a!biological!indicator.

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