Caracterización de la Calidad de Sedimentos Afectados por

Caracterización de la Calidad de Sedimentos Afectados por

Caracterización de la Calidad de
Sedimentos Afectados por
Vertidos de Petróleo
Comparación entre Casos de Vertidos
Accidentales (Impacto Agudo) frente a
Derrames Continuos (Impacto Crónico)
TESIS DOCTORAL
Carmen Morales Caselles
UNIVERSIDADȱDEȱCÁDIZȱ
FACULTADȱDEȱCIENCIASȱDELȱMARȱYȱAMBIENTALESȱ
CARACTERIZACIÓNȱDEȱLAȱCALIDADȱDEȱSEDIMENTOSȱAFECTADOSȱ
PORȱVERTIDOSȱDEȱPETRÓLEO:ȱCOMPARACIÓNȱENTREȱCASOSȱDEȱ
VERTIDOSȱACCIDENTALESȱ(IMPACTOȱAGUDO)ȱFRENTEȱAȱDERRAMESȱ
CONTINUOSȱ(IMPACTOȱCRÓNICO)ȱ
CarmenȱMoralesȱCasellesȱ
Cádiz,ȱ2007ȱ
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EstaȱTesisȱDoctoralȱhaȱsidoȱrealizadaȱdentroȱdelȱGrupoȱdeȱ
Investigaciónȱ delȱ Planȱ Andaluzȱ deȱ Investigaciónȱ
OceanografíaȱLitoralȱyȱEcofisiologíaȱ(nºȱRNMȱ0144),ȱdeȱlaȱ
Facultadȱ deȱ Cienciasȱ delȱ Marȱ yȱ Ambientalesȱ deȱ laȱ
UniversidadȱdeȱCádizȱyȱdelȱInstitutoȱdeȱCienciasȱMarinasȱ
deȱ Andalucíaȱ delȱ Consejoȱ Superiorȱ deȱ Investigacionesȱ
Científicas,ȱ enȱ elȱ ámbitoȱ deȱ laȱ Unidadȱ Asociadaȱ UCAȬ
CSICȱ‘CalidadȱAmbientalȱyȱPatología’ȱenȱelȱmarcoȱdeȱlasȱ
actividadesȱ deȱ investigaciónȱ deȱ laȱ cátedraȱ SantanderȬ
UNESCOȱ UNITWIN/WiCopȱ enȱ suȱ áreaȱ deȱ calidadȱ
ambiental.ȱ
Elȱ trabajoȱ queȱ seȱ resumeȱ enȱ estaȱ Memoriaȱ haȱ sidoȱ
principalmenteȱ financiadoȱ porȱ losȱ siguientesȱ proyectos:ȱ
“Caracterizaciónȱ deȱ laȱ calidadȱ ambientalȱ deȱ ecosistemasȱ
costerosȱafectadosȱporȱvertidosȱdeȱpetróleo:ȱcomparaciónȱ
entreȱ casosȱ deȱ vertidosȱ accidentalesȱ (impactoȱ agudo)ȱ
frenteȱ aȱ derramesȱ continuosȱ (impactoȱ crónico)”ȱ
(VEM2003Ȭ20563/INTER),ȱ Ministerioȱ deȱ Cienciaȱ yȱ
Tecnología;ȱ“PlanȱNacionalȱdeȱI+D+I.ȱȱDesarrolloȱyȱmejoraȱ
delȱanálisisȱintregradoȱparaȱlaȱevaluaciónȱȱ deȱ laȱ calidadȱ
deȱ sedimentosȱ litorales,ȱ incluidosȱ losȱ materialesȱ deȱ
dragadoȱ portuario.ȱ CTM2005Ȭ07282ȬC03Ȭ01/TECNO.”ȱ
Ministerioȱ deȱ Educaciónȱ yȱ Ciencia;ȱ “Asistenciaȱ Técnicaȱ
paraȱlaȱEvaluaciónȱyȱseguimientoȱdeȱlosȱdañosȱderivadosȱ
delȱvertidoȱdelȱȱPrestigeȱenȱelȱParqueȱNacionalȱdeȱlasȱIslasȱ
Atlánticasȱ yȱ enȱ otrosȱ espaciosȱ protegidosȱ deȱ relevanciaȱ
comunitaria”ȱMinisterioȱdeȱMedioȱAmbiente.ȱ
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D.ȱ T.ȱ ÁNGELȱ DELVALLSȱ CASILLASȱ Profesorȱ Titularȱ delȱ Departamentoȱ deȱ
QuímicaȬFísicaȱdeȱlaȱUniversidadȱdeȱCádiz,ȱDña.ȱINMACULADAȱRIBAȱLÓPEZȱ
Investigadoraȱ contratadaȱ delȱ Institutoȱ deȱ Cienciasȱ Marinasȱ deȱ Andalucíaȱ
(ICMANȬCSIC)ȱ yȱ Dña.ȱ CARMENȱ SARASQUETEȱ REIRIZȱ Profesoraȱ deȱ
Investigaciónȱ delȱ Institutoȱ deȱ Cienciasȱ Marinasȱ deȱ Andalucíaȱ (ICMANȬCSIC)ȱ
comoȱsusȱdirectoresȱ
HACENȱCONSTAR:ȱ
Queȱestaȱmemoria,ȱtituladaȱ“Caracterizaciónȱdeȱlaȱcalidadȱ
deȱ sedimentosȱ afectadosȱ porȱ vertidosȱ deȱ petróleo:ȱ
comparaciónȱ entreȱ casosȱ deȱ vertidosȱ accidentalesȱ
(impactoȱ agudo)ȱ frenteȱ aȱ derramesȱ continuosȱ (impactoȱ
crónico)”,ȱ presentadaȱ porȱ Dña.ȱ Carmenȱ Moralesȱ Caselles,ȱ
resumeȱ suȱ trabajoȱ deȱ Tesisȱ Doctoralȱ y,ȱ considerandoȱ queȱ
reúneȱ todosȱ losȱ requisitosȱ legales,ȱ autorizanȱ suȱ
presentaciónȱ yȱ defensaȱ paraȱ optarȱ alȱ gradoȱ deȱ Doctorȱ enȱ
CienciasȱdelȱMarȱporȱlaȱUniversidadȱdeȱCádiz.ȱ
Cádiz,ȱSeptiembreȱdeȱ2007ȱ
Dr.ȱT.ÁngelȱDelVallsȱ
ȱDra.ȱCarmenȱSarasqueteȱȱȱȱȱȱȱȱȱȱȱDra.ȱInmaculadaȱRibaȱ
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Memoriaȱpresentadaȱparaȱoptarȱalȱtítuloȱdeȱ
DoctorȱenȱCienciasȱdelȱMarȱ
CarmenȱMoralesȱCasellesȱ
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Prólogoȱ
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Fueȱ duranteȱ losȱ últimosȱ añosȱ deȱ laȱ carreraȱ cuandoȱ meȱ diȱ cuentaȱ queȱ loȱ
queȱ meȱ gustabaȱ eraȱ laȱ investigación.ȱ Entoncesȱ meȱ incorporéȱ comoȱ alumnaȱ
colaboradoraȱalȱdepartamentoȱdeȱQuímicaȱFísicaȱdeȱlaȱfacultadȱdeȱCienciasȱdelȱ
Marȱ yȱ Ambientales,ȱ dondeȱ meȱ acogieronȱ conȱ cariño.ȱ Aȱ partirȱ deȱ ahí,ȱ estuveȱ
aprendiendoȱ conȱ yȱ graciasȱ aȱ ellos,ȱ losȱ profesoresȱ D.ȱ Ángelȱ delȱ Valls,ȱ D.ȱ Joséȱ
AntonioȱRubio,ȱD.ȱAbelardoȱGómezȱParra,ȱD.ȱEduardoȱGonzálezȱMazo,ȱD.ȱJesúsȱ
Forja,ȱ ȱ yȱ losȱ compañerosȱ queȱ estabanȱ conȱ laȱ tesisȱ reciénȱ leídaȱ oȱ enȱ proceso,ȱ
Inma,ȱDori,ȱRocío,ȱVictor,ȱMónica,ȱQuique,ȱIdoia,ȱMerche,ȱLoli,ȱLaura,ȱNatalia,ȱ
Pablo,ȱCarmen,ȱBibianȱyȱDiana.ȱȱ
Enȱ losȱ dosȱ últimosȱ veranosȱ deȱ laȱ carreraȱ elȱ Centroȱ Oceanográficoȱ deȱ
Murciaȱ delȱ Institutoȱ Españolȱ deȱ Oceanografíaȱ meȱ ofrecióȱ laȱ posibilidadȱ deȱ
trabajarȱ conȱ ellos.ȱ Elȱ directorȱ delȱ centroȱ D.ȱ Julioȱ Más,ȱ misȱ tutoresȱ allíȱ Joséȱ
BenedictoȱAlbladejoȱ(Nané)ȱyȱConcepciónȱMartínezȱ(Concha)ȱyȱmisȱcompañerosȱ
(especialmenteȱInés,ȱAntonio,ȱBeaȱyȱJuan)ȱmeȱrecibieronȱyȱmeȱenseñaronȱenȱmiȱ
primerȱpasoȱhaciaȱelȱestudioȱdeȱlaȱcontaminaciónȱmarina.ȱȱ
AlȱpocoȱdeȱfinalizarȱlaȱcarreraȱmarchéȱaȱItaliaȱconȱunaȱbecaȱArgoȱqueȱmeȱ
permitióȱ continuarȱ miȱ formaciónȱ trabajandoȱ enȱ elȱ Centroȱ Ricercheȱ Ambientaliȱ
diȱ Marinaȱ diȱ Ravenna.ȱ Allíȱ deȱ nuevoȱ tuveȱ laȱ posibilidadȱ deȱ aprenderȱ nuevasȱ
cosasȱ referentesȱ aȱ laȱ investigación,ȱ graciasȱ aȱ Antonellaȱ Iacondini,ȱ Federicaȱ
AbbondanziȱyȱTizianaȱCampisi,ȱiȱcapi,ȱmaravilliose.ȱMisȱcompañerosȱallíȱfueronȱ
estupendosȱ especialmenteȱ Agustina,ȱ Amaya,ȱ Ángelesȱ (Angelita),ȱ Alfredo,ȱ
Marcoȱ (Racco),ȱ Juan,ȱ Luca…ȱ Tambiénȱ conocíȱ aȱ genteȱ queȱ meȱ hizoȱ laȱ estanciaȱ
másȱalegre,ȱlosȱhermanosȱFrezzatiȱ(AnnalisaȱyȱPietro),ȱElena,ȱCristina,ȱGiorgia,ȱ
losȱchicosȱdelȱ“AnimalȱHouse”ȱ(Cecco,ȱMatte,ȱCaprix,ȱDanielle,ȱGallo,ȱPaco…)ȱyȱ
tantosȱotrosȱamig@sȱdeȱRavenna.ȱȱȱȱȱ
AȱlaȱvueltaȱdeȱItaliaȱcompletéȱlosȱcursosȱdeȱdoctoradoȱyȱelȱprofesorȱÁngelȱ
delȱVallsȱdepositóȱsuȱconfianzaȱenȱmíȱparaȱcomenzarȱconȱesteȱproyecto,ȱlaȱtesis,ȱ
enȱunȱtemaȱqueȱdesdeȱelȱprincipioȱmeȱentusiasmó.ȱSiempreȱagradeceréȱsuȱapoyoȱ
queȱfueȱmuyȱimportanteȱenȱlosȱinicios,ȱyȱloȱsiguióȱsiendoȱenȱelȱdesarrolloȱdeȱmiȱ
trabajo.ȱ Laȱ doctoraȱ Inmaculadaȱ Riba,ȱ meȱ haȱ prestadoȱ siempreȱ suȱ ayudaȱ asíȱ
comoȱ suȱ comprensiónȱ yȱ afecto,ȱ graciasȱ Inma.ȱ Miȱ agradecimientoȱ aȱ laȱ doctoraȱ
Carmenȱ Sarasquete,ȱ quienȱ meȱ haȱ ofrecidoȱ suȱ conocimientoȱ yȱ experienciaȱ asíȱ
comoȱsuȱayudaȱenȱtodoȱmomento.ȱEllosȱsonȱmisȱdirectoresȱdeȱtesis,ȱlosȱqueȱenȱ
definitiva,ȱ hanȱ hechoȱ posibleȱ esteȱ proyecto,ȱ graciasȱ porȱ serȱ misȱ tutores,ȱ porȱ
orientarme,ȱenseñarme,ȱyȱporȱtantasȱcosas;ȱtodoȱmiȱcariñoȱparaȱvosotros.ȱ
Hayȱmuchasȱpersonasȱȱaȱquienȱagradecerȱsuȱayudaȱduranteȱelȱdesarrolloȱ
deȱestaȱtesisȱdoctoral.ȱEspecialmenteȱaȱMiguelȱyȱaȱtodosȱlosȱcompañerosȱdelȱCISȱ
(Rosina,ȱ Carlos,ȱ Pablo,ȱ Nazaret…)ȱ porȱ laȱ ayudaȱ prestadaȱ enȱ laȱ tomaȱ yȱ
procesamientoȱ deȱ muestrasȱ enȱ Galicia;ȱ graciasȱ tambiénȱ porȱ vuestraȱ
hospitalidad.ȱ
Graciasȱ alȱ personalȱ delȱ Institutoȱ deȱ Cienciasȱ Marinasȱ deȱ Andalucíaȱ –ȱ
CSIC,ȱ enȱ especialȱ aȱ Juliánȱ Blasco;ȱ graciasȱ aȱ laȱ genteȱ deȱ administraciónȱ porȱ suȱ
paciencia,ȱ aȱ losȱ técnicos,ȱ compañerosȱ yȱ aȱ aquellaȱ genteȱ queȱ teȱ animaȱ porȱ losȱ
pasillosȱ yȱ laboratorios,ȱ haciendoȱ queȱ losȱ problemasȱ noȱ loȱ seanȱ tanto;ȱ graciasȱ aȱ
misȱ compañerosȱ deȱ congresoȱ Olivia,ȱ Miriamȱ yȱ Manolo.ȱ Aȱ misȱ compañerosȱ
Pabloȱ Vidalȱ yȱ Antonioȱ Moreno,ȱ queȱ meȱ hanȱ hechoȱ disfrutarȱ deȱ losȱ
interminablesȱ muestreos,ȱ conȱ losȱ queȱ meȱ heȱ reídoȱ tantoȱ yȱ hanȱ supuestoȱ unaȱ
ráfagaȱdeȱaireȱfrescoȱenȱmuchosȱmomentos.ȱ
Enȱ laȱ facultadȱ tambiénȱ haȱ habidoȱ muchaȱ gente,ȱ queȱ comoȱ decíaȱ antes,ȱ
haceȱ queȱ lasȱ dificultadesȱ seȱ achiquen;ȱ aȱ ellosȱ gracias.ȱ Primordialȱ haȱ sidoȱ laȱ
presenciaȱyȱlaȱayudaȱdeȱmisȱcompañerosȱdeȱdepartamento,ȱsobretodoȱaquellosȱ
queȱ comoȱ yoȱ estabanȱ “empantanados”ȱ conȱ susȱ tesis.ȱ Natalia,ȱ Carmenȱ yȱ Lauraȱ
graciasȱ porȱ vuestraȱ paciencia,ȱ porȱ vuestrasȱ conversaciones,ȱ porȱ escucharmeȱ yȱ
porȱserȱasíȱdeȱúnicas;ȱJudit,ȱMariaȱJoséȱyȱDianaȱFdez,ȱgraciasȱporȱestarȱsiempreȱ
ahíȱ yȱ habermeȱ ayudadoȱ enȱ tantasȱ ocasiones,ȱ soisȱ geniales;ȱ Sara,ȱ graciasȱ porȱ
hacermeȱ reírȱ tantasȱ veces,ȱ porȱ haberȱ sidoȱ miȱ “compi”ȱ deȱ pisoȱ yȱ porȱ olerȱ aȱ
fumarolaȱ sinȱ problemas.ȱ Nuria,ȱ muchasȱ graciasȱ porȱ compartirȱ conmigoȱ laȱ
primeraȱimpresiónȱdeȱlasȱgusanas,ȱyȱenseñarlasȱaȱhablarȱporȱteléfono…ȱGraciasȱ
tambiénȱaȱErku,ȱDianaȱyȱPablo,ȱyȱaȱlaȱgenteȱqueȱhaȱidoȱllegando,ȱJulia,ȱAraceli,ȱ
Pilar…GraciasȱaȱQuiqueȱporȱsuȱayuda,ȱaȱManolo,ȱAugustoȱyȱRodrigo.ȱȱ
Laȱ beca/contratoȱ FPUȱ meȱ haȱ permitidoȱ realizarȱ tresȱ estanciasȱ enȱ otrosȱ
centrosȱduranteȱelȱdesarrolloȱdeȱlaȱtesis.ȱGraciasȱaȱCarlosȱValeȱporȱrecibirmeȱenȱ
IPIMARȱ (Lisboa),ȱ dondeȱ Anaȱ Mariaȱ Ferreira,ȱ Cristinaȱ Micaeloȱ eȱ Isabellinaȱ
Santosȱ meȱ prestaronȱ suȱ ayuda.ȱ Tambiénȱ agradecerȱ aȱ Tamaraȱ Gallowayȱ elȱ
permitirmeȱrealizarȱlaȱestanciaȱenȱlaȱUniversidadȱdeȱPlymouthȱdondeȱrecibíȱelȱ
apoyoȱdeȱCeri,ȱTrevor,ȱChris,ȱJo,ȱAlan,ȱWas,ȱMarie...graciasȱtambiénȱaȱCarenȱporȱ
suȱ acogida,ȱ yȱ aȱ todaȱ laȱ genteȱ queȱ meȱ hizoȱ conocerȱ laȱ vidaȱ inglesaȱ duranteȱ
aquellosȱ meses.ȱ Laȱ últimaȱ estanciaȱ realizadaȱ duranteȱ laȱ tesisȱ fueȱ enȱ elȱ Centroȱ
Oceanográficoȱ deȱ Murcia,ȱ deȱ nuevoȱ agradecerȱ aȱ suȱ genteȱ porȱ elȱ tiempoȱ entreȱ
ellos.ȱ
Aunqueȱ enȱ algunosȱ momentosȱ parecíaȱ imposibleȱ porȱ faltaȱ deȱ tiempo,ȱ
conseguíȱ realizarȱ elȱ “Masterȱ Erasmusȱ Mundusȱ inȱ Waterȱ andȱ Coastalȱ
Management”.ȱGraciasȱaȱmisȱcompañerosȱporȱhaberȱpasadoȱjuntosȱlosȱbuenosȱyȱ
noȱ tanȱ buenosȱ momentos,ȱ Oscar,ȱ Natalia,ȱ Diana,ȱ Carmen,ȱ Maria,ȱ Rodrigoȱ yȱ
todosȱlosȱdemásȱaventureros.ȱGraciasȱtambiénȱaȱCarmenȱLópez.ȱ
Duranteȱestosȱañosȱheȱdisfrutadoȱdeȱgenteȱmaravillosaȱdentroȱyȱfueraȱdeȱ
laȱ ciencia.ȱ Creoȱ queȱ heȱ tenidoȱ muchaȱ suerteȱ enȱ eseȱ sentido,ȱ porȱ esoȱ tambiénȱ
quieroȱ agradecerȱ aȱ aquellaȱ genteȱ queȱ haȱ supuestoȱ unaȱ víaȱ deȱ escape,ȱ tanȱ aȱ
menudoȱ necesariaȱ duranteȱ laȱ tesis.ȱ Esaȱ peñitaȱ cupuleraȱ yȱ demásȱ compisȱ deȱ laȱ
universidad,ȱ queȱ yaȱ seanȱ delȱ Norteȱ oȱ delȱ Sur,ȱ seȱ hicieronȱ gaditanosȱ oȱ
puertorrealeñosȱ porȱ unosȱ añosȱ yȱ yaȱ laȱ llevanȱ consigoȱ alláȱ dondeȱ vayan.ȱ Soisȱ
tantos,ȱqueȱnoȱvoyȱaȱescribirȱlosȱnombresȱporqueȱtendríaȱqueȱredactarȱunaȱlistaȱ
enorme,ȱperoȱosȱguardoȱparaȱmí,ȱesperoȱqueȱloȱsepáisȱcomprenderȱyȱosȱdoyȱlasȱ
graciasȱaȱmogollón,ȱconȱunȱabrazoȱenȱelȱqueȱcabéisȱtod@sȱapretujaillos.ȱȱ
GraciasȱaȱlaȱgenteȱqueȱmeȱquedóȱdeȱMadrid,ȱmisȱniñas,ȱqueȱaunqueȱnosȱ
veamosȱ deȱ muchoȱ enȱ mucho,ȱ pareceȱ queȱ elȱ tiempoȱ noȱ pasa.ȱ Yȱ misȱ isleñosȱ
queridos,ȱ misȱ amigosȱ deȱ laȱ infanciaȱ yȱ deȱ siempre,ȱ osȱ echoȱ deȱ menosȱ peroȱ
tambiénȱséȱqueȱpuedoȱcontarȱconȱvosotros,ȱgraciasȱdeȱcorazón.ȱȱ
Adri,ȱsinȱdarteȱcuentaȱmeȱhasȱdadoȱunȱmontónȱdeȱenergíaȱespecialmenteȱ
enȱ algunosȱ momentosȱ deȱ laȱ tesisȱ cuandoȱ flaqueabanȱ lasȱ fuerzas;ȱ graciasȱ porȱ
tantasȱcosas,ȱeresȱunȱsol.ȱȱ
ȱYȱ porȱ último,ȱ miȱ másȱ tiernoȱ agradecimientoȱ paraȱ miȱ gente,ȱ miȱ familia,ȱ
queȱ siempreȱ haȱ estadoȱ ahíȱ paraȱ todo,ȱ fueraȱ buenoȱ oȱ regular.ȱ Graciasȱ aȱ misȱ
hermanosȱqueȱsonȱlosȱmejores,ȱyȱqueȱsonȱsinȱdudarloȱloȱmásȱvaliosoȱqueȱtengoȱyȱ
queȱconvencieronȱaȱmisȱpadresȱparaȱdejarmeȱirȱaȱestudiarȱaȱCádiz.ȱAȱmisȱpadresȱ
graciasȱporȱnoȱhacerȱcasoȱdeȱlosȱqueȱcreenȱqueȱaúnȱsigoȱestudiandoȱlaȱcarrera,ȱ
graciasȱporȱcomprenderȱelȱcaminoȱqueȱheȱelegido,ȱgraciasȱporȱvuestroȱapoyoȱyȱ
porȱ todoȱ loȱ queȱ meȱ habéisȱ enseñado.ȱ Graciasȱ Abuȱ porȱ serȱ tanȱ estupendaȱ yȱ
cuidarmeȱ siempreȱ aunqueȱ estuvieraȱ lejos;ȱ yȱ graciasȱ aȱ losȱ pequeñosȱ porȱ esaȱ
alegríaȱqueȱdesprendéis,ȱtanȱpuraȱyȱtanȱcontagiosa.ȱ
Aȱtodos,ȱgracias.ȱ
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Loȱqueȱsabemosȱesȱunaȱgotaȱdeȱagua,ȱloȱ
queȱignoramosȱesȱelȱocéanoȱȱ
(IsaacȱNewton)ȱ
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Aȱmiȱfamiliaȱ
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ÍndiceȱdeȱContenidosȱ
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Prólogo
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Índice de Contenidos
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CAPÍTULOȱ1.ȱINTRODUCCIÓN,ȱZONASȱDEȱESTUDIO,ȱOBJETIVOSȱ
YȱORGANIZACIÓNȱDEȱLAȱTESISȱ
1.ȱIntroducción………………………………………………………………ȱȱȱ
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2.ȱZonasȱdeȱestudio………………………………………………………….ȱ
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3.ȱObjetivosȱeȱhipótesis……………………………………………………...ȱ
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4.ȱEstructuraȱdeȱlaȱtesis……………………………………………………..ȱ
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5.ȱBibliografía………………………………………………………………...ȱ
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CAPÍTULOȱ2.ȱANÁLISISȱDEȱLAȱCONTAMINACIÓNȱYȱ
EVALUACIÓNȱȱDEȱLAȱTOXICIDADȱAGUDAȱMEDIANTEȱ
ENSAYOSȱENȱLABORATORIOȱ
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I.ȱAcuteȱtoxicityȱofȱresidualȱfuelȱoilȱfromȱtheȱtankerȱ“Prestige”ȱusingȱ
amphipods.......................................................................................................ȱ
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II.ȱUsingȱtheȱpolychaeteȱArenicolaȱmarinaȱtoȱdetermineȱtoxicityȱandȱ
bioaccumulationȱofȱPAHsȱboundȱtoȱsediments.........................................ȱ
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III.ȱComparingȱsedimentȱqualityȱinȱSpanishȱlittoralȱareasȱaffectedȱbyȱ
acuteȱ(Prestige,ȱ2002)ȱandȱchronicȱ(BayȱofȱAlgeciras)ȱoilȱspills................ȱȱ
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IV.ȱSedimentȱcontamination,ȱbioavailabilityȱandȱtoxicityȱofȱsedimentsȱ
affectedȱbyȱanȱacuteȱoilȱspill.ȱFourȱyearsȱafterȱtheȱsinkingȱofȱtheȱtankerȱ
Prestigeȱ(2002)………………………………………………………………..ȱ
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CAPÍTULOȱ3.ȱESTUDIOȱDEȱEFECTOSȱSUBLETALESȱENȱ
ORGANISMOSȱBAJOȱCONDICIONESȱDEȱLABORATORIOȱ
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V.ȱEcotoxicityȱofȱsedimentsȱcontaminatedȱbyȱtheȱoilȱspillȱassociatedȱ
withȱtheȱtankerȱ‘‘Prestige’’ȱUsingȱJuvenilesȱofȱtheȱfishȱSparusȱaurata.....ȱ
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VI.ȱKineticȱofȱbiomarkerȱresponsesȱinȱjuvenilesȱofȱtheȱfishȱSparusȱ
aurataȱexposedȱtoȱcontaminatedȱsediments................................................ȱ
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VII.ȱRoleȱofȱbiomarkersȱtoȱassessȱoilȬcontaminatedȱsedimentȱqualityȱ
usingȱtoxicityȱtestsȱwithȱclamsȱandȱcrabs....................................................ȱȱ
117
VIII.ȱVitellogeninȱvariationȱinȱtheȱcrabȱCarcinusȱmaenasȱexposedȱtoȱ
sedimentsȱaffectedȱbyȱoilȱspillsȱ(Spain).......................................................ȱ
141
IX.ȱAȱmultibiomarkerȱapproachȱusingȱtheȱpolychaeteȱArenicolaȱmarinaȱ
toȱassessȱoilȱcontaminatedȱsediments..........................................................ȱ
155
ȱ
CAPÍTULOȱ4.ȱEVALUACIÓNȱDEȱEFECTOSȱSUBLETALESȱINȱSITUȱ
181
X.ȱSublethalȱresponsesȱinȱcagedȱorganismsȱexposedȱtoȱsedimentsȱ
affectedȱbyȱoilȱspills........................................................................................ȱ
187
XI.ȱKineticȱofȱbiomarkersȱinȱtheȱclamȱRuditapesȱphilippinarum.................ȱ
211
XII.ȱAȱkineticȱapproachȱinȱtheȱPAHȱdetoxificationȱsystemȱinȱaȱmarineȱ
invertebrateȱspecie:ȱtheȱcrabȱCarcinusȱmaenas.............................................ȱ
229
XIII.ȱAȱcomparativeȱanalysisȱofȱmacrobenthicȱcommunityȱstructureȱinȱ
relationȱtoȱdifferentȱoilȱcontaminatedȱsediments:ȱtheȱGalicianȱCoastȱ
(acute,ȱPrestigeȱoilȱspill)andȱtheȱBayȱofȱAlgecirasȱ(chronicȱoilȱspills)......ȱ
ȱ
ȱ
249
CAPÍTULOȱ5.ȱAPLICACIÓNȱDEȱUNȱMÉTODOȱINTEGRADOȱPARAȱ
LAȱCARACTERIZACIÓNȱDEȱSEDIMENTOSȱAFECTADOSȱPORȱ
273
VERTIDOSȱDEȱPETRÓLEOȱ
XIV.ȱUsingȱaȱclassicalȱweightȬofȬevidenceȱapproachȱforȱ4Ȭyearsȱ
monitoringȱofȱtheȱimpactȱofȱanȱaccidentalȱoilȱspillȱonȱsedimentȱqualityȱ
279
XV.ȱAȱweightȱofȱevidenceȱapproachȱforȱqualityȱassessmentȱinȱ
sedimentsȱimpactedȱbyȱanȱoilȱspill:ȱtheȱroleȱofȱaȱnewȱlineȱofȱevidenceȱ
usingȱaȱsetȱofȱbiomarkers...............................................................................ȱ
307
XVI.ȱTheȱapplicationȱofȱaȱweightȱofȱevidenceȱapproachȱtoȱcompareȱtheȱ
qualityȱofȱcoastalȱsedimentsȱaffectedȱbyȱacuteȱ(Prestigeȱ2002)ȱandȱ
chronicȱ(BayȱofȱAlgeciras)ȱoilȱspills..............................................................ȱ
331
ȱ
CAPÍTULOȱ6.ȱCONCLUSIONESȱȱ
355
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
Capítuloȱ1.ȱ
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱ
organizaciónȱdeȱlaȱtesisȱ
ȱ
1.ȱIntroducciónȱ
Enȱ laȱ actualidad,ȱ elȱ petróleoȱ yȱ susȱ derivadosȱ hanȱ llegadoȱ aȱ serȱ
imprescindiblesȱ comoȱ fuenteȱ deȱ energíaȱ yȱ paraȱ laȱ fabricaciónȱ deȱ múltiplesȱ
productosȱdeȱlaȱindustriaȱquímica,ȱfarmacéutica,ȱalimenticia,ȱetc.ȱSinȱembargo,ȱ
alrededorȱdelȱȱ0,2%ȱdeȱlaȱproducciónȱmundialȱdeȱpetróleoȱacabaȱvertidoȱalȱmarȱ
(FundaciónȱSantiagoȱReyȱFernándezȬLaTorre.ȱ2003).ȱȱDeȱmaneraȱgeneralȱseȱdiceȱ
queȱ elȱ crudoȱ delȱ petróleoȱ estáȱ compuestoȱ principalmenteȱ porȱ hidrocarburos,ȱ
aunqueȱ realmenteȱ esȱ unaȱ mezclaȱ muyȱ complejaȱ deȱ hidrocarburosȱ yȱ deȱ
derivadosȱ delȱ azufre,ȱ nitrógeno,ȱ oxígenoȱ yȱ complejosȱ organometálicos,ȱ queȱ
abarcaȱ desdeȱ compuestosȱ volátilesȱ deȱ bajoȱ pesoȱ molecular,ȱ comoȱ elȱ metano,ȱ
hastaȱ compuestosȱ pesadosȱ noȱ volátiles,ȱ comoȱ losȱ asfaltenos.ȱ Estosȱ compuestosȱ
seȱdistribuyenȱenȱfamiliasȱdeȱhidrocarburosȱsaturadosȱ(alcanosȱyȱcicloalcanos),ȱ
aromáticos,ȱ cicloalcanosȱ parcialmenteȱ aromatizadosȱ (naftenȬaromáticos)ȱ yȱ
derivadosȱ heteroaromáticosȱ (Fundaciónȱ Santiagoȱ Reyȱ FernándezȬLaTorre.ȱ
2003).ȱ Elȱ mayorȱ oȱ menorȱ porcentajeȱ deȱ hidrocarburosȱ ligerosȱ oȱ pesados,ȱ asíȱ
comoȱ lasȱ variacionesȱ importantesȱ enȱ concentraciónȱ deȱ lasȱ diversasȱ estructurasȱ
moleculares,ȱhaceȱqueȱexistanȱgrandesȱdiferenciasȱentreȱlosȱcrudosȱproducidosȱaȱ
loȱlargoȱdeȱlaȱgeografíaȱmundialȱ(CSIC,ȱ2003a).ȱLaȱteoríaȱmásȱaceptadaȱsobreȱlaȱ
formaciónȱdelȱcrudoȱdeȱpetróleoȱafirmaȱqueȱesȱelȱproductoȱdeȱlaȱdegradación,ȱaȱ
-1-
Capítuloȱ1
travésȱdeȱgrandesȱpresionesȱyȱtemperaturas,ȱdeȱlaȱmateriaȱorgánicaȱprocedenteȱ
deȱrestosȱdeȱanimalesȱyȱplantas.ȱUnaȱvezȱextraídoȱelȱpetróleo,ȱgeneralmenteȱseȱ
someteȱ aȱ destilaciónȱ paraȱ separarȱ susȱ componentesȱ yȱ poderȱ utilizarseȱ
(FundaciónȱSantiagoȱReyȱFernándezȬLaTorre.ȱ2003).ȱDadoȱqueȱlosȱcomponentesȱ
delȱ petróleoȱ cubrenȱ unȱ amplioȱ margenȱ deȱ puntosȱ deȱ ebullición,ȱ seȱ puedenȱ
separarȱunaȱserieȱdeȱfraccionesȱdeȱinterésȱeconómicoȱ(gasolina,ȱqueroseno,ȱgasȬ
oil,ȱ aceitesȱ lubricantes,ȱ etc),ȱ quedandoȱ unȱ residuoȱ deȱ menorȱ valor,ȱ queȱ seȱ
destinaȱ aȱ combustibleȱ industrialȱ (fuelȬoil)ȱ oȱ aȱ asfaltoȱ (Fundaciónȱ Santiagoȱ Reyȱ
FernándezȬLaTorre.ȱ2003).ȱȱȱ
Característicasȱdeȱlosȱhidrocarburosȱaromáticosȱpolicíclicosȱ
Losȱ hidrocarburos,ȱ talȱ yȱ comoȱ suȱ nombreȱ indica,ȱ estánȱ formadosȱ porȱ
átomosȱ deȱ carbonoȱ eȱ hidrógeno.ȱ Losȱ hidrocarburosȱ másȱ importantesȱ sonȱ losȱ
alcanos,ȱ alquenos,ȱ alquinosȱ yȱ compuestosȱ aromáticos.ȱ Losȱ hidrocarburosȱ
aromáticosȱ policíclicosȱ (PAHs)ȱ estánȱ compuestosȱ porȱ átomosȱ deȱ Cȱ eȱ Hȱ
ordenadosȱenȱformaȱdeȱdosȱoȱmásȱanillosȱdeȱbenceno.ȱHayȱmilesȱdeȱPAHs,ȱcadaȱ
unoȱconȱunȱnúmeroȱyȱunaȱdisposiciónȱparticularȱdeȱlosȱanillosȱaromáticosȱyȱdeȱ
losȱ sustituyentesȱ (Simsȱ yȱ Overcash,ȱ 1983).ȱ Lasȱ propiedadesȱ físicoȱ químicasȱ deȱ
losȱ PAHsȱ varíanȱ generalmenteȱ conȱ elȱ pesoȱ molecular.ȱ Alȱ aumentarȱ elȱ pesoȱ
molecular,ȱlaȱsolubilidadȱenȱaguaȱdisminuye,ȱyȱelȱpuntoȱdeȱfusión,ȱelȱpuntoȱdeȱ
ebulliciónȱ yȱ elȱ logȱ Kowȱ (coeficienteȱ deȱ particiónȱ octanolȬagua)ȱ aumentanȱ
suponiendoȱunȱaumentoȱdeȱlaȱsolubilidadȱenȱlasȱgrasas,ȱunaȱdisminuciónȱenȱlaȱ
resistenciaȱaȱlaȱoxidaciónȬreducción,ȱyȱunaȱdisminuciónȱdeȱlaȱpresiónȱdeȱvaporȱ
(tablaȱ2.1).ȱPorȱello,ȱlosȱPAHsȱvaríanȱsuȱcomportamientoȱenȱelȱmedioȱambienteȱ
enȱ funciónȱ deȱ suȱ pesoȱ molecularȱ (Eisler,ȱ 2000).ȱ Así,ȱ porȱ ejemplo,ȱ elȱ
Benzo[a]pirenoȱdebidoȱaȱsuȱbajaȱsolubilidadȱyȱpresiónȱdeȱvaporȱyȱelevadoȱKowȱ
seȱvaȱaȱincorporarȱprincipalmenteȱalȱsueloȱyȱaȱlosȱsedimentos,ȱquedandoȱmenosȱ
delȱ1%ȱenȱelȱrestoȱdeȱlosȱcompartimentosȱambientalesȱ(agua,ȱbiota,ȱaireȱyȱsólidosȱ
-2-
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱorganizaciónȱdeȱlaȱtesis
enȱsuspensión).ȱOtrosȱPAHs,ȱcomoȱlosȱdeȱ2ȱyȱ3ȱanillosȱ(Naftaleno,ȱAcenafteno,ȱ
Antraceno,ȱFluoreno,ȱAcenaftilenoȱyȱFenantreno),ȱsuelenȱenȱcambioȱencontrarseȱ
predominantementeȱenȱelȱaireȱ(Baekȱetȱal.,ȱ1991).ȱAsí,ȱseȱhaȱpodidoȱcomprobarȱ
comoȱlaȱbajaȱsolubilidadȱdeȱlosȱPAHsȱyȱsuȱaltaȱafinidadȱporȱelȱcarbonoȱorgánicoȱ
haceȱqueȱenȱelȱmedioȱacuáticoȱmásȱdeȱ2/3ȱseȱencuentrenȱasociadosȱaȱpartículas,ȱ
eliminándoseȱ losȱ deȱ medioȱ yȱ altoȱ pesoȱ molecularȱ porȱ adsorciónȱ alȱ sedimentoȱ
principalmenteȱyȱlosȱdeȱbajoȱpesoȱporȱvolatilizaciónȱyȱbiodegradación.ȱȱ
Tablaȱ 2.1.ȱ Algunasȱ deȱ lasȱ propiedadesȱ químicasȱ delȱ Naftaleno,ȱ
Antraceno,ȱBenzo[a]antraceno,ȱBenzo[a]pireno,ȱBenzo[g,h,i]perileno.ȱ
Solubilidadȱ
Logȱ
enȱaguaȱ
Kowȱ
(mgȱLȬ1)ȱ
Nºȱ
anillos
Pesoȱ
molecularȱ
Puntoȱdeȱ
fusiónȱ(ºC)ȱ
Naftalenoȱ
2ȱ
128ȱ
80ȱ
30.0ȱ
3.37ȱ
Antracenoȱ
3ȱ
178ȱ
216ȱ
0.07ȱ
4.45ȱ
Benzo[a]antracenoȱ
4ȱ
228ȱ
158ȱ
0.014ȱ
5.61ȱ
Benzo[a]pirenoȱ
5ȱ
252ȱ
179ȱ
0.0038ȱ
6.04ȱ
Benzo[g,h,i]perilenoȱ
6ȱ
276ȱ
222ȱ
0.00026ȱ
7.23ȱ
Compuestoȱ
ȱ
Elȱ origenȱ deȱ estosȱ hidrocarburosȱ aromáticosȱ policíclicosȱ enȱ elȱ medioȱ
ambienteȱ esȱ muyȱ diverso,ȱ aunqueȱ básicamenteȱ seȱ puedenȱ identificarȱ tresȱ
fuentesȱdistintasȱ(Eisler,ȱ2000):ȱȱ
ȬȱOrigenȱpirolítico,ȱprocedenteȱdeȱlaȱcombustiónȱincompletaȱdeȱlaȱmateriaȱ
orgánica,ȱ recienteȱ oȱ fósil,ȱ bienȱ porȱ causasȱ naturalesȱ (incendiosȱ deȱ bosques,ȱ
erupcionesȱ volcánicas,ȱ etc.)ȱ oȱ antropogénicasȱ (utilizaciónȱ deȱ combustiblesȱ
fósiles,ȱ incineraciónȱdeȱresiduos,ȱ emisionesȱ deȱvehículos,ȱprocesosȱindustrialesȱ
deȱgasificaciónȱyȱlicuefacciónȱdelȱcarbón,ȱȱetc.).ȱȱ
Ȭ 3ȱȬ
Capítuloȱ1
ȬȱOrigenȱpetrogénico,ȱproducidoȱporȱvertidosȱaccidentalesȱoȱintencionadosȱ
deȱderivadosȱdelȱpetróleo.ȱSeȱcaracterizanȱporȱserȱmezclasȱcomplejasȱformadasȱ
porȱcompuestosȱconȱcadenasȱalquílicasȱdeȱhastaȱ5ȱoȱ6ȱátomosȱdeȱcarbono.ȱ
Ȭȱ Origenȱ diagenéticoȱ deȱ laȱ materiaȱ orgánicaȱ sedimentaria,ȱ laȱ cualȱ puedeȱ
sufrirȱ unaȱ serieȱ deȱ procesosȱ geoquímicosȱ naturales,ȱ comoȱ sonȱ laȱ
descarboxilación,ȱ aromatización,ȱ desfuncionalización,ȱ etc.ȱ paraȱ convertirseȱ enȱ
PAHsȱ deȱ origenȱ natural.ȱ Entreȱ éstosȱ encontraríamosȱ ciertosȱ derivadosȱ delȱ
crisenoȱyȱdelȱpiceno,ȱasíȱcomoȱelȱretenoȱyȱelȱperileno,ȱaunqueȱestosȱdosȱúltimosȱ
tambiénȱpuedenȱtenerȱunȱorigenȱpirolítico.ȱ
Aȱ pesarȱ deȱ queȱ losȱ PAHsȱ seȱ encuentranȱ deȱ formaȱ naturalȱ enȱ elȱ medioȱ
ambiente,ȱlaȱmayorȱparteȱdeȱestosȱcompuestosȱpresentesȱenȱlaȱnaturalezaȱtienenȱ
origenȱantropogénico,ȱllegandoȱaȱdescargarseȱenȱelȱmedioȱacuáticoȱalrededorȱdeȱ
230000ȱtoneladasȱdeȱPAHsȱalȱañoȱ(tablaȱ2.2).ȱ
Tablaȱ1.1.ȱPrincipalesȱfuentesȱdeȱPAHsȱenȱelȱmedioȱacuático.ȱ
PAHsȱtotalesȱ
Toneladas/añoȱ
Vertidosȱdeȱpetróleoȱ
170000ȱ
Deposiciónȱatmosféricaȱ
50000ȱ
Aguasȱresidualesȱ
4400ȱ
Escorrentíaȱȱ
2940ȱ
Biosíntesisȱ
2700ȱ
Comportamientoȱdeȱlosȱhidrocarburosȱenȱelȱmedioȱmarino.ȱ
Talȱ yȱ comoȱ seȱ haȱ vistoȱ anteriormente,ȱ elȱ crudoȱ deȱ petróleoȱ estáȱ
compuestoȱ principalmenteȱ porȱ hidrocarburosȱ queȱ varíanȱ entreȱ muyȱ volátiles,ȱ
-4-
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱorganizaciónȱdeȱlaȱtesis
queȱsonȱsustanciasȱligerasȱcomoȱelȱpropanoȱyȱelȱbenceno,ȱyȱcompuestosȱpesadosȱ
comoȱlosȱasfaltenosȱyȱresinas.ȱUnaȱvezȱqueȱlosȱhidrocarburosȱseȱviertenȱalȱmar,ȱ
éstosȱ tiendenȱ aȱ volatilizarseȱ oȱ esparcirseȱ enȱ elȱ medio,ȱ enȱ funciónȱ deȱ suȱ
composición.ȱ Esteȱ comportamientoȱ deȱ losȱ hidrocarburosȱ enȱ elȱ mar,ȱ vaȱ aȱ
determinarȱ queȱ susȱ componentesȱ seȱ distribuyanȱ enȱ losȱ distintosȱ
compartimentosȱ ambientales.ȱ ȱ Laȱ formaȱ enȱ queȱ laȱ capaȱ deȱ hidrocarburosȱ seȱ
rompeȱyȱseȱdisipaȱenȱelȱmar,ȱvaȱaȱdependerȱenȱgranȱparteȱdeȱlaȱpersistenciaȱdelȱ
fuelȱ vertido.ȱLosȱ productosȱligerosȱcomoȱelȱqueroseno,ȱ tiendenȱ aȱevaporarseȱyȱ
seȱ disipanȱ rápidamente.ȱ Estosȱ tiposȱ deȱ fuelȱ seȱ denominanȱ “noȱ persistentes”.ȱȱ
Porȱotraȱparte,ȱlasȱsustanciasȱ“persistentes”,ȱcomoȱlaȱmayoríaȱdeȱlosȱcrudosȱdeȱ
petróleo,ȱseȱdisipanȱmásȱlentamente.ȱLaȱInternationalȱTankerȱOwnersȱPollutionȱ
Federation,ȱ ITOPFȱ (http://www.itopf.com/index.html)ȱ haȱ realizadoȱ unaȱ
clasificaciónȱ deȱ losȱ procesosȱ queȱ puedenȱ sufrirȱ losȱ hidrocarburosȱ unaȱ vezȱ
vertidosȱenȱelȱmedioȱmarino:ȱ
A)ȱ Esparcimiento.ȱ Tanȱ prontoȱ comoȱ elȱ fuelȱ esȱ vertidoȱ enȱ elȱ mar,ȱ ésteȱ
comienzaȱ aȱ esparcirseȱ porȱ laȱ superficieȱ delȱ agua.ȱ Laȱ velocidadȱ aȱ laȱ queȱ tieneȱ
lugarȱ esteȱ proceso,ȱ dependeȱ enȱ granȱ parteȱ deȱ laȱ viscosidadȱ delȱ fuel.ȱ Aquellosȱ
queȱseanȱfluidos,ȱconȱbajaȱviscosidadȱseȱvanȱaȱesparcirȱmásȱrápidamenteȱqueȱlosȱ
queȱpresentanȱviscosidadȱalta.ȱLaȱcapaȱdeȱfuelȱvaȱaȱromperseȱporȱlaȱacciónȱdelȱ
viento,ȱ lasȱ olas,ȱ yȱ lasȱ turbulencias,ȱ deȱ formaȱ queȱ lasȱ llamadasȱ “manchasȱ deȱ
petróleo”ȱ seȱ vanȱ aȱ disponerȱ paralelamenteȱ aȱ laȱ direcciónȱ delȱ viento.ȱ Laȱ
velocidadȱ aȱ laȱ queȱ seȱ vanȱ aȱ esparcirȱ lasȱ manchasȱ deȱ petróleo,ȱ tambiénȱ vaȱ aȱ
dependerȱ deȱ otrosȱ factoresȱ comoȱ laȱ temperatura,ȱ lasȱ corrientes,ȱ laȱ mareaȱ yȱ laȱ
velocidadȱdelȱviento.ȱCuantoȱmásȱseverasȱseanȱlasȱcondicionesȱmeteorológicas,ȱ
másȱrápidamenteȱseȱvaȱaȱproducirȱelȱesparcimientoȱyȱrupturaȱdeȱlasȱmanchasȱdeȱ
petróleo.ȱ
Ȭ 5ȱȬ
Capítuloȱ1
B)ȱȱEvaporación.ȱAquellosȱcompuestosȱmásȱligerosȱseȱvanȱaȱevaporarȱaȱlaȱ
atmósfera.ȱLaȱcantidadȱdeȱhidrocarburoȱqueȱseȱevaporeȱyȱlaȱvelocidadȱconȱqueȱ
loȱ haga,ȱ vaȱ aȱ dependerȱ deȱ loȱ volátilesȱ queȱ seanȱ losȱ compuestosȱ queȱ loȱ
componen.ȱAsí,ȱporȱejemplo,ȱsiȱseȱtrataȱdeȱunȱfuelȱpesadoȱsóloȱseȱevaporaráȱunaȱ
pequeñaȱ parte.ȱ Deȱ maneraȱ general,ȱ seȱ puedeȱ decirȱ queȱ aquellosȱ componentesȱ
delȱ fuelȱ cuyoȱ puntoȱ deȱ ebulliciónȱ seaȱ menorȱ queȱ 200ºCȱ vanȱ aȱ tenderȱ aȱ
evaporarseȱ enȱ lasȱ primerasȱ 24ȱ horas.ȱ Elȱ fenómenoȱ deȱ evaporaciónȱ seȱ vaȱ aȱ
incrementarȱ alȱ aumentarȱ elȱ áreaȱ superficialȱ deȱ laȱ capaȱ deȱ fuel.ȱ Lasȱ altasȱ
temperaturas,ȱ laȱ altaȱ velocidadȱ delȱ viento,ȱ etc.,ȱ vanȱ aȱ aumentarȱ laȱ tasaȱ deȱ
evaporaciónȱyȱlaȱproporciónȱdeȱfuelȱqueȱsufreȱesteȱproceso.ȱȱȱ
C)ȱ Dispersión.ȱ Lasȱ olasȱ yȱ laȱ turbidezȱ enȱ laȱ superficieȱ delȱ marȱ puedenȱ
provocarȱqueȱlaȱmanchaȱdeȱfuelȱseȱfragmenteȱenȱtrozosȱdeȱdistintoȱtamaño.ȱEstoȱ
haceȱ queȱ muchosȱ deȱ estosȱ trozosȱ deȱ fuelȱ lleguenȱ aȱ losȱ nivelesȱ másȱ altosȱ deȱ laȱ
columnaȱ deȱ agua.ȱ Algunosȱ deȱ losȱ trozosȱ másȱ pequeñosȱ seȱ mantendránȱ
suspendidosȱenȱlaȱcolumnaȱdeȱagua,ȱmientrasȱqueȱaquellosȱmásȱgrandesȱvanȱaȱ
tenderȱaȱȱvolverȱaȱlaȱsuperficie,ȱdondeȱpuedenȱunirseȱconȱotrosȱtrozosȱyȱvolverȱaȱ
formarȱunaȱfinaȱcapaȱsuperficial.ȱElȱfuelȱqueȱquedaȱenȱlaȱcolumnaȱdeȱaguaȱtieneȱ
unaȱelevadaȱáreaȱsuperficialȱtrasȱelȱprocesoȱdeȱdispersión,ȱloȱqueȱinduceȱaȱqueȱ
seȱ denȱ otrosȱ procesosȱ naturalesȱ comoȱ laȱ disolución,ȱ biodegradaciónȱ yȱ
sedimentación.ȱȱLaȱvelocidadȱaȱlaȱqueȱunȱfuelȱseȱdispersa,ȱvaȱaȱdependerȱdeȱlaȱ
naturalezaȱ delȱ mismoȱ yȱ delȱ estadoȱ deȱ laȱ mar,ȱ incrementándoseȱ siȱ elȱ fuelȱ esȱ
ligero,ȱtieneȱbajaȱviscosidad,ȱyȱsiȱlaȱsuperficieȱdelȱmarȱseȱencuentraȱalterada.ȱLaȱ
adiciónȱ deȱ dispersantesȱ químicosȱ puedeȱ acelerarȱ elȱ procesoȱ naturalȱ deȱ
dispersión.ȱ
D)ȱ Emulsificación.ȱ Unaȱ emulsiónȱ seȱ formaȱ cuandoȱ seȱ combinanȱ dosȱ
líquidos,ȱdeȱformaȱqueȱunoȱdeȱlosȱdosȱquedaȱsuspendidoȱenȱelȱotro.ȱSeȱhablaȱdeȱ
emulsificaciónȱ delȱ fuelȱ cuandoȱ lasȱ gotasȱ deȱ aguaȱ seȱ suspendenȱ conȱ él.ȱ Estoȱ
ocurreȱ porȱ mezclaȱ físicaȱ deȱ ambosȱ componentesȱ yȱ vieneȱ determinadaȱ porȱ laȱ
-6-
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱorganizaciónȱdeȱlaȱtesis
turbulenciaȱenȱlaȱsuperficieȱdelȱagua.ȱLaȱemulsiónȱformadaȱsueleȱserȱviscosaȱyȱ
másȱ persistenteȱ queȱ elȱ fuelȱ original,ȱ yȱ haceȱ queȱ elȱ volumenȱ delȱ contaminanteȱ
aumenteȱ entreȱ tresȱ oȱ cuatroȱ veces.ȱ Estoȱ ralentizaȱ yȱ retrasaȱ otrosȱ procesosȱ queȱ
permitiríanȱ laȱ disipaciónȱ delȱ fuel.ȱ Aquellosȱ tiposȱ deȱ fuelȱ conȱ unȱ contenidoȱ enȱ
asfaltenosȱ mayorȱ delȱ 0.5%ȱ tiendenȱ aȱ formarȱ emulsionesȱ establesȱ queȱ puedenȱ
persistirȱvariosȱmesesȱdespuésȱdelȱvertido.ȱSiȱunaȱvezȱenȱlaȱcosta,ȱlasȱemulsionesȱ
aumentanȱ suȱ temperaturaȱ debidoȱ aȱ laȱ incidenciaȱ deȱ laȱ luzȱ solar,ȱ éstasȱ vanȱ aȱ
poderȱromperseȱseparandoȱdeȱnuevoȱelȱfuelȱyȱelȱagua.ȱ
E)ȱDisolución.ȱLosȱcompuestosȱsolublesȱdelȱfuelȱvanȱaȱpoderȱdisolverseȱ
enȱelȱagua.ȱEstoȱdependeȱdeȱlaȱcomposiciónȱyȱdelȱestadoȱdelȱfuel,ȱyȱocurreȱmásȱ
rápidamenteȱ cuandoȱelȱ fuelȱseȱ encuentraȱdispersoȱenȱlaȱcolumnaȱdeȱagua.ȱLosȱ
componentesȱmásȱsolublesȱenȱelȱaguaȱdeȱmarȱsonȱlosȱhidrocarburosȱaromáticosȱ
ligerosȱ comoȱ elȱ bencenoȱ yȱ elȱ tolueno.ȱ Sinȱ embargo,ȱ estosȱ compuestosȱ sonȱ
aquellosȱqueȱseȱvanȱaȱperderȱenȱprimerȱlugarȱporȱevaporación,ȱunȱprocesoȱqueȱ
esȱ10ȱ–ȱ100ȱvecesȱmásȱrápidoȱqueȱlaȱdisolución.ȱȱ
F)ȱOxidación.ȱElȱfuelȱvaȱaȱreaccionarȱquímicamenteȱconȱelȱoxígeno,ȱbienȱ
rompiéndoseȱ enȱ fraccionesȱ solubles,ȱ oȱ bienȱ formandoȱ compuestosȱ persistentesȱ
queȱ recibenȱ elȱ nombreȱ deȱ “alquitrán”.ȱ Esteȱ procesoȱ estáȱ favorecidoȱ porȱ laȱ luzȱ
solarȱ (fotooxidación)ȱ yȱ vaȱ aȱ dependerȱ delȱ tipoȱ deȱ fuelȱ yȱ deȱ laȱ formaȱ queȱ seȱ
encuentraȱ expuestoȱ aȱ laȱ luz.ȱ Sinȱ embargo,ȱ esȱ unȱ procesoȱ muyȱ lento.ȱ Laȱ
formaciónȱ delȱ alquitránȱ seȱ daȱ porȱ laȱ oxidaciónȱ deȱ capasȱ gruesasȱ deȱ fuelȱ muyȱ
viscosoȱ oȱ porȱ laȱ oxidaciónȱ deȱ emulsiones.ȱ Esteȱ procesoȱ formaȱ unaȱ protecciónȱ
externaȱparaȱlosȱcomponentesȱpesadosȱloȱqueȱhaceȱaumentarȱlaȱpersistenciaȱdelȱ
fuel.ȱ Lasȱ conocidasȱ “galletasȱ deȱ alquitrán”ȱ queȱ seȱencuentranȱ normalmenteȱ enȱ
laȱcosta,ȱsonȱproductosȱdeȱesteȱprocesoȱdeȱoxidación.ȱ
G)ȱ Sedimentación.ȱ ȱ Algunosȱ componentesȱ pesadosȱ tienenȱ densidadesȱ
mayoresȱqueȱelȱaguaȱdulceȱporȱloȱqueȱsedimentan.ȱSinȱembargo,ȱelȱaguaȱdeȱmarȱ
Ȭ 7ȱȬ
Capítuloȱ1
tieneȱ unaȱ densidadȱ mayorȱ queȱ elȱ aguaȱ dulceȱ yȱ muyȱ pocosȱ crudosȱ deȱ petróleoȱ
sonȱ loȱ suficientementeȱ densosȱ comoȱ paraȱ hundirseȱ enȱ elȱ medioȱ marino.ȱ Laȱ
sedimentaciónȱ ocurreȱ normalmenteȱ porȱ laȱ adhesiónȱ delȱ fuelȱ conȱ partículasȱ deȱ
sedimentoȱoȱmateriaȱorgánica.ȱGeneralmente,ȱlasȱaguasȱsomerasȱestánȱcargadasȱ
deȱsólidosȱenȱsuspensiónȱfavoreciendoȱelȱprocesoȱdeȱsedimentación.ȱCuandoȱelȱ
fuelȱ llegaȱ aȱ costasȱ arenosasȱ sueleȱ mezclarseȱ conȱ arenaȱ yȱ otrosȱ sedimentos.ȱ Siȱ
posteriormente,ȱ estaȱ mezclaȱ vuelveȱ alȱ mar,ȱ probablementeȱ seȱ hunda.ȱ Además,ȱ
siȱ elȱ fuelȱ seȱ quemaȱ trasȱ serȱ vertido,ȱ losȱ residuosȱ puedenȱ volverseȱ loȱ
suficientementeȱdensosȱyȱllegarȱaȱsedimentar.ȱȱȱ
H)ȱBiodegradación.ȱElȱaguaȱdeȱmarȱposeeȱunaȱserieȱdeȱmicroorganismosȱ
queȱ puedenȱ degradarȱ parcialmenteȱ oȱ completamenteȱ elȱ fuelȱ enȱ compuestosȱ
solublesȱenȱagua,ȱhastaȱCO2ȱyȱagua.ȱCadaȱtipoȱdeȱmicroorganismosȱesȱcapazȱdeȱ
degradarȱ unȱ grupoȱ deȱ compuestosȱ particulares.ȱ Sinȱ embargo,ȱ algunosȱ
compuestosȱdeȱhidrocarburosȱsonȱresistentesȱaȱlaȱdegradación.ȱLaȱeficienciaȱdeȱ
laȱ biodegradaciónȱ vaȱ aȱ dependerȱ deȱ variosȱ factores:ȱ losȱ nivelesȱ deȱ nutrientesȱ
(nitrógenoȱ yȱ fósforo)ȱ enȱ elȱ agua,ȱ laȱ temperaturaȱ yȱ laȱ presenciaȱ deȱ oxígeno.ȱ
Comoȱlaȱbiodegradaciónȱprecisaȱdeȱoxígenoȱparaȱpoderȱrealizarse,ȱesteȱprocesoȱ
sóloȱvaȱaȱpoderȱllevarseȱaȱcaboȱenȱlaȱinterfaseȱfuelȬagua,ȱyaȱqueȱelȱfuelȱnoȱposeeȱ
oxígeno.ȱ Porȱ tanto,ȱ laȱ biodegradaciónȱ seráȱ mayorȱ alȱ aumentarȱ laȱ relaciónȱ
superficie/volumenȱdelȱfuelȱ(porȱejemplo,ȱtrasȱprocesosȱdeȱdispersión).ȱ
Losȱprocesosȱdeȱesparcimiento,ȱevaporación,ȱdispersión,ȱemulsificaciónȱyȱ
disolución,ȱ sonȱ losȱ másȱ importantesȱ duranteȱ lasȱ primerasȱ fasesȱ delȱ vertido,ȱ
mientrasȱ queȱ laȱ oxidación,ȱ laȱ sedimentaciónȱ yȱ laȱ biodegradaciónȱ cobranȱ másȱ
importanciaȱ despuésȱ deȱ pasadoȱ unȱ tiempoȱ yȱ determinanȱ elȱ destinoȱ finalȱ delȱ
fuel.ȱ
-8-
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱorganizaciónȱdeȱlaȱtesis
Efectosȱdeȱlosȱhidrocarburosȱenȱelȱmedioȱmarinoȱ
Losȱ efectosȱ deȱ losȱ vertidosȱ deȱ hidrocarburosȱ enȱ elȱ medioȱ ambienteȱ
marinoȱ puedenȱ estarȱ causadosȱ porȱ laȱ naturalezaȱ físicaȱ delȱ fuelȱ oȱ bienȱ porȱ laȱ
composiciónȱquímicaȱdeȱéste.ȱDesdeȱelȱpuntoȱdeȱvistaȱfísico,ȱlaȱimpregnaciónȱdeȱ
losȱorganismosȱqueȱentranȱenȱcontactoȱdirectoȱconȱelȱfuelȱvaȱaȱsuponerȱunaȱdeȱ
lasȱ causasȱ letalesȱ másȱ comunes,ȱ asíȱ comoȱ elȱ agotamientoȱ yȱ laȱ ingestaȱ deȱ
hidrocarburos.ȱLaȱȱmortalidadȱseȱproduceȱalȱimpedirȱlaȱrespiración,ȱfotosíntesis,ȱ
oȱ alȱ modificarȱ laȱ resistenciaȱ térmicaȱ (porȱ ejemploȱ lasȱ aves).ȱ Desdeȱ elȱ puntoȱ deȱ
vistaȱquímico,ȱcabeȱdestacarȱlosȱefectosȱtóxicosȱagudosȱyȱcrónicos.ȱAdemás,ȱlasȱ
alteracionesȱ aȱ nivelȱ deȱ organismo,ȱ yȱ susȱ consecuenciasȱ demográficas,ȱ puedenȱ
desembocarȱ enȱ cambiosȱ enȱ laȱ estructuraȱ deȱ lasȱ comunidadesȱ ecológicasȱ y,ȱ porȱ
tanto,ȱ enȱ unaȱ alteraciónȱ deȱ laȱ redȱ deȱ lasȱ interaccionesȱ existentesȱ (Fundaciónȱ
SantiagoȱReyȱFernándezȬLaTorre,ȱ2003).ȱȱȱ
Losȱcomponentesȱmásȱtóxicosȱdelȱpetróleoȱsonȱaquellosȱhidrocarburosȱdeȱ
bajoȱ pesoȱ molecularȱ queȱ seȱ vanȱ aȱ perderȱ rápidamenteȱ porȱ evaporación,ȱ deȱ
formaȱ queȱ noȱ suelenȱ llegarȱ aȱ concentrarseȱ loȱ suficienteȱ paraȱ provocarȱ efectosȱ
letalesȱenȱlosȱorganismos.ȱPorȱotraȱparte,ȱunaȱexposiciónȱprolongadaȱalȱfuelȱoȱaȱ
susȱ componentesȱ puedeȱ producirȱ efectosȱ subletalesȱ queȱ puedenȱ afectarȱ aȱ lasȱ
funcionesȱ vitalesȱ deȱ losȱ organismosȱ comoȱ laȱ reproducción,ȱ crecimientoȱ oȱ
alimentaciónȱ(ITOPF,ȱhttp://www.itopf.com/index.html).ȱ
Laȱ toxicidadȱ deȱ losȱ hidrocarburosȱ deȱ petróleoȱ resideȱ básicamenteȱ enȱ elȱ
potencialȱ cancerígenoȱ deȱ losȱ hidrocarburosȱ aromáticosȱ policíclicosȱ (PAHs).ȱ
Entreȱ ellosȱ destacanȱ losȱ 16ȱ compuestosȱ incluidosȱ enȱ laȱ listaȱ deȱ contaminantesȱ
prioritariosȱ deȱ laȱ Environmentalȱ Protectionȱ Agencyȱ deȱ losȱ Estadosȱ Unidos:ȱ
Naftaleno,ȱ Acenaftileno,ȱ Acenafteno,ȱ Fluoreno,ȱ Fenantreno,ȱ Antraceno,ȱ
Fluoranteno,ȱ
Pireno,ȱ
Criseno,ȱ
Benzo[a]antraceno,ȱ
Ȭ 9ȱȬ
Benzo[k]fluoranteno,ȱ
Capítuloȱ1
Benzo[a]pireno,ȱ
Dibenzo[ah]antraceno,ȱ
Indeno[123Ȭcd]pireno,ȱ
Benzo[ghi]perilenoȱ(USEPA,ȱ2000).ȱȱ
UnaȱdeȱlasȱcaracterísticasȱdeȱlosȱPAHsȱqueȱlosȱhaceȱmásȱpeligrososȱesȱsuȱ
acumulaciónȱ enȱ losȱ organismosȱ acuáticosȱ desdeȱ elȱ agua,ȱ elȱ sedimentoȱ oȱ elȱ
alimento.ȱ Asíȱ seȱ hanȱ encontradoȱ valoresȱ deȱ factorȱ deȱ bioacumulaciónȱ (BCF)ȱ
entreȱ 10ȱ yȱ 10000ȱ paraȱ pecesȱ yȱ crustáceos,ȱ correspondiendoȱ losȱ valoresȱ másȱ
elevadosȱ aȱ losȱ PAHsȱ deȱ mayorȱ pesoȱ molecularȱ (Eislerȱ etȱ al.,ȱ 2000).ȱ Estasȱ
sustanciasȱ sinȱ embargo,ȱ puedenȱ serȱ rápidamenteȱ metabolizadasȱ porȱ losȱ
organismosȱ (deȱ 2ȱ aȱ 9ȱ díasȱ enȱ peces),ȱ loȱ cualȱ evitaȱ laȱ biomagnificaciónȱ peroȱ
puedeȱpresentarȱunȱproblemaȱaúnȱmayorȱalȱgenerarseȱmetabolitosȱcarcinógenosȱ
yȱmutagénicos.ȱȱ
Enȱ teoría,ȱtodosȱlosȱorganismosȱposeenȱencimasȱdeȱbiotransformaciónȱ oȱ
detoxificaciónȱ queȱ conviertenȱ lasȱ sustanciasȱ xenobióticasȱ lipofílicosȱ enȱ
metabolitosȱsolublesȱenȱaguaȱqueȱpuedenȱserȱexcretadosȱ(Neff,ȱ1979).ȱEnȱlaȱFaseȱ
Iȱdeȱlosȱprocesosȱmetabólicos,ȱlosȱPAHsȱsonȱtransformadosȱenȱvariosȱproductosȱ
comoȱ epóxidos,ȱ fenoles,ȱ quinónes,ȱ dihidrodioles,ȱ epóxidos,ȱ tetrahidrotriolesȱ yȱ
tetrahidrotetroles.ȱ Losȱ metabolitosȱ intermedios,ȱ hanȱ sidoȱ identificadosȱ comoȱ
agentesȱmutagénicos,ȱcarcinógenosȱyȱteratógenosȱ(SimsȱandȱOvercash,ȱ1983).ȱLaȱ
activaciónȱ deȱ estosȱ mecanismosȱ tóxicosȱ ocurreȱ porȱ hidroxilaciónȱ oȱ porȱ
producciónȱ deȱ epóxidosȱ inestablesȱ deȱ PAHsȱ queȱ dañanȱ elȱ ADN,ȱ iniciandoȱ losȱ
procesosȱcancerígenosȱ(JackimȱyȱLake,ȱ1978;ȱSchnitzȱyȱO’Connor,ȱ1992).ȱȱȱȱ
Importanciaȱdeȱlosȱsedimentosȱenȱestudiosȱdeȱcontaminaciónȱ
Cualquierȱ sustanciaȱ queȱ seȱ encuentreȱ enȱ unȱ medioȱ enȱ concentracionesȱ
superioresȱ aȱ losȱ nivelesȱ naturales,ȱ puedeȱ alterarȱ elȱ equilibrioȱ delȱ ecosistema,ȱ
llegandoȱ inclusoȱ aȱ destruirȱ elȱ biotopo,ȱ aȱ limitarȱ laȱ explotaciónȱ deȱ losȱ recursosȱ
biológicosȱyȱaȱponerȱenȱpeligroȱlaȱsaludȱhumana.ȱLaȱpresenciaȱdeȱunaȱsustanciaȱ
- 10 -
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱorganizaciónȱdeȱlaȱtesis
enȱ elȱ medioȱ enȱ concentracionesȱ mayoresȱ aȱ lasȱ naturalesȱ seȱ denominaȱ
contaminación,ȱ yȱ elȱ fenómenoȱ porȱ elȱ queȱ esteȱ enriquecimientoȱ deȱ
contaminantesȱproduceȱefectosȱbiológicos,ȱseȱdefineȱcomoȱpolución.ȱ
Muchosȱ deȱ losȱ contaminantesȱ orgánicosȱ eȱ inorgánicosȱ queȱ seȱ originanȱ
conȱ laȱ actividadȱ humana,ȱ sonȱ transportados,ȱ tantoȱ enȱ disoluciónȱ comoȱ
adsorbidosȱ sobreȱ elȱ materialȱ particuladoȱ enȱ suspensión,ȱ desdeȱ lasȱ zonasȱ
continentalesȱhaciaȱlosȱocéanos.ȱLasȱzonasȱcosteras,ȱyȱespecialmenteȱlosȱsistemasȱ
másȱ someros,ȱ sonȱ losȱ receptoresȱ principalesȱ deȱ lasȱ sustanciasȱ contaminantesȱ
vertidasȱaȱlosȱocéanos.ȱDadoȱelȱcarácterȱactivoȱdeȱestosȱcompuestos,ȱduranteȱelȱ
transporteȱ seȱ incorporanȱ alȱ sedimentoȱ aȱ travésȱ deȱ diferentesȱ procesosȱ deȱ
adsorciónȱoȱreaccionesȱquímicasȱ(Salomonsȱetȱal.,ȱ1987).ȱȱ
Además,ȱ seȱ haȱ demostradoȱ queȱ losȱ contaminantesȱ orgánicosȱ menosȱ
polaresȱ yȱ losȱ elementosȱ trazaȱ másȱ tóxicosȱ reaccionanȱ fuertementeȱ conȱ elȱ
materialȱ particuladoȱ yȱ seȱ acumulanȱ enȱ elȱ sedimentoȱ enȱ órdenesȱ deȱ magnitudȱ
másȱ altosȱ queȱ enȱ soluciónȱ (DelVallsȱ etȱ al.,ȱ 2002).ȱ Losȱ sedimentosȱ actúan,ȱ porȱ
tanto,ȱ comoȱ “trampa”ȱ deȱ contaminantes,ȱ deȱ formaȱ queȱ éstosȱ reflejanȱ deȱ unaȱ
formaȱ muyȱ eficazȱ elȱ gradoȱ deȱ contaminaciónȱ deȱ unȱ áreaȱ determinada.ȱ Esteȱ
hechoȱ haȱ llevadoȱ aȱ numerososȱ autoresȱ aȱ considerar,ȱ desdeȱ unȱ puntoȱ deȱ vistaȱ
ecológico,ȱ aȱ losȱ sedimentosȱ comoȱ unȱ elementoȱ deȱ unaȱ importanciaȱ
trascendentalȱdelȱhábitatȱacuáticoȱ(Chapman,ȱ1989;ȱLuoma,ȱ1983;ȱ1989;ȱLuomaȱyȱ
Ho,ȱ1992).ȱȱȱȱ
Losȱestudiosȱdeȱcontaminaciónȱenȱsedimentosȱseȱbasanȱgeneralmenteȱenȱ
laȱ comparaciónȱ deȱ losȱ nivelesȱ deȱ contaminantesȱ medidosȱ enȱ unaȱ zonaȱ
determinadaȱ conȱ aquellosȱ nivelesȱ deȱ contaminantesȱ medidosȱ enȱ unaȱ zonaȱ deȱ
referenciaȱ queȱ seȱ consideraȱ noȱ contaminada.ȱ Enȱ muchosȱ casosȱ losȱ estudiosȱ deȱ
esteȱ tipoȱ permitenȱ identificarȱ elȱ origenȱ deȱ laȱ contaminaciónȱ (Luomaȱ yȱ Philips,ȱ
1988;ȱ Philipsȱ etȱ al.,ȱ 1992;ȱ French,ȱ 1993)ȱ eȱ inclusoȱ estimarȱ suȱ biodisponibilidadȱ
Ȭ 11ȱȬ
Capítuloȱ1
(Harveyȱ yȱ Luoma,ȱ 1985;ȱ Luomaȱ yȱ Bryan,ȱ 1978;ȱ Arjonillaȱ etȱ al.,ȱ 1994).ȱ Estaȱ
metodología,ȱ sinȱ embargo,ȱ noȱ permiteȱ obtenerȱ informaciónȱ sobreȱ elȱ fenómenoȱ
deȱpolución.ȱ
Unaȱgranȱvariedadȱdeȱorganismosȱquedanȱexpuestosȱaȱlosȱcontaminantesȱ
presentesȱ enȱ losȱ sedimentos.ȱ Losȱ animalesȱ queȱ ingierenȱ sedimentoȱ oȱ detritusȱ
particuladoȱcomoȱalimento,ȱseȱencuentranȱdirectamenteȱafectados,ȱdeȱformaȱqueȱ
estosȱcontaminantesȱentranȱaȱformarȱparteȱdeȱlaȱcadenaȱtrófica,ȱporȱloȱqueȱotrosȱ
organismosȱ puedenȱ verseȱ tambiénȱ afectadosȱ yȱ seȱ puedenȱ darȱ fenómenosȱ deȱ
biomagnificaciónȱdelȱcontaminanteȱenȱlosȱsucesivosȱnivelesȱtróficos.ȱ
Laȱcontaminaciónȱdeȱlosȱsedimentosȱnoȱsóloȱafectaȱaȱlosȱorganismosȱdeȱlaȱ
zonaȱcontaminadaȱsinoȱqueȱademásȱpuedeȱextenderseȱaȱecosistemasȱalejadosȱdeȱ
lasȱfuentesȱmedianteȱlaȱresuspensiónȱyȱtransporteȱdeȱpartículasȱ(Luoma,ȱ1990).ȱ
Laȱdefiniciónȱdeȱtoxicidadȱdelȱsedimento,ȱenȱsuȱsentidoȱmásȱamplio,ȱestáȱ
referidaȱ aȱ losȱ cambiosȱ ecológicosȱ yȱ biológicosȱ causadosȱ porȱ losȱ contaminantesȱ
queȱ seȱ encuentranȱ incorporadosȱ enȱ losȱ sedimentos.ȱ Enȱ términosȱ toxicológicos,ȱ
quedaȱ definidaȱ comoȱ laȱ respuestaȱ adversaȱ queȱ seȱ observaȱ enȱ unoȱ oȱ enȱ variosȱ
organismosȱ sometidosȱ aȱ unaȱ pruebaȱ dondeȱ seȱ lesȱ exponeȱ aȱ sedimentosȱ
contaminados.ȱEstaȱrespuestaȱadversaȱseȱevalúaȱdeȱmaneraȱobjetivaȱcuandoȱlosȱ
animalesȱ seȱ exponenȱ aȱ sedimentosȱ limpiosȱ aȱ losȱ queȱ lesȱ hanȱ sidoȱ añadidasȱ
concentracionesȱ conocidasȱ deȱ contaminanteȱ (“Spikedȱ Sediment”),ȱ oȱ bienȱ
cuandoȱ sonȱ expuestosȱ aȱ sedimentosȱ contaminadosȱ recogidosȱ enȱ elȱ medio.ȱ
(ChapmanȱyȱMorgan,ȱ1983;ȱOakdenȱetȱal.,ȱ1984;ȱKempȱyȱSwartz,ȱ1988;ȱSwartzȱetȱ
al.,ȱ1989,ȱSwartzȱetȱal.,ȱ1990;ȱMeador,ȱ1990;ȱMcGeeȱetȱal.,ȱ1993;ȱDelValls,ȱ1994).ȱ
ȱ
Laȱ dificultadȱ delȱ estudioȱ deȱ laȱ toxicidadȱ deȱ losȱ sedimentosȱ marinosȱ seȱ
basaȱenȱqueȱlosȱcontaminantesȱpresentesȱenȱellosȱformanȱunaȱmezclaȱcomplejaȱ
- 12 -
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱorganizaciónȱdeȱlaȱtesis
deȱ sustanciasȱ queȱ puedenȱ darȱ lugarȱ aȱ efectosȱ biológicosȱ sinérgicosȱ oȱ
antagónicos.ȱ Quizásȱ porȱ ello,ȱ enȱ laȱ actualidad,ȱ aúnȱ existeȱ ciertoȱ
desconocimientoȱsobreȱlaȱtoxicidadȱdeȱmuchasȱsustanciasȱvertidasȱalȱmedio.ȱDeȱ
hecho,ȱ seȱ consideraȱ queȱ sóloȱ delȱ 5ȱ alȱ 10%ȱ deȱ ellas,ȱ hanȱ sidoȱ probadasȱ enȱ
laboratorioȱparaȱevaluarȱsuȱtoxicidad.ȱDeȱeseȱnúmero,ȱmenosȱdelȱ1%ȱloȱhanȱsidoȱ
utilizandoȱorganismosȱmarinosȱ(Martellȱetȱal.,ȱ1988).ȱȱ
Algunosȱ gobiernosȱ hanȱ diseñadoȱ programasȱ paraȱ evaluarȱ laȱ calidadȱ
ambientalȱdeȱlosȱsedimentos,ȱyȱhanȱrealizadoȱunasȱguíasȱnuméricasȱdeȱcalidadȱ
deȱ losȱ sedimentosȱ (Sedimentȱ Qualityȱ Guidelinesȱ ȬSQGsȬ)ȱ paraȱ cadaȱ
contaminanteȱ (Longȱ andȱ Buchman,ȱ 1989;ȱ NOAA,ȱ 1999;ȱ Environmentȱ Canada,ȱ
2003)ȱbasándoseȱenȱestudiosȱpreviosȱdeȱtoxicidad.ȱEstasȱguíasȱseȱdefinenȱcomoȱ
lasȱ concentracionesȱ deȱ contaminantesȱ presentesȱ enȱ sedimentosȱ queȱ estánȱ
asociadosȱ oȱ noȱ aȱ efectosȱ biológicosȱ enȱ laboratorioȱ oȱ campoȱ (DelVallsȱ yȱ
Chapman,ȱ 1998).ȱ Lasȱ guíasȱ permitenȱ compararȱ elȱ gradoȱ deȱ poluciónȱ entreȱ
distintasȱ zonas,ȱ deȱ formaȱ queȱ puedeȱ resultarȱ unaȱ herramientaȱ útilȱ enȱ estudiosȱ
deȱcalidadȱambiental.ȱ
2.ȱZonasȱdeȱestudioȱ
Paraȱllevarȱaȱcaboȱelȱpresenteȱtrabajoȱseȱescogieronȱdosȱzonasȱdelȱlitoralȱ
españolȱafectadasȱporȱvertidosȱdeȱpetróleo.ȱAsimismoȱseȱseleccionóȱunȱáreaȱdeȱ
referenciaȱparaȱelȱestudio.ȱ
LaȱcostaȱdeȱGalicia:ȱelȱvertidoȱdelȱPrestigeȱ
Aȱfinalesȱdelȱ2002ȱelȱaccidenteȱdelȱpetroleroȱmonocascoȱPrestigeȱprovocóȱ
unȱ vertidoȱ deȱ 63000ȱ toneladasȱ deȱ fuelȱ oilȱ pesadoȱ queȱ seȱ esparcióȱ enȱ manchas,ȱ
másȱoȱmenosȱcompactasȱyȱqueȱsupusoȱunaȱdeȱlasȱ‘mareasȱnegras’ȱmásȱdañinasȱ
Ȭ 13ȱȬ
Capítuloȱ1
deȱ lasȱ ocurridasȱ enȱ Galiciaȱ enȱ losȱ últimosȱ añosȱ juntoȱ aȱ lasȱ causadasȱ porȱ elȱ
Urquiolaȱ (1976)ȱ yȱ elȱ Aegeanȱ Seaȱ (1992).ȱ Además,ȱ seȱ trataȱ deȱ laȱ terceraȱ mareaȱ
negraȱ deȱ fuelȱ pesadoȱ enȱ aguasȱ europeasȱ enȱ menosȱ deȱ 4ȱ años,ȱ despuésȱ deȱ lasȱ
provocadasȱ porȱ losȱ petrolerosȱ Erikaȱ (1999)ȱ yȱ Balticȱ Carrierȱ (2001).ȱ Laȱ costaȱ
gallegaȱfueȱlaȱzonaȱmásȱafectadaȱporȱelȱvertidoȱyȱlosȱefectosȱdeȱlaȱcatástrofeȱseȱ
dejaronȱsentirȱenȱelȱmedioȱambienteȱyȱenȱlaȱeconomíaȱyȱsupusoȱunȱgranȱimpactoȱ
ecológicoȱyȱsocial.ȱ
Laȱ costaȱ deȱ Galiciaȱ haȱ vividoȱ muchosȱ naufragiosȱ enȱ susȱ aguasȱ yȱ esȱ
consideradaȱunaȱzonaȱdeȱaltoȱriesgoȱparaȱlosȱnavíosȱ(Bulot,ȱ2003).ȱAȱloȱlargoȱdeȱ
laȱhistoria,ȱGaliciaȱhaȱsufridoȱenȱvariasȱocasionesȱlaȱllegadaȱdeȱhidrocarburosȱaȱ
susȱcostas.ȱUnaȱdeȱlasȱprimerasȱalertasȱdeȱcontaminaciónȱporȱhidrocarburosȱseȱ
produjoȱelȱ27ȱdeȱfebreroȱdeȱ1961,ȱcuandoȱelȱpetroleroȱcanadienseȱAndrosȱFortune,ȱ
colisionóȱ conȱ unȱ carguero.ȱ Sinȱ embargo,ȱ enȱ estaȱ ocasión,ȱ elȱ vertidoȱ fueȱ muyȱ
reducido,ȱ yaȱ queȱ sóloȱ seȱ vertióȱ alȱ marȱ elȱ combustibleȱ previstoȱ paraȱ elȱ
funcionamientoȱ deȱ lasȱ máquinasȱ (Bulot,ȱ 2003).ȱ Elȱ 6ȱ deȱ mayoȱ deȱ 1970,ȱ elȱ
petroleroȱ noruegoȱ Polycommander,ȱ tocóȱ fondoȱ alȱ salirȱ delȱ puertoȱ deȱ Vigoȱ
vertiendoȱ unasȱ 15000ȱ toneladasȱdeȱcrudoȱligeroȱenȱlaȱbahíaȱdeȱVigo.ȱSeisȱañosȱ
después,ȱelȱpetroleroȱespañolȱUrquiola,ȱcolisionóȱconȱunaȱagujaȱsumergidaȱenȱelȱ
accesoȱ alȱ puertoȱ deȱ Aȱ Coruña.ȱ Seȱ vertieronȱ 110000ȱ toneladasȱ deȱ crudoȱ (Bulot,ȱ
2003).ȱȱElȱ31ȱdeȱdiciembreȱdeȱ1978,ȱelȱpetroleroȱgriegoȱAndrosȱpatria,ȱsufrióȱunaȱ
importanteȱbrechaȱliberandoȱunasȱ60000ȱtoneladasȱdeȱpetróleoȱqueȱmancharonȱ
unosȱ50ȱkmȱdeȱlaȱcostaȱdeȱLugoȱ(Bulot,ȱ2003).ȱFueȱcasiȱcatorceȱañosȱdespués,ȱelȱ3ȱ
deȱdiciembreȱdeȱ1992,ȱcuandoȱelȱAegeanȱSeaȱprovocóȱunaȱnuevaȱmareaȱnegraȱenȱ
lasȱcostasȱdeȱGalicia,ȱvertiendoȱ66800ȱtoneladasȱdeȱcrudoȱligeroȱjuntoȱaȱlaȱTorreȱ
deȱ Hérculesȱ enȱ Aȱ Coruña.ȱ Esteȱ episodioȱ haȱ servidoȱ deȱ baseȱ paraȱ realizarȱ
estudiosȱposterioresȱenȱelȱcasoȱdelȱPrestigeȱ(CSIC,ȱ2003a).ȱȱ
Sinȱ embargo,ȱ elȱ comportamientoȱ delȱ fuelȱ enȱ elȱ medioȱ marinoȱ depende,ȱ
enȱgranȱparte,ȱdeȱsuȱcomposición.ȱSabiendoȱqueȱlaȱcargaȱdelȱPrestigeȱconsisteȱenȱ
- 14 -
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱorganizaciónȱdeȱlaȱtesis
unȱ fuelȱ pesado,ȱ cabeȱ analizarȱ losȱ accidentesȱ deȱ petrolerosȱ queȱ hanȱ originadoȱ
esteȱ tipoȱ deȱ vertido,ȱ deȱ formaȱ queȱ nosȱ podamosȱ acercarȱ aȱ losȱ posiblesȱ efectosȱ
ocasionadosȱsobreȱelȱmedioȱmarino.ȱAsí,ȱporȱejemplo,ȱlaȱexperienciaȱadquiridaȱ
conȱelȱvertidoȱdeȱ20000ȱtoneladasȱdeȱfuelȱpesadoȱdelȱErikaȱ(12ȱdeȱdiciembreȱdeȱ
1999)ȱfrenteȱaȱlasȱcostasȱfrancesas,ȱhaȱpermitidoȱavanzarȱenȱlasȱactuacionesȱyȱlosȱ
estudiosȱ delȱ Prestigeȱ (Hoefer,ȱ T.,ȱ 2003;ȱ LeȬCedre,ȱ http://www.leȬcedre.fr/;ȱ
Ifremer,ȱhttp://www.ifremer.fr).ȱȱȱȱȱȱȱ
Losȱejemplosȱdeȱvertidosȱaccidentalesȱdeȱunȱproductoȱpetrolíferoȱpesadoȱ
enȱelȱmar,ȱdeȱunȱpetróleoȱcrudoȱoȱdeȱunȱproductoȱrefinadoȱcuyaȱdensidadȱvaríaȱ
entreȱ0.95ȱyȱ1.00ȱgȱmȬ3,ȱsonȱnumerososȱ(LeȬCedre,ȱhttp://www.leȬcedre.fr).ȱEnȱlaȱ
tablaȱ 2.3ȱ seȱ resumenȱ algunosȱ deȱ estosȱ accidentes.ȱ Esteȱ tipoȱ deȱ vertidoȱ (fuelȱ
pesado)ȱ produceȱ extensasȱ manchas,ȱ galletasȱ yȱ bolasȱ deȱ hidrocarburos,ȱ
originadasȱ porȱ laȱ fragmentaciónȱ delȱ productoȱ duranteȱ suȱ derivaȱ marina.ȱ Casiȱ
siempre,ȱ elȱ impactoȱ sobreȱ lasȱ avesȱ yȱ losȱ mamíferosȱ marinosȱ impregnadosȱ deȱ
petróleoȱ esȱ muyȱ elevado.ȱ Elȱ vertidoȱ deȱ esteȱ fuelȱ pesado,ȱ alȱ presentarȱ unosȱ
componentesȱevaporablesȱyȱbiodegradablesȱminoritariosȱ(menosȱdelȱ10ȱ%ȱdeȱsuȱ
masa),ȱsueleȱ exigirȱ unosȱ ampliosȱ trabajosȱdeȱlimpieza,ȱqueȱseȱvuelvenȱdifícilesȱ
debidoȱaȱlaȱextremadaȱviscosidadȱdelȱproducto.ȱEnȱcompensación,ȱésteȱdifundeȱ
enȱelȱaguaȱescasosȱcomponentesȱtóxicosȱy,ȱunaȱvezȱqueȱlaȱlimpiezaȱseȱhaȱllevadoȱ
aȱ cabo,ȱ losȱ efectosȱ aȱ largoȱ plazoȱ sonȱ mínimosȱ (LeȬCedre,ȱ http://www.leȬ
cedre.fr).ȱȱ
Porȱ otraȱ parte,ȱ losȱ fenómenosȱ deȱ sedimentaciónȱ delȱ fuelȱ pesadoȱ vanȱ aȱ
suponerȱ unaȱ entradaȱ deȱ contaminantesȱ aȱ losȱ sedimentos,ȱ queȱ másȱ adelanteȱ
puedenȱ volverȱ aȱ laȱ columnaȱ deȱ agua.ȱ Esteȱ fenómenoȱ seȱ haȱ comprobadoȱ enȱ
estudiosȱ previos,ȱ dondeȱ seȱ detectaronȱ nuevosȱ picosȱ deȱ contaminaciónȱ mesesȱ
despuésȱ delȱ vertido,ȱ enȱ elȱ casoȱ delȱ Erikaȱ (Burgeot,ȱ 2001)ȱ debidoȱ aȱ efectosȱ
Ȭ 15ȱȬ
Capítuloȱ1
mareales,ȱyȱaȱlaȱremociónȱdeȱfondosȱporȱlaȱ extracciónȱdeȱarenas,ȱenȱelȱcasoȱdeȱ
AegeanȱSeaȱ(MMA,ȱ1993).ȱȱ
Tablaȱ 1.2.ȱ Accidentesȱ deȱ petrolerosȱ queȱ hanȱ vertidoȱ alȱ marȱ fuelȱ pesadoȱ
enȱlosȱúltimosȱañosȱ(LeȬCedre,ȱhttp://www.leȬcedre.fr).ȱ
Añoȱ
Petroleroȱ
2001ȱ BalticȱCarrierȱ
Cantidadȱvertidaȱ
(toneladas)ȱ
Lugarȱ
Dinamarcaȱ
2700ȱ
Franciaȱ
20000ȱ
Japónȱ
6.20ȱ
1997ȱ Katjaȱ
Franciaȱ
0.19ȱ
1988ȱ Nestuccaȱ
EEUUȱ
11ȱ
1984ȱ Mobiloilȱ
EEUUȱ
0.64ȱ
1980ȱ Tanioȱ
Franciaȱ
6500ȱ
1976ȱ Bohlenȱ
Franciaȱ
6500ȱ
1976ȱ ArgoȱMerchantȱ
EEUUȱ
5.70ȱ
1972ȱ Tamanoȱ
EEUUȱ
0.40ȱ
Canadáȱ
12ȱ
GranȱBretañaȱ
0.64ȱ
1999ȱ Erikaȱ
1997ȱ Nakhodkaȱ
1970ȱ Arrowȱ
1969ȱ HamiltonȱTraderȱ
ȱ
Losȱ análisisȱ químicos,ȱ revelaronȱ unaȱ composiciónȱ delȱ fuelȱ delȱ Prestigeȱ
cercanaȱaȱlaȱdelȱErika.ȱLosȱnivelesȱdeȱconcentracionesȱenȱPAHsȱenȱlaȱmuestraȱdeȱ
fuelȱ delȱ Prestigeȱ sonȱ próximosȱ aȱ aquellosȱ delȱ fuelȱ delȱ Erikaȱ recogidasȱ enȱ lasȱ
playas.ȱSinȱembargo,ȱpareceȱserȱqueȱlaȱmuestraȱdeȱreferenciaȱErikaȱdeȱlaȱrefineriaȱ
deȱDunkerqueȱesȱmásȱricaȱenȱPAHs.ȱLaȱequivalenciaȱtóxicaȱenȱBenzo(a)pyreneȱ
pareceȱ indicarȱ potencialesȱ tóxicosȱ similaresȱ entreȱ losȱ dosȱ fuelsȱ (Ifremer,ȱ
http://www.ifremer.fr/envlit/prestige/indexsp.htm).ȱ
- 16 -
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱorganizaciónȱdeȱlaȱtesis
TranscurridosȱtresȱañosȱdelȱnaufragioȱdelȱErika,ȱlosȱanálisisȱquímicosȱenȱ
losȱ mariscos,ȱ mostraronȱ queȱ aúnȱ seȱ encuentranȱ huellasȱ delȱ Erikaȱ enȱ ciertosȱ
lugares.ȱ Estoȱ muestraȱ laȱ persistenciaȱ deȱ ciertosȱ componentesȱ deȱ eseȱ fuelȱ enȱ elȱ
aguaȱ yȱ enȱ losȱ sedimentosȱ (Ifremerȱ http://www.ifremer.fr/)ȱ loȱ queȱ poneȱ deȱ
manifiestoȱlaȱimportanciaȱdelȱestudioȱdeȱlaȱpoluciónȱaȱloȱlargoȱdelȱtiempo.ȱ
ȱȱ
ȱ
Bahíaȱdeȱ
ȱ
CormeȬLaxeȱ
Galiciaȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ParqueȱNacionalȱ
deȱlasȱislasȱ
Atlánticasȱ
ȱ
ȱ
Figuraȱ 1.1.ȱ Áreasȱ deȱ estudioȱ enȱ lasȱ costasȱ gallegasȱ afectadasȱ porȱ elȱ
vertidoȱdelȱpetroleroȱPrestigeȱ(imágenesȱtomadasȱdeȱGoogleTMȱEarth).ȱ
Trasȱ elȱ accidenteȱ delȱ petroleroȱ Prestige,ȱ losȱ archipiélagosȱ deȱ Cíes,ȱ Onsȱ yȱ
Salvora,ȱdeclaradosȱenȱjulioȱdelȱ2002ȱ(Leyȱ15/2002)ȱParqueȱNacionalȱdeȱlasȱIslasȱ
Atlánticas,ȱ fueronȱ laȱ barreraȱ naturalȱ queȱ frenóȱ laȱ entradaȱ delȱ fuelȱ enȱ lasȱ Ríasȱ
BajasȱGallegas.ȱElȱdirectorȱdeȱParquesȱNaturalesȱreconocióȱqueȱenȱunaȱprimeraȱ
Ȭ 17ȱȬ
Capítuloȱ1
oleadaȱ deȱ mareaȱ negra,ȱ elȱ 85%ȱ delȱ Parqueȱ Nacionalȱ deȱ lasȱ Islasȱ Atlánticasȱ
resultóȱ afectado,ȱ elevándoseȱ esteȱ porcentajeȱ aȱ másȱ delȱ 90%ȱ trasȱ oleadasȱ
posterioresȱdeȱfuelȱrecibidasȱporȱelȱParque.ȱȱ
ElȱvertidoȱdelȱpetroleroȱPrestigeȱtambiénȱafectóȱdeȱmaneraȱimportanteȱaȱlaȱ
Bahíaȱ deȱ CormeȬLaxeȱ laȱ cualȱ eraȱ consideradaȱ unaȱ zonaȱ conȱ bajaȱ influenciaȱ
antropogénicaȱ yȱ unaȱ moderadaȱ actividadȱ industrial,ȱ destacandoȱ sobretodoȱ lasȱ
tareasȱagropecuariasȱyȱpesquerasȱdeȱlaȱzona.ȱElȱpolígonoȱdeȱviverosȱdeȱCormeȱ
tieneȱ bateasȱ experimentalesȱ dedicadasȱ aȱ laȱ producciónȱ deȱ mejillón,ȱ vieira,ȱ
zamburiñaȱyȱostraȱplana.ȱCormeȱLaxeȱpresentaȱunaȱinfluenciaȱoceánicaȱgrandeȱ
yȱelȱmayorȱriesgoȱestáȱenȱelȱtráficoȱmarítimoȱexteriorȱqueȱtransportaȱmercancíasȱ
peligrosasȱ yȱ estáȱ expuestoȱ aȱ sufrirȱ unȱ accidenteȱ marítimoȱ (Diarioȱ oficialȱ deȱ
Galicia,ȱ2004).ȱLosȱtiposȱdeȱhidrocarburosȱsusceptiblesȱdeȱserȱvertidosȱenȱlaȱzonaȱ
sonȱprincipalmenteȱelȱgasoilȱdeȱconsumoȱdeȱlosȱbarcos,ȱgasolinas,ȱfuelȬoilȱyȱgasȬ
oilȱ deȱ losȱ transportesȱ terrestresȱ yȱ estacionesȱ deȱ suministroȱ deȱ combustible,ȱ
residuosȱ oleososȱ procedentesȱ deȱ reparacionesȱ enȱ puertoȱ yȱ talleresȱ deȱ
automoción.ȱPorȱotraȱparte,ȱelȱríoȱAnllóns,ȱqueȱdesembocaȱenȱlaȱría,ȱhaȱsufridoȱ
episodiosȱdeȱcontaminaciónȱporȱvertidosȱdeȱpurinasȱprocedentesȱdeȱgranjas.ȱ
ElȱGolfoȱdeȱCádiz:ȱLaȱBahíaȱdeȱAlgecirasȱyȱlaȱBahíaȱdeȱCádizȱ
Aȱ partirȱ deȱ losȱ añosȱ 60,ȱ laȱ franjaȱ costeraȱ deȱ laȱ Bahíaȱ deȱ Algecirasȱ seȱ haȱ
vistoȱpaulatinamenteȱ sometidaȱaȱdrásticasȱtransformacionesȱprovocadasȱporȱelȱ
crecienteȱ desarrolloȱ socioeconómicoȱ deȱ laȱ zona.ȱ Así,ȱ enȱ laȱ actualidad,ȱ enȱ susȱ
márgenesȱ seȱ asientaȱ unȱ importanteȱ poloȱ industrialȱ integradoȱ porȱ plantasȱ
petroquímicas,ȱ centralesȱ térmicas,ȱ industriasȱ siderometalúrgicas,ȱ papeleras,ȱ
astilleros,ȱ ademásȱ deȱ unaȱ intensaȱ actividadȱ portuaria,ȱ unaȱ crecienteȱ presiónȱ
demográficaȱ yȱ elȱ elevadoȱ nivelȱ deȱ transformaciónȱ deȱ laȱ costaȱ porȱ distintasȱ
construccionesȱ(Conradiȱetȱal.,ȱ1995).ȱȱ
- 18 -
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱorganizaciónȱdeȱlaȱtesis
Existenȱpequeñosȱcaucesȱfluvialesȱqueȱdesembocanȱalȱinteriorȱdeȱlaȱbahíaȱ
deȱ Algeciras,ȱ siendoȱ losȱ ríosȱ Palmonesȱ yȱ Gaudarranqueȱ losȱ másȱ importantesȱ
respectoȱalȱcaudalȱ(Conradiȱetȱal.,ȱ1995).ȱLaȱhidrologíaȱgeneralȱdeȱlaȱbahíaȱseȱveȱ
afectadaȱ porȱ laȱ circulaciónȱ generalȱ deȱ lasȱ masasȱ deȱ aguaȱ atlánticasȱ yȱ
mediterráneasȱ aȱ travésȱ delȱ Estrechoȱ deȱ Gibraltar,ȱ aunqueȱ lasȱ corrientesȱ queȱ
afectanȱlaȱbahíaȱestánȱcausadasȱprincipalmenteȱporȱcambiosȱdeȱdirecciónȱenȱlaȱ
corrienteȱ deȱ marea,ȱ vientos,ȱ presiónȱ atmosféricaȱ yȱ laȱ corrienteȱ mediterráneaȱ
superficialȱ (Conradiȱ etȱ al.,ȱ 1995).ȱ Estaȱ bahíaȱ presentaȱ unaȱ altaȱ tasaȱ deȱ
renovaciónȱ deȱ susȱ aguas,ȱ debidoȱ aȱ suȱ proximidadȱ alȱ Estrecho,ȱ yȱ aȱ lasȱ fuertesȱ
corrientesȱ deȱ aguas,ȱ loȱ queȱ daȱ lugarȱ aȱ queȱ losȱ efectosȱ deȱ losȱ vertidosȱ
contaminantesȱ seȱ veanȱ notablementeȱ reducidos,ȱ alȱ dispersarseȱ enȱ unaȱ granȱ
masaȱdeȱaguaȱ(ConsejeríaȱdeȱMedioȱAmbiente,ȱ2007).ȱȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
Bahíaȱdeȱ
Cádizȱ
ȱ
Golfoȱdeȱ
Cádizȱ
ȱ
ȱ
Bahíaȱdeȱ
Algeciras
Figuraȱ 1.2.ȱ Áreasȱ deȱ estudioȱ seleccionadasȱ enȱ elȱ golfoȱ deȱ Cádizȱ
(imágenesȱtomadasȱdeȱGoogleTMȱEarth).ȱȱȱ
Ȭ 19ȱȬ
Capítuloȱ1
Lasȱ fuentesȱ potencialesȱ deȱ contaminaciónȱ enȱ elȱ entornoȱ deȱ laȱ Bahíaȱ deȱ
Algecirasȱsonȱmuyȱdiversas,ȱaunqueȱseȱvinculanȱaȱlasȱindustriasȱasentadasȱenȱlaȱ
zona.ȱ Asimismoȱ hayȱ queȱ considerarȱ laȱ contaminaciónȱ difusaȱ yȱ losȱ lixiviadosȱ
procedentesȱdeȱlaȱintensaȱactividadȱurbanaȱeȱindustrialȱqueȱseȱdesarrollaȱenȱelȱ
área,ȱ asíȱ comoȱ unaȱ intensaȱ actividadȱ portuariaȱ yȱ tráficoȱ marítimo.ȱ Todoȱ elloȱ
haceȱ queȱ laȱ Bahíaȱ deȱ Algecirasȱ sufraȱ deȱ formaȱ rutinariaȱ vertidosȱ deȱ petróleo,ȱ
aportesȱ deȱ gasesȱ yȱ materialȱ particuladoȱ aȱ laȱ atmósfera,ȱ metalesȱ pesadosȱ yȱ
partículasȱ enȱ suspensiónȱ enȱ lasȱ aguas,ȱ queȱ posteriormenteȱ seȱ transfierenȱ aȱ losȱ
sedimentos,ȱyȱenȱmuchosȱcasosȱalcanzanȱlosȱsuelosȱdeȱlaȱzonaȱ(CSIC,ȱ2003b).ȱLaȱ
mitadȱ deȱ estosȱ vertidosȱ sonȱ urbanosȱ eȱ industriales,ȱ unȱ 19ȱ %ȱ provienenȱ deȱ lasȱ
actividadesȱ deȱ mantenimientoȱ deȱ losȱ buquesȱ comoȱ elȱ “bunkering”ȱ realizadosȱ
tantoȱporȱparteȱdelȱpuertoȱdeȱAlgecirasȱcomoȱGibraltar,ȱyȱunȱ5ȱ%ȱdeȱlaȱentradaȱ
deȱcontaminantesȱȱcorrespondeȱaȱvertidosȱaccidentales.ȱȱ
Losȱ aportesȱ urbanos,ȱ correspondenȱ fundamentalmenteȱ aȱ lasȱ aguasȱ
residualesȱ deȱ laȱ poblaciónȱ asentadaȱ enȱ elȱ Campoȱ deȱ Gibraltar.ȱ Entreȱ estosȱ
núcleosȱdeȱpoblaciónȱcabeȱdestacar:ȱAlgeciras,ȱLaȱLínea,ȱSanȱRoqueȱyȱlosȱBarriosȱ
(CSIC,ȱ 2003b).ȱ Porȱ otroȱ lado,ȱ losȱ vertidosȱ industrialesȱ deȱ laȱ zonaȱ estánȱ
constituidosȱporȱemisionesȱdeȱgasesȱyȱmaterialȱparticuladoȱyȱefluentesȱlíquidosȱ
producidosȱ porȱ lasȱ industriasȱ situadasȱ enȱ elȱ Campoȱ deȱ Gibraltar,ȱ dedicadasȱ
fundamentalmenteȱaȱlaȱproducciónȱquímica:ȱrefinoȱdeȱpetróleoȱyȱpetroquímica,ȱ
asíȱ comoȱ aquellosȱ asociadosȱ aȱ lasȱ actividadesȱ delȱ tráficoȱ portuarioȱ (CSIC,ȱ
2003b).ȱ Lasȱ principalesȱ empresasȱ seȱ agrupanȱ enȱ tornoȱ aȱ losȱ siguientesȱ núcleosȱ
deȱpoblación:ȱ
ȬȱAlgeciras:ȱIndustriasȱpapeleras:ȱTorraspapel,ȱC.L.HȱyȱCentralȱTérmica.ȱ
Ȭȱ Sanȱ Roque:ȱ Polígonoȱ petroquímico:ȱ Cepsa,ȱ Interquisa,ȱ Eastmanȱ
Chemical,ȱPetresa.ȱ
ȬȱLosȱBarrios:ȱCentralȱTérmica,ȱAcerinox,ȱEndesaȱPuertos.ȱ
- 20 -
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱorganizaciónȱdeȱlaȱtesis
Organizacionesȱ noȱ gubernamentales,ȱ aseguranȱ queȱ alrededorȱ deȱ 4000ȱ yȱ
5000ȱ deȱ losȱ 100000ȱ barcosȱ queȱ cruzanȱ elȱ estrechoȱ cadaȱ año,ȱ sonȱ petroleros,ȱ yȱ
muchosȱ entranȱ enȱ laȱ bahíaȱ deȱ Algecirasȱ aȱ realizarȱ operacionesȱ cargaȱ deȱ
combustibleȱ ȱ (“bunkering”).ȱ Enȱ elȱ casoȱ deȱ Gibraltar,ȱ elȱ bunkeringȱ seȱ realizaȱ
desdeȱunȱbuqueȱ–ȱelȱȇVemabalticȇ,ȱcapacidad:ȱ107544ȱToneladasȱȬȱfondeadoȱenȱlaȱ
mismaȱ bahía,ȱ yȱ seȱ trataȱ delȱ únicoȱ lugarȱ deȱ laȱ Uniónȱ Europeaȱ dondeȱ seȱ sigueȱ
realizandoȱestaȱoperaciónȱdesdeȱunȱbuqueȱfondeadoȱ(LópezȱdeȱUralde,ȱ2007).ȱ
ȱ
7%
ȱ
2%
1%
7%
2%
ȱ
ȱ
57%
21%
ȱ
ȱ
ȱ
Aceite de oliva y sus fracciones
Alcoholes acíclicos y derivados
Fuel-Oil
Gas-Oil
Hidrocarburos acíclicos
Lubricantes
Abonos minerales
Alcohol etílico sin desnaturalizar
Flúor, cloro, bromo y yodo
Aceites y Productos Destilación
Crudos de petróleos
Gas de petróleo
Gasolina, Keroseno y Petróleo Refinado
Hidrocarburos cíclicos
Aceite de oliva y sus fracciones
Ácidos policarboxílicos
Eteres y derivados halogenados
Polímeros de etileno
ȱ
Figuraȱ 1.3.ȱ Mercancíasȱ peligrosasȱ cargadasȱ yȱ descargadasȱ enȱ elȱ Puertoȱ
BahíaȱdeȱAlgecirasȱ(MartínȬDíazȱyȱDelValls,ȱcomunicaciónȱpersonal)ȱȱ
Estaȱ evidenteȱ influenciaȱ antropogénicaȱ enȱ laȱ zonaȱ yȱ losȱ posiblesȱ efectosȱ
adversosȱ enȱ elȱ ecosistemaȱ deȱ laȱ bahíaȱ noȱ hanȱ transcendidoȱ tantoȱ comoȱ porȱ
ejemploȱ elȱ vertidoȱ delȱ petroleroȱ Prestigeȱ enȱ Galicia,ȱ aunqueȱ cadaȱ vezȱ hayȱ másȱ
interésȱ yȱ preocupaciónȱ porȱ losȱ posiblesȱ dañosȱ deȱ losȱ vertidosȱ enȱ laȱ Bahíaȱ deȱ
Algeciras.ȱ
Ȭ 21ȱȬ
Capítuloȱ1
OtraȱbahíaȱsituadaȱtambiénȱenȱelȱGolfoȱdeȱCádizȱfueȱescogidaȱdeȱmaneraȱ
complementariaȱ paraȱ llevarȱ aȱ caboȱ esteȱ estudio;ȱ unaȱ zonaȱ queȱ noȱ presentaȱ
fuentesȱ deȱ contaminaciónȱ importantes:ȱ laȱ bahíaȱ deȱ Cádiz.ȱ Laȱ Consejeríaȱ deȱ
MedioȱAmbienteȱ(2007)ȱhaȱdeclaradoȱqueȱestaȱbahíaȱpresentaȱunaȱbuenaȱcalidadȱ
ambiental,ȱyȱademás,ȱelȱáreaȱseleccionadaȱhaȱsidoȱampliamenteȱcaracterizadaȱyȱ
susȱsedimentosȱhanȱsidoȱanalizadosȱyȱevaluadosȱbajoȱelȱpuntoȱdeȱvistaȱquímicoȱ
yȱecotoxicológicoȱresultandoȱserȱaptosȱparaȱsuȱusoȱcomoȱestaciónȱdeȱreferenciaȱ
enȱesteȱtrabajo.ȱȱȱ
3.ȱObjetivosȱeȱhipótesisȱ
Laȱ hipótesisȱ deȱ partidaȱ consideraȱ queȱ unȱ ecosistemaȱ queȱ recibeȱ deȱ
maneraȱ continuaȱ moderadasȱ dosisȱ deȱ vertidosȱ deȱ hidrocarburosȱ duranteȱ unȱ
largoȱ periodoȱ deȱ tiempoȱ (impactoȱ crónico)ȱ resultaȱ másȱ dañadoȱ yȱ presentaȱ
mayorȱ poluciónȱ (contaminaciónȱ masȱ efectos)ȱ queȱ enȱ elȱ casoȱ deȱ unȱ ecosistemaȱȱ
queȱ recibeȱ enȱ unȱ cortoȱ periodoȱ deȱ tiempoȱ unȱ vertidoȱ deȱ grandesȱ dimensionesȱ
(impactoȱ agudo).ȱ Elȱ objetivoȱ generalȱ deȱ estaȱ tesisȱ doctoralȱ consisteȱ enȱ
determinarȱ yȱ compararȱ laȱ calidadȱ deȱ losȱ sedimentosȱ deȱ dosȱ zonasȱ delȱ litoralȱ
españolȱ afectadasȱ porȱ vertidosȱ deȱ petróleo,ȱ laȱ costaȱ gallegaȱ yȱ laȱ Bahíaȱ deȱ
Algecirasȱ frenteȱ aȱ unaȱ zonaȱ consideradaȱ noȱ afectadaȱ porȱ esteȱ tipoȱ deȱ
contaminación.ȱ Considerandoȱ laȱ metodologíaȱ queȱ seȱ haȱ seguido,ȱ seȱ pretendíaȱ
realizarȱ estaȱ aportaciónȱ aȱ travésȱ deȱ laȱ consecuciónȱ deȱ losȱ siguientesȱ objetivosȱ
concretos:ȱȱ
1.ȱ Determinarȱ elȱ gradoȱ deȱ contaminaciónȱ porȱ losȱ principalesȱ
contaminantesȱ (metalesȱ yȱ PAHs)ȱ queȱ seȱ encontrabanȱ enȱ losȱ vertidosȱ deȱ
hidrocarburosȱ enȱ losȱ sedimentosȱ delȱ Parqueȱ Nacionalȱ deȱ lasȱ Islasȱ
Atlánticasȱ yȱ Bahíaȱ deȱ CormeȬLaxeȱ (costaȱ deȱ Galicia)ȱ yȱ enȱ laȱ
- 22 -
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱorganizaciónȱdeȱlaȱtesis
desembocaduraȱ deȱ losȱ ríosȱ Guadarranqueȱ yȱ Palmonesȱ enȱ laȱ Bahíaȱ deȱ
AlgecirasȱasíȱcomoȱestablecerȱsusȱnivelesȱenȱlaȱBahíaȱdeȱCádiz.ȱȱ
2.ȱ Establecerȱ elȱ efectoȱ adversoȱ deȱ estosȱ contaminantesȱ unaȱ vezȱ queȱ seȱ
incorporanȱaȱlosȱsedimentosȱmedianteȱdiseñoȱyȱaplicaciónȱdeȱensayosȱdeȱ
laboratorioȱ bajoȱ condicionesȱ controladasȱ yȱ enȱ exposicionesȱ deȱ tipoȱ
aguda,ȱ letalesȱ yȱ bioacumulaciónȱ utilizandoȱ poblacionesȱ deȱ laȱ bacteriaȱ
Vibrioȱfischeri,ȱdeȱlosȱanfípodosȱCorophiumȱvolutatorȱyȱAmpeliscaȱbrevicornis,ȱ
yȱdelȱpoliquetoȱArenícolaȱmarinaȱ
3.ȱ Caracterizarȱ losȱ posiblesȱ efectosȱ adversosȱ deȱ losȱ contaminantesȱ bajoȱ
condicionesȱ deȱ laboratorioȱ yȱ campoȱ medianteȱ ensayosȱ deȱ laboratorioȱ
crónicoȱyȱmedidasȱdelȱefectoȱsubletal,ȱqueȱincorporanȱdeterminacionesȱdeȱ
biomarcadoresȱ deȱ exposiciónȱ (actividadȱ EROD,ȱ Metalotioneinas,ȱ
actividadȱ GST,ȱ GPX,ȱ GR,ȱ FRAP,ȱ vitelogenina,ȱ TBARS)ȱ yȱ deȱ efectoȱ
(Alteraciónȱ deȱ comportamiento,ȱ histopatologíaȱ yȱ dañoȱ deȱ ADN)ȱ
utilizandoȱ juvenilesȱ deȱ laȱ especieȱ comercialȱ delȱ pezȱ Sparusȱ aurataȱ
(dorada),ȱelȱcangrejoȱCarcinusȱmaenas,ȱlaȱalmejaȱRuditapesȱphilippinarumȱyȱ
elȱpoliquetoȱArenicolaȱmarina.ȱ
4.ȱ Determinaciónȱ deȱ lasȱ alteracionesȱ bentónicasȱ “inȱ situ”ȱ queȱ permitenȱ
evaluarȱ elȱ posibleȱ impactoȱ deȱ losȱ contaminantesȱ sobreȱ elȱ ecosistemaȱ
marino,ȱ aȱ travésȱ delȱ estudioȱ deȱ parámetrosȱ poblacionalesȱ (númeroȱ deȱ
especies,ȱ diversidad,ȱ riquezaȱ específica,ȱ dominanciaȱ yȱ presenciaȱ deȱ losȱ
taxonesȱprincipales).ȱ
5.ȱ Identificarȱ lasȱ sustanciasȱ contaminantesȱ queȱ producenȱ elȱ efectoȱ
adversoȱ medianteȱ laȱ integraciónȱ deȱ losȱ resultadosȱ deȱ contaminaciónȱ
(fisicoquímicos)ȱ yȱ deȱ susȱ efectosȱ (toxicidadȱ agudaȱ yȱ crónica)ȱ bajoȱ
condicionesȱ deȱ laboratorioȱ yȱ campo,ȱ asíȱ comoȱ laȱ alteraciónȱ bentónica,ȱ
Ȭ 23ȱȬ
Capítuloȱ1
determinandoȱ laȱ calidadȱ deȱ losȱ sedimentosȱ delȱ Parqueȱ Nacionalȱ deȱ lasȱ
IslasȱAtlánticas,ȱlaȱBahíaȱ deȱ CormeȬLaxe,ȱyȱlaȱBahíaȱdeȱ Algeciras,ȱzonasȱ
afectadasȱenȱmayorȱoȱmenorȱmedidaȱporȱvertidosȱdelȱpetróleo.ȱ
6.ȱEstablecerȱlosȱnivelesȱdeȱpoluciónȱenȱcadaȱunaȱdeȱlasȱzonasȱestudiadasȱ
determinandoȱlaȱseveridadȱdeȱlosȱimpactosȱproducidosȱporȱcadaȱtipoȱdeȱ
vertidoȱ estudiadosȱ (agudos,ȱ vertidoȱ Prestigeȱ enȱ zonaȱ Gallega;ȱ crónicos,ȱ
vertidosȱ continuosȱ Bahíaȱ deȱ Algeciras),ȱ yȱ siempreȱ porȱ comparaciónȱ
frenteȱaȱunaȱestaciónȱdeȱreferencia,ȱlocalizadaȱenȱlaȱBahíaȱdeȱCádiz.ȱ
4.ȱEstructuraȱdeȱlaȱtesisȱ
Estaȱtesisȱdoctoralȱseȱhaȱestructuradoȱenȱseisȱcapítulos:ȱelȱprimeroȱconstaȱ
deȱ introducción,ȱ descripciónȱ deȱ losȱ objetivosȱ deȱ laȱ tesis,ȱ asíȱ comoȱ laȱ
presentaciónȱ deȱ lasȱ áreasȱ deȱ estudio,ȱ mientrasȱ queȱ enȱ losȱ cuatroȱ capítulosȱ
siguientesȱseȱpresentaȱlaȱmemoriaȱenȱsí,ȱfinalizandoȱconȱunȱúltimoȱcapítuloȱenȱelȱ
queȱ seȱ muestranȱ lasȱ conclusionesȱ obtenidasȱ enȱ elȱ estudio.ȱ Cadaȱ unoȱ deȱ losȱ
cuatroȱcapítulosȱcentralesȱconstaȱdeȱunaȱintroducciónȱyȱdescripciónȱresumidaȱenȱ
españolȱyȱlosȱtrabajosȱdeȱinvestigaciónȱescritosȱenȱinglésȱpublicados,ȱaceptados,ȱ
oȱbienȱenviadosȱaȱdistintasȱrevistasȱinternacionales.ȱDeȱestaȱformaȱenȱelȱcapítuloȱ
2ȱseȱincluyenȱlosȱtrabajosȱI,ȱII,ȱIIIȱyȱIVȱqueȱdescribenȱlosȱresultadosȱdeȱtoxicidadȱ
agudaȱ obtenidosȱ deȱ laȱ realizaciónȱ deȱ losȱ bioensayosȱ conȱ dilucionesȱ deȱ fuelȱ
extraídoȱ delȱ petroleroȱ Prestigeȱ (trabajosȱ Iȱ yȱ II),ȱ asíȱ comoȱ experimentosȱ conȱ
muestrasȱambientalesȱdeȱsedimentoȱ(trabajosȱIIIȱyȱIV).ȱElȱcapítuloȱ3ȱpresentaȱlosȱ
resultadosȱ deȱ laȱ evaluaciónȱ deȱ losȱ efectosȱ subletalesȱ bajoȱ condicionesȱ deȱ
laboratorio.ȱ Aȱ loȱ largoȱ deȱ 5ȱ trabajosȱ seȱ describenȱ losȱ estudiosȱ deȱ toxicidadȱ
realizadosȱ conȱ 4ȱ especiesȱ marinasȱ yȱ enȱ losȱ queȱ seȱ hanȱ obtenidoȱ respuestasȱ
subletalesȱdeȱtoxicidadȱtrasȱexponerȱlosȱorganismosȱaȱlosȱsedimentosȱdeȱestudio.ȱ
Elȱ capítuloȱ 4ȱ incluyeȱ 4ȱ trabajosȱ enȱ losȱ queȱ seȱ llevaȱ aȱ caboȱ laȱ evaluaciónȱ deȱ losȱ
- 24 -
Introducción,ȱzonasȱdeȱestudio,ȱobjetivosȱyȱorganizaciónȱdeȱlaȱtesis
efectosȱ subletalesȱ deȱ losȱ contaminantesȱ bajoȱ condicionesȱ deȱ campo.ȱ Enȱ esteȱ
ámbito,ȱ losȱ trabajosȱ X,ȱ XIȱ yȱ XIIȱ muestranȱ losȱ resultadosȱ deȱ bioensayosȱ
realizadosȱ conȱ dosȱ especiesȱ marinasȱ medianteȱ laȱ instalaciónȱ enȱ jaulasȱ enȱ losȱ
puntosȱ deȱ muestreo,ȱ mientrasȱ queȱ enȱ elȱ trabajoȱ XIIIȱ seȱ comparanȱ lasȱ
alteracionesȱ bentónicasȱ deȱ lasȱ áreasȱ deȱ estudio.ȱ Enȱ elȱ capítuloȱ 5ȱ seȱ realizaȱ laȱ
integraciónȱdeȱlosȱresultadosȱmostradosȱaȱloȱlargoȱdeȱestaȱmemoria,ȱyȱconstaȱdeȱ
3ȱ trabajos.ȱ Elȱ trabajoȱ XIVȱ realizaȱ unȱ estudioȱ deȱ laȱ evoluciónȱ deȱ laȱ calidadȱ
ambientalȱdeȱlosȱsedimentosȱdeȱGaliciaȱaȱloȱlargoȱdeȱlosȱúltimosȱaños,ȱmedianteȱ
elȱempleoȱdeȱunaȱmetodologíaȱTRIADȱclásica.ȱEnȱelȱtrabajoȱXVȱseȱdesarrollaȱunaȱ
nuevaȱ metodologíaȱ dentroȱ deȱ unȱ “weightȱ ofȱ evidenceȱ approach”ȱ enȱ laȱ queȱ seȱ
incorporaȱunaȱnuevaȱlíneaȱdeȱevidenciaȱdeȱefectosȱsubletalesȱparaȱlaȱevaluaciónȱ
deȱ laȱ calidadȱ deȱ losȱ sedimentosȱ enȱ elȱ áreaȱ deȱ Galicia.ȱ Enȱ elȱ últimoȱ trabajo,ȱ elȱ
XVI,ȱ seȱ llevaȱ aȱ caboȱ unaȱ integraciónȱ conjuntaȱ deȱ losȱ datosȱ obtenidosȱ enȱ losȱ
diferentesȱ estudiosȱ deȱ losȱ sedimentosȱ deȱ laȱ zonaȱ deȱ Galiciaȱ yȱ laȱ Bahíaȱ deȱ
Algeciras.ȱ ȱ ȱ Finalmente,ȱ enȱ elȱ capítuloȱ 6ȱ deȱ estaȱ memoria,ȱ seȱ establecenȱ lasȱ
conclusionesȱ obtenidasȱ trasȱ laȱ consecuciónȱ deȱ losȱ objetivosȱ propuestosȱ enȱ estaȱ
tesisȱdoctoral.ȱ
5.ȱBibliografíaȱ
ȱȱȱdelȱ Fuel.ȱ Ensayosȱ sobreȱ elȱ <<Prestige>>.ȱ
Fundaciónȱ Santiagoȱ Reyȱ FernándezȬ
LaTorre:ȱ10Ȭ41.ȱ
Arjonilla,ȱ M.,ȱ Forja,ȱ J.M.,ȱ GómezȬParra,ȱ A.ȱ
1994.ȱSedimentȱanalysisȱdoesȱnotȱprovideȱ
aȱ goodȱ measureȱ ofȱ heavyȱ metalȱ
bioavalibilityȱ toȱ Cerastodermaȱ glaucumȱ
(Mollusca:ȱ Bivalvia)ȱ inȱ confinedȱ coastalȱ
sediments.ȱ Bull.ȱ Environ.ȱ Contam.ȱ
Toxicol.ȱ52:ȱ810Ȭ817.ȱ
Burgeot,ȱT.ȱ2001.ȱAnȱoverviewȱofȱstatusȱandȱ
monitoringȱ ofȱ theȱ Erikaȱ Hillȱ spill.ȱ Reportȱ
WgbecȬIcesȬcm(Warnemünde,ȱGermany).ȱ
Chapman,ȱ P.M,ȱ Morgan,ȱ J.D.ȱ 1983.ȱ
Sedimentȱ bioassayȱ withȱ Oysterȱ Larvae.ȱ
Bull.ȱ Environ.ȱ Contam.ȱ Toxicol.ȱ 31:ȱ 438Ȭ
444.ȱ
Baek,ȱ S.O.,ȱ Goldstone,ȱ M.E.,ȱ Kirk,ȱ P.W.W.,ȱ
Lester,ȱ J.N.,ȱ Perry,ȱ R.ȱ 1991.ȱ Phaseȱ
distributionȱandȱparticleȱsizeȱdependencyȱ
ofȱ polycyclicȱ aromaticȱ hydrocarbonsȱ inȱ
theȱ urbanȱ atmosphere.ȱ Chemosphereȱ
22(5Ȭ6):ȱ503Ȭ520. Bulot,ȱJ.ȱ2003.ȱLaȱHuellaȱȱ
ȱ
Ȭ 25ȱȬ
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Chapman,ȱ P.M.ȱ 1989.ȱ Currentȱ approachesȱ
toȱ developingȱ sedimentȱ qualityȱ criteria.ȱ
Environ.ȱToxicol.ȱChem.ȱ8:ȱ589Ȭ599.ȱ
Fundaciónȱ Santiagoȱ Reyȱ FernándezȬ
LaTorre.ȱ 2003.ȱ Laȱ Huellaȱ delȱ Fuel.ȱ
Ensayosȱsobreȱelȱ<<Prestige>>.ȱ278pp.ȱ
Conradi,ȱ M,ȱ etȱ al.ȱ 1995.ȱ Estudioȱ biológicoȱ
deȱ lasȱ comunidadesȱ bentónicasȱ deȱ laȱ
bahíaȱdeȱAlgeciras.ȱI:ȱSinopsisȱgeneralȱdeȱ
resultadosȱ yȱ conclusiones.ȱ Universidadȱ
deȱSevilla.ȱ
Harvey,ȱ R.W.,ȱ Luoma,ȱ S.N.ȱ 1985.ȱ Effectȱ ofȱ
adherentȱ
bacteriaȱ
andȱ
bacterialȱ
extracelularȱ polymersȱ uponȱ assimilationȱ
byȱ Macomaȱ balthicaȱ ofȱ sedimentȬȱ boundȱ
Cd,ȱ Znȱ andȱ Ag.ȱ Mar.ȱ Ecol.ȱ Prog.ȱ Ser.ȱ 22:ȱ
281Ȭ289.ȱȱ
Consejeríaȱ deȱ Medioȱ Ambiente.ȱ 2007.ȱ
Informeȱ 2006ȱ Medioȱ Ambienteȱ enȱ
Andalucía.ȱJuntaȱdeȱAndalucía.ȱ
Hoefer,ȱ T.ȱ 2003.ȱ Tankerȱ safetyȱ andȱ coastalȱ
environment:ȱ Prestige,ȱ Erika,ȱ andȱ whatȱ
else?.ȱ Environmentalȱ Scienceȱ andȱ
PollutionȱResearchȱInternationalȱ10ȱ(1):ȱ1Ȭ
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CSIC.ȱ 2003a.ȱ Impactoȱ deȱ unȱ vertidoȱ deȱ
petróleoȱ sobreȱ losȱ organismosȱ marinos.ȱ
Algunasȱ leccionesȱ delȱ vertidoȱ delȱ Aegeanȱ
Sea.ȱInformeȱnúmeroȱ15.ȱ3ȱpp.ȱȱ
Jackim,ȱ E.,ȱ Lake,ȱ C.ȱ 1978.ȱ Polynuclearȱ
aromaticȱ hydrocarbonsȱ inȱ estuarineȱ andȱ
nearshoreȱ
environments.ȱ
Estuarineȱ
Interactions.ȱ Academicȱ Press,ȱ Newȱ York.ȱ
415Ȭ428pp.ȱ
CSIC.ȱ 2003b.ȱ Informeȱ diciembreȱ 2003:ȱ
Diagnósticoȱ sobreȱ laȱ situaciónȱ ambientalȱ
delȱ entornoȱ delȱ Campoȱ deȱ Gibraltar.ȱ 194ȱ
pp.ȱ
Kemp,ȱ P.F.,ȱ Swartz,ȱ R.C.ȱ 1998.ȱ Acuteȱ
toxicityȱ ofȱ interstitialȱ andȱ particleȬboundȱ
cadmiumȱtoȱaȱmarineȱinfaunalȱamphipod.ȱ
Mar.ȱEnviron.ȱRes.ȱ26ȱ:ȱ135Ȭ153.ȱ
DelValls,ȱ T.A.ȱ 1994.ȱ Aplicaciónȱ deȱ unȱ
métodoȱ integradoȱ paraȱ laȱ medidaȱ deȱ laȱ
calidadȱambientalȱenȱecosistemasȱlitoralesȱ
delȱ golfoȱ deȱ Cádiz.ȱ Tesisȱ doctoral.ȱ
UniversidadȱdeȱCádiz.ȱ389pp.ȱ
Long,ȱ E.R.,ȱ Buchman,ȱ M.F.ȱ 1989.ȱ Anȱ
evaluationȱ ofȱ candidateȱ measuresȱ ofȱ
biologicalȱ effectsȱ forȱ theȱ Nationalȱ Statusȱ
andȱ Trendsȱ Program.ȱ NOAAȱ Technicalȱ
Memorandumȱ NOSȱ OMAȱ 45.ȱ Nationalȱ
Oceanicȱ
andȱ
Atmosphericȱ
Administration.ȱ Seattle,ȱ Washington.ȱ
105pp.ȱ
DelValls,ȱT.A.,ȱChapman.ȱ1998.ȱSiteȬspecificȱ
sedimentȱ qualityȱ valuesȱ forȱ theȱ gulaȱ ofȱ
Cádizȱ (Spain)ȱ andȱ Sanȱ Franciscoȱ Bayȱ
(USA),ȱ usingȱ theȱ sedimentȱ qualityȱ triadȱ
andȱ multivariateȱ análisis.ȱ Cienciasȱ
Marinas,ȱ24(3):ȱ313Ȭ336. ȱ
DelValls,ȱ .T.A.,ȱ Forjaȱ J.M.,ȱ GómezȬParraȱ A.ȱ
2002.ȱSeasonalityȱofȱcontamination,ȱȱ
Luoma,ȱ S.N.,ȱ Bryan,ȱ G.W.ȱ 1978.ȱ Factorsȱ
controllingȱ theȱ availabilityȱ ofȱ sedimentsȬ
boundȱ leadȱ toȱ theȱ estuarineȱ bivalveȱ
Scrobiculariaȱplana.ȱJ.ȱMar.ȱBiol.ȱAss.ȱU.K.ȱ
58:ȱ793Ȭ802.ȱ
ȱȱȱȱtoxicity,ȱ andȱ qualityȱ valuesȱ inȱ sedimentsȱ
fromȱ littoralȱ ecosystemsȱ inȱ theȱ Gulfȱ ofȱ
Cádizȱ(SWȱSpain).ȱChemosphereȱ46ȱ1033–
1043.ȱ
Luoma,ȱ S.N.ȱ 1983.ȱ Bioavailabilityȱ ofȱ traceȱ
metalsȱ toȱ aquaticȱ organismsȱ ȬȬȱ Aȱ review.ȱ
Sci.ȱTotalȱEnviron.ȱ28:ȱ1Ȭ22.ȱ
Eisler,ȱ R.ȱ 2000.ȱ Handbookȱ ofȱ chemicalȱ riskȱ
assessment.ȱLewisȱpublishers.ȱChapterȱ25.ȱ
Luoma,ȱ S.N.,ȱ Philips,ȱ D.H.J.ȱ 1988.ȱ
Distribution,ȱ variability,ȱ andȱ impactsȱ ofȱ
traceȱelementsȱinȱSanȱFranciscoȱBay.ȱMar.ȱ
Pollut.ȱBull.ȱ19(9):ȱ413Ȭ425.ȱ
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Luoma,ȱ S.N.ȱ 1989.ȱ Canȱ weȱ determineȱ theȱ
biologicalȱ availabilityȱ ofȱ sedimentȱ boundȱ
traceȱ elements?ȱ Hidrobiologiaȱ 176/177:ȱ
79Ȭ396.ȱ
NOAA.ȱ 1999.ȱ Sedimentȱ Qualityȱ Guidelinesȱ
developedȱ forȱ theȱ Nationalȱ Statusȱ andȱ
TrendȱPrograms.ȱȱȱ
Neff,ȱ J.M.ȱ 1979.ȱ Polyciclicȱ Aromaticȱ
Hydrocarbonsȱ
inȱ
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aquaticȱ
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London.ȱ262ȱpp.ȱ
Luoma,ȱ S.N.ȱ 1990.ȱ Processȱ affectingȱ metalȱ
concentrationsȱ inȱ estuarineȱ andȱ coastalȱ
marineȱ sediments.ȱ Heavyȱ metalsȱ inȱ theȱ
marineȱ environment.ȱ CRCȱ Press,ȱ Inc.ȱ
BocaȱRaton,ȱFlorida,ȱ51Ȭ66.ȱ
Oakden,ȱ J.M.,ȱ Bejda,ȱ A.J.,ȱ Pearson,ȱ W.H.ȱ
1984.ȱ EDTAȱ chelationȱ andȱ zincȱ
antagonismȱ withȱ cadmiumȱ inȱ sediment:ȱ
effectsȱonȱtheȱbehaviourȱandȱmortalityȱofȱ
twoȱ infaunalȱ amphipods.ȱ Mar.ȱ Biol.ȱ 85:ȱ
125Ȭ130.ȱ
Luoma,ȱS.N.,ȱHo,ȱK.T.ȱ1992.ȱTheȱappropiateȱ
usesȱ ofȱ marineȱ andȱ estuarineȱ sedimentȱ
bioassays.ȱ
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226.ȱ
Salomons,ȱW.,ȱdeȱRooij,ȱN.M.,ȱKerdjik,ȱBrill,ȱ
J.ȱ 1987.ȱ Sedimentsȱ asȱ aȱ sourceȱ forȱ
contaminants?ȱHidrobiologiaȱ149:ȱ13Ȭ30.ȱ
Martell,ȱ F.L.,ȱ Motekaitis,ȱ R.T.,ȱ Smith,ȱ R.M.ȱ
1988.ȱ StructureȬstabilityȱ relationshipsȱ ofȱ
metalȱ complexesȱ andȱ metalȱ speciationȱ inȱ
environmentalȱ
aqueousȱ
solutions.ȱ
Environ.ȱToxicol.ȱChem.ȱ7:ȱ417Ȭ434
Schnitz,ȱ A.R.,ȱ O’Connor,ȱ J.M.ȱ 1992.ȱ Inȱ vivoȱ
DNA/RNAȱ
adductionȱ
ofȱ
7,12Ȭ
dimethylbenz(a)anthraceneȱ (DMBA)ȱ andȱ
benzo(a)pyreneȱ (BaP)ȱ inȱ theȱ liverȱ ofȱ
rainbowȱ troutȱ (Oncorhynchusȱ mykiss).ȱ
Jour.ȱ Environ.ȱ Pathol.ȱ Toxicol.ȱ Oncol.ȱ
11:229Ȭ233.ȱȱȱ
MartínȬDíaz,ȱ L.,ȱ DelValls,ȱ T.A.ȱ Presionesȱ
sobreȱ elȱ medioȱ ambienteȱ deȱ laȱ Bahíaȱ deȱ
Algecirasȱ debidoȱ aȱ lasȱ actividadesȱ
asociadasȱ
alȱ
tráficoȱ
marítimo.ȱ
Presentaciónȱoral.ȱ
Sims,ȱ R.C,ȱ Overcash,ȱ R.ȱ 1983.ȱ Fateȱ ofȱ
Polynuclearȱaromaticȱcompoundsȱ(PNAs)ȱ
inȱ soilȬplantȱ systems.ȱ Residueȱ Rev.ȱ 88:ȱ 1Ȭ
68.ȱ
McGee,ȱ B.L.,ȱ Schlekat,ȱ Ch.E.,ȱ Reinharz,ȱ E.ȱ
1993.ȱ Assesingȱ sublethalȱ levelsȱ ofȱ
sedimentsȱ contaminationȱ usingȱ theȱ
estuarineȱ
amphipodȱ
Leptocheirusȱ
plumosus.ȱ Environ.ȱ Toxicol.ȱ Chem.ȱ 12:ȱ
577.587.ȱ
Swartz,ȱ R.C.,ȱ Kemp,ȱ P.F.,ȱ Schults,ȱ D.W.,ȱ
Ditsworth,ȱG.R.,ȱOzretich,ȱR.J.ȱ1989.ȱAcuteȱ
toxicityȱofȱsedimentsȱfromȱEagleȱHarbour,ȱ
Washington,ȱ toȱ theȱ infaunalȱ amphipodȱ
Rhepoxyniousȱ abronius.ȱ Environ.ȱ Toxicol.ȱ
Chem.ȱ8:ȱ212Ȭ222.ȱ
Meador,ȱ J.P.,ȱ Ross,ȱ B.D.,ȱ Dinnel,ȱ P.A.,ȱ
Picquelle,ȱ S.J.ȱ 1990.ȱ Anȱ analysisȱ ofȱ theȱ
relationshipȱ betweenȱ aȱ SandȬDollarȱ
embryoȱ elutriateȱ assayȱ andȱ sedimentȱ
contaminantsȱ fromȱ stationsȱ inȱ anȱ urbanȱ
embaymentȱofȱPugetȱSound,ȱWashington.ȱ
Mar.ȱEnviron.ȱRes.ȱ30:ȱ251Ȭ272.ȱ
Swartz,ȱ R.C.,ȱ Schults,ȱ D.W.,ȱ DeWitt,ȱ T.H.,ȱ
Ditsworth,ȱ G.R.,ȱ Lamberson,ȱ J.O.ȱ 1990.ȱ
Toxicityȱ ofȱ fluorantheneȱ inȱ sedimentȱ toȱ
marineȱ amphipods:ȱ aȱ testȱ ofȱ theȱ
equilibriumȱ partitioningȱ approachȱ toȱ
sedimentȱ qualityȱ criteria.ȱ Environ.ȱ
Toxicol.ȱChem.ȱȱ9:ȱ1071Ȭ1080.ȱ
MMA.ȱ 1993.ȱ Seguimientoȱ deȱ lasȱ
contaminacionesȱ producidasȱ porȱ elȱ
accidenteȱ delȱ buqueȱ Aegeanȱ Sea.ȱ Ed.ȱ
Centroȱ Publ.ȱ Sec.ȱ G.ȱ Técnica.ȱ Ministerioȱ
deȱMedioȱAmbiente.ȱMadrid.ȱ
USEPA,ȱ 2000.ȱ Toxicȱ Releaseȱ Inventoryȱ
PublicDataRelease,hppt://www.epa.gov/t
riinter/tridata/index.htm,ȱ
Officeȱ
ofȱ
EnvironmentalȱInformation,ȱUnitedȱStatesȱ
Ȭ 27ȱȬ
ȱ
- 28 -
ȱ
Capítuloȱ2.ȱ
Análisisȱdeȱlaȱcontaminaciónȱyȱevaluaciónȱȱdeȱlaȱ
toxicidadȱagudaȱmedianteȱensayosȱenȱlaboratorioȱ
Laȱpropiedadȱqueȱtieneȱunaȱsustanciaȱdeȱproducirȱefectosȱadversosȱaȱunȱ
sistemaȱ biológicoȱ seȱ denominaȱ toxicidad.ȱ Sinȱ embargo,ȱ elȱ hechoȱ deȱ queȱ unaȱ
sustanciaȱtóxicaȱestéȱpresenteȱenȱelȱmedioȱnoȱimplicaȱnecesariamenteȱqueȱvayaȱaȱ
causarȱ efectosȱ tóxicos.ȱ Unaȱ sustanciaȱ presenteȱ enȱ elȱ medioȱ ambienteȱ aȱ
concentracionesȱ superioresȱ deȱ lasȱ naturalesȱ conllevaȱ unȱ episodioȱ deȱ
contaminación,ȱ yȱ enȱ elȱ casoȱ deȱ queȱ tambiénȱ presenteȱ efectosȱ tóxicosȱ sobreȱ laȱ
biotaȱestaremosȱhablandoȱdelȱfenómenoȱdeȱpolución.ȱEnȱesteȱúltimoȱcaso,ȱdosisȱ
bajasȱdeȱcontaminanteȱpuedenȱproducirȱalteracionesȱenȱlasȱfuncionesȱvitalesȱdeȱ
losȱ organismos,ȱ mientrasȱ queȱ dosisȱ altasȱ deȱ laȱ mismaȱ sustanciaȱ puedenȱ serȱ
letales.ȱȱȱ
Tradicionalmenteȱ seȱ haȱ evaluadoȱ laȱ calidadȱ ambientalȱ deȱ unȱ sedimentoȱ
medianteȱelȱanálisisȱdeȱlasȱconcentracionesȱdeȱcontaminantesȱyȱlaȱcomparaciónȱ
conȱ guíasȱ numéricasȱ (SQGs).ȱ Deȱ esteȱ modoȱ seȱ pretendíaȱ evaluarȱ elȱ riesgoȱ
potencialȱdeȱlosȱcontaminantesȱasociadosȱalȱsedimentoȱ(CasadoȬMartínez,ȱ2006).ȱ
Enȱ laȱ actualidadȱ seȱ hanȱ propuestoȱ determinacionesȱ basadasȱ enȱ análisisȱ
químicosȱ juntoȱ conȱ ensayosȱ deȱ toxicidadȱ enȱ laboratorioȱ conȱ elȱ finȱ deȱ llevarȱ aȱ
caboȱ unȱ estudioȱ deȱ losȱ procesosȱ deȱ poluciónȱ enȱ ecosistemasȱ costeros.ȱ Losȱ
bioensayosȱ deȱ toxicidadȱ sonȱ instrumentosȱ queȱ seȱ empleanȱ paraȱ determinarȱ laȱ
ȱ
Ȭȱ29ȱȬȱ
Capítuloȱ2
ecotoxicidadȱ yȱ biodisponibilidadȱ queȱ producenȱ losȱ compuestosȱ químicosȱ delȱ
sedimentoȱ enȱ losȱ organismosȱ bentónicos.ȱ ȱ Enȱ esteȱ tipoȱ deȱ experimentos,ȱ losȱ
organismosȱ seȱ exponenȱ aȱ muestrasȱ deȱ sedimentoȱ yȱ trasȱ unȱ periodoȱ deȱ
exposiciónȱ seȱ mideȱ unaȱ respuestaȱ biológica.ȱ Estaȱ respuestaȱ haȱ deȱ serȱ sensible,ȱ
relevanteȱyȱfácilȱdeȱestandarizarȱ(Stebbingȱetȱal.,ȱ1980).ȱȱ
Laȱ toxicidadȱ agudaȱ haceȱ referenciaȱ alȱ efectoȱ nocivoȱ resultanteȱ deȱ unaȱ
exposiciónȱ relativamenteȱ cortaȱ aȱ unaȱ sustanciaȱ tóxica.ȱ Estosȱ efectosȱ sobreȱ elȱ
organismoȱ suelenȱ desarrollarseȱ rápidamenteȱ yȱ suelenȱ dejarȱ deȱ aparecerȱ enȱ elȱ
momentoȱ queȱ cesaȱ laȱ dosis.ȱ Losȱ ensayosȱ deȱ toxicidadȱ agudaȱ seȱrealizanȱ enȱ unȱ
periodoȱ deȱ tiempoȱ queȱ puedeȱ variarȱ deȱ minutosȱ aȱ variosȱ días,ȱ yȱ vanȱ aȱ
proporcionarȱ respuestasȱ puntualesȱ (mortalidad,ȱ inhibiciónȱ delȱ crecimiento,ȱ
inhibiciónȱdeȱlaȱbioluminiscencia,ȱetc.)ȱ
Enȱesteȱcapítuloȱseȱpresentanȱcuatroȱtrabajosȱenȱlosȱqueȱseȱllevanȱaȱcaboȱ
distintosȱ ensayosȱ deȱ toxicidadȱ agudaȱ conȱ cuatroȱ especiesȱ deȱ invertebradosȱ
marinos.ȱ Enȱ losȱ primerosȱ dosȱ trabajosȱ (Iȱ yȱ II)ȱ seȱ llevaȱ aȱ caboȱ unȱ estudioȱ
preliminarȱparaȱdeterminarȱlaȱtoxicidadȱdelȱfuelȱdelȱpetroleroȱPrestige.ȱParaȱelloȱ
seȱ realizaronȱ unaȱ serieȱ deȱ dilucionesȱ deȱ fuelȱ extraídoȱ delȱ barcoȱ hundidoȱ conȱ
sedimentoȱlimpioȱprocedenteȱdeȱlaȱBahíaȱdeȱCádiz,ȱdeȱformaȱqueȱseȱobtuvieronȱ
distintasȱ concentracionesȱ deȱ fuelȱ enȱ sedimento.ȱ Enȱ elȱ trabajoȱ Iȱ seȱ efectuaronȱȱ
exposicionesȱconȱelȱanfípodoȱAmpeliscaȱbrevicornisȱyȱtrasȱunȱperiodoȱdeȱdiezȱdíasȱ
seȱ contabilizóȱ laȱ mortalidadȱ enȱ cadaȱ unaȱ deȱ lasȱ diluciones.ȱ Losȱ resultadosȱ
obtenidosȱpermitieronȱllevarȱaȱcaboȱelȱcálculoȱdelȱLC50,ȱparámetroȱtoxicológicoȱ
queȱindicaȱlaȱconcentraciónȱdeȱsustanciaȱcapazȱdeȱproducirȱunaȱmortalidadȱdelȱ
50ȱ%.ȱLosȱanfípodosȱsonȱutilizadosȱdeȱmaneraȱhabitualȱenȱtestsȱdeȱtoxicidadȱyȱseȱ
correlacionanȱpositivamenteȱconȱcambiosȱenȱlasȱcomunidadesȱbentónicas.ȱȱ
Enȱelȱsegundoȱtrabajoȱ(II)ȱseȱllevóȱaȱcaboȱunaȱexposiciónȱsimilarȱaunqueȱ
estaȱ vezȱ conȱ elȱ poliquetoȱ Arenicolaȱ marinaȱ yȱ seȱ calculóȱ elȱ LC50ȱ trasȱ 10ȱ díasȱ deȱ
- 30 -
Contaminaciónȱyȱtoxicidadȱagudaȱȱ
exposiciónȱyȱunȱsegundoȱLC50ȱpasadosȱ21ȱdías;ȱademásȱseȱdeterminóȱelȱfactorȱ
deȱ bioacumulaciónȱ (BCF)ȱ paraȱ PAHsȱ aȱ partirȱ deȱ laȱ relaciónȱ entreȱ laȱ
concentraciónȱ deȱ contaminanteȱ enȱ elȱ organismoȱ frenteȱ aȱ laȱ concentraciónȱ delȱ
mismoȱenȱelȱsedimento.ȱElȱpoliquetoȱArenicolaȱmarinaȱmostróȱserȱmenosȱsensibleȱ
frenteȱaȱlaȱcontaminaciónȱorgánicaȱqueȱelȱanfípodoȱAmpeliscaȱbrevicornisȱaunqueȱ
aȱsuȱvezȱpresentaȱlaȱcapacidadȱdeȱhabitarȱenȱzonasȱpolucionadasȱofreciendoȱlaȱ
posibilidadȱdeȱllevarȱaȱcaboȱestudiosȱdeȱbioacumulación.ȱ
Tablaȱ 2.1.ȱ Relaciónȱ deȱ bioensayosȱ agudosȱ realizadosȱ paraȱ laȱ evaluaciónȱ
deȱlaȱcalidadȱdeȱlosȱsedimentosȱ(adaptadoȱdeȱCasadoȬMartínezȱetȱal.,ȱ2006).ȱ
Bioensayoȱ
Especieȱ
Medidaȱfinalȱ
Rutaȱdeȱ
exposiciónȱ
Tiempoȱdeȱ
exposiciónȱ
Lixiviadoȱ
5ȱȬȱ30ȱ
minutosȱ
Inhibiciónȱdeȱlaȱ
Microtox®ȱȱȱȱ Vibrioȱfischeriȱ
bioluminescenciaȱ
(IC50)ȱ
Anfípodosȱ
Ampeliscaȱbrevicornisȱ
supervivenciaȱ
Faseȱsólidaȱ
10ȱdíasȱ
Anfípodosȱ
Corophiumȱvolutatorȱ
supervivenciaȱ
Faseȱsólidaȱ
10ȱdíasȱ
Poliquetosȱ
Arenicolaȱmarinaȱ
Supervivenciaȱyȱ
bioacumulaciónȱ
Faseȱsólidaȱ
10ȱ–ȱ15ȱdíasȱ
Elȱ tercerȱtrabajoȱ (III)ȱ muestraȱlaȱprimeraȱaproximaciónȱalȱestudioȱdeȱlosȱ
sedimentosȱdeȱlaȱBahíaȱdeȱAlgecirasȱyȱelȱParqueȱNacionalȱdeȱlasȱIslasȱAtlánticasȱ
enȱ Galicia.ȱ 14ȱ puntosȱ deȱ muestreosȱ fueronȱ evaluadosȱ juntoȱ aȱ dosȱ controlesȱ
(negativoȱ yȱ positivo)ȱ medianteȱ elȱ usoȱ deȱ unaȱ caracterizaciónȱ físicoȱ químicaȱ deȱ
losȱ sedimentosȱ yȱ suȱ relaciónȱ conȱ resultadosȱ deȱ dosȱ ensayosȱ deȱ toxicidad:ȱ
Microtox®ȱ ȱ ȱ yȱ elȱ testȱ deȱ 10ȱ díasȱ conȱ elȱ anfípodoȱ Corophiumȱ volutator.ȱ Elȱ testȱ
Microtox®ȱseȱbasaȱenȱlaȱmediciónȱdeȱlaȱemisiónȱdeȱluzȱdeȱunaȱbacteriaȱmarinaȱ
Vibrioȱ fischeri,ȱ laȱ cualȱ seȱ exponeȱ aȱ unaȱ serieȱ deȱ dilucionesȱ deȱ unaȱ muestraȱ deȱ
sedimento.ȱ Laȱ bioluminiscenciaȱ esȱ directamenteȱ proporcionalȱ alȱ estadoȱ
metabólicoȱdeȱlaȱcélula;ȱcuandoȱlaȱbacteriaȱseȱexponeȱaȱunaȱsustanciaȱtóxica,ȱelȱ
Ȭ 31ȱȬ
Capítuloȱ2
cuerpoȱcelularȱsufreȱalgunosȱcambiosȱdeȱformaȱqueȱlaȱbioluminiscenciaȱemitidaȱ
disminuye.ȱElȱ testȱ evalúaȱlaȱ disminuciónȱdeȱlaȱbioluminiscenciaȱdespuésȱdeȱlaȱ
exposiciónȱ deȱ losȱ organismosȱ alȱ sedimento,ȱ deȱ formaȱ queȱ losȱ resultadosȱ
obtenidosȱ seȱ expresanȱ enȱ términosȱ deȱ laȱ concentraciónȱ efectivaȱ deȱ unaȱ
determinadaȱ sustanciaȱ presenteȱ enȱ elȱ medioȱ queȱ produceȱ unaȱ reducciónȱ deȱ laȱ
emisiónȱdelȱluzȱdelȱmicroorganismoȱdelȱ50%ȱ(IC50).ȱLaȱNormativaȱCanadienseȱ
estableceȱqueȱunaȱmuestraȱesȱtóxicaȱcuandoȱelȱvalorȱdeȱIC50ȱesȱmenorȱdeȱ1000ȱ
mgȱ LȬ1ȱ pesoȱ secoȱ yȱ noȱ tóxicaȱ cuandoȱ elȱ medidoȱ esȱ mayorȱ deȱ 1000ȱ mgȱ LȬ1ȱ pesoȱ
seco.ȱPorȱsuȱparte,ȱelȱCentroȱdeȱEstudiosȱyȱExperimentaciónȱdeȱObrasȱPúblicasȱ
(CEDEX)ȱdefineȱunaȱmuestraȱcomoȱtóxicaȱcuandoȱIC50ȱesȱmenorȱdeȱ750mgȱLȬ1ȱ
pesoȱ secoȱ yȱ noȱ tóxicaȱ cuandoȱ ésteȱ esȱ mayorȱ deȱ 750ȱ mgȱ LȬ1.ȱ Tambiénȱ existenȱ
distintosȱ criteriosȱ paraȱ considerarȱ aȱ unaȱ muestraȱ comoȱ tóxicaȱ enȱ funciónȱ delȱ
resultadoȱ obtenidoȱ enȱ elȱ testȱ agudoȱ realizadoȱ conȱ anfípodos.ȱ Todosȱ estosȱ
criteriosȱ utilizanȱ elȱ valorȱ delȱ porcentajeȱ deȱ mortalidadȱ deȱ anfípodosȱ queȱ seȱ
obtieneȱ restandoȱ aȱ 100ȱ elȱ valorȱ delȱ porcentajeȱ deȱ supervivenciaȱ observado.ȱ Enȱ
esteȱsentidoȱsonȱdiversosȱlosȱpaísesȱqueȱincluyenȱesteȱtestȱdentroȱdeȱlasȱbateríasȱ
paraȱ establecerȱ laȱ nocividadȱ deȱ muestrasȱ deȱ sedimentosȱ y/oȱ materialȱ deȱ
dragado.ȱ Entreȱ ellosȱ cabeȱ destacarȱ laȱ Normativaȱ Holandesa,ȱ establecidaȱ porȱ elȱ
MinisterioȱdeȱfomentoȱHolandésȱqueȱconsideraȱqueȱunaȱmuestraȱdeȱsedimentoȱ
esȱtóxicaȱsiȱlaȱmortalidadȱdeȱlaȱespecieȱCorophiumȱvolutatorȱesȱigualȱoȱmayorȱdelȱ
25%.ȱPorȱsuȱparte,ȱlaȱNormativaȱInglesaȱconsideraȱque,ȱparaȱestaȱmismaȱespecieȱ
deȱanfípodo,ȱlaȱmortalidadȱdeȱlaȱpoblaciónȱexpuestaȱhaȱdeȱserȱigualȱoȱmayorȱalȱ
40%ȱ paraȱ queȱ laȱmuestraȱ deȱ sedimentoȱ seaȱconsideradaȱ tóxica.ȱ ȱ Laȱ Normativaȱ
paraȱelȱáreaȱdeȱHongȱKongȱutilizaȱunaȱespecieȱdeȱanfípodosȱnoȱdetalladaȱenȱlosȱ
testȱdeȱtoxicidad,ȱyȱproponeȱqueȱlaȱmortalidadȱdeȱlosȱindividuosȱhaȱdeȱserȱigualȱ
oȱ mayorȱ alȱ 30%ȱ paraȱ considerarȱ queȱ existeȱ toxicidad.ȱ Porȱ otraȱ parte,ȱ existenȱ
normativasȱdondeȱnoȱseȱproponenȱunȱúnicoȱvalorȱindicativoȱsinoȱqueȱseȱutilizaȱ
unȱ dobleȱ criterio,ȱ incluyendoȱ unȱ valorȱ observadoȱ yȱ unȱ criterioȱ estadístico.ȱ Enȱ
esteȱ sentido,ȱ laȱ Normativaȱ Estadounidense,ȱ elaboradaȱ porȱ laȱ Environmentalȱ
- 32 -
Contaminaciónȱyȱtoxicidadȱagudaȱȱ
Protectionȱ Agencyȱ (USEPA)ȱ yȱ elȱ cuerpoȱ deȱ ingenierosȱ delȱ Ejércitoȱ Americano,ȱ
utilizaȱ laȱ especieȱ Ampeliscaȱ abditaȱ yȱ estableceȱ comoȱ criterioȱ deȱ toxicidadȱ unaȱ
mortalidadȱ superiorȱ alȱ 20%ȱ conȱ respectoȱ alȱ sedimentoȱ deȱ referenciaȱ yȱ ademásȱ
significativamenteȱ diferenteȱ (p<0.05)ȱ delȱ control.ȱ Elȱ Centroȱ deȱ Estudiosȱ yȱ
ExperimentaciónȱdeȱObrasȱPúblicasȱ(CEDEX)ȱproponeȱesteȱmismoȱcriterioȱdobleȱ
paraȱ clasificarȱ lasȱ muestrasȱ deȱ sedimentoȱ comoȱ tóxicasȱ oȱ noȱ tóxicasȱ paraȱ elȱ
litoralȱ español.ȱ Losȱ resultadosȱ deȱ esteȱ trabajoȱ muestranȱ lasȱ diferenciasȱ entreȱ
ambasȱ zonasȱ deȱ estudioȱ yȱ determinaȱ queȱ laȱ Bahíaȱ deȱ Algecirasȱ presentaȱ unaȱ
degradaciónȱambientalȱmayorȱqueȱaquellaȱobservadaȱenȱlasȱcostasȱgallegas.ȱ
Enȱelȱúltimoȱtrabajoȱ(IV)ȱdeȱesteȱsegundoȱcapítulo,ȱseȱaplicanȱlosȱtestȱdeȱ
toxicidadȱanteriormenteȱdescritos:ȱanfípodos,ȱMicrotox®ȱyȱArenicolaȱmarinaȱparaȱ
evaluarȱ laȱ toxicidadȱ deȱ sedimentosȱ muestreadosȱ enȱ lasȱ costasȱ gallegasȱ cuatroȱ
añosȱ despuésȱ delȱ vertidoȱ delȱ petroleroȱ Prestige.ȱ Parteȱ deȱ esteȱ estudio,ȱ enȱ
concretoȱlosȱanálisisȱdeȱbioacumulaciónȱdeȱPAHsȱenȱelȱpoliquetoȱA.ȱmarina,ȱseȱ
realizaronȱduranteȱunaȱestanciaȱenȱelȱcentroȱIPIMARȱenȱLisboa.ȱLosȱresultadosȱ
indicanȱ unaȱ disminuciónȱ deȱ laȱ contaminaciónȱconȱrespectoȱaȱestudiosȱprevios,ȱ
asíȱ comoȱ unaȱ desapariciónȱ deȱ laȱ toxicidadȱ agudaȱ aunqueȱ unaȱ importanteȱ
bioacumulaciónȱ deȱ PAHsȱ fueȱ detectadaȱ principalmenteȱ enȱ elȱ áreaȱ deȱ CormeȬ
Laxe,ȱloȱqueȱsugiereȱqueȱaȱpesarȱdeȱqueȱlaȱcalidadȱambientalȱseȱhaȱrecuperadoȱ
deȱformaȱnotable,ȱexisteȱlaȱposibilidadȱdeȱefectosȱsubletalesȱenȱlaȱbiota.ȱȱȱȱ
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CEDEX.ȱ 2003.ȱ Investigaciónȱ conjuntaȱ sobreȱ
laȱ selecciónȱ yȱ puestaȱ aȱ puntoȱ deȱ ensayosȱ
biológicosȱ paraȱ laȱ caracterizaciónȱ deȱ
materialȱ deȱ dragado.ȱ Informeȱ finalȱ deȱ laȱ
UniversidadȱdeȱCádiz.128pp.ȱ
USEPA,ȱ 2000.ȱ Toxicȱ Releaseȱ Inventoryȱ
Publicȱ
Dataȱ
Release,ȱ
hppt://www.epa.gov/triinter/tridata/index.htm,ȱ
Officeȱ ofȱ Environmentalȱ Information,ȱ
Unitedȱ Statesȱ Environmentalȱ Protectionȱ
Agency,ȱWashington,ȱD.C.ȱ
Environmentalȱ Canada.ȱ 2003.ȱ Canadianȱ
EnvironmentalȱQualityȱGuidelines.ȱ
Long,ȱ E.R.,ȱ Buchman,ȱ M.F.ȱ 1989.ȱ Anȱ
evaluationȱ ofȱ candidateȱ measuresȱ ofȱ
biologicalȱ effectsȱ forȱ theȱ Nationalȱ Statusȱ
andȱ Trendsȱ Program.ȱ NOAAȱ Technicalȱ
Memorandumȱ NOSȱ OMAȱ 45.ȱ Nationalȱ
Oceanicȱ
andȱ
Atmosphericȱ
Administration.ȱ Seattle,ȱ Washington.ȱ
105pp.ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
- 34 -
Acuteȱtoxicityȱofȱresidualȱfuelȱoilȱfromȱtheȱtankerȱ“Prestige”ȱusingȱ
amphipodsȱ
MoralesȬCaselles,ȱC.ȱ1,2,*,ȱRiba,ȱI.ȱ1,2;ȱSarasquete,ȱC.1,ȱDelValls,ȱT.A.1,2ȱ
1
ȱUnidadȱAsociadaȱdeȱCalidadȱAmbientalȱyȱPatologíaȱ(CSICȱ&ȱUCA)ȱinstitutoȱdeȱ
CienciasȱMarinasȱdeȱAndalucíaȱ(ICMANȬCSIC),ȱAvda.ȱRepúblicaȱSaharauiȱs/n,ȱPuertoȱ
Realȱ11510,ȱCádiz,ȱSpainȱ
UNESCOȱUNITWIN/WiCop,ȱFacultadȱdeȱCienciasȱdelȱMarȱyȱAmbientales,ȱ
2ȱ
UniversidadȱdeȱCádiz,ȱPolígonoȱRíoȱSanȱPedroȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱSpain.ȱ
Abstractȱ
Inȱ Novemberȱ 2002,ȱ theȱ simpleȱ hullȱ tankerȱ “Prestige”ȱ sinkedȱ inȱ theȱ
GalicianȱCoastȱ(NWȱSpain)ȱandȱspiltȱ63,000ȱTonsȱofȱfuelȱoil.ȱTwoȱyearsȱafterȱtheȱ
spill,ȱ theȱ remaindingȱ oilȱ hasȱ beenȱ extractedȱ fromȱ theȱ hullȱ andȱ hasȱ beenȱ
physicochemicallyȱ andȱ ecotoxicologicallyȱ characterized.ȱ Anȱ acuteȱ bioassayȱ
usingȱ theȱ amphipodȱ Ampeliscaȱ brevicornisȱ hasȱ beenȱ carriedȱ outȱ inȱ orderȱ toȱ
determineȱ toxicityȱ associatedȱ withȱ theȱ contaminantsȱ presentsȱ inȱ theȱ fuel.ȱ Theȱ
bioassayȱ wasȱ conductedȱ byȱ exposingȱ duringȱ 10ȱ daysȱ theȱ individualsȱ ofȱ
Ampeliscaȱtoȱcleanȱsedimentȱmixedȱwithȱdifferentȱproportionsȱofȱfuelȱoilȱ(0.1%,ȱ
0.5%,ȱ 2%,ȱ 8%,ȱ 16%ȱ andȱ 32%).ȱ Resultsȱ wereȱ linkedȱ withȱ theȱ chemicalȱ dataȱ inȱ
orderȱ toȱ determineȱ theȱ sensitivityȱ ofȱtheȱamphipodȱtoȱtheȱfuelȱoilȱcompounds.ȱ
TheȱLC50ȱvalueȱobtainedȱforȱAmpeliscaȱbrevicornisȱ(1.37%ȱ±ȱ0.33)ȱandȱtheȱPAHsȱ
concentrationsȱ measuredȱ inȱ theȱ fuelȱ oilȱ haveȱ permittedȱ toȱ calculateȱ theȱ
Sedimentȱ Qualityȱ Valuesȱ (SQVs)ȱ thatȱ areȱ similarȱ fromȱ thoseȱ obtainedȱ fromȱ
previousȱ studiesȱ corroboratingȱ thatȱ theȱ acuteȱ toxicityȱ ofȱ theȱ fuelȱ oilȱ itȱ wasȱ
mainlyȱassociatedȱwithȱtheȱconcentrationȱofȱPAHs.ȱ
Keywords:ȱ PAHs,ȱ Ampeliscaȱ brevicornis,ȱ qualityȱ values,ȱ bioassay,ȱ sedimentȱ toxicity,ȱ
sedimentȱdilution.ȱ
EnvironmentalȱToxicologyȱ(enviado)ȱ
- 35 -
1.ȱIntroductionȱ
Onȱ 13thȱ Novemberȱ 2002ȱ theȱ tankerȱ Prestigeȱ brokedownȱ inȱ theȱ Galicianȱ
Coastȱ(NWȱSpain)ȱandȱsankȱsixȱdaysȱlaterȱinȱwaterȱ3,500ȱmetresȱdeep.ȱInȱall,ȱitȱisȱ
estimatedȱthatȱtheȱPrestigeȱspiltȱ63,000ȱtonnesȱofȱheavyȱfuelȱoilȱleadingȱtoȱoneȱofȱ
theȱ greatestȱ ecologicalȱ catastropheȱ inȱ Spain.ȱ Onȱ Septemberȱ 2004ȱ theȱ 58,000ȱ
tonnesȱofȱremainingȱfuelȱthatȱwereȱstillȱinȱtheȱtankerȱwereȱfinallyȱcollected.ȱȱ
Theȱ compositionȱ ofȱ thisȱ fuelȱ wasȱ aȱ mixtureȱ ofȱ saturatedȱ hydrocarbons,ȱ
aromaticȱ hydrocarbons,ȱ resinsȱ andȱ asphaltenes,ȱ beingȱ mostȱ ofȱ theȱ polycyclicȱ
aromaticȱ hydrocarbonsȱ ȬPAHsȬȱ ofȱ anȱ intermediumȬhighȱ molecularȱ weightȱ
(Blancoȱetȱal.,ȱinȱpress).ȱTheȱphysicochemicalȱcharacteristicsȱofȱtheȱoilȱspilledȱbyȱ
theȱ tankerȱ Prestigeȱ showȱ thatȱ theȱ solubleȱ fractionȱ isȱ lowȱ andȱ theȱ kineticȱ ofȱ
degradationȱ isȱ slowȱ underȱ naturalȱ conditionsȱ soȱ itȱ isȱ expectedȱ toȱ beȱ
accumulatedȱ inȱ sedimentsȱ (CSICȱ 2003).ȱ Theȱ biologicalȱ effectsȱ associatedȱ withȱ
theȱ chemicalsȱ fromȱ theȱ oilȱ spillȱ willȱ beȱ dependentȱ onȱ theȱ natureȱ ofȱ theȱ
ecosystemȱthatȱacceptsȱthemȱandȱtheȱorganismsȱlivingȱinȱitȱ(DelVallsȱ2003).ȱTheȱ
firstȱresearchȱnotesȱaboutȱtheȱearlyȱimpactȱsupportȱthatȱtheȱacuteȱtoxicityȱofȱtheȱ
weatheredȱ fuelȱ (MariñoȬBalsaȱ 2003),ȱ veryȱ richȱ inȱ highȱ molecularȱ weightȱ
compounds,ȱwasȱrelativelyȱlowȱforȱtheȱorganismsȱtestedȱ(clamsȱandȱmicroalga).ȱ
Theȱ Polycyclicȱ aromaticȱ hydrocarbonsȱ (PAHs)ȱ withȱ intermediateȱ toȱ highȱ
molecularȱ weightȱ doȱ notȱ usuallyȱ showȱ severeȱ toxicityȱ withinȱ theirȱ solubilityȱ
limitsȱ inȱ water.ȱ However,ȱ itȱ isȱ necessaryȱ toȱ noteȱ thatȱ someȱ PAHsȱ canȱ becomeȱ
moreȱdangerousȱdueȱtoȱtheirȱphotomodification.ȱThisȱisȱparticularlyȱdangerousȱ
forȱorganismsȱlivingȱinȱtheȱintertidalȱzoneȱorȱnearȱtheȱwaterȱsurfaceȱ(Carballeiraȱ
2003).ȱReportedȱresponsesȱofȱinfaunaȱafterȱanȱoilȱspillȱincludeȱveryȱhighȱinitialȱ
mortalitiesȱ inȱ speciesȱ sensitiveȱ toȱ hydrocarbons,ȱ suchȱ asȱ crustaceansȱ andȱ
especiallyȱamphipods,ȱandȱtheirȱsubsequentȱdisappearanceȱ(Pearsonȱetȱal.ȱ1978;ȱ
Sandersȱetȱal.ȱ1980;ȱGlémarecȱandȱHussenotȱ1982;ȱGrayȱandȱPearsonȱ1982).ȱ
- 36 -
Sedimentȱ toxicityȱ testsȱ provideȱ informationȱ onȱ theȱ toxicityȱ ofȱ
contaminatedȱsedimentsȱthatȱcanȱbeȱneitherȱderivedȱfromȱchemicalȱanalysisȱnorȱ
fromȱecologicalȱsurveysȱperformedȱaloneȱ(ChapmanȱandȱLongȱ1983;ȱLongȱandȱ
Chapmanȱ 1985).ȱ Theȱ speciesȱ ofȱ organismsȱ usedȱ inȱ theȱ sedimentȱ toxicityȱ testsȱ
shouldȱprovideȱanȱappropriateȱindicationȱofȱtheȱhazardsȱofȱchemicalȱstressorsȱinȱ
theȱsedimentȱ(Chapmanȱetȱal.ȱ2002).ȱAmphipodsȱareȱgenerallyȱacknowledgedȱasȱ
theȱ organism’sȱ choiceȱ forȱ manyȱ sedimentȱ toxicityȱ assessments,ȱ andȱ amphipodȱ
toxicityȱ testȱ resultsȱ canȱ correlateȱ positivelyȱ withȱ changesȱ inȱ benthicȱ
communitiesȱ(Longȱetȱal.ȱ2001;ȱMarínȬGuiraoȱetȱal.ȱ2005).ȱ
TheȱaimȱofȱthisȱworkȱisȱtoȱassesȱtheȱsensibilityȱofȱtheȱamphipodȱAmpeliscaȱ
brevicornisȱtoȱtheȱcontaminationȱassociatedȱwithȱtheȱremainentȱfuelȱoilȱcollectedȱ
fromȱtheȱtankerȱPrestigeȱonȱseptemberȱ2004ȱandȱtoȱstablishȱtheȱusefulnessȱofȱtheȱ
toxicȱresponseȱmeasuredȱforȱfurtherȱmanagementȱofȱsedimentsȱcontaminatedȱbyȱ
theȱ oilȱ spillȱ andȱ inȱ generalȱ byȱ organicȱ pollutants.ȱ Inȱ orderȱ toȱ reachȱ theseȱ
objetives,ȱ aȱ bioassayȱ wasȱ conductedȱ exposingȱ aȱ populationȱ ofȱ theȱ amphipodȱ
Ampeliscaȱbrevicornisȱtoȱdifferentȱdilutionsȱofȱfuelȱoilȱwithȱcleanȱsediment.ȱ
2.ȱMaterialȱandȱmethodsȱ
2.1.ȱApproachȱ
Theȱ presentȱ studyȱ wasȱcarriedȱoutȱusingȱsixȱdifferentȱ sedimentȱdilutionȱ
ofȱ fuelȱ oil.ȱ Theȱ oilȱ usedȱ wasȱ extractedȱ fromȱ theȱ remainingȱ fuelȱ ofȱ theȱ tankerȱ
Prestigeȱ(Septemberȱ2004)ȱandȱwasȱmixedȱwithȱcleanȱsedimentȱfromȱtheȱBayȱofȱ
Cádizȱ(0.1%,ȱ0.5%,ȱ2%,ȱ8%,ȱ16%ȱandȱ32ȱ%ȱȬȱdryȱweightȱofȱfuelȱoilȬ)ȱthatȱwasȱalsoȱ
usedȱasȱnegativeȱcontrolȱ(BC).ȱCleanȱsedimentȱwasȱcollectedȱinȱaȱpristineȱareaȱofȱ
theȱBayȱofȱCádizȱ(Ribaȱetȱal.ȱ2003)ȱandȱwasȱfilteredȱ(0.6ȱmm)ȱpriorȱtoȱtheȱtoxicityȱ
testȱ inȱ orderȱ toȱ removeȱ meansȱ interferencesȱ asȱ shells,ȱ predatorsȱ andȱ otherȱ
- 37 -
residues.ȱ Theseȱ sedimentsȱ wereȱ driedȱ andȱ homogenizedȱ atȱ roomȱ temperatureȱ
priorȱtoȱchemicalȱanalysis.ȱ
IndividualsȱofȱtheȱspecieȱAmpeliscaȱbrevicornisȱusedȱinȱtheȱbioassayȱwereȱ
collectedȱfromȱtheȱcleanȱsedimentȱ(negativeȱcontrol)ȱlocatedȱinȱtheȱintermarealȱ
zoneȱofȱ theȱBayȱ ofȱ Cádiz,ȱbyȱsievingȱtheȱsedimentȱthroughȱaȱ0.6ȱmmȱ mesh,ȱasȱ
reportedȱ byȱ Ribaȱ etȱ al.ȱ (2003).ȱ Theyȱ wereȱ inmediatelyȱ transportedȱ toȱ theȱ
laboratoryȱ whereȱ theyȱ wereȱ placedȱ inȱ 11ȱ litersȱ aquariumsȱ withȱ cleanȱ seawaterȱ
andȱsievedȱsedimentȱfromȱtheȱsameȱlocation.ȱAirationȱwasȱprovidedȱandȱnaturalȱ
photoperiodȱwasȱselected.ȱDuringȱacclimatationȱtheȱorganismsȱwereȱfedȱtwiceȱaȱ
weekȱ withȱ aȱ specialȱ foodȱ forȱ invertebratesȱ (mixtureȱ madeȱ ofȱ aminoacidsȱ andȱ
organicȱparticles)ȱandȱwaterȱwasȱreplaced.ȱ
2.2.ȱChemicalȱanalysisȱofȱsedimentsȱ
Forȱ traceȱ metalȱ analysisȱ (Ni,ȱ V,ȱ Cd,ȱ Pb,ȱ Cr,ȱ Co)ȱ theȱ sedimentȱ wasȱ
digestedȱ asȱ describedȱ byȱ Loringȱ andȱ Rantalaȱ (1992).ȱ Traceȱ metalsȱ wereȱ
measuredȱ byȱ graphiteȱ furnaceȱ atomicȱ absorptionȱ spectrophotometryȱ (Perkin–
Elmerȱ4100ȱZL)ȱ(CobeloȬGarcíaȱetȱal.ȱ2005)ȱResultsȱareȱexpressedȱasȱmgkgȬ1ȱdryȱ
sediment.ȱ Theȱ analyticalȱ proceduresȱ wereȱ checkedȱ usingȱ referenceȱ materialȱ
(MESSȬ1ȱ NRCȱ andȱ CRMȱ 277ȱ BCR)ȱ andȱ showedȱ agreementȱ withȱ theȱ certifiedȱ
valuesȱhigherȱthanȱ90%.ȱ
Polycyclic aromatic hydrocarbons (Fluorene, Acenaphthene, Naphthalene,
Phenanthrene, Anthracene, Fluoranthene, Pyrene, Benzo[a]anthracene, Chrysene,
Benzofluoranthene, Benzo[e]pyrene, Benzo[a]pyrene, Perilene, Dibenzo[ah]anthracene,
Indene[123-cd]pyrene,
Benzo[ghi]perilene)
were
analyzed
by
using
a
gas
chromatography equipped with an electron capture detector (GC/MS) (U.S.
Environmental Protection Agency SW-846 Method 8270) (USEPA 1984). Briefly, dried
samples were soxhlet extracted with n-hexane for 18 h, and the extracts were isolated by
column chromatography on Florisil-alumino-silica. PAHs were eluted and their
- 38 -
fractions were dried in a rotatory evaporator and re-dissolved in isooctane. Aromatic
fractions were analyzed on a Hewlett–Pakard (HP) 5890 Series II gas chromatograph
coupled with HP 5970 mass spectrometer. Chromatographic resolution was achieved
with a 30 m × 0.250 mm DB-5 capillary column, which has a 0.25 µm film thickness,
with helium as carrier gas. Quality control was carried out using NRC-CNRC HS-6
sediment reference material. The analytical procedure allow agreement with the
certified values higher than 90%.ȱ
2.3.ȱToxicityȱtestȱ
Theȱtoxicityȱtestȱwasȱperformedȱexposingȱindividualsȱofȱtheȱamphipodsȱ
Ampeliscaȱbrevicornisȱtoȱbulkȱsedimentȱusingȱtheȱpercentageȱofȱsurvivalȱafterȱtenȱ
daysȱ ofȱ exposureȱ asȱ theȱ endȱ pointȱ (ASTMȱ 1993).ȱ Theȱ dilutionsȱ withȱ cleanȱ
sedimentȱandȱfuelȱoilȱ(0.1%,ȱ0.5%,ȱ2%,ȱ8%,ȱ16%ȱandȱ32%ȱȬdryȱweightȱofȱfuelȱoilȬ)ȱ
andȱtheȱnegativeȱcontrolȱ(200ȱg)ȱwereȱplacedȱinȱ2ȱLȱglassȱbeakersȱandȱaboutȱ800ȱ
mLȱ ofȱ cleanȱ seawaterȱ wereȱ added.ȱ Whenȱ theȱ sedimentȱ settledȱ downȱ inȱ theȱ
beakers,ȱaerationȱwasȱprovided,ȱandȱ12ȱhoursȱafterȱtheȱindividualsȱwereȱsievedȱ
fromȱtheȱacclimatizationȱaquariumsȱandȱ20ȱadultsȱ(3Ȭ5ȱmm)ȱofȱAmpeliscaȱwhereȱ
placedȱ inȱ eachȱ replicate.ȱ Noȱ foodȱ wasȱ providedȱ duringȱ theȱ experiment.ȱ Theȱ
containersȱwhereȱkeptȱinȱanȱincubatorȱwithȱphotoperiodȱ12hȬlight/12hȬdarkȱandȱ
mantainedȱ atȱ 19ȱ ±ȱ 1ȱ ºCȱ duringȱ theȱ 10ȱ daysȱ ofȱ exposure.ȱ Afterȱ thatȱ time,ȱ theȱ
beakersȱwhereȱsievedȱandȱtheȱsurvivalȱwasȱcountedȱinȱeachȱreplicate.ȱ
ȱ
2.4.ȱDataȱcalculationȱ
TheȱmortalityȱofȱAmpeliscaȱbrevicornisȱmeasuredȱafterȱ10ȱdaysȱofȱexposureȱ
timeȱ wasȱ usedȱ toȱ deriveȱ aȱ toxicȱ parameterȱ (LC50)ȱ associatedȱ withȱ theȱ fuelȱ oil.ȱ
Fromȱ theȱ toxicȱ responsesȱ (mortality)ȱ obtainedȱ duringȱ theȱ exposureȱ toȱ theȱ
differentȱ dilutionsȱ (0.1%,ȱ 0.5%,ȱ 2%,ȱ 8%,ȱ 16%ȱ andȱ 32%)ȱ wasȱ definedȱ theȱ
concentrationȱ(percentageȱofȱdryȱweightȱofȱfuelȱoil)ȱthatȱprovokesȱtheȱmortalityȱ
- 39 -
ofȱ theȱ 50%ȱ ofȱ theȱ Ampeliscaȱ brevicornisȱ populationȱ exposed.ȱ Theȱ LC50ȱ wasȱ
calculatedȱbyȱlinearȱregressionsȱofȱlogȱtoxicantȱdilutionȱofȱfuelȱoilȱonȱdecliningȱ
probitȱvaluesȱ(probitȬanalysisȬprogram,ȱversionȱ1.5).ȱȱ
Sedimentȱqualityȱvaluesȱ(SQVs)ȱhaveȱbeenȱcalculatedȱinȱorderȱtoȱidentifyȱ
theȱ concentrationȱ ofȱ PAHsȱ (Fluorene,ȱ Acenaphthene,ȱ Naphthalene,ȱ
Phenanthrene,ȱ
Anthracene,ȱ
Fluoranthene,ȱ
Pyrene,ȱ
Benzo[a]anthracene,ȱ
Chrysene,ȱ Benzofluoranthene,ȱ Benzo[e]pyrene,ȱ Benzo[a]pyrene,ȱ Perilene,ȱ
Dibenzo[ah]anthracene,ȱ Indene[123Ȭcd]pyrene,ȱ Benzo[ghi]perilene)ȱ responsibleȱ
ofȱtheȱtoxicityȱassociatedȱwithȱtheȱfuelȱoilȱfromȱtheȱPrestigeȱspill.ȱTheȱSQVsȱhaveȱ
beenȱ calculatedȱ usingȱ theȱ LC50ȱ valuesȱ obtainedȱ andȱ theȱ concentrationȱ ofȱ
individualsȱ PAHsȱ measuredȱ inȱ theȱ fuelȱ oil.ȱ Thus,ȱ theȱ SQVsȱ areȱ definedȱ asȱ theȱ
concentrationȱofȱindividualȱandȱtotalȱPAHsȱassociatedȱwithȱtheȱmortalityȱofȱ50%ȱ
ofȱtheȱtotalȱpopulationȱofȱamphipodsȱafterȱ10ȱdaysȱofȱexposureȱtoȱfuelȱoil.ȱȱȱȱ
3.ȱResultsȱ
Tableȱ1ȱshowsȱsummarizedȱresultsȱofȱcontaminantsȱȬtotalȱandȱindividualȱ
PAHsȱ andȱ traceȱ metalsȱ (Ni,ȱ V,ȱ Cd,ȱ Pb,ȱ Cr,ȱ Co)ȱ expressedȱ asȱ mgȱ KgȬ1ȱ dryȱ
sedimentȬȱthatȱwereȱmeasuredȱinȱtheȱdilutionsȱofȱfuelȱoilȱȱ(0.1%,ȱ0.5%,ȱ2%,ȱ8%,ȱ
16%ȱandȱ32%ȱȬdryȱweightȱofȱfuelȱoilȬ)ȱandȱinȱtheȱnegativeȱcontrolȱ(BC).ȱ
Theȱ pureȱ fuelȱ oilȱ extractedȱ fromȱ theȱ tankerȱ Prestigeȱ (Septemberȱ 2004)ȱ
presentsȱaȱconcentrationȱofȱtotalȱPAHsȱofȱ1443ȱmgȱKgȬ1ȱȬdryȱweightȬ,ȱwhereasȱinȱ
theȱnegativeȱcontrolȱ(BC)ȱPAHsȱwereȱnotȱdetected;ȱtherefore,ȱtheȱconcentrationȱ
ofȱPAHsȱinȱtheȱdilutionsȱdependsȱonlyȱinȱtheȱpresenceȱofȱPAHsȱinȱtheȱfuelȱoil.ȱ
Regardingȱtoȱtheȱcontentȱofȱtraceȱmetalsȱinȱtheȱfuelȱoilȱresultsȱshowȱthatȱlevelsȱofȱ
theseȱcontaminantsȱareȱnotȱhighȱexceptȱforȱNiȱ(55ȱmgȱkgȬ1)ȱandȱVȱ(170mgȱkgȬ1).ȱȱ
- 40 -
ItȱasȱbeenȱobservedȱthatȱtheȱindividualsȱPAHsȱpredominantsȱinȱtheȱfuelȱ
oilȱareȱNaphtaleneȱ(395ȱmgȱkgȬ1),ȱPhenantreneȱ(385ȱmgȱkgȬ1),ȱPyreneȱ(111ȱmgȱkgȬ
1
),ȱChryseneȱ(mgȱkgȬ1),ȱFluoreneȱ(99.3ȱmgȱkgȬ1)ȱandȱAcenaphtheneȱ(75.3ȱmgȱkgȬ1).ȱȱ
Tableȱ1.ȱTotalȱPAHsȱandȱmetalȱconcentrationȱinȱmgȱKgȬ1ȱȬdryȱsedimentȬȱ
measuredȱinȱtheȱnegativeȱcontrolȱandȱinȱtheȱfuelȱoilȱdilutions.ȱ
ȱ
ȱȱ
Oilȱ
BCȱ
0.10%ȱ
0.50%ȱ
2%ȱ
8%ȱ
16%ȱ 32%
PAHsȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
TotalȱPAHsȱ
Fluoreneȱ
Acenaphtheneȱ
Naphthaleneȱ
Phenanthreneȱ
Anthraceneȱ
Fluorantheneȱ
Pyreneȱ
Benzo[a]anthraceneȱ
Chryseneȱ
Benzofluorantheneȱ
Benzo[e]pyreneȱ
Benzo[a]pyreneȱ
Perileneȱ
Dibenzo[ah]anthraceneȱ
Indene[123Ȭcd]pyreneȱ
Benzo[ghi]perileneȱ
1443ȱ
99.3ȱ
75.3ȱ
395ȱ
385ȱ
51.4ȱ
28.5ȱ
111ȱ
55.9ȱ
102ȱ
16.0ȱ
45.7ȱ
29.7ȱ
11.4ȱ
5.70ȱ
5.23ȱ
17.1ȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
n.dȱ
0.72ȱ
0.05ȱ
0.04ȱ
0.20ȱ
0.19ȱ
0.03ȱ
0.01ȱ
0.06ȱ
0.03ȱ
0.05ȱ
0.01ȱ
0.02ȱ
0.01ȱ
0.01ȱ
0.00ȱ
0.00ȱ
0.01ȱ
3.61ȱ
0.25ȱ
0.19ȱ
0.99ȱ
0.96ȱ
0.13ȱ
0.07ȱ
0.28ȱ
0.14ȱ
0.22ȱ
0.04ȱ
0.11ȱ
0.07ȱ
0.03ȱ
0.01ȱ
0.01ȱ
0.04ȱ
14.4ȱ
0.99ȱ
0.75ȱ
3.95ȱ
3.85ȱ
0.51ȱ
0.29ȱ
1.11ȱ
0.56ȱ
9.98ȱ
0.16ȱ
0.46ȱ
0.30ȱ
0.11ȱ
0.06ȱ
0.04ȱ
0.17ȱ
57.7ȱ
3.97ȱ
3.01ȱ
15.8ȱ
15.4ȱ
2.05ȱ
1.14ȱ
4.43ȱ
2.24ȱ
3.63ȱ
0.64ȱ
1.83ȱ
1.19ȱ
0.46ȱ
0.23ȱ
0.16ȱ
0.68ȱ
115ȱ
7.95ȱ
6.03ȱ
31.6ȱ
30.8ȱ
4.11ȱ
2.28ȱ
8.86ȱ
4.48ȱ
7.12ȱ
1.28ȱ
3.65ȱ
2.37ȱ
0.91ȱ
0.46ȱ
0.32ȱ
1.37ȱ
231ȱ
15.9
12.1
63.2
61.6
8.22
4.57
17.7
8.95
14.3
2.56
7.31
4.75
1.83
0.91
0.65
2.74
Metalsȱ
ȱ
ȱ
ȱ
ȱ
ȱ
Niȱ
Vȱ
Cdȱ
Pbȱ
Crȱ
Coȱ
55ȱ
170ȱ
n.dȱ
n.dȱ
0.31ȱ
n.dȱ
14.1
80.0
n.dȱ
23.0
31.0
3.40
7.07ȱ
40.1ȱ
n.dȱ
11.5ȱ
15.5ȱ
1.70ȱ
7.15ȱ
40.2ȱ
n.dȱ
11.4ȱ
15.4ȱ
1.69ȱ
7.46ȱ
40.9ȱ
n.dȱ
11.3ȱ
15.2ȱ
1.67ȱ
8.69ȱ
43.6ȱ
n.dȱ
10.6ȱ
14.3ȱ
1.56ȱ
10.3ȱ
47.2ȱ
n.dȱ
9.7ȱ
13.0ȱ
1.43ȱ
13.6
54.4
n.dȱ
7.80
10.6
1.16
Figureȱ1ȱshowsȱsummarizedȱresultsȱofȱtheȱamphipodsȱmortalityȱ(%)ȱafterȱ
10ȱdaysȱofȱexposureȱtoȱtheȱdifferentȱdilutionsȱofȱfuelȱoil.ȱMeanȱsurvivalȱinȱallȱtheȱ
replicatesȱ ofȱ cleanȱ sedimentȱ fromȱ theȱ Bayȱ ofȱ Cádizȱ (control)ȱ wasȱ higherȱ thanȱ
95%.ȱ Theȱ mortalityȱ ofȱ Ampeliscaȱ increasesȱ withȱ theȱ %ȱ ofȱ theȱ fuelȱ oilȱ inȱ theȱ
dilutions.ȱ Resultsȱ obtainedȱ atȱ theȱ lowestȱ dilutionȱ ofȱ fuelȱ oilȱ (0.1%)ȱ showȱ aȱ
survivalȱ rangedȱ betweenȱ 80Ȭ100%ȱ duringȱ theȱ tenȱ daysȱ ofȱ exposure.ȱ Allȱ
- 41 -
replicatesȱ inȱ theȱ concentrationȱ ofȱ 0.5%ȱ ofȱ fuelȱ oilȱ indicateȱ aȱ survivalȱ ofȱ 80%ȱ inȱ
thisȱdilution.ȱ Theȱ mortalityȱregisteredȱforȱtheȱdilutionȱ ofȱ2%ȱofȱfuelȱoilȱrangedȱ
betweenȱ70Ȭ100%ȱwhereasȱnoȱsurvivalȱwasȱdetectedȱinȱtheȱhighestȱdilutionsȱofȱ
%ȱMortality
fuelȱoilȱ(8%,ȱ16ȱ%ȱandȱ32%)ȱafterȱtheȱexposureȱperiod.ȱȱ
150
100
50
0
BC
0.1%
0.5%
2%
8%
16%
32%
Dilutions
ȱ
Figureȱ1.ȱAverageȱandȱstandardȱdeviationsȱofȱtheȱpercentageȱofȱmortalityȱ
ofȱ theȱ amphipodȱ Ampeliscaȱ brevicornisȱ afterȱ 10ȱ daysȱ ofȱ exposureȱ toȱ eachȱ
dilutionȱofȱfuelȱoil.ȱȱ
Fromȱ theȱ mortalityȱ obtainedȱ duringȱ theȱ exposureȱ ofȱ theȱ amphipodsȱ toȱ
theȱ differentȱ dilutionsȱ itȱ hasȱ beenȱ calculatedȱ theȱ percentageȱ ofȱ dryȱ weightȱ ofȱ
fuelȱ oilȱ thatȱ provokesȱ theȱ mortalityȱ ofȱ theȱ 50%ȱ ofȱ theȱ Ampeliscaȱ brevicornisȱ
populationȱ exposed.ȱ Theȱ lethalȱ concentrationȱ (LC50)ȱ ofȱ dryȱ weightȱ ofȱ fuelȱ oilȱ
associatedȱ withȱ theȱ toxicȱ responsesȱ calculatedȱ inȱ Ampeliscaȱ brevicornisȱ isȱ
1.37±0.33ȱ(percentageȱofȱdryȱweightȱofȱfuelȱoil).ȱ
Theseȱ LC50ȱ valueȱ obtainedȱ forȱ Ampeliscaȱ brevicornisȱ andȱ theȱ PAHsȱ
concentrationsȱ measuredȱ inȱ theȱ fuelȱ oilȱ andȱ inȱ theȱ negativeȱ controlȱ (BC)ȱ haveȱ
permittedȱ toȱ calculateȱ theȱ ȱ Sedimentȱ Qualityȱ Valuesȱ (SQVs)ȱ shownȱ inȱ tableȱ 2.ȱ
TheȱSQVsȱobtainedȱforȱtheȱdifferentȱcontaminantsȱareȱcomparedȱtoȱotherȱvaluesȱ
- 42 -
ȱȱ
PAHsȱ(ΐgȱkgȬ1)ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
Metals(mgȱkgȬ1)ȱ
ȱ
ȱ
ȱ
ȱ
ȱȱ
ȱȱ
TotalȱPAHsȱ
Fluoreneȱ
Acenafphtheneȱ
Naphthaleneȱ
Phenanthreneȱ
Anthraceneȱ
Fluorantheneȱ
Pyreneȱ
Benzo[a]anthraceneȱ
Chryseneȱ
Benzofluorantheneȱ
Benzo[e]pyreneȱ
Benzo[a]pyreneȱ
Perileneȱ
Dibenzo[ah]anthracene
Indene[123Ȭcd]pyreneȱ
Benzo[ghi]perileneȱ
Niȱ
Vȱ
Cdȱ
Pbȱ
Crȱ
Coȱ
Ampeliscaȱbrevicornisȱ
19790.7ȱ
1362ȱ
1033ȱ
5417ȱ
5276ȱ
705ȱ
391ȱ
1519ȱ
767ȱ
1398ȱ
219ȱ
626ȱ
407ȱ
157ȱ
78ȱ
71.7ȱ
235ȱ
7.3ȱ
40.6ȱ
0.0ȱ
11.3ȱ
15.3ȱ
1.7ȱ
ERM PugetȱSoundȱAET CommencementȱBay
44792
11752±14548ȱ
16771ȱ
540ȱ
1000ȱ
707±1341ȱ
500ȱ
2000ȱ
654±1049ȱ
2100ȱ
2400ȱ
1564±1735ȱ
1500ȱ
5400ȱ
2838±4603ȱ
1100ȱ
1900ȱ
476.2±549.2ȱ
5100ȱ
n.a.ȱ
n.a.ȱ
2600ȱ
4300ȱ
1820±2252ȱ
1600ȱ
1600ȱ
931±1322.8ȱ
2800ȱ
2800ȱ
1363±1970ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
1600ȱ
2400ȱ
890±1322ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
260ȱ
260ȱ
183±344ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
51.6ȱ
n.a.ȱ
41±32ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
9.6ȱ
93ȱ
63.2±148ȱ
218ȱ
660ȱ
170.8±192ȱ
370ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
- 43 -
ERL
4022
19ȱ
16ȱ
160ȱ
240ȱ
85ȱ
600ȱ
665ȱ
261ȱ
384ȱ
n.a.ȱ
n.a.ȱ
430ȱ
n.a.ȱ
63ȱ
n.a.ȱ
n.a.ȱ
20.9
n.a.ȱ
1.2ȱ
46.7
81ȱ
n.a.ȱ
CommencementȱBay,ȱTetraȱTechȱ(1985);ȱSanȱFranciscoȱBay,ȱLongȱetȱal.ȱ(1989)).
SanȱFranciscoȱBayȱȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
n.a.ȱ
510ȱ
1100ȱ
n.a.ȱ
2600ȱ
1100ȱ
2100ȱ
n.a.ȱ
n.a.ȱ
432±344ȱ
n.a.ȱ
80±88ȱ
n.a.ȱ
n.a.ȱ
99±35ȱ
n.a.ȱ
70ȱ
95.7±93ȱ
141.8±86.5ȱ
n.a.ȱ
dataȱ(ERL=ȱEffectsȱRangeȬLowȱandȱERM=ȱEffectsȱRangeȬMedian,ȱNOAAȱ(1999);ȱPugetȱSoundȱAET,ȱSwartzȱetȱal.ȱ(1985,ȱ1986);ȱ
Ampeliscaȱ brevicornisȱ usedȱ inȱ theȱ sedimentȱ toxicityȱ test.ȱ Sedimentȱ Qualityȱ Guidelinesȱ wereȱ derivedȱ usingȱ previousȱ studiesȱ
ȱTableȱ 2.ȱ Sedimentȱ qualityȱ valuesȱ forȱ PAHsȱ (ΐgȱ kgȬ1ȱ –dryȱ weightȬ)ȱ areȱ obtainedȱ fromȱ theȱ LC50ȱ andȱ calculatedȱ forȱ theȱ
proposedȱ byȱ previousȱ studiesȱ (NOAAȱ 1999)ȱ whichȱ showȱ aȱ compilationȱ ofȱ
SedimentȱQualityȱGuidelinesȱthatȱhaveȱbeenȱcalculatedȱbasedȱonȱaȱwideȱrangedȱ
dataȱbaseȱ(LongȱandȱMorganȱ1991;ȱLongȱetȱal.ȱ1995).ȱNOAAȱ(1999)ȱexplainsȱtheȱ
10thȱ percentileȱ valuesȱ namedȱ theȱ ERLȱ (Effectsȱ RangeȬLow)ȱ asȱ theȱ
concentrationsȱ belowȱ whichȱ adverseȱ effectsȱ rarelyȱ occur,ȱ whereasȱ theȱ 50thȱ
percentilesȱ namedȱ ERMȱ (Effectsȱ RangeȬMedian)ȱ values,ȱ areȱ representativeȱ ofȱ
concentrationsȱ aboveȱ whichȱ effectsȱ frequentlyȱ occurȱ (NOAAȱ 1999).ȱ ȱ Someȱ
examplesȱfromȱSQVsȱobtainedȱfromȱpreviousȱstudiesȱ(tableȱ2)ȱwithȱamphipodsȱȬ
Pugetȱ Soundȱ AET,ȱ Commencementȱ Bayȱ andȱ Sanȱ Franciscoȱ BayȬȱ (Longȱ andȱ
Morganȱ 1991)ȱ areȱ similarȱ toȱ thoseȱ recordedȱ withȱ theȱ presentȱ studyȱ forȱ A.ȱ
brevicornis.ȱȱ
4.ȱDiscussionȱ
Resultsȱ obtainedȱ fromȱ theȱ chemicalȱ analysisȱ showȱ aȱ contentȱ ofȱ totalȱ
PAHsȱ ofȱ 1443ȱ mgȱ KgȬ1ȱ Ȭdryȱ weightȬȱ fromȱ whichȱ moreȱ thanȱ 27%ȱ isȱ Naphtaleneȱ
(395ȱmgȱKgȬ1ȱ ȬdryȱweightȬ).ȱPreviousȱstudiesȱ(AlbaigésȱandȱBayonaȱ2003)ȱshowȱ
thatȱ Naphtaleneȱ mayȱ significantlyȱ accumulateȱ inȱ theȱ biotaȱ andȱ thatȱ
concentrationsȱ ofȱ Naphtaleneȱ inȱ sedimentsȱ higherȱ thanȱ 34.6ȱ mgȱ KgȬ1ȱ Ȭdryȱ
weightȬ)ȱ mayȱ produceȱ negativeȱ effectsȱ onȱ theȱ benthicȱ organisms.ȱ Specifically,ȱ
oilsȱ spillsȱ suchȱ asȱ theȱ Seaȱ Empressȱ haveȱ involvedȱ aȱ declineȱ inȱ theȱ amphipodȱ
faunaȱ withȱ theȱ generaȱ Ampeliscaȱ particularlyȱ affectedȱ (Nikitikȱ andȱ Robinsonȱ
2003).Theȱ LC50ȱ obtainedȱ fromȱ theȱ acuteȱ bioassayȱ usingȱ Ampeliscaȱ brevicornisȱ
showsȱthatȱtheȱfuelȱoilȱisȱtoxicȱatȱconcentrationsȱequalȱorȱlowerȱthanȱ1.37%.ȱThisȱ
valueȱ andȱ theȱ concentrationȱ ofȱ chemicalsȱ onȱ theȱ fuelȱ oilȱ andȱ cleanȱ sediment,ȱ
allowsȱcalculatingȱtheȱSedimentȱQualityȱValuesȱ(SQVs)ȱforȱeachȱcontaminant.ȱ
Resultsȱ ofȱ SQVsȱ forȱ metalsȱ areȱ lowerȱ thanȱ theȱ NOAAȱ guidelinesȱ whatȱ
explainsȱthatȱtheȱtoxicityȱitȱisȱnotȱprobablyȱassociatedȱwithȱtheȱconcentrationȱofȱ
- 44 -
metalsȱinȱtheȱfuelȱoilȱdilutions.ȱOnȱtheȱotherȱhandȱPAHsȱseemȱtoȱbeȱresponsibleȱ
forȱtheȱacuteȱtoxicityȱmeasuredȱatȱtheȱendȱofȱtheȱtestȱandȱtheȱSQVsȱobtainedȱforȱ
totalȱ PAHsȱ (19790.7ȱ ΐgȱ kgȬ1)ȱ areȱ inȱ theȱ sameȱ rangeȱ thanȱ thoseȱ previouslyȱ
reportedȱ forȱ theseȱ contaminantsȱ byȱ differentȱ authorsȱ (4022ȱ ΐgȱ kgȬ1ȱ (ERL)ȱ –ȱȱ
44792ȱ ΐgȱ kgȬ1ȱ (ERM)ȱ (NOAAȱ 1999);ȱ 11752±14548ȱ ΐgȱ kgȬ1ȱ ȱ (Pugetȱ Soundȱ AET)ȱ
(Swartzȱ etȱ al.ȱ 1985;ȱ Swartzȱ etȱ al.ȱ 1986);ȱ 16771ȱ ΐgȱ kgȬ1ȱ ȱ (Commencementȱ Bay)ȱ
(Tetraȱ Techȱ 1985)).ȱ Mostȱ ofȱ theȱ SQVsȱ ofȱ individualsȱ PAHsȱ calculatedȱ inȱ theȱ
presentȱ studyȱ surpassȱ theȱ rangesȱ orȱ theyȱ areȱ inȱ theȱ sameȱ rangeȱ asȱ theȱ SQGsȱ
recordedȱinȱpreviousȱstudiesȱ(NOAAȱ1999;ȱSwartzȱetȱal.ȱ1985;ȱSwartzȱetȱal.ȱ1986;ȱ
TetraȱTechȱ1985;ȱLongȱandȱBuchmanȱ1989).ȱNaphtaleneȱshowsȱtheȱhighestȱvalueȱ
ofȱSQVȱofȱtheȱtotalȱPAHsȱ(5417ȱΐgȱkgȬ1)ȱwhichȱisȱhigherȱthanȱtheȱdataȱrecordedȱ
inȱ previousȱ studiesȱ (2400ȱ ΐgȱ kgȬ1ȱ (Pugetȱ Soundȱ AET);ȱ 1564±1735ȱ ΐgȱ kgȬ1ȱ
(CommencentȱBay);ȱ160ȱΐgȱkgȬ1ȱ(ERL)ȱȬȱ2100ȱΐgȱkgȬ1ȱ(ERM)(NOAAȱ1999)).ȱTheȱ
SQVȱcalculatedȱfromȱtheȱA.ȱbrevicornisȱtoxicityȱtestȱforȱFluoreneȱ(1362ȱΐgȱkgȬ1),ȱisȱ
alsoȱhigherȱthanȱthoseȱSQGsȱfromȱotherȱstudiesȱasȱitȱisȱshownȱinȱtableȱ2,ȱbutȱitȱisȱ
closedȱtoȱtheȱvalueȱcalculatedȱforȱCommencentȱBayȱ(707±1341ΐgȱkgȬ1)ȱandȱPugetȱ
Soundȱ AETȱ (1000ΐgȱ kgȬ1)ȱ forȱ thisȱ individualȱ PAH.ȱ Acenaphtheneȱ presentsȱ aȱ
SQVȱ(1033ȱΐgȱkgȬ1)ȱclosedȱtoȱtheȱSQGȱforȱCommencementȱBayȱ(654±1049ȱΐgȱkgȬ
1
).ȱ Theȱ SQVȱ calculatedȱ forȱ theȱ individualȱ PAHȱ Phenanthreneȱ (5276ȱ ΐgȱ kgȬ1)ȱ isȱ
closedȱ toȱ thoseȱ SQGsȱ fromȱ Commencentȱ Bayȱ (2838±46031ΐgȱ kgȬ1)ȱ andȱ Pugetȱ
SoundȱAETȱ(5400ΐgȱkgȬ1)ȱwhereasȱtheȱSQVȱforȱAnthraceneȱderivedȱfromȱtheȱA.ȱ
brevicornisȱtoxicityȱtestȱ(705ȱΐgȱkgȬ1)ȱisȱinȱtheȱrangeȱofȱtheȱsameȱvaluesȱproposedȱ
byȱ NOAAȱ (1999)ȱ (85ȱ ΐgȱ kgȬ1ȱ (ERL)ȱ Ȭȱ 1100ȱ ΐgȱ kgȬ1)ȱ andȱ itȱ isȱ closedȱ toȱ theȱ SQGȱ
derivedȱforȱSanȱFranciscoȱBayȱ(1100ΐgȱkgȬ1).ȱSimilarȱcomparisonsȱȱareȱforȱSQVsȱ
ofȱ
Pyreneȱ
(1519ΐgkgȬ1),ȱ
Benzo[a]anthraceneȱ
(767ȱ
ΐgȱ
kgȬ1)ȱ
andȱ
Dibenzo[ah]anthracene(78ȱΐgȱkgȬ1)ȱwhichȱareȱinȱtheȱrangeȱofȱNOAAȱ(1999)ȱ(665ȱ
ΐgȱkgȬ1ȱ(ERL)ȱȬȱ2600ȱΐgȱkgȬ1;ȱȱ261ȱΐgȱkgȬ1ȱ(ERL)ȱȬȱ1600ȱΐgȱkgȬ1;ȱ63ȱΐgȱkgȬ1ȱ(ERL)ȱȬȱ
- 45 -
260ȱΐgȱkgȬ1,ȱrespectively),ȱwhereasȱBenzo[a]anthraceneȱisȱalsoȱclosedȱtoȱtheȱSQGȱ
inȱSanȱFranciscoȱBayȱ(1100ΐgȱkgȬ1).ȱTheȱSQVȱobtainedȱforȱChryseneȱ(1519ȱΐgȱkgȬ
1
)ȱisȱquiteȱclosedȱtoȱtheȱSQGȱdeterminedȱȱforȱCommencementȱBayȱ(1363±1970ȱΐgȱ
kgȬ1)ȱandȱisȱinȱtheȱrangeȱdeterminedȱbyȱNOAAȱ(1999)ȱ(ȱ384ȱΐgȱkgȬ1ȱ(ERL)ȱȬȱ2800ȱ
ΐgȱ kgȬ1).ȱ Theȱ dataȱ obtainedȱ forȱ Fluorantheneȱ (391ΐgȱ kgȬ1)ȱ andȱ Benzo[a]pyreneȱ
(407ΐgȱ kgȬ1)ȱ areȱ lowerȱ thanȱ thoseȱ obtainedȱ inȱ previousȱ studiesȱ (tableȱ 2),ȱ
althoughȱtheȱSQVȱofȱBenzo[a]pyreneȱisȱclosedȱtoȱtheȱERLȱofȱNOAAȱ(1999)ȱ(430ȱ
ΐgȱkgȬ1).ȱȱ
Fromȱ theȱ comparisonsȱ carriedȱ out,ȱ weȱ canȱ sayȱ thatȱ theȱ SQVsȱ obtainedȱ
fromȱ theȱ A.ȱ brevicornisȱ acuteȱ toxicityȱ test,ȱ areȱ similarȱ toȱ thoseȱ reportedȱ inȱ
previousȱstudiesȱforȱtheȱsameȱcontaminants.ȱThisȱcorroboratesȱtheȱfactȱthatȱtheȱ
toxicityȱ measuredȱ inȱ theȱ testȱ isȱ dueȱ toȱ theȱ presenceȱ ofȱ PAHsȱ inȱ theȱ fuelȱ oil.ȱ
Benzofluoranthenes,ȱ Perilene,ȱ Dibenzo[ah]anthracene,ȱ Indene[123Ȭcd]pyreneȱ
andȱ Benzo[ghi]perileneȱ showȱ theȱ lowestȱ SQVsȱ obtainedȱ fromȱ theȱ experimentȱ
whatȱmeansȱthatȱtheseȱPAHsȱareȱpotentiallyȱtheȱmostȱtoxicȱagreeingȱwithȱotherȱ
studiesȱ (NOAAȱ 1999;ȱ Swartzȱ etȱ al.ȱ 1985;ȱ Swartzȱ etȱ al.ȱ 1986;ȱ Tetraȱ Techȱ 1985;ȱ
Longȱ andȱ Buchmanȱ 1989;ȱ Batelleȱ 2000;ȱ Leeȱ etȱ al.ȱ 2001).ȱ Howeverȱ theseȱ higherȱ
molecularȱweightȱPAHsȱprobablyȱareȱtightlyȱboundȱtoȱparticlesȱwhatȱdecreasesȱ
theirȱbioavailability.ȱ
Furtherȱ studiesȱ wouldȱ beȱ needȱ toȱ establishȱ theȱ toxicityȱ effectȱ ofȱ eachȱ ofȱ
theȱ individualȱ PAHsȱ presentedȱ inȱ theȱ fuelȱ oilȱ ofȱ theȱ tankerȱ Prestige,ȱ speciallyȱ
whenȱ theyȱ reachȱ theȱ environmentȱ whereȱ theyȱ canȱ sufferȱ changesȱ inȱ theȱ
proportionȱ ofȱ individualsȱ PAHsȱ andȱ becomeȱ moreȱ dangerousȱ dueȱ toȱ
photomodificationȱ(DelVallsȱ2003;ȱCarballeiraȱ2003).ȱPreviousȱstudiesȱ(Pelletierȱ
etȱal.ȱ1997;ȱPelletierȱetȱal.ȱ2000)ȱhaveȱshownȱthatȱAnthracene,ȱFlourantheneȱandȱ
Pyreneȱincreaseȱtheirȱtoxicityȱonȱlarvaeȱandȱembryosȱofȱmarineȱinvertebrates.ȱInȱ
thisȱsense,ȱstudiesȱcarriedȱoutȱwithȱenvironmentalȱsedimentȱsamplesȱaffectedȱbyȱ
- 46 -
theȱ oilȱ spillȱ willȱ beȱ ableȱ toȱ establishȱ theȱ toxicologicalȱ effectȱ ofȱ theȱ individualsȱ
PAHsȱonceȱtheyȱhaveȱbeenȱmodifiedȱbyȱtheȱenvironmentalȱconditionsȱbeingȱtheȱ
workȱhereȱ presentedȱ aȱfirstȱstepȱtoȱaddressȱtheȱpotentialȱadverseȱeffectsȱofȱtheȱ
contaminantsȱ presentedȱ inȱ theȱ originalȱ fuelȱ whenȱ dilutedȱ withȱ naturalȱ andȱ
cleanȱlittoralȱsediments.ȱȱȱ
5.ȱConclusionsȱȱ
Previousȱstudiesȱ(Ribaȱetȱal.ȱ2003)ȱhaveȱshownȱthatȱtheȱamphipodȱspecieȱ
Ampeliscaȱbrevicornisȱisȱaȱsensitiveȱorganismȱvalidȱtoȱassessȱtoxicityȱofȱsedimentsȱ
contaminatedȱ byȱ traceȱ metals.ȱ Resultsȱ obtainedȱ inȱ thisȱ studyȱ showȱ thatȱ thisȱ
specieȱ canȱ beȱ alsoȱ usedȱ toȱ assessȱ sedimentȱ toxicityȱ associatedȱ withȱ samplesȱ
contaminatedȱ byȱ PAHs,ȱ andȱ sedimentȱ bioassaysȱ withȱ Ampeliscaȱ brevicornisȱ
couldȱ beȱ includedȱ inȱ theȱ assessmentȱ ofȱ oilȱ spillsȱ toȱ determineȱ acuteȱ toxicityȱ
responsesȱandȱtoȱderiveȱSedimentȱQualityȱGuidelines.ȱȱ
Inȱthisȱsense,ȱtheȱamphipodȱAmpeliscaȱbrevicornisȱhasȱbeenȱvalidatedȱasȱanȱ
appropriateȱ organismȱ toȱ conductȱ sedimentȱ toxicityȱ testsȱ withȱ metalsȱ andȱ
hydrocarbonsȱ contaminatedȱ sedimentsȱ andȱ makesȱ thisȱ specieȱ suitableȱ forȱ theȱ
assessmentȱ ofȱ hazardousȱ materialsȱ thatȱ mayȱ accumulateȱ inȱ sedimentsȱ andȱ
produceȱeffectsȱonȱtheȱbiota.ȱ
6.ȱAknowledgmentsȱ
Theȱ workȱ describedȱ wasȱ partlyȱ supportedȱ byȱ theȱ projectsȱ VEM2003Ȭ
20563/INTER,ȱ andȱ CTM2005Ȭ07282ȬC03Ȭ01/TECNOȱ financedȱ byȱ theȱ Spanishȱ
Educationȱ andȱ Scienceȱ Ministryȱ andȱ byȱ CISȱ fundedȱ byȱ theȱ Ministryȱ ofȱ
Environment.ȱ Carmenȱ MoralesȬCasellesȱ thanksȱ theȱ Ministryȱ ofȱ Educationȱ andȱ
Scienceȱ forȱ fundingȱ herȱ researchȱ fellowshipȱ (FPU).ȱ Dr.ȱ Ribaȱ thanksȱ theȱ I3pȱ
- 47 -
programȱ forȱ supportingȱ herȱ participationȱ inȱ thisȱ research.ȱ ȱ Inmaculadaȱ Ribaȱ
thanksȱ theȱ CSICȱ forȱ herȱ I3Pȱ contract.ȱ Weȱ thankȱ theȱ supportȱ andȱ helpȱ ofȱ theȱ
membersȱofȱtheȱCIS.ȱ
7.ȱReferencesȱȱȱȱ
Albaigésȱ J,ȱ Bayonaȱ JM.ȱ 2003.ȱ Laȱ Huellaȱ delȱ Fuel,ȱ in:ȱ Ensayosȱ sobreȱ elȱ <<Prestige>>.ȱ
(Eds)ȱFundaciónȱSantiagoȱReyȱFernándezȬLaTorre,ȱAȱCoruñaȱ(Spain)ȱ
AmericanȱSocietyȱforȱTestingȱandȱMaterials.ȱ1993.ȱStandardȱguideȱforȱconductingȱ10Ȭ
dayȱ staticȱ sedimentȱ toxicityȱ testsȱ withȱ theȱ marineȱ andȱ estuarineȱ amphipods.ȱ
Publ.ȱE.ȱPhiladelphia,ȱ1367Ȭ1392ȱȱ
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Environ Monit Assess
DOI 10.1007/s10661-007-9926-5
Using the polychaete Arenicola marina to determine toxicity
and bioaccumulation of PAHS bound to sediments
Carmen Morales-Caselles & Julia Ramos &
Inmaculada Riba & T. Ángel DelValls
Received: 27 February 2007 / Accepted: 27 August 2007
# Springer Science + Business Media B.V. 2007
Abstract The present study was conducted to evaluate
a sediment toxicity and bioavailability test with the
polychaete Arenicola marina as a potential tool to
assess sediments contaminated by oil spills. A bioassay
using the lugworm Arenicola marina was carried out
in order to determine toxicity and bioaccumulation
associated with the contaminants present in the fuel oil
extracted from a sank tanker. After 10 and 21 days of
exposure to sediments with different proportions of
fuel oil (0.5, 1, 2, 4 and 8%) polychaetes were sampled
to determine the mortality and the levels of individual
PAHs in the organisms. During the experiment,
mortality was recorded and the concentration (percentage of fuel oil) that provokes the mortality of the 50%
of the Arenicola marina population exposed was
calculated for both sampling dates (LC50(10)=6.4%;
LC50(21)=2.4%). Bioaccumulation was mainly produced for fluoranthene, pyrene, benzo(b)fluoranthene
and benzo(k)fluoranthene, whereas phenantrene and
anthracene where initially accumulated and then
metabolized. The results obtained in the present study
suggest Arenicola marina can be a suitable species for
assessing PAHs toxicity and bioaccumulation as part of
oil spill management.
Keywords LC50 . Quality values . Sediment toxicity .
Oil spill
Introduction
C. Morales-Caselles : I. Riba
Instituto de Ciencias Marinas de Andalucía (CSIC),
Polígono Río San Pedro s/n.,
11510 Puerto Real (Cádiz), Spain
The biological effects associated with the chemicals
from the oil spill depend on the nature of the affected
ecosystem (DelValls 2003). Petroleum can adversely
affect organisms by physical action (smothering,
reduced light), habitat modification (altered pH,
decreased dissolved oxygen, decreased food availability) and toxic action (Albers 2003). The remaining
fuel from an important oil spill in the North of Spain
(Prestige, 2002) that was still in the tanker was
eventually extracted in 2004; the composition of this
heavy fuel-oil (type M-100) was a mixture of
saturated hydrocarbons, aromatic hydrocarbons, resins and asphaltenes, being most of the polycyclic
aromatic hydrocarbons – PAHs – of an intermedium-
J. Ramos : I. Riba : T. Á. DelValls
UNESCO/UNITWIN/Wicop, Facultad de Ciencias
del Mar y Ambientales, Universidad de Cádiz,
Polígono Río San Pedro s/n,
11510 Puerto Real (Cádiz), Spain
C. Morales-Caselles (*) : T. Á. DelValls
Unidad Asociada Universidad de Cádiz-Calidad
Ambiental y Patología (UCA-CSIC),
Avenida Saharaui s/n,
11510 Puerto Real (Cádiz), Spain
e-mail: carmen.morales@uca.es
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Environ Monit Assess
high molecular weight (Alzaga et al. 2004; Blanco
et al. 2006). PAHs are a ubiquitous group of contaminants and can accumulate and persist in marine
sediments (Neff 1979), and affect organisms through
toxic action (Albers 2003).
Sediment bioassays (toxicity and bioaccumulation)
are instruments used to test the toxicity and bioavailability of chemical compounds in sediments to
benthic organisms. In the present study we selected
Arenicola marina, in order to test the toxicity and
bioaccumulation of the fuel oil extracted from the
tanker. A. marina is a bulk sediment feeding polychaete worm that lives in a U-shaped burrow. This
species was chosen for the bioassay because it: (a) is
continuously exposed to contaminants in the sediment, which it ingest while feeding; (b) is available
all the year round, often in reasonably high densities;
(c) tolerates a wide range of particle sizes and
salinities, (d) has a broad geographic range; and (e)
supposes an important species in coastal food chain
(Bat and Raffaeli 1997). In addition this species has
been recommended by Oslo-Paris Commission (1995)
as monitoring organism.
Accumulation of hydrophobic organic contaminants by benthic organisms can occur either from
aqueous phase or dietary exposure (Lamoureux and
Brownawell 1998). Uptake of hydrophobic compounds from ingested material has been reported as
a major contributor to an animal’s total body burden
of toxicants (Kaag et al. 1996; Kaag et al. 1998;
Penry and Weston 1998; Selck et al. 2003). Also
bioaccumulation studies have shown that lugworms
accumulate organic contaminants to higher concentrations than filter feeding animals (Kaag et al. 1997).
The ability of organisms to metabolize and excrete
PAHs also has been shown to be related to bioaccumulation (Rust et al. 2004a, 2004b), where species
with limited metabolic ability tend to accumulate
higher PAHs concentrations in their tissue (Varanari
et al. 1985; Driscoll and McElroy 1996; Rust et al.
2004b). Even though the presence of a PAH metabolising system in A. marina (Christensen et al. 2002)
has been strongly suggested, invertebrates tend to
excrete metabolites more slowly than vertebrate species (Rust et al. 2004b). Metabolites have been found
to be eliminated at rates either greater or lower than
those of the parent compound (Spacie and Hamelink
1995). Therefore, toxicity will depend on a combination of relative retention time and relative toxicity of
parent versus metabolites (Selck et al. 2003). Previous
studies agree that A. marina appears to be an
appropriate choice as indicator species for PAH
bioaccumulation (Rust et al. 2004b), and a suitable
organism to monitor PAH pollution.
The aim of this study is to asses the sensitivity of the
polychaete A. marina to the contamination associated
with PAHs from oil spills by using the remaining fuel
oil extracted from a tanker and to determine the
bioavailability of PAHs present in the fuel oil by
measuring the bioaccumulation in the exposed organisms. The results obtained will permit the calculation of
the Sediment Quality Guidelines (SQGs) for this group
of PAHs and will help to predict toxicity and bioaccumulation of the fuel oil in invertebrates. In order to
reach these objectives, a bioassay was conducted
exposing a population of the polychaete Arenicola
marina to different dilutions of fuel oil with clean
sediment.
Material and methods
Toxicity test
Intertidal clean sediment from the Bay of Cádiz
(South of Spain) was mixed with fuel oil extracted
from the tanker (0.5, 1, 2, 4 and 8% dry weight). The
sediment was filtered (0.6 mm) prior to the toxicity to
remove inorganic and organic debris and benthic
organisms capable of preying A. marina (Riba et al.
2003). These sediments were dried and homogenized
at room temperature prior to chemical analysis.
The A. marina lugworms were sampled in field by
hand-digging and immediately transported to laboratory in containers with sea water. Once there, lugworms
were placed in aquariums with sieved sediment from
the Bay of Cádiz (5 cm thick) and acclimated for
10 days; air was provided and water was replaced three
times per day. Water temperature was kept at 18°C and
natural photoperiod was selected. The dilutions of fuel
oil (0.5, 1, 2, 4 and 8% dry weight) in sediment and
the clean sediment without fuel oil (2 kg) were placed
in replicates (three) in 11 L tanks and clean sea water
was added. Lugworms were put into the tanks (six per
tank) which were covered to avoid evaporation. The
experiment lasted 21 days. Mortality was daily
recorded and after 10 and 21 days of exposure
sampling was performed by transferring individuals
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Environ Monit Assess
to aerated clean sea water without sediment, where
they were held for approximately 4 h to empty the
sediment of the body. Organisms were then frozen
at −20°C.
(Fraction I: aliphatic hydrocarbons) and finally 40 ml
of hexane/methane 4:1 (Fraction II: PAHs). Flasks
with Fraction II were dried in a rotatory evaporator and
re-dissolved in hexane (final volume 10 ml). These
extracts were evaporated until a final volume of
0.5 ml. Aromatic fractions were analyzed with a gas
chromatograph coupled with a mass spectrometer
(Finnigan Mat, GCQ tm). Chromatographic resolution
was achieved with a 30 m×0.250 mm DB-5 capillary
column, which has a 0.25 μm film thickness, with
helium as carrier gas.
Chemical analysis
Sediment was digested as described by Loring and
Rantala (1992) for trace metal analysis (Ni, V, Cd, Pb,
Cr, Co). Measurement was performed by graphite
furnace atomic absorption spectrophotometry (PerkinElmer 4100 ZL) (USEPA 1984), et al. Results are
expressed as milligrams per kilogram of dry sediment.
The analytical procedures were checked using reference material (MESS-1 NRC and CRM 277 BCR)
and showed a recovery greater than 90% of the
certified concentration.
Polycyclic aromatic hydrocarbons (fluorene, acenaphthene, naphthalene, phenanthrene, anthracene,
fluoranthene, pyrene, benzo[a]anthracene, chrysene,
benzofluoranthene, benzo[e]pyrene, benzo[a]pyrene,
perylene, dibenzo[ah]anthracene, indene[123-cd]pyrene, benzo[ghi]perilene) were analyzed by using a gas
chromatograph equipped with an electron capture
detector (GC/MS) (US Environmental Protection
Agency 1984). Briefly, dried samples were soxhlet
extracted with n-hexane for 18 h, and the extracts
were isolated by column chromatography on Florisil–
alumino–silica. PAHs were eluted and their fractions
were dried in a rotatory evaporator and re-dissolved in
isooctane. Aromatic fractions were analyzed on a
Hewlett-Packard (HP) 5890 Series II gas chromatograph coupled with HP 5970 mass spectrometer.
Chromatographic resolution was achieved with a
30 m×0.250 mm DB-5 capillary column, which has
a 0.25 μm film thickness, with helium as carrier gas.
Quality control was carried out using NRC-CNRC
HS-6 sediment reference material. The analytical
procedure showed a recovery greater than 90% of
the certified concentration.
The individuals of each treatment were put
together and liophilized for the PAHs analysis.
Briefly, the samples were soxhlet extracted with
hexane/acetone 1:1 during 24 h; then, the extracts
were transferred into tubes and dissolved with hexane
until a final volume of 20 ml. Samples of each tube
were evaporated to 2 ml. These extracts were isolated
by column chromatography on alumino–silica using
20 ml hexane, then 30 ml hexane/methane 9:1
Data calculations
The toxic parameter associated with the fuel oil
(LC50) was obtained from the mortality data of
Arenicola marina measured after 10 and 21 days of
exposure to the fuel dilutions (0.5, 1, 2, 4 and 8%).
LC50 was defined as the concentration (percentage
of fuel oil) that provokes the mortality of 50% of
the Arenicola marina population exposed. The LC50
was calculated by linear regressions of log toxicant
dilution of fuel oil on declining probit values (ProbitAnalysis-Program, version 1.5). Sediment Quality
Values were calculated basing in the LC50 results.
Results and discussion
Acute toxicity
The mortality of the lugworm Arenicola marina in
each treatment was recorded after 10 and 21 days of
exposure. Figure 1 shows how mortality in control
(clean sediment from the Bay of Cádiz) was 0 after
10 days of exposure, while after 21 days 5.6% of the
organisms exposed died. However, mean survival in
all the replicates of clean sediment from the control
(Ca1) was higher than 94% after 10 and 21 of
exposure. Death of test organisms was positively
related to dose at time of exposure. No survival was
detected in the highest dilutions of fuel oil (8%) after
21 days of exposure. For all treatments, the day 21 of
exposure shows higher mortality of Arenicola than the
day 10. Survival results for Arenicola show substantial variability among replicates which has been
already observed in previous studies (Matthiessen
et al. 1998). Other authors obtained differences in
tolerance to toxicants, including PAHs, depending on
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Environ Monit Assess
Fig. 1 Average and
standard deviations of the
percentage of mortality of
the polychaete Arenicola
marina after 10 (dotted
bars) and 21 (striped bars)
days of exposure to each
dilution
the polychaete specie (Bach et al. 2005) whereas low
percentages of oil contaminated sediment inhibited
Arenicola feeding almost completely (Grant and
Briggs 2002). The mortality of individuals of A.
marina exposed to dredged material demonstrated a
slight correlation with the organic contaminants
(PAHs and PCBs) even though these correlations
were not significant (Casado-Martínez 2007). Results
obtained in this study show a toxicity related to time
of exposure, what was previously confirmed by
previous studies (Rossi and Neff 1978).
Despite the variability of mortality results in the
Arenicola bioassays the estimation of LC50 values can
be performed. The mortality data were used to
calculate two LC50s: LC50(10) to describe toxicity
after 10 days of exposure and LC50(21) to describe
toxicity after 21 days of exposure. The LC50 value and
the concentration of PAHs or metals in the sediment
permits calculation of the Sediment Quality Values
(SQVs) for each contaminant (Table 1). The LC50(10)
value of fuel oil associated with the toxic responses
for Arenicola marina is 6.4% of fuel oil, which
corresponds with a concentration of 92.42 mg kg−1 of
total PAHs ([PAH]oil*6.4/100) (SQV1). On the other
hand the LC50(21) of fuel oil associated with toxicity
for this polychaete is 2.4%, which accounts for a
concentration of 34.52 mg kg−1 of total PAHs ([PAH]
oil*2.4/100) (SQV2). Previously, a 10-day exposure
study with the amphipod Corophium volutator and
fuel oil from the tanker produced an LC50 of 1.37%
(Morales-Caselles, ICMAN-CSIC, personal observa-
tions) which suggests that Arenicola presents lower
sensitivity to the fuel than Corophium. Previous
studies have demonstrated that Arenicola marina
shows markedly lower acute toxicity to hydrocarbon
and other contaminants bound to sediments than
Corophium (Matthiessen et al. 1998; Grant and
Briggs 2002).
The values of total PAHs concentration obtained
from the LC50s calculations (SQV1 and SQV2) may be
compared with international Sediment Quality Guidelines (SQGs). National Oceanic and Atmospheric
Administration (1999) explains the 10th percentile
values named the ERL (Effects Range-Low) as the concentrations below which adverse effects rarely occur,
whereas the 50th percentiles named ERM (Effects
Range-Median) values are representative of concentrations above which effects frequently occur. SQV1 for
total PAHs (92.42 mg kg−1) is higher than the
guidelines ERL and ERM calculated (4,022 μg kg−1
and 44,792 μg kg−1 respectively) while SQV2
(34.52 mgkg−1) keeps higher than ERL and lower
than ERM. The justification about why the SQVs
obtained for Arenicola marina are higher than the
ERM could be because of the fact that this polychaete
species presents lower sensitivity to the PAHs toxicity
than other marine organisms, which allows Arenicola
to survive in an environment highly contaminated by
these compounds. On the other hand results of SQVs
for metals are lower than the National Oceanic and
Atmospheric Administration guidelines, hence metals
are probably not a toxicity factor.
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Environ Monit Assess
Table 1 Total PAHs and metal concentration measured in the negative control (Ca1) and in the fuel oil
−1
PAHs (μg kg )
Metals (mg kg−1)
Total PAHs
Fluorene
Acenaphthene
Naphthalene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
Benzofluoranthene
Benzo[e]pyrene
Benzo[a]pyrene
Perilene
Dibenzo[ah]anthracene
Indene[123-cd]pyrene
Benzo[ghi]perilene
Ni
V
Cd
Pb
Cr
Co
Fuel
Ca1
ERL
ERM
SQV1
SQV2
1443
99.3
75.3
395
385
51.4
28.5
111
55.9
102
16.0
45.7
29.7
11.4
5.70
5.23
17.1
55
170
n.d
n.d
0.31
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
14.1
80.0
n.d
23.0
31.0
3.40
4022
19
16
160
240
85
600
665
261
384
n.a.
n.a.
430
n.a.
63
n.a.
n.a.
20.9
n.a.
1.2
46.7
81
n.a.
44792
540
500
2100
1500
1100
5100
2600
1600
2800
n.a.
n.a.
1600
n.a.
260
n.a.
n.a.
51.6
n.a.
9.6
218
370
n.a.
92424
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
8.3
42.7
n.d
10.8
14.6
1.6
34517
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
7.5
41.1
n.d
11.2
15.1
1.7
Sediment quality values for PAHs are obtained from the LC50 and calculated for the Arenicola marina used in the sediment toxicity
test. Sediment Quality Guidelines were derived using previous studies data (ERL= Effects Range-Low and ERM= Effects RangeMedian, National Oceanic and Atmospheric Administration (1999) (n.d, not detected; n.a., not analyzed).
the highest amount of fuel oil. This trend has been shown
in previous studies on other invertebrates (Landrum
et al. 2003) where, in general, the uptake coefficient
declined with increasing PAHs concentration, especially with pyrene. Also, lower accumulation factors were
found to correspond to treatments for which significant
mortality was observed (Rust et al. 2004a).
The BCF calculated for the day 10 of exposure is
higher for phenanthrene, anthracene, fluoranthene,
pyrene and benzofluoranthenes (Fig. 2); after 21 days
of exposure the highest levels of bioaccumulation
were for fluoranthene, pyrene and benzofluoranthenes
but not for the lower molecular weight compounds
phenantrene and anthracene. The decreased of the
BCF for phenantrene and anthracene could be due to
the fact that after 21 days the organisms have been
able to metabolize the PAHs with lower molecular
weight. On the other hand, during long-term contact
between PAHs and sediment particles, PAHs become
tightly bound to organic phases in the sediment,
reducing their bioavailability (Neff 2002).
Bioaccumulation
The polychaetes ingest sediment and thus are exposed
to PAHs in solution in the interstitial water and those
adsorbed to sediment particles (Neff 2002).
Organisms were sampled the day 10 and 21 of the
fuel oil exposure experiment in order to analyze the
content of PAHs in their bodies. A biota/sediment
bioaccumulation factor (BCF) was defined to interpret
the results obtained. This factor accounts for the
concentration of PAHs in the organisms (Co) related
to the concentration of that contaminant in the sediment
(Cs) (BCF = Co/Cs). BCF results are shown in Fig. 2.
In general, bioaccumulation decreases when the percentage of fuel in the sediment sample increases. This
behaviour could be due to the fact that toxicity increases
with the content of fuel. Casts were not found in those
tanks with higher concentrations of hydrocarbons, so
probably feeding was inhibited by the presence of
contaminants in the sediment; this fact could lead to
lower levels of bioaccumulation in those treatments with
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Environ Monit Assess
Fig. 2 Bioaccumulation factors (BCF) calculated for Arenicola
marina after 10 and 21 days of exposure to the dilutions of fuel
oil (0.5, 1, 2, 4 and 8%). ANA Acenaphtene, F fluorene, P
phenantrene, A anthracene, FL fluoranthene, PY pyrene, BA
benzoanthrazene, C chrysene, BBF + BKF benzo(b)fluoran-
thene and Benzo(k)fluoranthene, BEP benzo[e]pyrene, BAP
benzo[a]pyrene, IN indene[123-cd]pyrene, DBA dibenzo[ah]
anthracene, BPE benzo[ghi]perilene, TOTAL sum of individual
PAHs
Levels of BCF confirm that those PAHs that
present logKow values of 5–6 show the highest
accumulation potential as reported in previous studies
(Kaag et al. 1997; kaag et al. 1998; Rust et al. 2004a).
Fluoranthene presents high bioaccumulation potential
relative to smaller or larger PAHs and it is known to be
highly toxic to benthic invertebrates (Selck et al. 2003;
Landrum 1989; Swartz et al. 1990). This compound
may also possess genotoxic (mutagenic and carcinogenic) properties, though these effects are not associated directly with the parent compound, but arise largely
as a result of biotransformation processes that lead to
the formation of reactive intermediates (Rastetter et al.
1982; Babson et al. 1986; Bach et al. 2005). Pyrene
presents a high bioaccumulation factor and associates
strongly to sediment particles (Landrum 1989). Results
of BCF for pyrene increase from day 10 to day 21, in
contrast with other authors that found that the fraction
of unmetabolized pyrene in tissues of A. marina was
unaffected by the duration of exposure (Christensen
et al. 2002). Benzo(b)fluoranthene and benzo(k)fluoranthene present similar values of BCF for the day 10
and 21 of exposure, which suggests that these PAHs
with high molecular weight were initially bound to the
organism tissues and were not metabolized probably
due to their low solubility. On the other hand this could
be as a result of the fact that threshold effect has been
achieved and the availability of the high molecular
weight compounds decreases as a consequence of tight
organic bonding in the sediment.
Conclusions
In the present study the sensitivity of the polychaete
Arenicola marina to the fuel oil from a tanker
(Prestige 2002) has been tested, and in spite of the
variability in mortality results, it showed a clear doserelated mortality but more endurance than other
organisms. Bioaccumulation was mainly produced
for fluoranthene, pyrene, benzo(b)fluoranthene and
benzo(k)fluoranthene whereas phenantrene and anthracene where initially accumulated and then probably metabolized.
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Environ Monit Assess
Although PAHs do not biomagnify through trophic
levels (Neff 2002), A. marina, which is often used as a
bite, is able to live in PAH-contaminated environments
and accumulate PAHs. Although A. marina is less
sensitive than other species, it is likely to be available
even in at polluted sites, for studies of PAH bioaccumulation. Attending to this, we propose that Arenicola
marina should be used in the assessment of oil impacts
associated with spills included in a set of bioassays, in
order to determine acute and sublethal toxicity
responses; in addition further research towards including biomarkers in this species it is recommended.
Christensen, M., Andersen, O., & Banta, G. T. (2002). Metabolism
of pyrene by the polychaetes Nereis diversicolor and
Arenicola marina. Aquatic Toxicology, 58, 15–25.
DelValls, T. A. (2003). The oil spill produced by the tanker
Prestige (13/11/2002): Impact assessment of the northwest
coast of the Iberian Peninsula. Ciencias Marinas, 29(1),
i–iii.
Driscoll, S. K., & McElroy, A. E. (1996). Bioaccumulation and
metabolism of benzo[a]pyrene in three species of polychaete worms. Environmental Toxicology and Chemistry,
15, 1401–1410.
Grant, A., & Briggs, A. D. (2002). Toxicity of sediments from
around a North Sea oil platform: are metals or hydrocarbons responsible for ecological impacts? Marine
Environmental Research, 53, 95–116.
Kaag, N. H. B. M., Foekema, E. M., Scholten, M. C. Th., &
Van Straalen, N. M. (1996). Comparison of contaminant
accumulation in three species of marine invertebrates with
different feeding habits. Environmental Toxicology and
Chemistry, 16(5), 837–842.
Kaag, N. H. B. M., Foekema, E. M., Scholten, M. C. Th., &
Van Straalen, N. M. (1997). Comparison of contaminant
accumulation in three species of marine invertebrates with
different feeding habits. Environmental Toxicology and
Chemistry, 16, 837–842.
Kaag, N. H. B. M., Scholten, M. C. Th., & Van Straalen, N. M.
(1998). Factors affecting PAH residues in the lugworm
Arenicola marina a sediment feeding polychaete. Journal
of Sea Research, 40, 251–261.
Lamoureux, E. M., & Brownawell, B. J. (1998). Chemical and
biological availability of sediment-sorbed hydrophobic
organic contaminants. Environmental Toxicology and
Chemistry, 18(8), 1733–1741.
Landrum, P. F. (1989). Bioavailability of polycyclic aromatic
hydrocarbons sorbed to sediments for the amphipod
Pontoporeia hoyi. Environmental Science & Technology,
23, 588–595.
Landrum, P. F., Lotufo, G. R., Gossiaux, D. C., Gedeon, M. L.,
& Lee, J. H. (2003). Bioaccumulation and critical body
residue of PAHs in the amphipod, Diporeia spp.: Additional evidence to support toxicity additivity for PAHs
mixtures. Chemosphere, 51, 481–489.
Loring, D. H., & Rantala, R. T. T. (1992). Methods for the
geochemical analyses of marine sediments and suspended
particulate matter. Earth-Science Reviews, 32, 235–283.
Matthiessen, P., Bifield, S., Jarrett, F., Kirby, F., Law, R. J.,
McMinn, W. R., et al. (1998). An assessment of sediment
toxicity in the River Tyne Estuary, UK by means of
bioassays. Marine Environmental Research, 45, 1–15.
National Oceanic and Atmospheric Administration (1999).
Sediment quality guidelines developed for the national
status and trend programs.
Neff, J. M. (1979). Polycyclic aromatic hydrocarbons in the
aquatic environment: Source, fates, and biological effects.
London, UK: Applied Science.
Neff, J. M. (2002). Polyciclic aromatic hydrocarbons in the
ocean, in Bioaccumulation in Marine Organisms. New
York: Elsevier, p. 452.
Oslo-Paris Commission (1995). Report of the workshop on biological effects monitoring techniques. Aberdeen, Scotland,
2–6 October.
Acknowledgments The work described was supported by a
Grant funded by the Ministry of Education and Science
VEM2003-20563. Carmen Morales-Caselles thanks the Ministry of Education and Science for funding her research
fellowship (FPU). We are grateful for the support and help of
the members of the CIS (Spain) and IPIMAR (Portugal).
Special thanks are given to Carlos Vale, Ana Ferreira, Isabellina
Santos, Ana Cristina Micaelo and Judit Kalman.
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Environmental Pollution 146 (2007) 233e240
www.elsevier.com/locate/envpol
Comparing sediment quality in Spanish littoral areas affected by
acute (Prestige, 2002) and chronic (Bay of Algeciras) oil spills
C. Morales-Caselles a,c, J. Kalman a,c, I. Riba a,c, T.A. DelValls b,c,*
a
Instituto de Ciencias Marinas de Andalucı́a (ICMAN-CSIC), Avda. República Saharaui s/n, Puerto Real 11510, Cádiz, Spain
UNESCO UNITWIN/UNICOP, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Polı́gono Rı́o San Pedro s/n,
Puerto Real 11510, Cádiz, Spain
c
Unidad Asociada de Calidad Ambiental y Patologı́a (CSIC & UCA), Avda. República Saharaui s/n, Puerto Real 11510, Cádiz, Spain
b
Received 7 December 2005; received in revised form 6 April 2006; accepted 12 April 2006
Littoral sediments affected by low or moderated but continuous oil spills are more polluted than those
affected by accidental oil spills such as the Prestige.
Abstract
The quality of sediments collected from two areas of the Spanish coast affected by different sources of contaminants has been compared in
this study. The areas studied are the coast of Galicia affected by the oil spill from the tanker Prestige (November 2002) and the Gulf of Cádiz
which suffers continuous inputs of contaminants from industries located in the area and from oil spills. Contamination by several chemicals
(metals, PCBs and PAHs) that bind to sediments was analyzed, and two toxicity tests (Microtox and amphipod 10-day bioassay) were conducted. PAHs were identified as the compounds responsible for the toxic effects. Results show differences between an acute impact related
to the sinking of the tanker Prestige and the chronic impact associated with continuous oil spills associated with the maritime and industrial
activities in the Bay of Algeciras, this being the most polluted part of the two coastal areas studied in this work.
2006 Elsevier Ltd. All rights reserved.
Keywords: Amphipods; Microtox; PAHs; Toxicity; Contamination
1. Introduction
Sediments are an important part of the ecosystem and play
a key role in the distribution of contaminants in the aquatic environment; the study of the quality of sediments provides information about the ecosystem health. Human activities in
coastal areas usually involve an input of contaminants to the
natural environment that becomes evident in the decreased
quality of coastal sediments. Many authors agree that sediment
quality is best determined by integrating the information
* Corresponding author. UNESCO UNITWIN/UNICOP, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Polı́gono Rı́o San Pedro
s/n, Puerto Real 11510, Cádiz, Spain. Tel.: þ34 956 016794; fax: þ34 956
016040.
E-mail address: angel.valls@uca.es (T.A. DelValls).
0269-7491/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2006.04.042
-61-
obtained from measures of chemicals concentration and
from specific tests to determine sediment toxicity (DelValls
and Conradi, 2000; Chapman et al., 2002). The biological effects can be established based on laboratory tests that determine toxic responses. Sediment bioassays are usually
relatively simple tests that evaluate the responses of the tested
organism to contaminated sediments under controlled conditions (Riba et al., 2004a).
In the present study, we have selected two different tests
in order to determine sediment toxicity: the Microtox test
and an amphipod acute bioassay. Use of the commercial bioassay Microtox has increased in recent years since it detects
the ‘‘hot spots’’ of field contamination in the screening procedure (Mowat and Bundy, 2001; Stronkhorst et al., 2003;
Van Beelen, 2003); Microtox has also been used before
to assess the impact of oil spills and oil contaminated
234
C. Morales-Caselles et al. / Environmental Pollution 146 (2007) 233e240
sediments (Brohon et al., 2001; Kenneth et al., 2003; Pelletier et al., 2004). The other acute bioassay was carried out
with the amphipod Corophium volutator which is an important test organism for the ecotoxicological quality assessment
of marine and estuarine sediment samples (Peters and Ahlf,
2005).
The bioassay with C. volutator is integrated in test batteries
for dredged material management (Peters et al., 2002; Stronkhorst et al., 2003) and it is required for compliance with certain International Standardization Organisation quality
standards (ISO, 2003). This kind of bioassay has been used
in previous studies for the assessment of spills (Grant and
Briggs, 2002; Briggs et al., 2003).
In this study a comparison is made between the quality of
sediments sampled on two lengths of Spanish coast affected
by different sources of contaminants. In the coast of Galicia,
the study addresses the acute impact provoked by the oil spill
resulting from the break-up and sinking of the tanker Prestige (November 2002), whereas in the Gulf of Cádiz the sediments studied have suffered a chronic impact lasting several
decades, caused by the input of oil and other contaminants
from the various industries located in the area and from accidental spills and deliberate discharges from commercial
shipping activities.
The main objectives of this study are: (1) to characterise
the contamination by PAHs in the selected areas of study
on the Galician Coast and in the Gulf of Cádiz; (2) to establish the sediment toxicity caused by the presence of contaminants in the sediment samples; (3) to compare the sediment
quality of the various areas studied by linking contamination
and ecotoxicological data.
2. Materials and methods
2.2. Chemical analysis
Sediment aliquots for chemical analysis were dried at room temperature
and then gently homogenized. Total organic carbon (TOC) concentration
and sediment grain size (fines: % of dry sediment < 63 mm) were studied in
order to determine the geochemical matrix characteristics. Organic carbon
content was determined using the method of Gaudette et al. (1974) with the
El Rayis (1985) modification. For sediment grain size, an aliquot of wet sediment was analyzed using a Fristch laser particle sizer (model Analysette 22)
following the method reported by DelValls and Chapman (1998).
For trace metal analysis, the sediments were digested as described by Loring and Rantala (1992). Zn and Cu concentrations in the extracts were determined using a PerkineElmer 2100 flame atomic absorption
spectrophotometer. The other trace metals were measured by graphite furnace
atomic absorption spectrophotometry (PerkineElmer 4100 ZL). Concentrations of Hg were determined using a PerkineElmer MHS-FIAS coupled
with a PerkineElmer 4100 ZL spectrophotometer. Results are expressed as
mg kg1 dry sediment. The analytical procedures were checked using reference material (MESS-1 NRC and CRM 277 BCR) and showed agreement
with the certified values of more than 90%.
The analyses of PAHs and PCBs were carried out according to USEPA
SW-846 Method 827C78082. Briefly, following recommendations by Riba
et al. (2002), dried samples were Soxhlet extracted with n-hexane for 18 h,
and the extracts were isolated by column chromatography on Florisile
alumino-silica. PCBs and PAHs were eluted and their fractions were dried
in a rotating evaporator and re-dissolved in isooctane. Aromatic fractions
were analyzed on a HewlettePackard (HP) 5890 Series II gas chromatographer coupled with an HP 5970 mass spectrometer. Chromatographic resolution
was achieved with a 30 m 0.250 mm DB-5 capillary column, which has
a 0.25 mm film thickness, with helium as carrier gas. The 16 priority PAHs
considered by the US Environmental Protection Agency were analyzed by
GCeMS using selected ion monitoring (SIM). Quality control was carried
out using NRC-CNRC HS-6 sediment reference material. Analysis of PCBs
as AROCLOR 1242 and AROCLOR 1260 was performed using the same instrument with an electron capture detector (GC/ECD) and a 30 m 0.25 mm
MDN-5S capillary column. Quantification was performed by the external standard technique by comparison of peak areas in the sample with those obtained
by injecting a standard mixture of AROCLOR 1242 and 1260. Quality control
was carried out with NRC-CNRC HS-1 sediment reference material. For both
set of organic chemicals, PAHs and AROCLOR, the analytical procedure
showed agreement with the certified values of more than 90%.
2.1. Approach
Fig. 1 shows the seven sediment sampling stations located in the National
park of the Atlantic Islands that were selected in the area of Galicia, three stations in the island of Ons (D07, D09 and D18) and four stations in the Cı́es
archipelago (D60, D66, D79 and FIG). In the Gulf of Cádiz seven stations
were selected in the area of the Bay of Algeciras: three stations in the mouth
of the river Guadarranque (GR1, which is near an oil-fired electricity generating plant, GR30 and GR4, both near chemical processing plants), one station in
the mouth of the river Palmones (P4) and three stations in the Bay (AL1 and
AL2, both located in the port and near the city of Algeciras, and AL5, near
a chemical plant). All these sediments have suffered repeated impacts by moderate or small oil spills caused by maritime traffic and bunkering activities in
the area during recent decades. Clean sediment from the Bay of Cádiz was
used as the negative control (Ca1). An artificial sample (TM) was made by
mixing a toxic mud from an accidental mining spill in Spain (Aznalcóllar,
April 1998) with the same clean sediment and used as positive toxicity control
(Riba et al., 2003).
Sediments were collected with a 0.025 m2 Van Veen grab and transferred
to the cooler. When sufficient sediment had been collected from a particular
station, the cooler was transported to the laboratory. The contents of the cooler
were homogenized with a Teflon spoon until no colour or textural differences
could be detected. The samples were subsampled for physical characterization
and chemical quantification. After that, sediment samples were maintained in
the cooler at 4 C in the dark until they were used for sediment toxicity testing,
but no longer than 2 weeks.
2.3. Microtox bioassay
The commercial Microtox test is a bioassay that uses the bioluminescence of the bacteria Vibrio fischeri as an indicator of the quality of the sample
(liquid or solid phase) exposed; the bioluminescence of the bacteria is related
to its metabolism therefore, a diminution of the sediment quality will be reflected in the decrease of the quantity of light emitted. In the Microtox toxicity test, the measures of the IC50 (which is the concentration of dry sediment
that provokes a 50% inhibition of the light emitted by the bacteria) provide the
information about the sediment toxicity. The bioassay of bioluminescence inhibition with the bacteria V. fischeri was conducted on solid phase with the
commercial Microtox apparatus (model 500) by following the protocols
for the solid phase test (SPT) according to the standard operating procedure
(AZUR Environmental, 1998). Briefly, 7 g (0.01 g) of sediment were tested
as suspensions prepared with 35 mL of commercial Microtox Solid Phase
Test Diluent and diluted to a series of nine concentrations in the cuvettes.
The reconstituted bacteria were added to the dilutions which were incubated
for a period of 20 min at 15 C in a waterbath. Next the dilutions were filtered
through the filter columns and 500 mL of content of each cuvette of the bath
were transferred to its corresponding cuvette in the apparatus and bioluminescence was measured in the ‘‘read well’’. The modification of the basic solid
phase test (BSPT) reported by Campisi et al. (2005) was carried out and an
average value of the IC50 from using both protocols was obtained for each
sample.
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C. Morales-Caselles et al. / Environmental Pollution 146 (2007) 233e240
•D18
235
Galician
Coast
•D07
Spain
•D09
N
Gulf of
Cádiz
Ons
Bay of Algeciras
GR1• •GR3’
•GR4
•P4 •AL5
•FIG
Bay of Cádiz
•D79
•AL2
•AL1
•D66
•D60
•Ca1
Cíes
Fig. 1. Map of the coastal area of Galicia, the Bay of Algeciras and the Bay of Cádiz showing the general areas sampled and locations of the sampling stations. D(#)
refers to the stations located in Galicia and AL(#), P(#), and GR(#) to those in the Bay of Algeciras. Ca1 was the station selected as negative control in the Bay of
Cádiz.
2.4. Amphipod bioassay
Individuals of C. volutator were obtained from the field in a clean area located in the coast of Galicia (Morales-Caselles, 2005) by sieving mud through
a 1 mm mesh; when the organisms were isolated they were placed in 11 L capacity aquariums with clean seawater and sieved sediment (collected in the
same area as the organisms) and were maintained in the laboratory under controlled conditions for acclimation until the start of the test. Aeration was provided and natural photoperiod was selected. During acclimation (1 month) the
organisms were fed twice a week with food for invertebrates (‘‘Marine Invertebrate Diet’’ which is a mixture of amino acids and organic particles) and
water was replaced.
The toxicity test was conducted in replicate (5) by exposing individuals of
the amphipods C. volutator to bulk sediment using the percentage of survival
after 10 days of exposure as the end point (ASTM, 1993). Mortality is measured after the time of exposure and the results obtained have been correlated
positively with changes in benthic communities (Long et al., 2001).
Approximately, 250 g of sieved (1 mm) sediment was placed in 2 L glass
containers and then about 750 mL of clean seawater were added. Aeration was
provided after the sediment had settled down. The individuals of Corophium
were sieved and separated from sediment of the acclimatising tanks and
were placed in each replicate container (20 individuals per container). The
containers were covered in order to avoid water evaporation (a hole was
made in the lid to provide aeration), and maintained at 19 C during the 10
days of exposure. After that time, the contents from the different stations
with the various replicates were sieved and the organisms’ survival rate was
recorded.
2.5. Statistical analysis
ANOVA was performed in order to determine significant differences
( p < 0.05) in amphipod survival among the toxicity results obtained for the
control site and the other sampling sites. Also, contamination and toxicity
data were linked by factor analysis, using principal components analysis
(PCA) as the extraction procedure; this is a multivariate statistical technique
for exploring variable distributions (Riba et al., 2003). The original data set
-63-
used in the analysis included two acute toxicity responses (amphipod survival
and the IC50 measured in the Microtox bioassay), the sediment concentration
of different contaminants (PAHs, PCBs, Cd, Cu, Ni, Co, V, Pb, Zn, Hg), and
the geochemical matrix characteristics (including total organic carbon and
grain size distributions). The objective of PCA is to derive a reduced number
of new variables as linear combinations of the original variables. This provides
a description of the structure of the data with the minimum loss of information.
3. Results
3.1. Chemical analysis
Summarized results of chemical analyses in the sediments
used for both bioassays are shown in Table 1. In general, results do not show a prevailing tendency in the concentration
of metals among sediments from the different areas, although
the toxic mud presents the highest concentration of metals. On
the other hand, organic contaminants (PAHs and PCBs) seem
to be at higher levels in sediments collected in the Bay of Algeciras than in the area of Galicia. The negative control (Ca1)
shows the lowest levels of metals, and no organic contamination was found.
3.2. Microtox bioassay
The highest inhibition of bioluminescence, which corresponds to an IC50 < 600 mg L1 dry weight, is shown in the
samples collected at the Bay of Algeciras stations AL2
(69 mg L1), AL1 (208 mg L1), GR30 (235 mg L1), GR4
(249 mg L1) and GR1 (522 mg L1), and in the sediments
from the coast of Galicia D60 (358 mg L1), D79
(364 mg L1), D18 (390 mg L1) and D66 (486 mg L1).
C. Morales-Caselles et al. / Environmental Pollution 146 (2007) 233e240
236
Table 1
Average values of total organic carbon (%dry weight), fines (% of dry sediment < 63 mm) and the concentration of contaminants (metals (mg kg1 dry weight);
PAHs and PCBs (mg kg1 dry weight)) in sediment samples (negative control: Ca1; positive control: TM; Algeciras Bay: GR1, GR30 , GR4, P4, AL1, AL2, AL5;
Galicia: D07, D09, D18, D60, D66, D79 and FIG.) Not detected is expressed by n.d.
Ca1
GR1
GR30
GR4
P4
AL1
AL2
AL5
D07
D09
D18
D60
D66
D79
FIG
TM
TOC
Fines
Zn
Cd
Pb
Cu
Ni
Co
V
Hg
PAH
PCBs
1.07
3.12
2.15
3.19
2.09
2.35
3.22
1.22
3.79
4.61
2.42
3.56
0.37
3.58
2.10
1.00
1.04
75.1
89.2
39.5
34
92.3
90.8
3.2
45.6
59.9
12.9
60.9
11.3
70.2
2.12
10.1
21.3
44.8
138
35.3
50.4
137
54.0
23.0
85.3
107
55.5
101
14.0
114
76.2
2181
0.92
0.61
0.17
0.10
0.62
0.16
0.11
0.14
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
5.40
2.31
9.10
22.0
6.21
5.64
32.0
9.81
7.12
23.1
28.3
14.2
31.0
4.10
29.1
26
789
6.98
12.6
5.01
3.67
11.3
30.5
7.59
10.8
251
160
20.8
70.9
16.2
150
18.5
210
0.06
6.01
74.7
13.1
24.7
50.9
15.1
52.6
1.04
11.7
3.44
16.2
4.60
4.44
11.8
8.5
3.40
n.d.
12.8
5.59
1.11
n.d.
1.69
5.09
n.d.
n.d.
2.00
n.d.
0.30
n.d.
0.50
n.d.
80.0
6.69
26.1
n.d.
85.0
60.8
4.82
2.19
81.2
116
54.0
125
n.d.
13.7
n.d.
n.d.
n.d.
n.d.
1.04
0.25
0.11
1.11
0.81
0.26
0.08
0.07
0.04
0.12
0.06
0.09
0.04
5.61
n.d.
546
2961
802
21.4
1383
1376
1218
465
240
480
702
384
273
390
n.d.
n.d.
0.86
22.0
1.75
4.64
0.46
0.65
0.33
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
The positive control of toxicity TM shows a 50% inhibition of
bioluminescence at very low concentration (142 mg L1). The
sediment obtained from the Bay of Cádiz and used as the clean
reference (station Ca1), showed the highest value of IC50
(6013 mg L1) and confirms its validity as the negative toxicity control. Microtox results are shown in Fig. 2.
measured in the sediments from GR30 (100%), TM (100%),
GR4 (75%), D18 (45%), D66 (60%), P4 (33.3%) and AL5
(35%) was significantly different *p < 0.05 from the negative
control Ca1. The other stations did not present significant differences in the mortality results compared to the control station (Ca1).
3.3. Amphipod bioassay
3.4. Statistical analysis
Mean mortality results after the 10 days amphipod toxicity
test are shown in Fig. 3. The highest mortality measurements
(100%) were associated with sediments found in the Bay of
Algeciras at station GR30 and with the positive control of toxicity TM, while lowest mortality (3.3%) was associated with
sediments from the reference station Ca1. The mortality
To link the set of data obtained, the original variables from
chemical concentration and toxicity responses were analyzed
by factor analysis, using principal components analysis
(PCA) as the extraction procedure; this is a multivariate statistical technique (MAA) for exploring distributions of the variables (chemical concentration, n ¼ 14; toxicity data, n ¼ 2).
2000
1800
8000
IC50 (mgL-1 dry weight)
Not
Toxic
Toxic
1600
1400
1200
[1]
1000
[2]
800
6000
600
400
200
0
4000
D09 P4 D07 FIG GR1 D66 D18 D79 D69 GR4 GR3’ AL1 TM AL2
2000
0
Ca1 AL 5 D09
P4
D07 FIG GR1 D66 D18 D79 D60 GR4 GR3' AL1 TM AL2
Fig. 2. IC50 results obtained from the application of the Microtox test to sediment samples from the various stations. A zoom is provided for IC50 < 1800 mg L1
(dry weight). Lines displayed show the limits below which the sediment sample is considered toxic by the Canadian Standards (1000 mg L1 dry weight) and by
the proposed Spanish Standards (Casado-Martı́nez et al., in press), 750 mg L1 (dry weight).
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C. Morales-Caselles et al. / Environmental Pollution 146 (2007) 233e240
237
120.0
*
*
*
80.0
*
60.0
*
*
Toxic
Mortality (
)
100.0
*
40.0
Not
Toxic
20.0
0.0
Ca1 Gr1 Gr3' Gr4 P4 AL1 AL2 AL5 D07 D09 D18 D60 D66 D79 FIG TM
stations
Fig. 3. Mortality results after 10 days of exposing C. volutator to the sediment samples. Asterisks indicate significant differences between the amphipod mortality
rate in the treatments and the negative control (*p < 005). The line displayed shows the limits below which the sediment samples are considered toxic by the
proposed Spanish Standards (DelValls et al., 2004; Casado-Martı́nez et al., in press). Those samples where the mortality rate of the amphipods is 20% higher
than the mortality recorded in the negative control (Ca1) and show significantly different (*p < 005) results compared to those obtained in Ca1, are considered
as toxic.
The factor analysis was performed on the correlation matrix;
the variables were autoscaled (standardized) so as to be treated
with equal importance (Riba et al., 2004a). The application of
MAA to the original 14 variables indicates that they can be
grouped in three new factors. These factors explain 78.3%
of the total variance in the original data set. In the present
study, we decided to interpret a group of variables as those associated with a particular component where the loading was
0.30 or higher (Table 2). This approximates to Comreys’
cut-off of 0.55 (Comreys, 1973) for a good association between an original variable and a factor, and also takes into account discontinuities in the magnitudes of loadings
approximating the original variables.
The first principal factor, #1 is predominant and accounts
for 35.0% of the variance; it shows the toxicity to the bacteria
and the amphipods associated with the presence of trace
metals in sediments (Zn, Cd, Pb, Cu and Hg). The second
Table 2
Sorted rotated factor loadings (pattern) of 14 variables for the three principal
factors resulting from the multivariate analysis of results obtained from the
chemical analysis and the acute toxicity tests (Microtox and C. volutator
bioassay)
%Variance
TOC
Fines
Zn
Cd
Pb
Cu
Ni
Co
V
Hg
PAH
PCBs
Microtox
Corophium
Factor 1
Factor 2
Factor 3
35.01
28.76
14.54
e
e
0.98
0.93
0.98
0.51
e
e
e
0.98
e
e
0.43
0.59
e
0.40
e
e
e
e
0.86
0.88
e
e
0.94
0.88
0.30
0.52
0.86
0.84
e
e
e
0.42
e
e
0.41
e
e
e
e
e
factor, #2 accounts for 28.8% of the variance; it explains the
amphipods and bacteria toxicity associated with the chemical
concentrations of the metals Co and Ni, the organic contaminants PAHs and PCBs. The third factor, #3 accounts for 14.5%
of the variance; it shows the relationship between the grain
size and the total organic carbon in the sediments with the
presence of Cu and V, but toxicity does not contribute to
this factor.
The influence of the three factors at the 16 stations is reflected by the Factor score at these stations and is shown in
Fig. 4. The definition of Factor 1, with positive loading, is
the acute toxicity of the organisms to metals bound to sediment, it is mainly prevalent in the positive control TM
(3.65) followed by GR30 (0.02) and AL2 (0.01) with low prevalence. Factor 2 is defined as the lethal toxicity of the amphipods related to the concentration of metals Co and Ni and to
the organic compounds, mainly PAHs, bound to sediments;
this factor shows significant prevalence in the stations from
the Bay of Algeciras: GR4 (0.49), AL1 (0.49), AL2 (0.47),
AL5 (0.61) and mainly in GR30 (3.28). The definition of Factor 3, with negative loading, does not include information
about the toxicity of the contaminants, but indicates the association of geochemical features of the sediment, described by
the relationship between total organic carbon and grain size.
4. Discussion
The chemical data obtained in the analyses show how the
levels of PAHs are, in general, higher at the stations in the
Bay of Algeciras than in the stations selected on the coast of
Galicia. These chemical data can be compared to international
sediment quality guidelines (SQGs) that specify the levels of
chemical contaminants associated with biological effects
(DelValls et al., 2004). In this regard, the samples collected
at GR30 , AL1, AL2 and AL5 exceed the SQGs for PAHs defined by Dutch agencies (Tweede Kamer, vergaderjaar,
1994e1995); this implies that the sediments from these locations could be considered slightly or moderately polluted
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C. Morales-Caselles et al. / Environmental Pollution 146 (2007) 233e240
238
Factor 1
4
TM
GR3'
AL2
0
AL1
D07
GR4
GR1
D79
D09
D18
P4
D60 D66
FIG
AL5
Ca1
-1
4
GR3'
3
1
Factor 2
GR4
AL1 AL2
AL5
0
P4
GR1
-1
D18 D60 D66
Ca1
D07 D09
D79
FIG TM
-2
2
AL2
1
Factor 3
D09
D07
AL1
D60
D79
GR1 GR3'
0
GR4
P4
D18
TM
-1
FIG
Ca1
AL5
D66
-2
Fig. 4. Estimated factor scores for the three factors in each of the 16 cases. The
factor scores quantify the prevalence of each factor for every station and is
used to establish the definition of each factor.
according to the Dutch SQGs for PAHs. Following the recommendations described by MacDonald et al. (1996), the sediments from GR4 and D60 would also be considered as
slightly polluted by this contaminant and adverse effects could
be frequent. There are some stations in the Galician islands
(D07, D09, D18, D60, D79 and FIG) where sediment exceeds
the SQGs defined for Cu by international agencies and previous studies (CEDEX, 1994; Tweede Kamer, vergaderjaar,
1994e1995; MacDonald et al., 1996; NOAA, 1999). Although
contamination by copper was observed in the uppermost layer
in the Prestige shipwreck area of the Northeast Atlantic Ocean
(Prego and Cobelo-Garcı́a, 2004; Cobelo-Garcı́a et al., 2004),
this Cu contamination should not be related to the shipwreck,
because levels of Cu in the fuel oil carried by the Prestige
were relatively low (3.39 mg kg1) and previous studies
have shown that there are other sources of this metal in the
area (Carballeira et al., 1997). Also, there are some stations
in the Bay of Algeciras (GR30 , AL1 and AL5) where sediment
exceeds some international SQGs (CEDEX, 1994; Tweede
Kamer, vergaderjaar, 1994e1995; MacDonald et al., 1996;
NOAA, 1999) defined for Ni and for Hg (GR30 , GR4, AL1,
AL2 and AL5). The positive control TM exceeds almost all
the SQGs defined by international agencies and other authors
(CEDEX, 1994; Tweede Kamer, vergaderjaar, 1994e1995;
MacDonald et al., 1996; NOAA, 1999) for the metals Zn,
Cd, Pb, Hg and Cu.
In Fig. 2 the Microtox results are shown. The lines represent the values below which sediment toxicity is assumed by
different international agencies (Casado-Martı́nez et al., in
press). The Canadian Standards (Environment Canada, 2002)
considers the limit to be 1000 mg L1 (dry weight) while in
the Spanish Standards (DelValls et al., 2004; Casado-Martı́nez
et al., in press) this limit is associated with a concentration of
750 mg L1 (dry weight). In this case both guidelines agree
and the sediments from GR1, D66, D18, D79, D69, GR4,
GR30 , AL1, TM and AL2 would all be considered as toxic.
The line displayed in the graph for the Corophium mortality
(Fig. 3) shows the limit above which sediments are considered
toxic by the US Environmental Protection Agency (USEPA,
1994) and the Spanish Standards (DelValls et al., 2004; Casado-Martı́nez et al., in press). These agencies establish that
a sediment sample can be considered toxic when the mortality
rate recorded from the treatment is 20% higher than the mortality measured in the negative control sediment; it also shows
significantly different (*p < 0.05) mortality results compared
with those obtained in the negative control. In this case all
the samples that are significantly different (*p < 0.05) from
Ca1 would be considered toxic by these agencies because
they also have a mortality rate higher than 20% compared to
the control sediment (10%).
From the MMA performed to link together the chemical
and ecotoxicological data, we have obtained three factors
that account for all the variables and have a different influence
for each sampling site. Factor 1 accounts for the toxicity responses of the two bioassays due to the metals that bind to sediments. This factor is seen with most prevalence in the positive
control TM which presents the highest levels of metals in the
study, a low IC50 and a 100% mortality of Corophium after 10
days of exposure.
Regarding the toxicity due to the presence of organic contaminants in the sediments, Factor 2 is mainly prevalent at the
stations in the Bay of Algeciras: GR30 , GR4, AL1, AL2 and
AL5. This factor also includes toxicity due to the metals Ni
and Co and the toxicity is determined by the mortality of
the amphipods and the low IC50 measured with the bacteria
bioassay. The content of PCBs in the sediments studied does
not exceed the SQGs and the highest concentration of PCBs
is found in sediments from GR30 whereas in the Galician sediments, it was not found at all. Thus, the PAHs can be considered as the main organic contaminant producing the toxicity
measured in the study. The metals Ni and Co have been previously reported associated with PAHs in the oil spills occurring in the area of Algeciras (CSIC, 2005) caused both by the
industrial plants located in the area and by maritime activities,
and in other areas affected by oil spills (Massoud et al., 1998).
However, both of these metals (Co and Ni) could originate
from any of the various local activities, not only accidental
oil spills. The toxicity results from Microtox have a low loading in this factor, maybe because this is not the best test for
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C. Morales-Caselles et al. / Environmental Pollution 146 (2007) 233e240
organic compounds, due to the insoluble nature of most of the
oil compounds (Simon et al., 2004). However, it is important
to highlight that the Microtox is a screening bioassay and
it is suggested that the biotests alone may not be representative
in certain cases of the full impact of a given pollutant on an
ecosystem (Brohon et al., 2001). On the other hand, previous
studies have shown that the toxicity of sediments to Corophium is closely correlated with their hydrocarbon content
(Grant and Briggs, 2002).
In general, results obtained in the MAA suggest that the
sediments from the Bay of Algeciras and the Galician islands
are contaminated with PAHs and that toxic responses due to
these compounds occur in both places. However, toxicity is
mainly seen in sediments from the Bay of Algeciras, and toxic
sediments show a higher frequency in the stations located in
this area (GR1, GR30 , GR4, P4, AL1, AL2 and AL5) than in
those sampled on the coast of Galicia (D07, D09, D18, D60,
D66, D79 and FIG). This could be associated either with the
higher quantity of PAHs or with the presence of a more complex mixture of contaminants that has not been analyzed in
this study, in sediments from the Bay of Algeciras rather
than in those from Galicia. Also, based on the results obtained
in this study, the concentration of PAHs bound to sediments in
the Bay of Algeciras could be more bioavailable for the organisms studied and would produce more toxicity than that found
in Galicia. However, this greater bioavailability of PAHs
bound to sediments in the Bay of Algeciras is only a hypothesis
that needs to be taken into account in later work.
Sediment quality guidelines (benchmarks) for PAHs can be
determined with the information obtained in this study by
means of MAA using the Factor scores. The highest concentrations of PAHs measured at those stations where the value
of the Factor 2 score is 0 or below 0, define the concentration
of PAHs ‘‘not associated with the toxic effect’’ measured in
the study. It is determined at station D60 with a value of
702 mg kg1 (dry weight) (V1). The lowest concentration of
PAHs measured at the station where the value of Factor 2 score
is positive defines the concentration of PAHs ‘‘associated with
the toxic effect measured in this study’’. It is determined at station GR4 with a value of 802 mg kg1 (dry weight) (V2).
In general, our results have shown that the highest pollution
measured in the Gulf of Cádiz was determined in sediments
sampled from the Bay of Algeciras and especially at station
GR30 , related to chronic contamination, while moderate contamination and low toxicity was determined on the coast of
Galicia in the Cı́es archipelago. Previous studies (Riba et al.,
2004b) agree that moderate but chronic inputs of contaminants
can produce more pollution in coastal sediments than higher
but acute environmental impacts.
5. Conclusions
From the results obtained in the various analyses performed
in this study, we can conclude that PAHs are the main contaminant at the sites studied on the coast of Galicia and in the Bay
of Algeciras, while there is no such contamination in the sediments from the station located in the Bay of Cádiz; the
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239
concentrations of PAHs in sediments from the Bay of Algeciras are, in general, higher than in sediments from the coast of
Galicia. Toxicity is also higher in the Bay of Algeciras than in
the Galician islands, but no toxicity was detected in the sediment from the Bay of Cádiz station. Finally, it has been shown
that sediments from the Bay of Algeciras are chronically polluted by PAHs (V1: 702 mg kg1; V2: 802 mg kg1) while
those of the coast of Galicia can only be considered as moderately or not polluted. In the Bay of Cádiz no environmental
degradation was measured.
To sum up, with the present study we have shown that sediments found in the Bay of Algeciras, affected by chronic oil
spills, are more environmentally degradated (polluted) than
those found in the coast of Galicia, which was mainly affected
by the Prestige accidental oil spill.
Acknowledgements
The work described was partly supported by a Grant funded
by the Ministry of Education and Science VEM2003-20563
and by CIS funded by the Ministry of Environment. Carmen
Morales-Caselles thanks the Ministry of Education and Science for funding her research fellowship (FPU). We are grateful for the support and help of the members of the CIS. Special
thanks are given to Mercedes Conradi, Augusto César and
Carmen Casado-Martı́nez.
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con el vertido del petrolero Prestige. Master thesis, Universidad de Cádiz,
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Mowat, F.S., Bundy, K.J., 2001. Correlation of field-measured toxicity with
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Pelletier, E., Delille, D., Delille, B., 2004. Crude oil bioremediation in subAntarctic intertidal sediments: chemistry and toxicity of oiled residues.
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Prego, R., Cobelo-Garcı́a, A., 2004. Cadmium, copper and lead contamination
of the seawater column on the Prestige shipwreck (NE Atlantic Ocean).
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guidelines in the Guadalquivir estuary associated with the Aznalcóllar
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in littoral regions of the Gulf of Cádiz: a triad approach to address the
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Fuller, C.B., Autenrietha, R.L., 2004. Evaluation of two commercial bioaugmentation products for enhanced removal of petroleum from a wetland.
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-68-
Sedimentȱcontamination,ȱbioavailabilityȱandȱtoxicityȱofȱsedimentsȱ
affectedȱbyȱanȱacuteȱoilȱspill.ȱFourȱyearsȱafterȱtheȱsinkingȱofȱtheȱ
tankerȱprestigeȱ(2002).ȱ
C.ȱMoralesȬCaselles1,2,*,ȱJ.ȱKalman1,2,ȱA.C.ȱMicaelo3,ȱA.M.ȱFerreira3,ȱC.ȱVale3,ȱI.ȱ
Riba1,2,ȱT.A.ȱDelValls1,2ȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱ
1
ȱUniversidadȱdeȱCádizȬCalidadȱAmbientalȱyȱPatologíaȱ(UCAȬCSIC).ȱConsejoȱSuperiorȱ
deȱInvestigacionesȱCientíficasȱ(CSIC).ȱInstitutoȱdeȱCienciasȱMarinasȱdeȱAndalucíaȱ
(ICMAN).ȱPolígonoȱRíoȱSanȱPedroȱs/n.ȱ11510ȱPuertoȱReal.ȱCádiz.ȱ
ȱ2ȱUNITWIN/UNESCO/WiCoP.ȱDepartamentoȱdeȱQuímicaȱFísica.ȱFacultadȱdeȱ
CienciasȱdelȱMarȱyȱAmbientales.ȱPolígonoȱRíoȱSanȱPedroȱs/n.ȱ11510ȱPuertoȱReal.ȱCádizȱ
3
ȱDepartamentoȱdoȱAmbienteȱAquatico.ȱInstitutoȱdeȱInvestigaçaoȱdasȱPescasȱeȱdoȱMar,ȱ
(INIAPȬIPIMAR).ȱAV.ȱBrasilia,ȱ1400ȱLisboa,ȱPortugal.ȱ
Abstractȱ
Sedimentȱ contaminationȱ andȱ threeȱ bioassaysȱ wereȱ usedȱ toȱ determineȱ theȱ
sedimentȱqualityȱfourȱyearsȱafterȱanȱoilȱspillȱ(Prestige,ȱ2002):ȱtheȱMicrotox®ȱtest,ȱ
aȱ10ȬdayȱbioassayȱusingȱtheȱamphipodȱAmpeliscaȱbrevicornis,ȱandȱaȱpolychaeteȱ
10Ȭdayȱ toxicityȱ testȱ withȱ theȱ lugwormȱ Arenicolaȱ marina.ȱ Inȱ addition,ȱ
bioaccumulationȱ ofȱ PAHsȱ wasȱ examinedȱ inȱ theȱ polychaeteȱ afterȱ 10ȱ daysȱ ofȱ
exposure.ȱ Theȱ resultsȱ obtainedȱ fromȱ theȱ toxicityȱ testsȱ andȱ bioaccumulationȱ
analysesȱwereȱstatisticallyȱcomparedȱtoȱtheȱsedimentȱchemicalȱdata,ȱinȱorderȱtoȱ
assessȱ theȱ bioavailabilityȱ ofȱ theȱ contaminants,ȱ theirȱ effects,ȱ andȱ theirȱ
relationshipȱwithȱtheȱoilȱspill.ȱTheȱsedimentsȱstudiedȱwereȱfromȱtwoȱareasȱofȱtheȱ
GalicianȱCoastȱ(NWȱSpain):ȱtheȱBayȱofȱCormeȬLaxeȱandȱtheȱCíesȱIsland,ȱlocatedȱ
inȱ theȱ Atlanticȱ Islandȱ Nationalȱ Park.ȱ Theȱ resultsȱ pointȱ toȱ aȱ decreaseȱ inȱ
contaminationȱwithȱrespectȱtoȱpreviousȱstudiesȱandȱtoȱtheȱdisappearanceȱofȱtheȱ
acuteȱ toxicityȱ fourȱ yearsȱ afterȱ theȱ oilȱ spill.ȱ Howeverȱ anȱ importantȱ
bioaccumulationȱofȱPAHsȱwasȱdetectedȱinȱtheȱorganismsȱexposedȱtoȱsedimentsȱ
ȱChemosphereȱ(aceptadoȱconȱrevisiones)
- 69 -
fromȱ CormeȬLaxe,ȱ suggestingȱ thatȱ despiteȱ theȱ recoveryȱ ofȱ theȱ environmentalȱ
qualityȱofȱtheȱarea,ȱeffectsȱinȱtheȱbiotaȱmightȱbeȱoccuring.
Keywords:ȱPAHs,ȱtoxicity,ȱbioaccumulation,ȱMicrotox,ȱamphipod,ȱpolychaeteȱȱ
1.ȱIntroductionȱ
Theȱ informationȱ obtainingȱ byȱ integratingȱ theȱ chemicalȱ concentrationȱ inȱ
sedimentsȱandȱfromȱspecificȱsedimentȱbioassaysȱ(toxicityȱandȱbioaccumulation)ȱ
isȱ consideredȱ atȱ theȱ presentȱ timeȱ theȱ bestȱ approachȱ toȱ assessȱ theȱ sedimentȱ
qualityȱ andȱ toȱ testȱ theȱ toxicityȱ andȱ bioavailabilityȱ ofȱ chemicalȱ compoundsȱ inȱ
sedimentsȱtoȱbenthicȱorganismsȱ(CasadoȬMartínezȱetȱal.,ȱinȱpress;ȱDelVallsȱandȱ
Conradi,ȱ 2000;ȱ Chapmanȱ etȱ al.,ȱ 2002).ȱ Chemicalȱ analysesȱ performedȱ aloneȱ inȱ
bulkȱ sedimentȱ doȱ notȱ alwaysȱ reflectȱ theȱ toxicȱ fractionȱ sinceȱ theyȱ varyȱ
dependingȱonȱtheȱbioavailabilityȱandȱbioreactivityȱofȱtheȱtoxicȱcompounds.ȱAnȱ
approachȱtoȱthisȱinformationȱcanȱbeȱreachedȱbyȱrunningȱbioassays.ȱȱ
Theȱ useȱ ofȱ theȱ commercialȱ bioassayȱ Microtox®ȱ hasȱ increasedȱ inȱ recentȱ
years,ȱ dueȱ toȱ theȱ straightforwardȱ wayȱ toȱ detectȱ theȱ ‘‘hotȱ spots’’ȱ ofȱ fieldȱ
contaminationȱ inȱ theȱ screeningȱ procedureȱ (Mowatȱ andȱ Bundy,ȱ 2001;ȱ
Stronkhorstȱ etȱ al.,ȱ 2003;ȱ Vanȱ Beelen,ȱ 2003,ȱ CasadoȬMartinezȱ etȱ al,ȱ 2006a).ȱ
Microtox®ȱhasȱalsoȱbeenȱusedȱsuccessfullyȱinȱtheȱpast,ȱtoȱassessȱtheȱimpactȱofȱoilȱ
spillsȱandȱoilȱcontaminatedȱsedimentsȱ(Brohonȱetȱal.,ȱ2001;ȱKennethȱetȱal.,ȱ2003;ȱ
Pelletierȱetȱal.,ȱ2004;ȱMoralesȬCasellesȱetȱal.,ȱ2007).ȱPreviousȱstudiesȱhaveȱshownȱ
thatȱ theȱ amphipodȱ specieȱ Ampeliscaȱ brevicornisȱ isȱ aȱ sensitiveȱ organismȱ validȱ toȱ
assessȱtoxicityȱofȱcontaminatedȱsedimentsȱ(Ribaȱetȱal.ȱ2003;ȱCasadoȱMartinezȱetȱ
al.,ȱ 2006b;ȱ MoralesȬCaselles,ȱ etȱ al.ȱ submitted).ȱ Theȱ toxicityȱ testȱ accordingȱ toȱ
standardȱmethodologiesȱ(ASTMȱ1993)ȱhasȱbeenȱusedȱinȱpreviousȱstudiesȱforȱtheȱ
assessmentȱ ofȱ spillsȱ (Grantȱ andȱ Briggs,ȱ 2002;ȱ Briggsȱ etȱ al.,ȱ 2003).ȱ Theȱ 10Ȭdȱ
Arenicolaȱ bioassayȱ (ASTMȱ 1993)ȱ isȱ relevantȱ inȱ PAHȱ accumulationȱ studiesȱ
- 70 -
becauseȱA.ȱmarinaȱisȱaȱbulkȱsedimentȱfeedingȱpolychaeteȱwormȱthatȱlivesȱinȱaȱUȬ
shapedȱ burrowȱ andȱ isȱ consideredȱ anȱ indicatorȱ ofȱ PAHȱ pollutionȱ (Rustȱ etȱ al.,ȱ
2004b,ȱCasadoȬMartínezȱetȱal.,ȱinȱpress).ȱȱ
Inȱ Novemberȱ 13thȱ 2002ȱ theȱ oilȱ tankerȱ Prestigeȱ sufferedȱ anȱ accidentȱ 30ȱ
milesȱ offȱ theȱ Galicianȱ Coastȱ (NWȱ Spain).ȱ Fewȱ daysȱ later,ȱ theȱ tankerȱ sankȱ 150ȱ
milesȱoffshoreȱreleasingȱapproximatelyȱ60ȱ000ȱtonnesȱofȱheavyȱfuelȱoilȱintoȱtheȱ
surroundingȱwaters,ȱwhichȱcontaminatedȱmoreȱthanȱ1000ȱkmȱofȱcoastline.ȱTheȱ
physicochemicalȱ characteristicsȱ ofȱ theȱ fuelȱ oilȱ fromȱ theȱ tankerȱ wereȱ similarȱ toȱ
thoseȱfromȱtheȱErikaȱ(France,ȱ1999)ȱ(CSIC,ȱ2003)ȱ)ȱandȱshowedȱaȱlowȱsolubleȱandȱ
slowȱkineticsȱofȱdegradationȱunderȱnaturalȱconditions,ȱsoȱthatȱitȱwasȱexpectedȱtoȱ
beȱ accumulatedȱ inȱ sedimentsȱ (DelVallsȱ 2003).ȱ Thisȱ paperȱ presentsȱ theȱ firstȱ
attemptȱ toȱ assessȱ theȱ sedimentȱ contamination,ȱ toxicityȱ andȱ bioavailabilityȱ ofȱ
PAHsȱ inȱ theȱ coastȱ ofȱ Galiciaȱ fourȱ yearsȱ afterȱ theȱ sinkingȱ ofȱ theȱ tankerȱ Prestigeȱ
(2002).ȱȱ
2.ȱMaterialȱandȱmethodsȱ
2.1.ȱSamplingȱ
Afterȱ theȱ accidentȱ ofȱ theȱ tankerȱ Prestige,ȱ theȱ Cíiesȱ Islandȱ inȱ theȱ Atlanticȱ
IslandsȱNationalȱParkȱȱinȱGaliciaȱactedȱasȱaȱnaturalȱbarrierȱprotectingȱtheȱcoast.ȱ
Theȱ Bayȱ ofȱ CormeȬLaxe,ȱ consideredȱ anȱ areaȱ whichȱ developsȱ farming,ȱ fishing,ȱ
andȱshellȱfishingȱactivitiesȱ(Blancoȱetȱal.,ȱ2006)ȱwasȱalsoȱimportantlyȱaffectedȱbyȱ
theȱ spill.ȱ Surfaceȱ sedimentsȱ wereȱ collectedȱ inȱ ȱ 2005Ȭ2006ȱ withȱ aȱ 0.025ȱ m2ȱ Vanȱ
Veenȱ grabȱ nearȱ theȱ islandȱ ofȱ Cíesȱ Islandȱ inȱ theȱ Atlanticȱ Islandsȱ Nationalȱ Parkȱ
(stationsȱ A,ȱ Bȱ andȱ C)ȱ inȱ theȱ ´Bayȱ ofȱ CormeȬLaxe’ȱ (stationsȱ D,ȱ E,ȱ F),ȱ andȱ inȱ theȱ
Bayȱ ofȱ Cádizȱ (CA),ȱ theȱ latterȱ usedȱ asȱ referenceȱ stationȱ (Figureȱ 1).ȱ Sedimentȱ
samplesȱwereȱkeptȱinȱaȱcoolerȱ(4ºC)ȱandȱthenȱtransferredȱtoȱtheȱlaboratory.ȱEachȱ
- 71 -
sedimentȱ sampleȱ wasȱ homogenizedȱ withȱ aȱ Teflonȱ spoonȱ untilȱ noȱ colourȱ orȱ
texturalȱdifferencesȱcouldȱbeȱdetected,ȱandȱafterwardsȱsubsampledȱforȱphysicalȱ
characterization,ȱ chemicalȱ analysisȱ andȱ toxicityȱ tests.ȱ Sedimentȱ toxicityȱ testsȱ
wereȱrunȱnoȱlongerȱthanȱ2ȱweeksȱafterȱsampling.ȱ
ȱ
ȱ
ȱ
ƒE
ƒF
ƒD
ȱ
Ría de CormeLaxe
ȱ
Galician
Coast
ȱ
ȱ
Spain
ȱ
ȱ
ȱ
•CA
Cíes
•C •A
•B
Ría de
Vigo
N
ȱ
ȱ
Figureȱ1.ȱMapȱofȱtheȱcoastalȱareaȱofȱGaliciaȱshowingȱtheȱlocationsȱofȱtheȱ
samplingȱstations.ȱA,ȱBȱandȱCȱrefersȱtoȱtheȱstationsȱlocatedȱinȱtheȱCiesȱIslandȱinȱ
theȱAtlanticȱIslandȱNationalȱParkȱandȱD,ȱEȱandȱFȱtoȱthoseȱinȱtheȱBayȱofȱCormeȬ
Laxe.ȱ Theȱstationȱ CAȱ locatedȱ inȱtheȱBayȱofȱCadizȱcorrespondsȱtoȱtheȱsedimentȱ
usedȱasȱnegativeȱcontrol.ȱ
- 72 -
2.2ȱMicrotox®ȱtestȱ
InȱtheȱcommercialȱMicrotox®ȱtestȱtheȱdecreaseȱofȱtheȱbioluminescenceȱofȱ
theȱbacteriaȱVibrioȱfischeriȱisȱusedȱasȱaȱqualityȱindicatorȱofȱtheȱsampleȱexposed.ȱ
Measuresȱ ofȱ theȱ IC50ȱ (whichȱ isȱ theȱ dryȱ sedimentȱ concentrationȱ thatȱ provokesȱ
50%ȱ inhibitionȱ ofȱ theȱ lightȱ emittedȱ byȱ theȱ bacteria)ȱ provideȱ theȱ informationȱ
aboutȱ theȱ sedimentȱ toxicity.ȱ Theȱ bioassayȱ ofȱ bioluminescenceȱ inhibitionȱ withȱ
theȱ bacteriaȱ V.ȱ fischeriȱ wasȱ conductedȱ onȱ solidȱ phaseȱ withȱ theȱ commercialȱ
Microtox®ȱapparatusȱ(modelȱ500),ȱbyȱfollowingȱtheȱprotocolsȱforȱtheȱbasicȱsolidȱ
phaseȱ testȱ (BSPT),ȱ accordingȱ toȱ theȱ standardȱ operatingȱ procedureȱ (AZURȱ
Environmental,ȱ 1998)ȱ withȱ theȱ modificationsȱ reportedȱ byȱ Campisiȱ etȱ al.ȱ (2005)ȱ
andȱCasadoȬMartínezȱetȱal.ȱ(2006a).ȱȱȱ
2.3.ȱAmphipodȱtoxicityȱtestȱ
IndividualsȱofȱtheȱspecieȱAmpeliscaȱbrevicornisȱusedȱinȱtheȱbioassayȱwereȱ
collectedȱ byȱ sievingȱ theȱ sedimentȱ inȱ theȱ fieldȱ throughȱ aȱ 0.6ȱ mmȱ mesh,ȱ asȱ
reportedȱbyȱRibaȱetȱal.ȱ(2003)ȱandȱCasadoȬMartínezȱetȱal.ȱ(2006b).ȱTheȱcollectedȱ
biotaȱ wereȱ immediatelyȱ transportedȱ toȱ theȱ laboratoryȱ whereȱ theyȱ wereȱ placedȱ
inȱ 11ȬLȱ aquariumsȱ withȱ cleanȱ seawaterȱ andȱ sievedȱ sedimentȱ fromȱ theȱ sameȱ
location.ȱAerationȱwasȱprovidedȱandȱnaturalȱphotoperiodȱwasȱselected.ȱDuringȱ
acclimationȱ waterȱ wasȱ replacedȱ twiceȱ aȱ week,ȱ andȱ organismsȱ wereȱ fedȱ withȱ
specialȱ foodȱ forȱ invertebratesȱ (mixtureȱ madeȱ ofȱ aminoacidsȱ andȱ organicȱ
particles).ȱ
Theȱsedimentsȱwereȱfilteredȱ(1ȱmm)ȱpriorȱtoȱtheȱtoxicityȱtest,ȱinȱorderȱtoȱ
removeȱinorganicȱandȱorganicȱdebrisȱandȱbenthicȱorganismsȱcapableȱofȱpreyingȱ
Ampeliscaȱ brevicornis.ȱ Theȱ toxicityȱ testȱ wasȱ performedȱ byȱ exposingȱ individualȱ
amphipodsȱ toȱ theirȱ respectiveȱ sedimentȱ fromȱ theȱ differentȱ studyȱ sites.ȱ Theȱ
percentageȱ ofȱ survivalȱ afterȱ 10ȱ daysȱ ofȱ exposureȱ wasȱtheȱ measuredȱ endȱ point.ȱ
- 73 -
200ȱgȱofȱsedimentsȱwereȱplacedȱinȱ2ȱLȱglassȱbeakersȱandȱaboutȱ800ȱmLȱofȱcleanȱ
seawaterȱwasȱadded.ȱWhenȱtheȱsedimentȱsettledȱdownȱinȱtheȱbeakers,ȱaerationȱ
wasȱ provided,ȱ andȱ 12ȱ hoursȱ afterwardsȱ theȱ individualsȱ wereȱ sievedȱ fromȱ theȱ
acclimatizationȱaquariumsȱandȱ20ȱadultsȱ(3Ȭ5ȱmm)ȱofȱAmpeliscaȱwhereȱplacedȱinȱ
eachȱ replicate.ȱ Noȱ foodȱ wasȱ providedȱ duringȱ theȱ experiment.ȱ Theȱ containersȱ
whereȱ keptȱ inȱ anȱ incubatorȱ withȱ photoperiodȱ 12hȬlight/12hȬdarkȱ andȱ
maintainedȱ atȱ 19ȱ ±ȱ 1ȱ ºCȱ duringȱ theȱ 10ȱ daysȱ ofȱ exposure.ȱ Afterȱ thisȱ time,ȱ theȱ
beakersȱwhereȱsievedȱandȱtheȱsurvivalȱwasȱcountedȱinȱeachȱreplicate.ȱ
2.4.ȱPolychaeteȱbioassayȱ
TheȱA.ȱmarinaȱlugwormsȱwereȱsampledȱinȱtheȱfieldȱbyȱhandȬdiggingȱandȱ
immediatelyȱtransportedȱtoȱtheȱlaboratoryȱinȱcontainersȱwithȱseaȱwater.ȱOnceȱinȱ
theȱlaboratory,ȱlugwormsȱwereȱplacedȱinȱaquariumsȱwithȱsievedȱsedimentȱfromȱ
theȱsurveyȱareaȱ(5ȱcmȱthick)ȱandȱacclimatedȱforȱ7ȱd;ȱairȱwasȱprovidedȱandȱwaterȱ
wasȱ replacedȱ threeȱ timesȱ perȱ day.ȱ Waterȱ temperatureȱ wasȱ keptȱ atȱ 18ȱ ºCȱ andȱ
naturalȱphotoperiodȱwasȱselected.ȱFilteredȱsedimentsȱfromȱtheȱstudyȱsitesȱwereȱ
placedȱinȱreplicatesȱinȱ11ȱLȱtanksȱreachingȱaȱ5ȱcmȱthickȱlayerȱandȱcleanȱseaȱwaterȱ
wasȱadded.ȱLugwormsȱwereȱputȱintoȱtheȱtanksȱ(6ȱperȱtank)ȱwhichȱwereȱcoveredȱ
toȱ avoidȱ evaporation.ȱ Mortalityȱ wasȱ recordedȱ dailyȱ andȱ afterȱ 10ȱ daysȱ ofȱ
exposureȱ survivingȱ organismsȱ wereȱ transferredȱ toȱ aeratedȱ cleanȱ seaȱ waterȱ
withoutȱ sediment,ȱ whereȱ theyȱ wereȱ heldȱ forȱ approximatelyȱ 8ȱ hoursȱ toȱ induceȱ
gutȱemptying.ȱOrganismsȱwereȱthenȱfrozenȱatȱȬ20ȱºCȱforȱPAHsȱbioaccumulationȱ
analysis.ȱȱȱ
2.5.ȱChemicalȱanalysisȱ
Geochemicalȱ matrixȱ characteristicsȱ wereȱ examinedȱ byȱ analyzingȱ totalȱ
organicȱ carbonȱ concentrationȱ andȱ sedimentȱgrainȱsize.ȱ Organicȱcarbonȱ contentȱ
wasȱdeterminedȱusingȱtheȱmethodȱofȱGaudetteȱetȱal.ȱ(1974)ȱwithȱElȱRayis’ȱ(1985)ȱ
- 74 -
modification.ȱForȱsedimentȱgrainȱsize,ȱanȱaliquotȱofȱwetȱsedimentȱwasȱanalyzedȱ
usingȱaȱlaserȱparticleȱsizeȱFristchȱ(modelȱAnalysetteȱ22),ȱfollowingȱtheȱmethodȱ
reportedȱbyȱDelVallsȱetȱal.ȱ(1998).ȱ
TraceȱmetalȱanalysisȱwereȱanalyzedȱasȱdescribedȱbyȱCasadoȬMartínezȱetȱ
al.ȱ (2006c);ȱ briefly,ȱ 2.5ȱ gȱ ofȱ sedimentsȱ (<0.065ȱ mm)ȱ wereȱ placedȱ inȱ Teflonȱ
containersȱ andȱ wereȱdigestedȱ inȱ microwaveȱ(400W,ȱ15ȱmin,ȱtwice)ȱwithȱHNO3ȱ
2Nȱ inȱ orderȱ toȱ extractȱ theȱ fractionȱ ofȱ metalȱ withȱ theȱ greatestȱ potentialȱ toȱ beȱ
bioavailable.ȱTheȱextractsȱwereȱpurifiedȱbyȱpassingȱthroughȱaȱCȬ18ȱcolumnȱandȱ
metalsȱ analysesȱ wereȱ performedȱ byȱ anodicȱ voltamperimetryȱ (ȬZn,ȱ Cd,ȱ Pb,ȱ Ni,ȱ
Coȱ andȱ CuȬȱ Metrohmȱ Applicationȱ Bulletinȱ Nºȱ 147;ȱ Ȭȱ VȬȱ Metrohmȱ Applicationȱ
NoteȱNºȱVȬ81).ȱForȱHgȱtheȱcoldȱvapourȱtechniqueȱwasȱusedȱandȱwasȱquantifiedȱ
usingȱatomicȱabsorptionȱspectrometry.ȱTheȱanalyticalȱproceduresȱwereȱcheckedȱ
usingȱ referenceȱ materialȱ (MESSȬ1ȱ NRCȱ andȱ CRMȱ 277ȱ BCR)ȱ andȱ showedȱ aȱ
recoveryȱgreaterȱthanȱ90%ȱofȱtheȱcertifiedȱconcentration.ȱTheȱreasonȱwhyȱmetalsȱ
wereȱanalyzedȱinȱsedimentsȱisȱbecauseȱweȱareȱstudyingȱenvironmentalȱsamplesȱ
whichȱmayȱpresentȱaȱmixtureȱofȱcontaminantsȱandȱthisȱinformationȱmayȱhelpȱtoȱ
elucidateȱ ifȱ theȱ biologicalȱ effectsȱ areȱ dueȱ toȱ otherȱ contaminantsȱ apartȱ fromȱ
PAHs.ȱ
ȱ
Toȱ analyseȱ polycyclicȱ aromaticȱ hydrocarbonsȱ inȱ driedȱ sedimentsȱ andȱ
lugworms,ȱ samplesȱ wereȱ soxhletȱ extractedȱ withȱ nȬhexaneȱ forȱ 18ȱ h,ȱ andȱ withȱ
acetoneȱ /hexaneȱ (1:1)ȱ duringȱ 24h,ȱ respectively.ȱ Beforeȱ extractionȱ ofȱ organisms,ȱ
surrogateȱ PAHsȱ standardsȱ wereȱ addedȱ toȱ eachȱ sampleȱ (acenaphetheneȬd10,ȱ
phenanthreneȬd10,ȱchryseneȬd12ȱandȱperyleneȬd12)ȱwereȱdone.ȱCompoundsȱwereȱ
isolatedȱfromȱextractsȱbyȱcolumnȱchromatographyȱonȱFlorisilȬaluminoȬsilicaȱforȱ
sedimentsȱ orȱ silicaȬaluminaȱ forȱ lugworms.ȱ PAHsȱ wereȱ elutedȱ withȱ
- 75 -
dichlorometane/hexaneȱ (9:1ȱ andȱ 4:1).ȱ Aromaticȱ fractionsȱ inȱ sedimentsȱ wereȱ
analyzedȱonȱaȱHewlett–Packardȱ(HP)ȱ5890ȱSeriesȱIIȱgasȱchromatographȱcoupledȱ
withȱ HPȱ 5970ȱ massȱ spectrometer.ȱ PAHȱ concentrationsȱ inȱ organismsȱ wereȱ
determinedȱ inȱ aȱ GCȬMSȱ Finniganȱ Matȱ GCQȱ TMȱ inȱ theȱ selectedȱ ionȱ mode.ȱ
Chromatographicȱanalysisȱwasȱachievedȱwithȱaȱ30ȱmȱ×ȱ0.250ȱmmȱDBȬ5ȱcapillaryȱ
columnȱ (0.25ȱ ΐmȱ filmȱ thickness)ȱ withȱ heliumȱ asȱ carrierȱ gas.ȱ Injectionȱ wasȱ
performedȱinȱtheȱsplitlessȱmodeȱatȱ280ºC.ȱHeliumȱwasȱusedȱasȱcarrierȱgasȱwithȱaȱ
flowȱofȱ1ȱmlȱminȱ Ȭ1.ȱTheȱovenȱtemperatureȱwasȱprogrammedȱasȱfollows:ȱinitialȱ
temperatureȱwasȱ75°ȱCȱandȱthenȱrampedȱtoȱ130°Cȱatȱ20°/minȱandȱthenȱrampedȱ
toȱ 320°Cȱ atȱ theȱ rateȱ ofȱ 4°C/minȱ andȱ heldȱ forȱ 15ȱ minutes.ȱ Theȱ electronȱ impactȱ
ionizationȱmodeȱconditionsȱwereȱtheȱfollowing:ȱionȱenergyȱ70ȱeVȱandȱionȱsourceȱ
andȱ transferȱ lineȱ temperaturesȱ 220ºC.ȱ PAHȱ wereȱ identifiedȱ byȱ retentionȱ timeȱ
andȱ oneȱ characteristicȱ massȱ fragmentȱ ion.ȱ Quantificationȱ ofȱ 16ȱ PAHȱ
(acenaphethylene,ȱ fluorene,ȱ phenanthrene,ȱ anthracene,ȱ fluoranthene,ȱ pyrene,ȱ
chrysene,ȱ benz[a]anthracene,ȱ benzo[b]fluoranthene,ȱ benzo[k]fluoranthene,ȱ
benzo[a]pyrene,ȱ
benzo[e]pyrene,ȱ
perylene,ȱ
indeno[1,2,3Ȭcd]pyrene,ȱ
dibenz[a,h]anthraceneȱ andȱ benzo[g,h,i]ȱ perylene)ȱ wereȱ doneȱ usingȱ aȱ 9Ȭpointȱ
calibrationȱcurveȱforȱeachȱcompound.ȱQualityȱcontrolȱsamplesȱwereȱanalyzedȱinȱ
eachȱ batchȱ ofȱ 12ȱ samples,ȱ referenceȱ materialȱ NIST,ȱ SRMȱ 2977ȱ (mussels),ȱ NRCȬ
CNRCȱ HSȬ6ȱ (sediments)ȱ andȱ blanks.ȱ Theȱ analyticalȱ procedureȱ showedȱ aȱ
recoveryȱ greaterȱ thanȱ 90%ȱ forȱ sedimentȱ materialsȱ andȱ betweenȱ 75Ȭ125%ȱ forȱ
NISTȱ material.ȱ Detectionȱ limitsȱ rangedȱ fromȱ 1.0ȱ toȱ 5.0ȱ ngȱ gȬ1,ȱ dryȱ weight,ȱ forȱ
organismsȱandȱforȱsediments.ȱ
2.6.ȱStatisticalȱanalysisȱ
Resultsȱ obtainedȱ fromȱ theȱ bioassaysȱ andȱ theȱ chemicalȱ measurementsȱ
wereȱ linkedȱ byȱ factorȱ analysis,ȱ usingȱ principalȱ componentsȱ analysisȱ (PCA)ȱ asȱ
theȱ extractionȱ procedure,ȱ whichȱ isȱ aȱ multivariateȱ statisticalȱ techniqueȱ forȱ
- 76 -
exploringȱ variableȱ distributionsȱ (Ribaȱ etȱ al.,ȱ 2003).ȱ Theȱ objectiveȱ ofȱ PCAȱ isȱ toȱ
deriveȱaȱreducedȱnumberȱofȱnewȱvariablesȱasȱlinearȱcombinationsȱofȱtheȱoriginalȱ
variables.ȱThisȱ providesȱ aȱdescriptionȱofȱtheȱdataȱstructureȱwithȱtheȱminimumȱ
lossȱ ofȱ information.ȱ ANOVAȱ wasȱ performedȱ inȱ orderȱ toȱ determineȱ significantȱ
differencesȱ(p<0.05)ȱinȱsurvivalȱorganismsȱbetweenȱtheȱtoxicityȱresultsȱobtainedȱ
forȱtheȱreferenceȱsiteȱandȱtheȱotherȱsamplingȱsites.ȱ
3.ȱResultsȱandȱdiscussionȱ
3.1.ȱChemicalȱanalysisȱ
Tableȱ 1ȱ presentsȱ theȱ generalȱ characteristicsȱ (fineȱ particlesȱ andȱ organicȱ
carbon)ȱ andȱ theȱ levelsȱ ofȱ PAHȱ andȱ traceȱ elementsȱ inȱ sedimentsȱ fromȱ threeȱ
groupsȱofȱstations:ȱReferenceȱsiteȱ(CA),ȱAtlanticȱIslandsȱNationalȱParkȱ(A,ȱBȱandȱ
C)ȱandȱCormeȬLaxeȱ(D,ȱEȱandȱF).ȱȱȱȱSedimentsȱconsistedȱofȱlargeȱproportionȱofȱ
sandȱ withȱ lowȱ organicȱ carbonȱ contentȱ andȱ diminishedȱ metalȱ concentrations.ȱ
Onlyȱ Znȱ presentedȱ moderateȱ concentrationsȱ inȱ stationsȱ Aȱ andȱ F.ȱ Theȱ levelsȱ ofȱ
PAHsȱ variedȱ broadlyȱ amongȱ theȱ stations:ȱ belowȱ detectionȱ limitȱ inȱ CAȱ andȱ Cȱ
andȱ higherȱ valuesȱ inȱ Aȱ (108ȱ mgȱ kgȬ1)ȱ andȱ Fȱ (323ȱ mgȱ kgȬ1).ȱ Theȱ comparisonȱ ofȱ
PAHȱlevelsȱinȱ2005Ȭ06ȱwithȱsedimentsȱcollectedȱinȱ2003Ȭ04ȱ(MoralesȬCasellesȱetȱ
al.,ȱ2007)ȱandȱ2004Ȭ05ȱ(MoralesȬCasellesȱetȱal,ȱ2006)ȱshowedȱaȱgeneralȱdecreaseȱ
inȱ concentrationsȱ inȱ theȱ stationsȱ surveyedȱ atȱ AINPȱ andȱ CormeȬLaxeȱ (Tableȱ 2).ȱ
ThisȱcomparisonȱsuggestsȱaȱdecreaseȱofȱPAHsȱinȱtheȱsedimentsȱofȱtheȱtwoȱareasȱ
affectedȱbyȱtheȱPrestigeȱoilȱspill.ȱȱ
3.2.ȱMicrotox®ȱtestȱ
Figureȱ 2ȱ showsȱ theȱ IC50ȱ resultsȱ obtainedȱ throughȱ theȱ Microtox®ȱ testȱ
withȱtheȱsedimentsȱcollectedȱinȱtheȱthreeȱareasȱinȱ2005Ȭ06.ȱAllȱIC50ȱresultsȱwereȱȱ
- 77 -
Tableȱ 1.ȱ Concentrationȱ ofȱ PAHsȱ (ΐgȱ kgȬ1ȱ dryȱ weight),ȱ metalsȱ (mgȱ kgȬ1ȱ
dryȱweight),ȱorganicȱcarbonȱ(%dryȱweight)ȱandȱpercentageȱofȱfineȱparticlesȱ(<ȱ63ȱ
ΐmȱ ofȱ dryȱ sediment)ȱ inȱ sedimentȱ samples.ȱ Reference:ȱ CA;ȱ Atlanticȱ islandsȱ
Nationalȱ Park:ȱ A,ȱ Bȱ andȱ C;ȱ CormeȬLaxe:ȱD,ȱ Eȱ andȱ F.ȱ n.d.ȱ =ȱ notȱ detectedȱ valueȱ
(<0.005ȱΐgȱKgȬ1).
ȱȱ
ȱȱ
PAHȱ Znȱȱ Cd Pbȱ Cu
Niȱȱ Coȱ Vȱ
Hgȱ OCȱ fines
REFERENCEȱ CA
n.d.ȱ 21.3ȱ 0.9 2.3ȱ 7.0ȱ
0.1ȱ
0.05ȱ 1.1ȱ
1.0ȱ
ȱȱ
AINPȱ
ȱȱ
Aȱ
Bȱ
Cȱ
108ȱ 377ȱ 0.3 1.5ȱ 5.2ȱ 13.3ȱ 0.3ȱ 0.7ȱ 0.04ȱ 0.4ȱ
67ȱ 91ȱ 0.2 0.9ȱ 1.4ȱ 2.4ȱ 0.2ȱ 0.8ȱ 0.28ȱ 0.4ȱ
n.d.ȱ 164ȱ n.d 0.9ȱ 1.4ȱ 4.5ȱ 0.1ȱ 0.6ȱ 0.09ȱ 0.3ȱ
4.3ȱ
2.8ȱ
2.8ȱ
CormeȬ
Laxeȱ
Dȱ
Eȱ
Fȱ
38ȱ 25ȱ 0.2 3.7 0.7
52ȱ 19.9ȱ 0.1 7.3 0.4
323ȱ 271ȱ 0.2 5.9 4.2
3.8ȱ
5.5ȱ
6.0ȱ
1.7ȱ
1.5ȱ
5.7ȱ
3.4ȱ 80ȱ
0.3 2.0
0.4 2.1
0.4 3.4
0.16ȱ 0.3ȱ
0.08ȱ 0.4ȱ
0.08ȱ 0.7ȱ
ȱ
Tableȱ 2.ȱ Tableȱ 2.ȱ ȱ Concentrationȱ ofȱ PAHsȱ (ΐgȱ kgȬ1ȱ dryȱ weight)ȱ inȱ theȱ
followingȱ yearsȱ afterȱ theȱ Prestigeȱ oilȱ spillȱ (November,ȱ 2002).ȱ n.a:ȱ notȱ availableȱ
data;ȱn.d.=ȱnotȱdetectedȱvaluesȱ(<0.005ȱmgȱkgȬ1)ȱ
station
2003-2004
2004-2005
2005-2006
A
390
119
108
B
2120
366
67
C
420
239
n.d.
D
n.a
537
38
E
n.a
558
52
F
n.a
820
323
ȱ
ȱ
- 78 -
clearlyȱaboveȱtheȱlimitsȱassumedȱforȱsedimentȱtoxicityȱ(expressedȱonȱdryȱweightȱ
units):ȱ1000ȱmgȱLȬ1ȱ(CanadianȱStandardsȱinȱEnvironmentȱCanada,ȱ2002)ȱandȱ750ȱ
mgȱLȬ1ȱ(SpanishȱStandardsȱinȱDelVallsȱetȱal.,ȱ2004;ȱCasadoȬMartínezȱetȱal.,ȱ2006).ȱ
Theseȱresultsȱindicateȱthatȱtheȱtestedȱsedimentsȱhadȱnoȱacuteȱtoxicity.ȱ
ȱ
25000
ȱ
IC50 (mg L-1 dry weight)
ȱ
20000
ȱ
ȱ
10000
ȱ
ȱ5000
ȱ
ȱ
0
CA
A
B
ȱ
C
D
E
F
treatments
Figureȱ2.ȱIC50ȱresultsȱobtainedȱfromȱtheȱapplicationȱofȱtheȱMicrotox®ȱtestȱ
toȱ sedimentȱ samplesȱ fromȱ theȱ variousȱ stations.ȱ Theȱ lineȱ indicatesȱ theȱ limitsȱ
belowȱ whichȱ theȱ sedimentȱ sampleȱ isȱ consideredȱ toxicȱ byȱ theȱ Canadianȱ
Standardsȱ(1000ȱmgȱLȬ1ȱdryȱweight).ȱ
3.3.ȱAmphipodȱandȱpolychaeteȱtoxicityȱtestsȱ
Theȱ resultsȱ ofȱ theȱ 10Ȭdȱ bioassaysȱ withȱ amphipodsȱ andȱ polychaeteȱ areȱ
givenȱ inȱ Figureȱ 3.ȱ Meanȱ valuesȱ ofȱ mortalityȱ wasȱ lessȱ thanȱ 25ȱ ȱ andȱ 27%ȱ forȱ
Ampeliscaȱ andȱ Arenicolaȱ bioassays,ȱ respectively.ȱ Noneȱ ofȱ theȱ testedȱ sedimentsȱ
wereȱ significantlyȱ (p<0,05)ȱ differentȱ fromȱ theȱ referenceȱ sedimentȱ (CA),ȱ
suggestingȱ thatȱ sedimentsȱ areȱ notȱ toxicȱ inȱ accordanceȱ withȱ theȱ USȱ
- 79 -
Environmentalȱ Protectionȱ Agencyȱ (USEPA,ȱ 1994)ȱ andȱ guidelinesȱ reportedȱ byȱ
CasadoȬMartínezȱ etȱ al.ȱ (2006b).ȱ Aȱ highȱ variabilityȱ inȱ theȱ mortalityȱ resultsȱ wasȱ
observedȱinȱtheȱpolychaeteȱassayȱandȱnoȱcorrelationȱwasȱobservedȱbetweenȱtheȱ
amphipodȱ andȱ polychaeteȱ mortalityȱ results,ȱ whichȱ canȱ beȱ dueȱ toȱ theȱ differentȱ
susceptibilities.ȱThisȱfactȱhighlightsȱtheȱimportanceȱofȱusingȱdifferentȱorganismsȱ
inȱ thisȱ kindȱ ofȱ studies,ȱ especiallyȱ whereȱ diverseȱ knownȱ andȱ unknownȱ
contaminantsȱareȱpresentȱandȱsinergic/antagonicȱeffectsȱmayȱoccur.ȱ
ȱ
50
ȱ
Ampelisca brevicornis
Arenicola marina
Mortality (%)
40 ȱ
ȱ
30
ȱ
20 ȱ
ȱ
10
ȱ
ȱ
0
ȱ
ȱ
CA
A
B
C
D
E
F
treatments
Figureȱ 3.ȱ Averagedȱ mortalityȱ resultsȱ andȱ standardȱ deviationsȱ afterȱ 10ȱ
daysȱ ofȱ exposingȱ Ampeliscaȱ brevicornisȱ andȱ Arenicolaȱ marinaȱ toȱ theȱ sedimentȱ
samples.ȱ
ȱ
ȱ
- 80 -
3.4.ȱBioaccumulationȱassayȱ
Despiteȱ theȱ absenceȱ ofȱ acuteȱ toxicity,ȱ lugwormsȱ exposedȱ 10ȱ daysȱ toȱ
sedimentsȱfromȱtheȱGalicianȱCoastȱshowedȱaȱhigherȱaccumulationȱofȱPAHsȱthanȱ
thoseȱ exposedȱ toȱ sedimentsȱ fromȱ theȱ referenceȱ station.ȱ Theȱ concentrationsȱ ofȱ
PAHsȱ inȱ thoseȱ wormsȱ exposedȱ toȱ sedimentsȱ variedȱ fromȱ 3.2Ȭ3.9ȱ mgȱ kgȬ1ȱ
(CormeȬLaxe)ȱ toȱ 2.6Ȭ2.7ȱ mgȱ kgȬ1ȱ (Atlanticȱ Islandȱ Nationalȱ Park).ȱ Aȱ PAHsȱ
bioaccumulationȱ indexȱ BI=Cx/CRȱ wasȱ definedȱ byȱ theȱ ratioȱ betweenȱ theȱ PAHȱ
concentrationȱinȱtheȱlugwormȱexposedȱtoȱsedimentȱXȱ(Cx)ȱandȱtoȱtheȱreferenceȱ
(CR).ȱ Theȱ BIȱ rangedȱ withinȱ theȱ narrowȱ intervalȱ ofȱ 1.0Ȭ1.6,ȱ meaningȱ thatȱ
lugwormsȱpresentedȱaȱnarrowȱintervalȱofȱaccumulationȱthanȱsediments.ȱȱ
ȱ
1.0
Zn
ȱ
ȱ
Ampelisca
0.6
ȱ
Factor 2
Cu
Ni 0.8
0.4
Microtox
0.2
ȱ
PAH
fines
ȱ
0.0
-1.0
-0.8
ȱ
Cd
Hg
ȱ
-0.6
-0.4
-0.2
0.0
0.2
-0.2
Arenicola
0.4
0.6
Co
0.8
1.0
1.2
Bioaccumulation
O.C.
-0.4
V
Pb
-0.6
ȱ
Factor 1
ȱ
Figureȱ 4.ȱ Factorȱ loadingsȱ ofȱ 15ȱ variablesȱ forȱ theȱ twoȱ principalȱ factorsȱ
resultingȱ fromȱ theȱ multivariateȱ analysisȱ ofȱ resultsȱ obtainedȱ fromȱ theȱ chemicalȱ
analysis,ȱtheȱacuteȱtoxicityȱtestsȱandȱtheȱbioaccumulationȱassay.ȱ
- 81 -
3.5.ȱPrincipalȱcomponentȱanalysisȱ
Aȱ principalȱ componentȱ analysisȱ (PCA)ȱ wasȱ performedȱ includingȱ
sedimentȱchemicalȱparameters,ȱtoxicityȱtestsȱandȱPAHsȱbioaccumulationȱindex.ȱ
Theȱ15ȱvariablesȱcanȱbeȱgroupedȱinȱ2ȱnewȱfactorsȱwhichȱexplainȱaȱ61.6ȱ%ȱofȱtheȱ
totalȱ variance.ȱ Figureȱ 4ȱ representsȱ theȱ loadingsȱ ofȱ theȱ variablesȱ inȱ eachȱ factor.ȱ
Forȱaȱgoodȱassociationȱbetweenȱvariableȱandȱaȱfactorȱweȱdecidedȱtoȱinterpretȱaȱ
groupȱ ofȱ variablesȱ asȱ thoseȱ associatedȱ toȱ aȱ particularȱ componentȱ whereȱ theȱ
loadingȱ wasȱ 0.50ȱ orȱ higherȱ whichȱ approximatesȱ toȱ Comreys’ȱ cutȬoffȱ ofȱ 0.55ȱ
(Comreys,ȱ 1973).ȱ ȱ Theȱ firstȱ factor,ȱ Factorȱ #1,ȱ accountsȱ forȱ 39.0ȱ %ȱ ofȱ theȱ totalȱ
varianceȱ andȱ linksȱ theȱ bioaccumulationȱ ofȱ PAHsȱ inȱ theȱ polychaeteȱ withȱ theȱ
presenceȱ ofȱ PAHs,ȱ Pb,ȱ Co,ȱ andȱ finesȱ inȱ theȱ sediments.ȱ Factorȱ #2ȱ accountsȱ forȱ
22.6ȱ%ȱofȱtheȱvarianceȱandȱshowsȱtheȱrelationshipȱbetweenȱmetalsȱZn,ȱCuȱandȱ
Niȱ withȱ theȱ lowȱ toxicityȱ (<25%ȱ ofȱ mortality)ȱ toȱ theȱ amphipodȱ Ampeliscaȱ
brevicornis.ȱ
Figureȱ5ȱshowsȱtheȱinfluenceȱofȱeachȱfactorȱinȱtheȱ7ȱstudyȱsites.ȱFactorȱ#1,ȱ
definedȱ asȱ theȱ PAHsȱ bioaccumulationȱ dueȱ toȱ theirȱ presenceȱ inȱ sedimentsȱ andȱ
theȱassociationȱwithȱPbȱandȱCo;ȱitȱhasȱmainlyȱprevalenceȱinȱtheȱsitesȱDȱ(0.35),ȱEȱ
(1.07)ȱ andȱ Fȱ (0.86)ȱ fromȱ CormeȬLaxe.ȱ Thisȱ meansȱ thatȱ theseȱ stationsȱ presentȱ aȱ
contaminationȱ byȱ PAHsȱ whichȱ areȱ ableȱ toȱ bioaccumulateȱ inȱ theȱ biotaȱ andȱ areȱ
accompaniedȱ byȱ theȱ metalsȱ Pbȱ andȱ Co.ȱ Howeverȱ itȱ doesȱ notȱ reflectȱ anȱ acuteȱ
toxicityȱ ofȱ theȱ benthicȱ organismsȱ exposedȱ toȱ theȱ sediments.ȱ Despiteȱ theȱ lowȱ
mortalityȱ withȱ theȱ 10Ȭdȱ bioassaysȱ (Figureȱ 3)ȱ theȱ PCAȱ suggestsȱ aȱ relationshipȱ
betweenȱsedimentȱquality,ȱbioaccumulationȱandȱtheȱslightȱmortality.ȱFactorȱ#2,ȱ
whichȱ relatesȱ aȱ slightȱ toxicityȱ withȱ theȱ metalsȱ Zn,ȱ Cuȱ andȱ Niȱ boundȱ toȱ
sediments,ȱpresentsȱaȱpositiveȱloadingȱinȱtheȱsitesȱAȱ(1.75),ȱCȱ(0.49)ȱandȱFȱ(0.34).ȱȱ
- 82 -
2.0
A
1.5
1.0
Factor 2
C
0.5
F
0.0
-2.5
-2.0
-1.5
-1.0
-0.5
B
0.0
0.5
1.0
1.5
-0.5
CA
-1.0
D
E
-1.5
Factor 1
Figureȱ 5.ȱ Estimatedȱ factorȱ scoresȱ forȱ theȱ twoȱ factorsȱ inȱ eachȱ ofȱ theȱ 7ȱ
studiedȱcases.ȱTheȱfactorȱscoresȱquantifyȱtheȱprevalenceȱofȱeachȱfactorȱforȱeveryȱ
stationȱandȱareȱusedȱtoȱestablishȱtheȱdefinitionȱofȱeachȱfactor.ȱ
3.6.ȱGeneralȱdiscussionȱ
Chemicalȱ analysesȱ ofȱ surfaceȱ sedimentsȱ fromȱ theȱ Galicianȱ coastȱ (Ríaȱ deȱ
CormeȬLaxeȱ andȱ Cíes)ȱ indicateȱ aȱ substantialȱ decreaseȱ inȱ theȱ contentȱ ofȱ PAHsȱ
fourȱ yearsȱ afterȱ theȱ oilȱ spill.ȱ Theȱ effectȱ ofȱ theȱ oilȱ spillȱ fromȱ Prestigeȱ probablyȱ
diminishedȱ dueȱ toȱ biotransformationȱ andȱ volatilizationȱ ofȱ compoundsȱ inȱ theȱ
sedimentsȱ (Albers,ȱ 2003).ȱ Itȱ shouldȱ notȱ beȱ ignoredȱ thatȱ hydrodynamicȱ
processes,ȱ namelyȱ sedimentȱ resuspension,ȱ mayȱ haveȱ influenceȱ inȱ thoseȱ
processesȱ(Neff,ȱ2002).ȱInȱaddition,ȱenhancedȱlevelsȱofȱVȱandȱNi,ȱwhichȱareȱoftenȱ
associatedȱ withȱ hydrocarbonȱ spills,ȱ suggestsȱ thatȱ theirȱ presenceȱ couldȱ beȱ
relatedȱ toȱ theȱ Prestigeȱ oilȱ spill.ȱ Additionalȱ sourcesȱ couldȱ contributeȱ toȱ theȱ
amountȱofȱZnȱandȱCuȱfoundȱinȱtheseȱareasȱ(CobeloȬGarcíaȱetȱal.,ȱ2004).ȱȱ
- 83 -
Apparently,ȱ theȱ linkȱ betweenȱ theȱ resultsȱ ofȱ theȱ 10Ȭdȱ bioassaysȱ andȱ theȱ
presenceȱofȱPAHsȱinȱtheȱsedimentsȱfromȱCormeȬLaxeȱisȱindicativeȱofȱsedimentȱ
toxicity.ȱ Thisȱ linkȱ isȱ howeverȱ insufficientȱ toȱ describeȱ thoseȱ sedimentsȱ asȱ toxic,ȱ
sinceȱvaluesȱwereȱlowerȱthanȱtheȱinternationalȱguidelinesȱemployedȱforȱthisȱtest.ȱ
Moreover,ȱtheȱresultsȱofȱtheȱMicrotoxȱtestȱwereȱunrelated.ȱDespiteȱtheȱdecreaseȱ
withȱ theȱ timeȱ (Tableȱ 2)ȱ ofȱ PAHȱ concentrationsȱ inȱ sedimentsȱ fromȱ theȱ Galicianȱ
areaȱ affectedȱ byȱ theȱ oilȱ spillȱ (Fernándezȱ etȱ al.,ȱ 2006;ȱ SorianoȬSanz,ȱ 2006;ȱ
MoralesȬCasellesȱ etȱ al.,ȱ 2007;ȱ MoralesȬCasellesȱ etȱ al.,ȱ accepted),ȱ PAHsȱ
accumulationȱ wasȱ identifiedȱ inȱ theȱ organismsȱ exposedȱ toȱ 10Ȭdȱ tests.ȱ Residuesȱ
stillȱremainȱinȱtissuesȱofȱbenthicȱorganisms,ȱprobablyȱindicatingȱthatȱPAHsȱareȱ
availableȱ toȱ theȱ foodȱ chain,ȱ andȱ thusȱ representingȱ aȱ potentialȱ riskȱ toȱ theȱ
wellbeingȱofȱtheȱecosystem.ȱTheȱfactȱthatȱnoȱacuteȱtoxicityȱwasȱdetectedȱinȱtheȱ
sedimentsȱandȱtheȱbioaccumulationȱproducedȱbyȱtheȱPAHsȱinȱCormeȬLaxeȱ(siteȱ
Eȱ andȱ F)ȱ suggestsȱ thatȱ thereȱ hasȱ beenȱ aȱ recoveryȱ ofȱ theȱ areaȱ affectedȱ byȱ theȱ
accidentalȱoilȱspill.ȱSimilarȱresultsȱwhereȱobtainȱyearsȱafterȱmajorȱtankerȱspills,ȱ
suchȱasȱtheȱExxonȱValdezȱ(USA,ȱ1989)ȱ(LeeȱandȱPageȱ1997),ȱtookȱplace.ȱHowever,ȱ
previousȱstudiesȱhaveȱshownȱtheȱrelationshipȱbetweenȱtheȱPrestigeȱoilȱspillȱandȱ
sublethalȱeffectsȱinȱtheȱorganismsȱexposedȱtoȱcontaminatedȱsedimentsȱfromȱtheȱ
Galicianȱ areaȱ (MartínezȬGómezȱ etȱ al.,ȱ 2006;ȱ Marigómezȱ etȱ al.,ȱ 2006;ȱ MoralesȬ
Casellesȱetȱal.,ȱ2006;ȱFernándezȱetȱal.,ȱ2006;ȱSorianoȬSanz,ȱ2006).ȱȱ
4.ȱConclusionsȱȱ
Theȱ resultsȱ obtainedȱ inȱ theȱ presentȱ studyȱ suggestȱ thatȱ thereȱ hasȱ beenȱ aȱ
recoveryȱofȱtheȱqualityȱofȱtheȱsedimentsȱaffectedȱbyȱtheȱPrestigeȱoilȱspillȱinȱtheȱ
GalicianȱCoast.ȱSedimentsȱfromȱCormeȬLaxeȱandȱtheȱAINPȱdoȱnotȱpresentȱacuteȱ
toxicityȱ althoughȱ theȱ presenceȱ ofȱ someȱ metalsȱ andȱ PAHsȱ inȱ theȱ sedimentsȱ isȱ
consideredȱ aȱ potentialȱ riskȱ inȱ thoseȱ areas;ȱ evenȱ thoughȱ PAHsȱ doȱ notȱ tendȱ toȱ
- 84 -
bioaccumulateȱalongȱtheȱtrophicȱchainȱ(Neff,ȱ2002),ȱtheȱaccumulationȱofȱPAHsȱ
inȱ theȱ polychaeteȱ Arenicolaȱ marinaȱ wasȱ relatedȱ toȱ theȱ presenceȱ ofȱ thisȱ
contaminantȱ inȱ theȱ Bayȱ ofȱ CormeȬLaxeȱ andȱ suggestsȱ theȱ possibilityȱ ofȱ
producingȱ sublethalȱ toxicȱ effectsȱ toȱ theȱ organismsȱ exposedȱ despiteȱ thereȱ wasȱ
notȱ acuteȱtoxicityȱ detected.ȱFurtherȱstudiesȱareȱrequired,ȱinȱorderȱtoȱfollowȱupȱ
theȱsublethalȱeffectsȱyearsȱafterȱtheȱoilȱspill.ȱ
5.ȱAcknowledgmentsȱ
Theȱ workȱ describedȱ wasȱ partlyȱ supportedȱ byȱ theȱ projectsȱ VEM2003Ȭ
20563/INTER,ȱ andȱ CTM2005Ȭ07282ȬC03Ȭ01/TECNOȱ financedȱ byȱ theȱ Spanishȱ
Educationȱ andȱ Scienceȱ Ministryȱ andȱ byȱ CISȱ fundedȱ byȱ theȱ Ministryȱ ofȱ
Environment.ȱ Carmenȱ MoralesȬCasellesȱ thanksȱ theȱ Ministryȱ ofȱ Educationȱ andȱ
Scienceȱ forȱ fundingȱ herȱ researchȱ fellowshipȱ (FPU).ȱ Dr.ȱ Ribaȱ thanksȱ theȱ I3pȱ
programȱ forȱ supportingȱ herȱ participationȱ inȱ thisȱ research.ȱ ȱ Inmaculadaȱ Ribaȱ
thanksȱtheȱCSICȱforȱherȱI3Pȱcontract.ȱWeȱareȱgratefulȱforȱtheȱsupportȱandȱhelpȱofȱ
theȱ membersȱ ofȱ theȱ CISȱ andȱ IPIMAR.ȱ Specialȱ thanksȱ areȱ givenȱ toȱ Isabelinaȱ
Santos.ȱ
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testȱ toȱ assessȱ theȱ impactȱ ofȱ inȱ situȱ oiledȱ shorelineȱ treatmentȱ options:ȱ naturalȱ
attenuationȱandȱsedimentȱrelocation.ȱSpill.ȱSci.ȱTechnol.ȱBull.ȱ8,ȱ273Ȭ284.ȱ
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- 90 -
ȱ
Capítuloȱ3.ȱ
Estudioȱdeȱefectosȱsubletalesȱenȱorganismosȱbajoȱ
condicionesȱdeȱlaboratorioȱ
Laȱimportanciaȱdeȱlosȱtestȱdeȱtoxicidadȱaȱnivelȱsubletalȱradicaȱenȱqueȱesteȱ
tipoȱdeȱestudioȱpermiteȱevaluarȱconȱunaȱmayorȱsensibilidadȱlaȱrespuestaȱdeȱunȱ
organismoȱ anteȱ unȱ tipoȱ deȱ contaminaciónȱ ademásȱ deȱ discriminarȱ puntosȱ conȱ
unaȱcontaminaciónȱmoderadaȱidentificandoȱlosȱefectosȱsubletales.ȱȱAntesȱdeȱqueȱ
seȱ produzcaȱ laȱ muerteȱ oȱ laȱ enfermedad,ȱ tantoȱ organismosȱ comoȱ poblacionesȱ
respondenȱ alȱ estrésȱ alterandoȱ diferentesȱ parámetrosȱ aȱ nivelȱ molecular,ȱ
histológico,ȱ inmunológicoȱ yȱ fisiológico,ȱ aȱ nivelȱ deȱ organismo,ȱ poblaciónȱ oȱ
ecosistemaȱ (Livingstone,ȱ 1993;ȱ LópezȬBarea,ȱ 1994).ȱ Losȱ biomarcadores,ȱ
permitenȱevaluarȱlosȱefectosȱdelȱestrésȱsubletalȱsobreȱlosȱorganismosȱexpuestosȱ
aȱ sustanciasȱ contaminantes,ȱ reflejandoȱ elȱ estadoȱ deȱ losȱ individuosȱ aȱ nivelȱ
molecularȱ oȱ celularȱ comoȱ respuestaȱ aȱ dichoȱ estrés.ȱ Además,ȱ estasȱ medidasȱ
puedenȱ identificarȱ rápidamenteȱ laȱ presenciaȱ deȱ sustanciasȱ tóxicas,ȱ ofreciendoȱ
unaȱ alertaȱ tempranaȱ antesȱ deȱ queȱ lasȱ alteracionesȱ lleguenȱ aȱ nivelesȱ deȱ
organizaciónȱ mayores.ȱ ȱ Losȱ biomarcadoresȱ hanȱ mostradoȱ serȱ lasȱ herramientasȱ
adecuadasȱ paraȱ caracterizarȱ elȱ estadoȱ deȱ losȱ organismosȱ presentesȱ enȱ zonasȱ
impactadasȱ dondeȱ seȱ daȱ laȱ presenciaȱ deȱ mezclasȱ complejasȱ deȱ contaminantesȱ
(Lafontaineȱetȱal.,ȱ2000;ȱMunnsȱetȱal.,ȱ2002;ȱGallowayȱetȱal.,ȱ2004;ȱMartínȬDíazȱetȱ
al.,ȱ2005,ȱMontserratȱetȱal.,ȱ2006).
ȱ
Ȭȱ91ȱȬȱ
Capítuloȱ3
Existenȱtresȱtiposȱgeneralesȱdeȱbiomarcadores:ȱ
Ȭ Losȱ biomarcadoresȱ deȱ exposición,ȱ determinan,ȱ dentroȱ deȱ losȱ organismosȱ
expuestosȱ aȱ contaminantes,ȱ metabolitosȱ derivadosȱ deȱ laȱ biotransformaciónȱ oȱ
productosȱdeȱsuȱreacciónȱconȱmoléculasȱbiológicas.ȱȱ
Ȭ Losȱ biomarcadoresȱ deȱ efecto,ȱ muestranȱ laȱ respuestaȱ delȱ organismoȱ
expuestoȱalȱagenteȱxenobióticoȱenȱparticularȱoȱalȱcomplejoȱdeȱmezcla.ȱȱ
Ȭ Losȱ biomarcadoresȱ aȱ nivelesȱ deȱ población,ȱ comunidadȱ yȱ ecosistema,ȱ
proporcionandoȱ informaciónȱ deȱ lasȱ alteracionesȱ aȱ mayoresȱ nivelesȱ deȱ
organización.ȱȱȱ
Unaȱ vezȱ realizadosȱ losȱ ensayosȱ agudosȱ bajoȱ condicionesȱ deȱ laboratorioȱ
talȱyȱcomoȱseȱdescribeȱenȱelȱcapítuloȱanterior,ȱenȱelȱpresenteȱcapítuloȱseȱrecogenȱ
cincoȱtrabajosȱenȱlosȱqueȱseȱdiscutenȱlosȱresultadosȱobtenidosȱenȱbioensayosȱdeȱ
tipoȱ subletalȱ realizadosȱ bajoȱ condicionesȱ deȱ laboratorioȱ conȱ cuatroȱ especiesȱ
marinas:ȱ elȱ pezȱ Sparusȱ aurata,ȱ elȱ cangrejoȱ Carcinusȱ maenas,ȱ laȱ almejaȱ Ruditapesȱ
philippinarumȱ yȱ elȱ poliquetoȱ Arenicolaȱ marina.ȱ Losȱ ensayosȱ subletales,ȱ
complementanȱyȱmejoranȱlaȱinformaciónȱobtenidaȱporȱlosȱexperimentosȱagudosȱ
yȱfueronȱrealizadosȱenȱperiodosȱdeȱtiempoȱqueȱvaríanȱentreȱlosȱ15ȱyȱ60ȱdías.ȱEnȱ
elȱprimerȱartículoȱ(V)ȱseȱpresentaȱunȱestudioȱdeȱdosȱmesesȱdeȱduraciónȱenȱelȱqueȱ
seȱ llevaronȱ aȱ caboȱ exposicionesȱ deȱ S.ȱ aurataȱ aȱ sedimentosȱ deȱ laȱ costaȱ gallegaȱ
afectadosȱporȱelȱvertidoȱdelȱpetroleroȱPrestige,ȱyȱseȱmidieronȱbiomarcadoresȱdeȱ
exposiciónȱ (actividadȱ ERODȱ yȱ metalotioneinas)ȱ yȱ efectoȱ (histopatología),ȱ
poniendoȱ deȱ manifiestoȱ laȱ importanciaȱ inicialȱ delȱ vertido.ȱ Enȱ elȱ trabajoȱ VIȱ seȱ
realizaȱ unaȱ valoraciónȱ deȱ laȱ cinéticaȱ deȱ lasȱ enzimasȱ implicadasȱ enȱ laȱ
detoxificaciónȱ deȱ PAHsȱ (actividadȱ EROD)ȱ enȱ S.ȱ aurataȱ yȱ seȱ relacionanȱ conȱ losȱ
dañosȱsobreȱlosȱtejidosȱcausadosȱporȱlosȱcontaminantesȱorgánicos.ȱEnȱelȱtrabajoȱ
VIIȱ seȱ muestranȱ losȱ resultadosȱ deȱ unȱ estudioȱ realizadoȱ conȱ C.ȱ maenasȱ yȱ R.ȱ
philippinarumȱ dondeȱ seȱ expusieronȱ losȱ organismosȱ aȱ sedimentosȱ deȱ Galiciaȱ yȱ
- 92 -
Estudioȱdeȱefectosȱsubletalesȱenȱorganismosȱbajoȱcondicionesȱdeȱlaboratorioȱ
Algeciras.ȱ Enȱ esteȱ estudioȱ seȱ seleccionóȱ unaȱ bateríaȱ deȱ biomarcadoresȱ deȱ
exposiciónȱrelacionadosȱconȱprocesosȱdeȱdetoxificaciónȱ(actividadȱERODȱcomoȱ
biomarcadorȱdeȱlaȱfaseȱIȱyȱGSTȱdeȱlaȱfaseȱII)ȱyȱactividadȱantioxidanteȱ(GPX,ȱGRȱ
yȱFRAP).ȱEstosȱbiomarcadoresȱseȱrelacionaronȱconȱlosȱcontaminantesȱanalizadosȱ
enȱ losȱ sedimentosȱ conȱ elȱ finȱ deȱ identificarȱ lasȱ sustanciasȱ causantesȱ delȱ estrés,ȱ
entreȱ lasȱ queȱ destacaronȱ losȱ PAHsȱ yȱ algunosȱ metales,ȱ principalmenteȱ enȱ laȱ
BahíaȱdeȱAlgecirasȱyȱCormeȬLaxeȱenȱGalicia.ȱ
Tablaȱ 3.1.ȱ Relaciónȱ deȱ bioensayosȱ subletalesȱ realizadosȱ paraȱ laȱ
evaluaciónȱdeȱlaȱcalidadȱdeȱlosȱsedimentos.ȱ
Especieȱ
Tiempoȱdeȱ
exposiciónȱ
Medidaȱfinalȱ
Sparusȱaurataȱ
60ȱdíasȱ
ActividadȱEROD,ȱMetalotioneinas,ȱ
Histopatología,ȱȱ
Carcinusȱmaenasȱ
28ȱdíasȱ
ActividadȱEROD,ȱGST,ȱGPX,ȱGR,ȱFRAP,ȱ
Vitelogenina,ȱHistopatologíaȱ
Ruditapesȱphilippinarumȱ
28ȱdíasȱ
ActividadȱEROD,ȱGST,ȱGPX,ȱGR,ȱFRAP,ȱ
Histopatologíaȱ
Arenicolaȱmarinaȱ
15ȱdíasȱ
alteraciónȱdelȱcomportamientoȱyȱ
alimentación,ȱGR,ȱGPX,ȱGST,ȱFRAP,ȱ
TBARS,ȱfagocitosis,ȱdañoȱdeȱADNȱ
Elȱ trabajoȱ VIIIȱ incluyeȱ unȱ estudioȱ deȱ laȱ variaciónȱ deȱ vitelogeninaȱ enȱ elȱ
cangrejoȱC.ȱmaenasȱtrasȱ28ȱdíasȱdeȱexposiciónȱaȱsedimentosȱrecogidosȱenȱGaliciaȱ
yȱ Algeciras.ȱ Losȱ resultadosȱ confirmanȱ laȱ relaciónȱ deȱ laȱ variaciónȱ deȱ esteȱ
biomarcadorȱ conȱ contaminantesȱ presentesȱ enȱ elȱ sedimento,ȱ principalmenteȱ
PAHsȱyȱalgunosȱmetalesȱqueȱvaríanȱenȱfunciónȱdeȱlaȱzonaȱdeȱestudio,ȱsiendoȱlaȱ
másȱafectadaȱlaȱBahíaȱdeȱAlgeciras.ȱEsteȱtrabajoȱfueȱpresentadoȱenȱelȱcongresoȱ
CEMEPE/SECOTOXȱ 2007ȱ yȱ obtuvoȱ elȱ premioȱ deȱ mejorȱ presentaciónȱ oralȱ deȱ
jóvenesȱ científicos.ȱ Paraȱ finalizarȱ esteȱ capítulo,ȱ elȱ trabajoȱ IX,ȱ realizadoȱ enȱ granȱ
parteȱ duranteȱ unaȱ estanciaȱ enȱ laȱ Universidadȱ deȱ Plymouthȱ (UK),ȱ presentaȱ losȱ
Ȭ 93ȱȬ
Capítuloȱ3
resultadosȱobtenidosȱtrasȱrealizarȱexposicionesȱdeȱquinceȱdíasȱdeȱduraciónȱconȱ
elȱpoliquetoȱArenicolaȱmarinaȱyȱanalizarȱunȱsetȱdeȱbiomarcadoresȱqueȱincluyen:ȱ
alteraciónȱdelȱcomportamientoȱyȱalimentación,ȱenzimasȱimplicadasȱenȱprocesosȱ
deȱ defensaȱ (GR,ȱ GPX,ȱ GST,ȱ FRAP,ȱ TBARSȱ yȱ fagocitosis),ȱ efectosȱ genotóxicosȱ
(dañoȱ deȱ ADN).ȱ Cabeȱ destacarȱ queȱ enȱ esteȱ estudioȱ seȱ determinóȱ porȱ primeraȱ
vezȱ laȱ capacidadȱ deȱ esteȱ invertebradoȱ marinoȱ deȱ activarȱ procesosȱ fagocíticosȱ
comoȱ respuestaȱ aȱ losȱ contaminantesȱ delȱ sedimento.ȱ Lasȱ exposicionesȱ seȱ
realizaronȱprincipalmenteȱconȱsedimentosȱprocedentesȱdeȱlaȱBahíaȱdeȱAlgecirasȱ
yȱ elȱ Parqueȱ Nacionalȱ deȱ lasȱ Islasȱ Atlánticasȱ yȱ losȱ resultadosȱ deȱ losȱ
biomarcadoresȱ seȱ relacionaronȱ conȱ losȱ contaminantesȱ ligadosȱ alȱ sedimento,ȱ
mostrando,ȱ deȱ nuevo,ȱ laȱ degradaciónȱ ambientalȱ enȱ laȱ Bahíaȱ deȱ Algecirasȱ yȱ laȱ
recuperaciónȱdeȱlasȱIslasȱCíesȱcuatroȱañosȱdespuésȱdelȱvertido.ȱ
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reviewȱ andȱ newȱ perspectives.ȱ Comp.ȱ
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Ȭȱ96ȱȬȱ
Arch. Environ. Contam. Toxicol. 51, 652–660 (2006)
DOI: 10.1007/s00244-005-0251-0
Ecotoxicity of Sediments Contaminated by the Oil Spill Associated with the
Tanker ‘‘Prestige’’ Using Juveniles of the Fish Sparus aurata
Carmen Morales-Caselles,1,3 Natalia Jimnez-Tenorio,2 M. Luisa Gonzlez de Canales,2,3 Carmen Sarasquete,1,3
T. ngel DelValls2,3
1
2
3
Instituto de Ciencias Marinas de Andaluca CSIC, Avda. Repfflblica Saharaui s/n. Puerto Real 11510, Cdiz, Spain
Facultad de Ciencias del Mar y Ambientales, University of Cdiz, Avda. Repfflblica Saharaui s/n Apdo, 40 Puerto Real 11510, Cdiz, Spain
Unidad Asociada de Calidad Ambiental y Patologa (CSIC & UCA), Spain
Received: 27 October 2005 /Accepted: 4 February 2006
Abstract. In November 2002, the oil spill from the tanker
Prestige in the Galician Coast caused an ecological catastrophe in Spain. The adverse effects associated with the contaminants bound to sediments were tested using juveniles of
the fish Sparus aurata (seabream). The approach evaluates
sediment quality by using an integrated assessment including
chemical and ecotoxicological data. Sediment samples were
physicochemically characterized, and the concentration of
contaminants (polycyclic aromatic hydrocarbons—(PAHs) and
metals) was measured. Different biomarkers of exposure
(metallothioneins and ethoxyresorufin O-deethylase activity
(EROD)) and biomarkers of effect (histopathology) were
analyzed along the time. A multivariate analysis approach was
used to correlate concentration of contaminants and sublethal
effects measured in individuals of fish. Results show that
increasing concentrations of PAHs in sediments were related
to increased EROD activities and histopathological lesions.
This is the first evidence showing adverse effects associated
with petroleum contamination of PAHs in sediments after this
spill, and it demonstrates the utility of the sublethal toxicity
tests for monitoring the impact of petroleum spills.
It has long been demonstrated that sediments can adsorb persistent and toxic chemicals to levels many times higher than
water column concentrations (DelValls et al. 2002), whereas the
sediment may become sufficiently polluted to disrupt natural
biological communities (Adams et al. 1992; Tolun et al. 2001).
For a better assessment of the pollution process in the marine
coastal environment, several authors have proposed determinations based on chemical measurements together with laboratory toxicity tests (Chapman 1988; Luoma and Ho 1992).
Sediment toxicity bioassays are instruments used to test the
ecotoxicity and bioavailability of chemical compounds in
sediments to benthic organisms. In this kind of bioassay, the
organisms are exposed to sediment samples collected in situ
Correspondence to: T. . DelValls; email: angel.valls@uca.es
-97-
and after the incubation period, a biological response is measured; this response must be sensitive, ecologically relevant,
and easy to standardize (Stebbing et al. 1980). Bioassays
provide information on the toxicity of contaminated sediments
that can be neither derived from chemical analysis nor from
ecological surveys performed alone (Chapman and Long 1983;
Long and Chapman 1985). Interest in the effects of environmental stressors on health and disease in fish and other marine
organisms has increased in recent years, and in particular,
histological and cellular alterations have been observed in
marine fish from polluted coastal waters and estuaries (Malins
et al. 1984; Stein et al. 1992). These sublethal responses have
been found to be a powerful tool to evaluate sediment toxicity
effects (DelValls et al. 1998a).
Biomarkers in fishes have been previously studied in the
assessments of oil spills such as Exxon Valdez (Varanasi et al.
1995; Jewett et al. 2002), Braer (Ritchie and OÕSullivan 1994),
and Sea Empress (Kirby et al. 1998). In the present study,
histopathology was conducted as a biomarker of effect in order
to measure the damage caused in the target tissues by the
presence of chemicals in the sediments. Two biomarkers of
exposure were selected to address the biological adverse effects associated with contaminants present in the studied
sediments. The toxicity of metals was assessed by metallothionein (MT) induction, whereas ethoxyresorufin O-deethylase activity (EROD) represents a good marker in MFO
(mixed-function oxygenase), which is the first mode of
detoxification of many organic pollutants (polycyclic aromatic
hydrocarbons (PAHs), polychlorinated biphenyls). The EROD
measurement in fish is considered a monitor of pollution
exposure and an indicator of potential future problems in the
health of fish populations (Carballeira 2003). Furthermore,
EROD induction can be documented in fish exposed to spilled
petroleum despite low tissues of PAHs (George et al. 1995;
Whyte et al. 2000); The reason for using metallothionein
induction is because we are studying a mixture of contaminants in the environment, and metallothioneins are one of the
main biomarkers. In addition, it has been proved that the
induction of this biomarker is not always just related to metals
(Stegeman et al. 1992; Muto et al. 1999; Van der Oost et al.
2003).
653
Tanker Oil Spill Toxicity Testing Using Sparus aurata
matrix characteristics were studied analyzing total organic carbon
concentration and sediment grain size. Organic carbon content was
determined using the method of Gaudette et al. (1974) with El Rayis
(1985) modification. For sediment grain size, an aliquot of wet sediment was analyzed using a laser particle size Fristch (model Analysette 22) following the method reported by DelValls and Chapman
(1998b).
For trace metal analysis, the sediments were digested as described
by Loring and Rantala (1992). Fe, Mn, Zn, and Cu concentrations in
the extracts were determined with a Perkin–Elmer 2100 flame atomic
absorption spectrophotometer. Concentrations of Hg and As were
determined by means of Perkin–Elmer MHS-FIAS coupled with a
Perkin–Elmer 4100 ZL spectrophotometer. The other trace metals
were measured by graphite furnace atomic absorption spectrophotometry (Perkin–Elmer 4100 ZL). Results are expressed as mg kg)1
dry sediment. The analytical procedures were checked using reference
material (MESS-1 NRC and CRM 277 BCR) and showed a 90–110
range.
PAHs were analyzed by using gas chromatography/mass spectrometry (U.S. Environmental Protection Agency SW-846 Method
8270); briefly, dried samples were Soxhlet extracted with n-hexane
for 18 h, and the extracts were isolated by column chromatography on
Florisil-alumino-silica. PAHs were eluted and their fractions were
dried in a rotatory evaporator and redissolved in isooctane. Aromatic
fractions were analyzed on a Hewlett–Packard (HP) 5890 Series II gas
chromatograph coupled with HP 5970 mass spectrometer. Chromatographic resolution was achieved with a 30 m · 0.250 mm DB-5
capillary column, which has a 0.25-lm film thickness, with helium as
carrier gas. Quality control was carried out using NRC-CNRC HS-6
sediment reference material. The analytical procedure allows agreement with the certified values in a 90–112 range.
The composition of the oil spilled by the tanker Prestige
was a mixture of saturated hydrocarbons, aromatic hydrocarbons, resins, and asphaltenes, with most of the PAHs being of
medium to high molecular weight (Albaigs and Bayona
2003). Furthermore, it presents some trace metals such as Ni,
V, Cu, Pb, and Zn (CSIC 2003; Albaigs and Bayona 2003;
Prego and Cobelo-Garca 2003; Prego and Cobelo-Garca
2004). The physicochemical characteristics of this fuel show
that the soluble fraction is low and the kinetics of degradation
are slow under natural conditions so it is expected to be
accumulated in sediments. The biological effects associated
with the chemicals from the oil spill will be dependent on the
nature of the ecosystem that accepts them and the organisms
living in it (DelValls 2003). The first research notes about the
early impact support the prediction that the acute toxicity of
the weathered fuel (MariÇo-Balsa 2003), very rich in high
molecular weight compounds, was relatively low for the
organisms tested (clams and microalgae). However, although
concentrations of individual PAHs in aquatic environments are
usually much lower than concentrations that are acutely toxic
to aquatic organisms, sublethal effects can be produced (Albers 2003).
The results presented in this work show the status of the
quality of the sediments 2 years after the accidental spill by
linking sublethal responses measured in the fish exposed to oilcontaminated sediments with chemical data determined in
sediments.
Materials and Methods
Sediment Bioassays
Approach
Toxicity tests were carried out using juveniles of Sparus aurata obtained from an aquaculture farm and transported to the laboratory
where the fish spent 1 month to acclimatize. S. aurata was selected
because is a common species along the Spanish coast. Its biology is
well known, having been used in previous pollution studies (DelValls
et al. 1998a), and it is easy to acclimatize to laboratory conditions.
The sea water used during the acclimatization period and the bioassay
was clean marine water. A baseline of 10 randomly chosen individuals
were weighed to provide data for feeding calculations. After the
acclimatization period, the fish had a weight that averaged 4 € 1 g.
Approximately 4 L of sediment from the negative control (BC) and
the other stations (Ga1, Ga2, Ga3, TM) were placed in replicate 25-L
glass tanks with clean sea water before the beginning of the experiment. After 24 h of particle settling, aeration was provided to maintain
adequate oxygen concentrations (greater than 80% saturation). At the
beginning of the test, another baseline group of 10 randomly chosen
individuals was measured, weighed, anesthetized, and processed for
biomarker responses (exposure and effect) to be used as the initial
cellular control. Twelve individuals were placed in every tank after
checking each tankÕs water quality and were fed 2 or 3 times per day
with commercial food (approximately 0.2 g per fish per day of ‘‘Mar
Perla T’’ 1.4–2.2 mm). The test was conducted over 2 months, during
which time no mortality was recorded. After the exposure period,
individuals from each station were anesthetized and processed for
histopathological, MTs, and EROD analysis. During the experiment
natural photoperiod was selected and constant temperature was
maintained (19 € 1C). The physicochemical parameters pH, temperature, oxygen, and salinity were recorded and controlled when
necessary to maintain quality control during the test. Water replacement was performed every day by renewing 33% of the water column
using a peristaltic pump.
The present study was carried out by using sediment samples collected along different littoral areas in the North and the South of
Spain. In the North, we chose sampling stations that have been affected by the oil spill in differing degrees and located along the
Galician Coast (Ga1, Ga2, Ga3). Another sample was located in the
South of Spain, in the Bay of Cdiz (BC) which is considered a
pristine area (Riba et al. 2004a) and was used as the negative control
reference. An artificial sample (TM) was made by mixing a toxic mud
from an accidental mining spill in Spain (Aznalcllar, April 1998)
with the clean sediment and used as positive control (Riba et al.
2003).
Sediment samples from each station were collected with a 0.025m2 Van Veen grab and placed in a cooler until a sufficient amount of
sediment was collected from a particular station (about 30 L). The
contents of the cooler were homogenized with a Teflon spoon until
no color or textural differences could be detected. The samples were
subsampled for physical characterization and chemical quantification.
After that, sediment samples were maintained in the cooler at 4C in
the dark until used in sediment toxicity tests. Testing occurred within
2 weeks of collection. Sediment was filtered (0.5 mm) prior to the
toxicity test in order to remove means interferences such as shells,
predators, and other residues.
Chemical Analysis
Sediment aliquots from each station were dried at room temperature
prior to chemical analysis and then gently homogenized. Geochemical
-98-
C. Morales-Caselles et al.
654
Histological Procedures
Organisms from the toxicity tests were analyzed to determine the
histopathological damages in different target tissues (liver and gills).
When the water was renewed, the survival rate for all tanks was
determined. Fish were removed from the tanks after 56 days of
exposure time and samples were collected. Fish were anesthetized
with 0.1% of 99% pure 2-phenoxyethanol during 5–10 min, then
weighed, measured for length, and externally examined. Target tissues
(liver and gills) from all of the organisms were obtained by dissection
and then fixed in phosphate-buffered 10% formaldehyde (pH 7.2) for
24 h and embedded in paraffin. The histological sections were stained
with hematoxylin–eosin and hematoxylin–VOF (Gutirrez 1967).
Sections were reviewed by light microscopy (Leitz Laborlux S) and
photographed (Sony DKC-CM30). Damage to the tissues was semiquantified by detecting the frequency of the lesions in each detected
alteration.
Biochemical Analysis
Fish were sampled for biochemical analysis, and after dissection, the
liver was kept at )80C prior to the homogenization. The samples
were homogenized following the procedure developed by Lafontaine
et al. (2000).
Metallothionein Concentration (MT)
Samples obtained to determine metallothionein content were centrifuged at 28,000g for 40 min. The supernatant was added to 0.9 ml of
NaCl (0.9%), heated to 95C for 4 min, and centrifuged at 10,000g for
15 min at 4C. Supernatant was stored at )80C prior to MT concentration determinations by Anodic Stripping Voltammetry (Olafson
and Olsson 1987) using purified rabbit metallothionein (Sigma-Aldrich). Total protein determination was carried out using the methodology described by Bradford (1976). Concentrations were
expressed as lg MT/mg total protein.
markers responses; the Tukey test was used as the post-hoc comparison. Also, contamination and toxicity data were linked by factor
analysis, and using principal components analysis (PCA) as the
extraction procedure, which is a multivariate statistical technique to
explore variable distributions (Riba et al. 2003). The original data set
used in the analysis included two biomarkers (EROD activity and
metallothionein induction), two histopathological indexes (lesions in
gills (LIG), and lesions in liver (LIL)), the concentration of different
contaminants (PAHs, Cd, Cr, Cu, Ni, Pb, Zn, Hg), and the geochemical matrix characteristics (including total organic carbon and
grain size distributions). The objective of PCA is to derive a reduced
number of new variables as linear combinations of the original variables. This provides a description of the structure of the data with the
minimum loss of information.
Results
Sediment Contamination
Summarized results of total organic carbon, grain size (percent
of fine grain <63 lm), concentration of metals and PAHs are
shown in Table 1. Of all the stations, the negative control (BC)
showed the lowest values of most of contaminants. In general,
it is observed that the concentration of PAHs in the area of
Galicia (Ga3>Ga2>Ga1) was higher than those measured in
the toxic mud and the sediments from the Bay of Cdiz (not
detected). The toxic mud, used as positive control, showed
high levels of metals in comparison to the other sample sites.
These chemical data can be compared to international sediment quality guidelines (SQGs) that account for the chemical
contaminants levels associated with biological effect (DelValls
et al. 2004). In Table 1, the contaminants that exceed any SQG
are highlighted. The letter that appears with the number indicates which SQG is surpassed.
Biomarker Responses
Mixed Function Oxidase Assay (EROD)
After homogenization of the samples, EROD samples were centrifuged at 10,000g for 30 min, and the supernatant was used for the
EROD activity determination and the total protein content described
by Bradford (1976). Mixed function oxygenase activity was measured
using the adapted EROD assay (Gagn and Blaise 1993). Briefly,
50 ll of supernatant (homogenate 10,000g for 30 min), 10 lM 7ethoxyresorufin, and 10 mM reduced NADPH in 100 mM KH2PO4
buffer (pH 7.4). The reaction was started by the addition of NADPH,
was allowed to proceed for 60 min at 30C, and stopped by the
addition of 100 ll of 0.1 M NaOH. The 7-hydroxyresorufin was
determined fluorometrically using 520 nm (excitation) and 590 nm
(emission) filters. 7-Hydroxyresorufin concentration in the samples
was achieved through a standard calibration curve developed with
concentrations of 7-hydroxyresorufin. Results were expressed as
pmol/mg total protein.
Statistical Analysis
Analysis of variance was performed in order to determine significant
differences (p < 0.05; p < 0.01) among sites in relation to the bio-
The basal level of metallothioneins measured in liver on day 0
of exposure was 20.1 lg)1 mg)1 and was lower than the levels
of this biomarker after 56 days of exposure; the measures show
that metallothionein levels in liver were significantly different
(p < 0.5; p < 0.01) between fishes exposed to control and those
exposed to other sediment (Figure 1). These differences were
more significant for the station Ga3 and TM (p < 0.01) than for
the other two stations located in the area of Galicia, Ga1, Ga2
(p < 0.05).
EROD activity determined in the liver of juveniles of S.
aurata on day 0 was 0.3 pmol mg)1 min)1. Results after 56
days of exposure are higher than basal levels and showed low
values in fish exposed to sediment samples with absence or
low levels of total PAHs (TM and BC, respectively) and were
significantly different (p < 0.05) from stations affected to a
different degree by the oil spill (Ga#) (Figure 1). The fish from
these stations present high values of EROD activity and high
levels of PAHs in their sediment (Ga1, Ga2, and Ga3). These
relationships increase when the concentration of PAHs in
sediments increases (Ga3 > Ga2 > Ga1). The EROD activity in
liver of fish exposed to sediments from station Ga2 and Ga3
showed a significant difference (p < 0.01) between the control
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655
Tanker Oil Spill Toxicity Testing Using Sparus aurata
Table 1. Values of total organic carbon (TOC) (% dry weight), fines (% dry weight), and the concentration of contaminants (polycyclic aromatic
hydrocarbons (PAHs) and metals) in sediment samples (concentrations are expressed in mg kg)1 dry weight)
)1
PAHs (mg kg )
Metals (mg kg)1)
Contaminant
BC
Ga1
Ga2
Ga3
TOC
Fines
Total PAHs
Fluorene
Acenaphthene
Naphthalene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
Benzofluoranthene
Benzo[e]pyrene
Benzo[a]pyrene
Perilene
Dibenzo[ah]anthracene
Indene[123-cd]pyrene
Benzo[ghi]perilene
Cd
Cr
Cu
Ni
Pb
Zn
Hg
1.07
1.04
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.92
0.10
6.98
0.06
2.28
21.3
n.d.
0.60
0.06
0.19
0.08
0.06
0.31
0.10
0.02
0.12
0.09
0.05
0.08
0.11
0.08
0.05
0.03
0.01
0.02
0.01
0.16
n.d.
12.8
1.71
2.73
14.7
n.d.
1.19
0.03
2.12
0.13
0.17
0.63
0.15
0.03
0.18
0.13
0.09
0.12
0.18
0.13
0.09
0.05
0.02
0.02
0.02
0.05
2.00
0.65
0.42
1.14
3.95
0.01
2.00
0.01
5.10
0.35
0.27
1.40
1.36
0.18
0.10
0.39
0.20
0.39
0.06
0.16
0.10
0.04
0.02
0.02
0.06
n.d.
1.51
1.19
0.66
1.26
6.45
n.d.
a,c
a,c
a,c
a,c
c
a,c
a,d
a,d
a,d
c
c
c
c
c
TM
a
a,d
a,d
a,d
a,d,e
a,c
c
a,c
c
c
1.00
10.1
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
5.40 a,d
3.28
210 a,d
8.50
790 b,d,e
2181b,d,e
5.61 b,d,e
a
ERL, Effect Range-Low (NOAA 1999).
ERM, Effect Range-Median (NOAA 1999).
c
ISQG, Interim sediment quality guideline (CCM 1999).
d
PEL, Probable effect level (CCM 1999).
e
SQG for San Francisco Bay (Long et al. 1989).
Notes: Not detected is expressed by n.d.
b
treatment. Also, those from station Ga1 were significantly
different from control treatment but with different value of the
statistical p (0.05).
For metallothioneins, Tukey test results set the five stations
in three homogeneous groups according to the differences of
averages among the sites. The first group includes only BC,
which is the negative control; the second group is constituted
by Ga1, Ga2, and Ga3; and the third group includes TM, which
is the positive control.
For EROD activity, there are three homogeneous groups set
by the Tukey test results. The first group includes BC and TM,
both negative and positive control, which do not present
significant differences; the second group is constituted by Ga1
(p < 0.05) and Ga2 (p < 0.01); and the third group includes
Ga3 (p < 0.01).
Histopathological Approaches
The organisms analyzed on day 0 did not present histopathological damages. Different alterations were observed in target
tissues (gills and liver) of fish exposed to sediment collected
after 56 days of exposure in the different stations, mainly in
gills, which showed shortening of secondary lamellae, hyper-
trophy, and hyperplasia, necrosis, and loss of epithelial cells in
Ga# and TM; fusion of the secondary lamellae above all in
Ga3 and TM; and presence of edematous areas in the distal
portion of lamellae in Ga#. Also, liver showed lesions: vacuolization of hepatocytes, necrosis, and decrease of the zymogen granules of the exocrine pancreas in Ga# and TM. In
general, an increase of cytoplasmic basophilia was detected in
the liver and exocrine pancreas of all exposed fish related to
the increase of PAHs. An example of some of these lesions is
shown in Figure 2.
These lesions have been previously recorded as related to
contaminants bound to sediments in S. aurata (DelValls et al.
1998a) and in other fish species such as Solea senegalensis
(Riba et al. 2004b, 2004c). As previously reported by DelValls
et al. (1998a) and based on the damage observed in the different tissues, histopathological alterations were evaluated
semiquantitatively in the fishes exposed to the different stations by ranking the frequency of lesions measured in a total
number of 6 individuals: – (0 individuals), +/) (1 individual),
+ (2 individuals), ++/+ (3 individuals), ++ (4 individuals),
+++/++ (5 individuals), and finally the maximum is associated
with the presence of a disease in the total number of individuals, +++ (6 individuals sampled). Gills were shown to be the
most damaged tissue, showing different lesions mainly in Ga#
-100-
C. Morales-Caselles et al.
Metalothioneins/Total proteins(μg/mg)
656
Discussion
120
**
100
80
**
60
*
*
Ga1
Ga2
40
20
0
BC
Ga3
T.M.
ERODactivity (pmol/mg/min )
3.0
**
2.5
2.0
**
1.5
*
1.0
0.5
0.0
BC
Ga1
Ga2
Ga3
T.M.
Fig. 1. Results of metallothionein concentration (mean and SD) in
lg/mg of protein and EROD activity (mean and SD) in pmol/mg/min
of protein in liver samples of S. aurata collected at 56 days of the
experiment in sediments sampled in the Bay of Cdiz (BC), Galicia
coast (Ga#), and toxic mud (TM) treatments. Asterisks indicate significant differences among the biomarker induction in the stations and
the negative control (**p < 001, *p < 005)
and TM. An average of this semiquantitative evaluation of the
frequency of the lesions measured from the different replicate
results is shown in Table 2.
General indexes of lesion (lesion index in gills [LIG] and
lesion index in liver [LIL]) were calculated for each tissue as
an average value of the fish damage semiquantified (Figure 3).
Fish exposure to sediment samples produced lesion damage
related to the increase of the concentrations of contaminants
(PAHs in Ga# and high levels of metals in TM) in the sediments selected in the bioassay. The lesions identified in all the
tissues analyzed were almost always present in animals exposed to sediments from stations Ga2 and Ga3. Evaluations of
histology of gills and liver revealed clear significant differences (p < 0.05) between the negative control of toxicity and
the Ga3 station and the toxic mud. The severity of the lesions
detected in the tissues of fish exposed to sediments collected in
Bay of Cdiz was lower than those measured in the area of
Galicia. Results show the lowest indexes in the Bay of Cdiz
(BC), which were significantly different (p < 0.05) from the
values from Galicia (Ga#) and toxic mud (TM). The index of
lesions measured for gills (LIG) in Galicia increase with the
presence of PAHs in the sediment samples (Ga3 > Ga2 > Ga1).
The LIL results show that TM has the highest index related to
liver lesions.
To link the set of data obtained, the original variables from
chemical concentration and sublethal responses were analyzed
by factor analysis, using PCA as the extraction procedure, which
is a multivariate statistical technique (MAA) to explore variable
(chemical concentration, n = 25; toxicity data, n = 4) distributions. The factor analysis was performed on the correlation
matrix, and the variables were autoscaled (standardized) so as to
be treated with equal importance (Riba et al. 2004a). The
applications of MAA to the original 29 variables indicate that
they can be grouped in two new factors. These factors explain
88.4% of the variance in the original data set. Negative values of
sorted rotated factor loadings (negative salience) are as important as positive values (positive salience). In the present study,
we selected to interpret a group of variables as those associated
with a particular component where loading was 0.40 or higher
(Table 3). This approximates ComreysÕ cutoff of 0.55 (Comreys
1973) for a good association between an original variable and a
factor, and also takes into account discontinuities in the magnitudes of loadings approximating the original variables.
The first principal factor, #1, is predominant and accounts
for 62.4% of the variance; it explains the toxicity of individual PAHs and combines the concentrations of PAHs
(fluorene, acenaphthene, naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene,
benzofluoranthene, benzo[e]pyrene, benzo[a]pyrene, perilene,
dibenzo[ah]anthracene, indene[123-cd]pyrene, benzo[ghi]perilene) in sediment, the total organic carbon, all the indexes of
histopathological lesions (gills, LIG; and liver, LIL), and the
EROD activity. The second factor, #2, accounts for 26.1% of
the variance; it explains the toxicity associated with the
metals in sediment combining, with negative loading, the
chemical concentrations of the metals Cd, Cr, Cu, Ni, Pb, Zn,
and Hg with the grain size with all the indexes of lesions
(gills, LIG; and liver, LIL) and with the induction of metallothioneins (MTs). Figure 4 shows the influence of both
factors in the five different stations. Factor 1, with positive
loading, is defined as the toxic responses of the fish to PAHs
bound to sediments; thus, station Ga2 (0.44) and especially
Ga3 (1.48) show the significant prevalence of this factor,
whereas this factor does not affect station Ga1, and both
controls positive (TM) and negative (BC). The definition of
Factor 2 as the toxic responses of the fish to metals bound to
sediment only has prevalence in the positive control (TM);
the negative loading of the toxic responses and the metals
concentration in sediment implies that the prevalence of these
factors in the station is associated with negative factor scores.
Furthermore, a linear relationship can be observed in the
scores of this factor from BC to Ga3, which confirms the
increase of toxicity when PAHs increase in sediments.
It is estimated that about 63,000 tons of heavy fuel oil were
lost from the single- hull tanker Prestige. Although a large
quantity of this fuel was collected and removed from the coast,
a large amount likely settled down at the bottom of the sea
covered with sediment reaching the littoral area of the Galician
coast after the first months of the spill (Albaiges and Bayona
2003). In the present study, we have aimed to assess the impact
of this enrichment in littoral sediments collected in different
affected areas and 2 years after the oil spill using juveniles of
-101-
657
Tanker Oil Spill Toxicity Testing Using Sparus aurata
Fig. 2. Example of histological
sections associated with
contaminants bound to sediments
used in the Sparus aurata sediment
toxicity test. (a) Gills from fish
exposed to referent sediment
showing primary lamellae and
secondary lamellae arising from
these, parallel with them and
perpendicular to the filament axis BC
(H & E ·10). (b) Hypertrophy and
hyperplasia of the secondary
lamellae Ga3 (H & E ·25). (c) Liver
from control fish showing the
exocrine pancreas around the blood
vessels. Parenchymatous distribution
of the hepatocytes in cords around
the sinusoids BC (H & E ·25). (d)
Hepatocytes and exocrine pancreas
alteration TM (H & VOF ·25). D:
decrease of the zymogen granules;
H: hypertrophy and hyperplasia; L:
loss of epithelial cells; V:
vacuolization of hepatocytes
Table 2. Frequency of lesions detected in microscopic abnormalities of individuals of juveniles of the fish Sparus aurata sampled in the Bay of
Cdiz (BC), Galicia coast (Ga#) and toxic mud (TM) treatments on day 56 of exposure
Samples zones
Organ
Histopathology
BC
Ga1
Ga2
Ga3
TM
Gills
Hypertrophy/hyperplasia
Fusion of secondary lamellae
Shortening of secondary lamellae
Edematous areas or aneurysm in distal portion of lamellae
Necrosis and lost of cells epithelial
Increase of lipid vacuoles in the hepatocytes
Increase of cytoplasmic basophilia of hepatocytes
Necrosis and decrease of the zymogen granules of exocrine pancreas
+/)
+
+
+/++
+
+/)
+/)
)
+++
+/++
+/++
++/+++
+/++
+
+
+/)
++
++
+/++
++/+++
+/++
+
++
+
+++
+/++
+/++
+++
++
+
+
+/)
+/++
+
+/++
+/++
++
++
+/)
+
Liver
(0 individuals), +/) (1 individual), + (2 individuals), ++/+ (3 individuals), ++ (4 individuals), +++/++ (5 individuals) and finally the maximum is
associated with the presence of a disease in the total number of individuals, +++ (6 individuals sampled).
the fish S. aurata by means of different sublethal endpoints
such as histopathological lesions, metallothionein induction,
and EROD activity.
Previous studies have shown how in water the toxicity of
individual PAHs increases as molecular weight (MW) increases up MW 202 and beyond it; solubility reduces and so
does lethal toxicity, but sublethal effects can result (Albers
2003). In the present study, it has been shown that the Galician
sediments (mainly Ga2 and Ga3) analyzed present levels of
PAHs with low MW (fluorene, acenaphthene, naphthalene,
phenanthrene, anthracene), medium MW (pyrene), and high
MW (benzo[a]anthracene, chrysene, benzo[a]pyrene, dibenzo[ah]anthracene), higher than some of the SQGs proposed by
international agencies. Furthermore, all individual PAHs seem
to have induced hepatic EROD and to produce histopathological damage; it is quite difficult to determine which of the
individual PAHs is the main pollutant that has caused the
biological effects (explained by factor 1 in the MAA);
however, it can be concluded that PAHs are the compounds
that are producing the adverse effects to the fishes.
The significant differences of EROD induction between Ga#
(Ga1, p < 0.05; Ga2 and Ga3, p < 0.01) show a strong relationship with the concentration of PAHs in the Galician sediments (Ga1, Ga2, and especially in Ga3)—impacted by the oil
spill—and the histopathological lesions in gills and liver,
studied in the MAA. Despite differences in the induction of
EROD among these Galician samples, the validity of this biomarker of contamination was shown.
twbIn the absence of fish mortality, other research on the
impact of the ‘‘Sea Empress’’ oil spill in the UK in 1996
(Edwards and White 1999) showed the possibility of sublethal and chronic effects using a variety of techniques such as
EROD activity. In these studies, there was evidence of high
levels of EROD activity in the sites exposed to oil constituents in comparison with the control sites. There are other
studies, carried out using biomarkers as EROD activity, that
-102-
C. Morales-Caselles et al.
658
Table 3. Sorted rotated factor loadings (pattern) of 29 variables for
the two principal factors resulting from the multivariate analysis of
results obtained from the bioassay with juveniles of Sparus aurata
1,4
1,2
*
LIG
1
*
*
*
0,8
0,6
0,4
0,2
0
BC
Ga1
Ga2
Ga3
TM
1,4
1,2
*
LIL
1
*
0,8
*
*
0,6
0,4
0,2
0
BC
Ga1
Ga2
Ga3
TM
Fig. 3. General indexes of lesions (mean and SD) measured in gills
(LIG) and liver (LIL) of Sparus aurata juveniles exposed to sediments
sampled in the Bay of Cdiz (BC), Galicia coast (Ga#), and toxic mud
(TM) treatments. Asterisks indicate significant differences among the
index value in the stations and the negative control (*p < 005)
support the conclusion of the persistent exposure of the
organisms to hydrocarbons after 10 years of the oil spill
caused by the tanker Exxon Valdez in Alaska in 1989 (Jewett
et al. 2002), emphasizing the potential for continuing oil
availability to biota. The ability of fish to metabolize many
PAHs makes the use of EROD induction for biomonitoring
purposes more beneficial than analytical measurements of
PAH uptake, providing a sensitive chemical exposure information many years after a contamination event (Whyte et al.
2000). The histopathological analysis showed histomorphological alterations that have been previously reported in this
organism when affected by sediment contamination caused
by metals and organic compounds (DelValls et al. 1998a;
Riba et al. 2004b, 2004c; Ortiz et al. 1999; Au 2004) such as
hyperplasia and hypertrophy of gills, and alterations in hepatocytes and exocrine pancreas (i.e., increased cytoplasmic
basophilia and vacuolization, necrosis, loss of zymogen
granules, etc.). An increase of cytoplasmic basophilia was
generally detected in liver and exocrine pancreas of all exposed fish. This fact could be related to a decreased protein
synthesis (Sarasquete and Gutirrez 2005), and possibly
related to necrotic focus. Moreover, loss of cytoplasmic hepatic glycogen is an early toxic response and may cause an
apparent increase in cytoplasmic basophilia (Vethaak and
Wester 1996). In general, contaminants can produce osmoregulatory, acid–base, or hemodynamic dysfunctions, and it
was proposed that such symptoms are secondary to toxin
interactions with specific transport steps or membrane-bound
receptors (Evans 1987).
Results show that LIG is always higher than LIL in all of the
stations; this could be explained by the affirmation that fish
gill is a multifunctional organ sensitive to chemicals in water,
because gill filaments and lamellae provide a very large sur-
% Variance
Factor 1 62.4
Factor 2 26.1
TOC
Fines
Fluorene
Acenaphthene
Naphthalene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
Benzofluoranthene
Benzo[e]pyrene
Benzo[a]pyrene
Perilene
Dibenzo[ah]anthracene
Indene[123-cd]pyrene
Benzo[ghi]perilene
Cd
Cr
Cu
Ni
Pb
Zn
Hg
MT
EROD
IGG
IGL
0.78
—
0.96
0.97
0.97
0.88
0.90
0.63
0.95
0.97
0.95
0.43
0.93
0.92
0.83
0.91
0.83
0.95
—
—
—
—
—
—
—
—
0.98
0.79
0.50
—
)0.93
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
)0.91
)0.88
)0.95
)0.95
)0.96
)0.96
)0.96
)0.97
—
)0.54
)0.74
face area for direct and continuous contact with contaminants
in water. Fish gill and liver are highly sensitive to pollutant
exposure; however, as previously indicated (Arellano et al.
1999), these pointed histopathological alterations are, in general, nonspecific effects, meaning that they are responsive to a
variety of pollutants, and therefore only indicative of the
general quality of the environment rather than specific types of
pollutants (Au 2004). The increase of lipid vacuoles (small
size) present in the hepatocytes can indicate an alteration of
lipid metabolism or a partial change in their morphology, or in
that of lysosomes (Arellano et al. 1999; Segner and Storch
1985). The cause–effect relationships and detailed mechanisms leading to the development of most pathological
symptoms are not generally clear. Nevertheless, certain hepatic lesions in fish have been well correlated with contaminant exposure (Au 2004). Lamellar fusion of gills could be a
protective effect for diminishing the amount of vulnerable gill
surface area (Mallat 1985).
The comparison between chemical analysis and the different
toxic response (biomarkers of exposure and of effect at different levels) is a useful tool to determine the quality of the
studied sediments. The importance of the use of chronic bioassays that provide long-term information on the effects of the
exposure to a toxic compound has been proved, because a
compound cannot reflect a considerable lethal toxicity, but it is
able to produce lesions at different levels to the organism
exposed.
-103-
659
Tanker Oil Spill Toxicity Testing Using Sparus aurata
2.0
1.0
Ga1
Ga1
Ga2
0.0
0.0
-1.0
BC
Ga3
Ga2
Score
Score
1.0
Ga3
-1.0
TM
BC
-2.0
-2.0
TM
Factor 2
Factor 1
Despite the repercussion of the spill in the biota, shown as a
decrease of the abundance of the microfauna (Junoy et al.
2005), previous studies have shown that there was not an
important toxic effect in different marine organisms (clams
and microalgae) exposed to samples of the sediments and their
elutriates associated with the spill caused by the tanker
‘‘Prestige’’ (MariÇo-Balsa et al. 2003). The bioassay using
juveniles of the fish S. aurata showed results sensitive enough
to determine the hazard associated with this oil-contaminated
sediment, displaying good correlation between the toxicity and
the contaminant levels using a sublethal set of measurements
including both biomarkers of exposure and effect. This study
demonstrates the necessity to monitor the impact of the spill on
sediment quality in the areas affected. Furthermore, it shows
that a subchronic test using a sensitive and sublethal endpoint
is a powerful tool to identify the risk associated with the
enrichment of PAHs in affected sediments. The higher sensitivity of this bioassay compared to the acute tests previously
used indicates the need to incorporate this kind of approach as
part of a more complete and integrated study based on a
weight-of-evidence approach, as previously recommended by
some authors (Carballeira 2003).
Acknowledgments. The described work was supported by a Grant
funded by the Ministry of Education and Science VEM2003-20563.
Carmen Morales-Caselles thanks the Ministry of Education and Science for funding her research fellowship (FPU). We thank the
members of the ICMAN-CSIC for their support and help.
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Environ Monit Assess (2007) 131:211–220
DOI 10.1007/s10661-006-9469-1
Kinetic of Biomarker Responses in Juveniles of the Fish
Sparus aurata Exposed to Contaminated Sediments
Carmen Morales-Caselles & Natalia Jiménez-Tenorio &
Inmaculada Riba & Carmen Sarasquete & T. Ángel DelValls
Received: 17 May 2006 / Accepted: 22 August 2006 / Published online: 14 December 2006
# Springer Science + Business Media B.V. 2006
Abstract Sediments in the National Park of the
Atlantic Islands (Galicia, Spain) were affected by the
spill of the tanker Prestige (November, 2002) and still
present high levels of Polycyclic aromatic hydrocarbons. The adverse effects associated with the
contaminants in sediments were tested using a chronic
bioassay, exposing juveniles of the fish Sparus aurata
(seabream). A toxicokinetic approach is proposed to
evaluate sediment quality by linking chemical and
C. Morales-Caselles : N. Jiménez-Tenorio : I. Riba :
C. Sarasquete
Consejo Superior de Investigaciones Científicas (CSIC),
Instituto de Ciencias Marinas de Andalucía (ICMAN),
Polígono Río San Pedro s/n,
11510 Puerto Real, Cádiz, Spain
C. Morales-Caselles : I. Riba : T. Á. DelValls
UNITWIN/UNESCO/WiCoP. Departamento de Química
Física, Facultad de Ciencias del Mar y Ambientales,
Polígono Río San Pedro s/n,
11510 Puerto Real, Cádiz, Spain
C. Morales-Caselles : C. Sarasquete : T. Á. DelValls
Unidad Asociada Universidad de Cádiz-Calidad Ambiental
y Patología (UCA-CSIC),
Polígono Río San Pedro s/n,
11510 Puerto Real, Cádiz, Spain
ecotoxicological data along the time. Sediment samples
were physicochemically characterized and the concentration of contaminants (Polycyclic aromatic hydrocarbons – PAHs – and metals) was measured. Fishes
were exposed to contaminated sediments, and samples
from different tissues were collected every 15 days
throughout the 60 days that lasted the experiment. A
biomarker of exposure (ethoxyresorufin O-deethylase
activity – EROD activity) and a biomarker of effect
(histopathology) were analyzed during the exposure
period. Results show a relationship between the
biomarkers and the concentrations in sediments of
polycyclic aromatic hydrocarbons—PAHs. Besides,
the toxicokinetic approach links biomarkers response
providing information about the relationship between
the detoxification process and the damages observed in
the different tissues. The frequency of the histological
damage is highest when the EROD activity slightly
decreases in accordance with the mechanism of
detoxification of this enzymatic system against PAHs
and other organic contaminants.
Keywords EROD activity . Histopathology . PAHs .
Prestige . Oil spill
1 Introduction
C. Morales-Caselles (*)
Departamento Química Física,
Facultad Ciencias del Mar y Ambientales,
Polígono Río San Pedro s/n,
11510 Puerto Real, Cádiz, Spain
e-mail: carmen.morales@uca.es
The heavy fuel oil spill from the tanker Prestige on
November 2002 affected more than 1,000 km of
coast, from the North of Portugal up to the South-east
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Environ Monit Assess (2007) 131:211–220
of France, being the Galician Coast the most damaged.
The composition of this fuel was a mixture of
saturated hydrocarbons, aromatic hydrocarbons, resins
and asphaltenes, being most of the Polycyclic aromatic
hydrocarbons—PAHs—of an intermedium-high molecular weight (Albaigés & Bayona, 2003; Blanco,
Prego, Azpíroz, Fernández-Domínguez, 2006). The
formation of the emulsions and the generation of tars
induced processes of sedimentation, so that 1 year
after the accident the marine sediments reached PAHs
concentrations which were 10 times higher than those
registered before the spill (IEO, 2003).
The use of biomarkers in fish which are indicative
of PAHs exposure may provide an early warning of
potential ecosystem degradation, contaminant bioavailability, and the defence responses of exposed
organisms (Goksøyr et al., 1996; Goksøyr & Förlin,
1992; Reynolds et al., 2003). Interest in the effects of
environmental stressors on health and alterations in
fish and other marine organisms has increased in
recent years, and in particular, histological and
cellular alterations have been observed in marine fish
from polluted coastal waters and estuaries (Malins
et al., 1984; Stein et al., 1992). The capacity of many
pollutants to alter different cells, tissues or organs has
led to design histopathological techniques in order to
evaluate the effects of contaminants (Lowe, 1988;
Sarasquete, Muñoz-Cueto, Arellano, & González de
Canales, 1997). On the other hand, the relation
between contaminated environments and fish alterations has been proved by different authors (Ortiz,
González de Canales, & Sarasquete, 2003; Husoy,
Myers, & Goksoyr, 1996; Martín-Díaz, Tuberty,
McKenney, Sales, & DelValls, 2005; Myers, Willis,
Husoy, Goksoyr, & Collier, 1995; Ortiz, González de
Canales, Sarasquete, 1999; Sarasquete et al., 2002).
The cytochromes P-450-1A (CYP1A) are of special
interest in ecotoxicology, due to their role in the biotransformation and bioactivation of different organic
xenobiotics (dioxins, PAHs, PCBs). The complex
CYP1A turns by monooxygenation, determined lipophilic xenobiotics, in more water-soluble metabolites,
helping its detoxification. The EROD measurement in
fish is considered a monitoring instrument of pollution
exposure and an indicator of potential future problems
in the health of fish populations (Carballeira, 2003).
According to other authors (Moore & Simpson,
1992; Pacheco & Santos, 2002), the information
provided by each biomarker individually is of limited
relevance, as there is a considerable likelihood of
misinterpretation; thus, biomarkers are best used as
selected batteries of tests rather than individually.
Furthermore, the study of the behaviour of various
biomarkers along the time (toxicokinetic approach)
may lead to a substantial improvement in the
knowledge of integrated fish toxic response (Pacheco
& Santos, 2002).
In the present study a bioassay using the fish
Sparus aurata was conducted by exposing the
individuals to environmental sediment samples collected in areas affected by the Prestige oil spill
(November 2002) in the National Park of the Atlantic
Islands two years after the Prestige oil spill. The main
objectives of this study were: (1) to characterize the
metals and PAHs contamination in sediments from the
selected areas in the Galician Coast and compare
them to a pristine area in the Gulf of Cádiz; (2) to
determine the sediment toxicity through the study of
the two biomarkers selected along the time; (3) to
determine and compare the sediment quality of the
different areas of the study by linking the contamination data and the biological effects, establishing a
mechanism of detoxification and proposing a toxicokinetic approach.
2 Material and Methods
2.1 Approach
The area selected to carry out this study was the
“Cíes” islands located in the national park of the
Atlantic Islands which has a high ecological value.
These islands played an important role during the
Prestige oil spill, since they operated as a natural
barrier against the entry of fuel in the “Rías Bajas”
(Galician Southern coast). Three stations (Ga1, Ga2
and Ga3), whose sediments were affected in different
degree by the oil spill of the tanker Prestige, were
selected in the internal face of the Archipelago
(Figure 1). Another sample was located in the South
of Spain, in the Bay of Cádiz (BC) which is considered
a clean area (Riba, Forja, Gómez-Parra, & DelValls,
2004b) and was used as the reference station.
Sediment samples from each of the stations were
collected with a 0.025 m2 Van Veen grab and were
homogenized with a Teflon® spoon until no colour or
textural differences could be detected. The samples
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Environ Monit Assess (2007) 131:211–220
213
Figure 1 Map of the locations of the area selected to
perform the study. Ga1,
Ga2 and Ga3 are located in
the Atlantic Islands in the
Galician Coast affected by
the oil spill related to the
Prestige tanker (November,
2002), whereas the reference station (BC) is located
in the Bay of Cádiz in the
South of Spain (not affected
by oil spills).
were subsampled for physical characterization and
chemical quantification. After that, sediment samples
were maintained at 4 °C in the dark until use in
sediment toxicity tests (no more than 2 weeks).
Sediment was filtered (1 mm) prior to the toxicity
test in order to remove means interferences as shells,
predators and other residues.
FIAS coupled with a Perkin-Elmer 4100 ZL spectrophotometer. Results are expressed as mg kg−1 dry
sediment. The analytical procedures were checked
using reference material (MESS-1 NRC and CRM
277 BCR) and comply with the certified values in
over a 90%.
Polycyclic aromatic hydrocarbons (PAHs) were
analyzed by using a gas chromatography equipped
with an electron capture detector (ECD) (U.S.
Environmental Protection Agency SW-846 Method
8270) (US EPA, 1984); briefly, dried samples were
soxhlet extracted with n-hexane for 18 h, and the
extracts were isolated by column chromatography on
Florisil-alumino-silica. PAHs were eluted and their
fractions were dried in a rotatory evaporator and
redissolved in isooctane. Aromatic fractions were
analyzed on a Hewlett-Packard (HP) 5890 Series II
gas chromatograph coupled with HP 5970 mass
spectrometer. Chromatographic resolution was
achieved with a 30 m×0.250 mm DB-5 capillary
column, which has a 0.25 μm film thickness, with
helium as carrier gas. Quality control was carried out
using NRC-CNRC HS-6 sediment reference material.
The analytical procedures comply with the certified
values in over a 90%.
2.2 Chemical analysis
Sediment aliquots from each station were dried at
room temperature prior to chemical analysis and then
gently homogenized. Geochemical matrix characteristics were studied analyzing organic carbon (TOC)
concentration and sediment grain size. For sediment
grain size an aliquot of wet sediment was analyzed
using a laser particle size Fristch (model Analysette
22) following the method reported by DelValls,
Blasco, Sarasquete, Forja, and Gómez-Parra (1998).
Organic carbon content was determined using the
method of Gaudette, Flight, Torner, and Folger (1974)
with El Rayis (1985) modification.
Sediments were digested for trace metal analysis,
as described by Loring and Rantala (1992). Zn, and
Cu concentrations in the extracts were determined
with a Perkin-Elmer 2100 flame atomic absorption
spectrophotometer. Cd, Cr, Ni and Pb were measured
by graphite furnace atomic absorption spectrophotometry (Perkin-Elmer 4100 ZL), while concentrations of
Hg were determined by means of Perkin-Elmer MHS-
2.3 Sediment bioassay
Juveniles of S. aurata were obtained in an aquaculture
farm and were transported to the laboratory where the
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Environ Monit Assess (2007) 131:211–220
fish spent one month to acclimatize before the
bioassay. S. aurata was selected because is a common
specie in the Spanish coast, its biology is well known,
has been used in previous pollution studies (DelValls
et al., 1998) and is easy to acclimatize to laboratory
conditions. Sediment (approximately 4 l) from the
negative control (BC) and the stations Ga1, Ga2, and
Ga3 were placed in replicate in 25-l glass tanks with
clean sea water before the beginning of the experiment. After 24 h of particle setting, aeration was
provided to maintain adequate oxygen concentrations
(higher than 80% saturation). A baseline group of 10
randomly chosen individuals were measured,
weighed, anaesthetized, and processed for biomarkers
responses (exposure and effect) to be used as the
initial cellular control. After checking the tanks water
quality, twelve individuals (with a weight averaged
4±1 g) were placed in every tank and were fed two or
three times per day. The test was conducted during
2 months, no mortality was recorded, and every
15 days six individuals from each station were
anaesthetized and processed for histopathological
and EROD analysis. During the experiment natural
photoperiod was selected and temperature was maintained constant (19±1 °C). Physicochemical parameters (ammonia, pH, temperature, oxygen and salinity)
were recorded and controlled when necessary to
maintain quality control during the test. Water
replacement was performed every day to avoid
increasing levels of ammonia, and the survival rate
for all tanks was determined.
General indexes of histological lesions were
calculated for each tissue (lesion index in gills [LIG]
and lesion index in liver [LIL]) as an average value of
the fish damage semi_quantified as previously
reported (Morales-Caselles, Jiménez-Tenorio, González
de Canales, Sarasquete, DelValls, 2006; Riba, CasadoMartínez, Blasco, DelValls, 2004a; Riba et al., 2004b;
Riba, González de Canales, Forja, & DelValls, 2004c).
The semiquantification was performed by ranking the
frequency of lesions measured in a total number of six
individuals: − (zero individuals), +/− (one individual),
+ (two individuals), ++/+ (three individuals), ++ (four
individuals), +++/++ (five individuals) and finally the
maximum is associated with the presence of alterations
in the total number of individuals, +++ (six individuals
sampled).
2.5 Biochemical analysis
Fish were sampled for biochemical analysis, and after
dissection, livers were kept at −80 °C prior to the
homogenization. The samples were homogenized
following the procedure developed by Lafontaine
et al. (2000). After homogenization of the samples,
EROD samples were centrifuged at 10,000×g for
30 min, and the supernatant was used for the EROD
activity determination and the total protein content
described by Bradford (1976). EROD assay was
performed following the methodology described by
Gagné and Blaise (1993). Briefly, 50 μl of supernatant (homogenate 10,000×g for 30 min), 10 μM 7ethoxyresorufin and 10 mM reduced NADPH in
100 mM KH2PO4 buffer (pH 7.4). The reaction was
started by the addition of NADPH, being allowed to
proceed for 60 min at 30 °C, and stopped by the
addition of 100 μl of 0.1 M NaOH. The 7hydroxyresorufin was determined fluorometrically
using 535 nm (excitation) and 580 nm (emission)
filters. 7-Hydroxyresorufin concentration in the samples was achieved through an standard calibration curve
developed with concentrations of 7-hydroxyresorufin.
Results were expressed as picomoles per milligram Total
protein (Martín-Díaz, 2004).
2.4 Histological procedures
Individual of the fish S. aurata proceeding from the
toxicity tests were analyzed to determine the histopathological damages in gills. Fish were removed
from the tanks at 15, 30, 45 and 60 days of exposure
time and samples were collected. Fish were anaesthetized with 0.1% 2-phenoxyethanol 99% during 5–
10 min; then weighed, measured in length and
externally examined. Liver and gills from all the
organisms were obtained by dissection and then fixed
in phosphate buffered 10% formaldehyde (pH 7.2) for
24 h and embedded in paraffin. The histological
sections were stained with Haematoxylin–Eosin and
Haematoxylin–VOF (Gutiérrez, 1967). Sections were
reviewed by light microscopy Leitz Laborlux S and
photographed (Sony DKC-CM30).
3 Results and Discussion
Table I shows the summarized results of total organic
carbon, grain size (% of fine grain <63 μm),
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Environ Monit Assess (2007) 131:211–220
Table I Values of total organic carbon (% dry
weight), fines (% dry
weight) and the concentration of contaminants (PAHs
and metals) in sediment
samples (concentrations are
expressed in mg kg−1 dry
weight)
215
Contaminant
−1
PAHs (mg kg )
Metals (mg kg−1)
Not detected is expressed by
n.d. Table adapted from
Morales-Caselles et al.
(2006).
TOC
Fines (<63 μm)
Total PAHs
Fluorene
Acenafphthene
Naphthalene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
Benzofluoranthene
Benzo[e]pyrene
Benzo[a]pyrene
Perilene
Dibenzo[ah]anthracene
Indene[123-cd]pyrene
Benzo[ghi]perilene
Cd
Cr
Cu
Ni
Pb
Zn
Hg
concentration of metals (Cd, Cr, Cu, Ni, Pb, Zn, Hg)
and PAHs (Fluorene, Acenafphthene, Naphthalene,
Phenanthrene, Anthracene, Fluoranthene, Pyrene,
Benzo[a]anthracene, Chrysene, Benzofluoranthene,
Benzo[e]pyrene, Benzo[a]pyrene, Perilene, Dibenzo
[ah]anthracene, Indene[123-cd]pyrene, Benzo[ghi]
perilene) in the different sediment used in the test
(Riba et al., 2004a). Sediments from the reference
station (BC) show low values of metals while PAHs
were not detected. The highest values of PAHs have
been measured in sediments from the station Ga3
(5.10 mg kg−1 dry weight) followed by the station
Ga2 (2.12 mg kg−1 dry weight) and Ga1 (0.19 mg
kg−1 dry weight). The concentration of metals in
sediments from the stations located in Galicia is
similar to those measured in the reference station
(BC). Previous studies pointed out the possible
amount of some metals concentration such as Ni, V,
Cu, Pb and Zn (Albaigés & Bayona, 2003; CSIC,
2003; Prego & Cobelo-García, 2003, 2004) from the
oil spill although they were not observed at high
levels in our study.
BC
Ga1
Ga2
Ga3
1.07
1.04
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.92
0.10
6.98
0.06
2.28
21.3
ND
0.60
0.06
0.19
0.08
0.06
0.31
0.10
0.02
0.12
0.09
0.05
0.08
0.11
0.08
0.05
0.03
0.01
0.02
0.01
0.16
ND
12.8
1.71
2.73
14.7
ND
1.19
0.03
2.12
0.13
0.17
0.63
0.15
0.03
0.18
0.13
0.09
0.12
0.18
0.13
0.09
0.05
0.02
0.02
0.02
0.05
2.00
0.65
0.42
1.14
3.95
0.01
2.00
0.01
5.10
0.35
0.27
1.40
1.36
0.18
0.10
0.39
0.20
0.39
0.06
0.16
0.10
0.04
0.02
0.02
0.06
ND
1.51
1.19
0.66
1.26
6.45
ND
Figure 2 shows the values of the EROD activity
measured in liver samples of the S. aurata exposed to
sediments treatments throughout 60 days. In general,
EROD activity increases with the presence of PAHs
in the sediment samples (Ga3>Ga2>Ga1>BC). Several studies agree that the use of EROD induction in
fish is particularly well suited for detection of PAH
exposure, because parent compounds may often not
be detected in tissues (Whyte, Jung, Schmitt, & Tillit,
2000).
The study of the behaviour of EROD activity
during the exposure period for Ga3 shows that EROD
activity increases significantly at the beginning of the
experiments until day number 15 (2.4 pmol/mg/min
of protein) and maximum levels are reached
(2.9 pmol/mg/min of protein) on day 30. The
measures of this biomarker in the liver of the
organisms exposed to sediments from Ga1 and Ga2
show a lower increase than in the case of exposure to
sediment in Ga3 and reach the maximum later than
Ga3, the day 45 (about 1.7 pmol/mg/min of protein
for both curves). In the course of the experiment the
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Environ Monit Assess (2007) 131:211–220
EROD activity (pmol mg-1 min-1)
BC
Ga1
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
0
15
30
45
60
0
15
EROD activity (pmol mg-1 min-1)
Ga2
30
45
60
45
60
Ga3
2.5
3.5
3.0
2.0
2.5
1.5
2.0
1.0
1.5
1.0
0.5
0.5
0.0
0.0
0
15
30
45
60
0
15
30
time (days)
time (days)
Figure 2 EROD activity in picomoles per milligram per minute of protein in liver samples of S. aurata exposed to sediments from
Galicia (Ga#) and control (BC) during the 60 days of bioassay.
EROD activity for Ga3 – which is the station with the
greatest amount of PAHs in their sediments, 5.1 mg
kg−1 dry weight – is always higher than the EROD
activity for Ga1 and Ga2. These sites (Ga1 and Ga2)
show a similar behaviour along the time. For all the
stations it is shown a slight decrease of the induction
of this biomarker of exposure after the day 30 for Ga3
and after day 45 for the other three stations (including
the reference station).
The histological alterations observed in target
tissues (gill and liver) of fish exposed to sediment
collected along the 60 days in the different stations
were mainly in gills, which showed shortening of
secondary lamellae, presence of edematous areas in
distal portion of lamellae, hypertrophy and hyperplasy, necrosis and lost of cells epithelial in the
organisms exposed to the Galician sediments; fusion
of the secondary was detected specially in organisms
exposed to sediments from Ga3. Liver showed lesions
such as vacuolization of hepatocytes, necrosis and
decrease of the zymogen granules of the exocrine
pancreas in the organisms exposed to the Galician
sediments. In general, an increase of cytoplasmic
basophilia was detected in the liver and exocrine
pancreas of all exposed fish that seems related to the
increase of PAHs.
In Figures 3 and 4 the summarized results of the
histopathological alteration are shown as the index of
lesions. The index for gills (LIG) increases with the
presence of PAHs in the sediment samples (Ga3>
Ga2>Ga1>BC) and, in general, LIG increases along
the time of exposure (Figure 3). The value of LIG is
maximum the day 60 of the experiment and the
highest frequency corresponds to the damages observed in the gills of the organisms exposed to
sediment from Ga3 (LIG=2.6), followed by Ga2
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Ga1
BC
3.0
4.0
2.5
3.0
LIG
2.0
1.5
2.0
1.0
1.0
0.5
0.0
0.0
0
15
30
45
0
60
15
Ga2
30
45
60
Ga3
4.0
3.0
2.5
3.0
LIG
2.0
2.0
1.5
1.0
1.0
0.5
0.0
0.0
0
15
30
45
60
0
time (days)
15
30
45
60
EROD activity (pmol mg-1 min-1)
217
EROD activity (pmol mg-1 min-1)
Environ Monit Assess (2007) 131:211–220
time (days)
Figure 3 The General Index of Lesions measured in the fish
Sparus aurata for gills (LIG) along the period of exposure to
the sediments are represented by bars. EROD activity in
picomoles per milligram per minute of protein in liver samples
of S. aurata along the duration of the experiment for the control
(BC) and Galician (Ga#) sites is represented by curves.
(LIG=1.9) and Ga1 (LIG=1.1). A similar behaviour
can be observed for the index of lesions determined in
liver (LIL), where Ga3 presents the highest value
(Ga3: LIL=1.3; Ga2: LIL=1.1; Ga1: LIL=0.8; all of
them evaluated at the end of the exposure period, after
60 days). The values of LIG were higher than LIL
throughout the whole bioassay for all the stations. Gill
is a multifunctional organ sensitive to chemicals in
water, since gill filaments and lamellae provide a very
large surface area for direct and continuous contact
with contaminants in water.
The EROD activity is used as a biomarker of
exposure to lipophilic organic contaminants and
measures the enzymatic activity of the phase I
catalyzed by the complex CYP1A; the complex
CYP1A transforms some lipophilic xenobiotics in
metabolites more water soluble, so that they are easier
to excrete. However, some of these new compounds
are highly reactive and more toxic than the original
contaminant, and they might interact with biological
macromolecules (Parkinson, 1995) producing lesions.
In the fishes exposed to sediments from the station
Ga3, it seems that there is a first phase where EROD
activity is induced (days 0–30) while there are some
histopathological damages. When the activity reaches
a maximum and begins to decrease (days 30–60), the
histopathological alterations continue increasing and
the frequency of the lesions is higher. This mechanism of induction of histopathological damages when
hepatic EROD decreases can be shown in the three
stations affected by the Prestige oil spill, although it is
produced faster and with higher intensity and frequency in the organisms exposed to sediments from
Ga3, which also shows the highest PAHs concentra-113-
Ga1
BC
3.0
2.5
2.5
2.0
LIL
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
0
15
30
45
0
60
15
LIL
Ga2
30
45
60
Ga3
2.5
3.0
2.0
2.5
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
0
15
30
45
60
0
time (days)
15
30
45
60
EROD activity (pmol mg-1 min-1)
Environ Monit Assess (2007) 131:211–220
EROD activity (pmol mg-1 min-1)
218
time (days)
Figure 4 The General Index of Lesions measured in the fish
Sparus aurata for liver (LIL) along the period of exposure to
the sediments are represented by bars. EROD activity in
picomoles per milligram per minute of protein in liver samples
of S. aurata along the duration of the experiment for the control
(BC) and Galician (Ga#) sites is represented by curves.
tion in sediment. This behaviour could be related to
the production of toxic metabolites as secondary
products in the detoxification process where the
EROD activates. It seems that when EROD activity
stabilizes or disappears from the cells, the tissues get
more defenceless to organic compounds (in this case
PAHs), and histopathological damages are caused
with more intensity and frequency.
Galicia were the chemicals responsible for the
measured adverse effects (biomarkers of exposure
and effect). The toxicokinetic approach used in this
study proposes a mechanism that can explain the
histopathological damage associated with the exposure of fish to environmental samples contaminated
by PAHs from an oil spill (Prestige, 2002). It gives us
the possibility to compare entire curves of behaviour
instead of numerical data (endpoint). Besides, it
permits to understand better the kinetic of the toxicity
based on the role of a detoxification system such as
the CYP1A complex. It has been proved the importance of the use of chronic bioassays which provide
long-term information of the effects of the exposure to
a toxic compound analyzed in environmental samples.
4 Conclusions
This study shows that the comparison between
chemical analysis and the different toxic responses
(biomarkers of exposure and effect) is a useful tool to
determine the quality of the studied sediments that
were affected by the oil spill. The results obtained
demonstrate that PAHs analyzed in sediments from
Acknowledgments The described work was partially supported by Grant funded by the Ministry of Education and
-114-
Environ Monit Assess (2007) 131:211–220
219
Science VEM2003-20563. Carmen Morales Caselles was
funded by the Ministerio de Ciencia y Tecnología (FPU) of
Spain. Thanks are due to the CIS from Santiago de Compostela
and to the members of the ICMAN-CSIC for their help during
sampling and analysis of the sediments.
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-116-
RoleȱofȱbiomarkersȱtoȱassessȱoilȬcontaminatedȱsedimentȱqualityȱ
usingȱtoxicityȱtestsȱwithȱclamsȱandȱcrabsȱȱ
CarmenȱMoralesȬCaselles§,§§§,ȱM.ȱLauraȱMartínȬDíaz§,§§§,ȱInmaculadaȱ
Riba§,§§§,ȱCarmenȱSarasquete§,ȱT.ȱÁngelȱDelValls§§,§§§ȱȱ
ȱ§ȱInstitutoȱdeȱCienciasȱMarinasȱdeȱAndalucíaȱ(ICMANȬCSIC),ȱAvda.ȱRepúblicaȱ
Saharauiȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱSpainȱ
§§ȱUNESCOȱUNITWIN/UNICOP,ȱFacultadȱdeȱCienciasȱdelȱMarȱyȱAmbientales,ȱ
UniversidadȱdeȱCádiz,ȱPolígonoȱRíoȱSanȱPedroȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱSpain.ȱ
§§§ȱUnidadȱAsociadaȱdeȱCalidadȱAmbientalȱyȱPatologíaȱ(CSICȱ&ȱUCA),ȱAvda.ȱ
RepúblicaȱSaharauiȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱSpain.ȱ
Abstractȱ
ȱAȱ 28Ȭdayȱ bioassayȱ wasȱ conductedȱ withȱ twoȱ invertebrateȱ speciesȱ withȱ
differentȱfeedingȱhabits,ȱtheȱclamȱRuditapesȱphilippinarumȱandȱtheȱshoreȱcrabȱ
Carcinusȱ maenas.ȱ Theȱ purposeȱ ofȱ theȱ studyȱ wasȱ toȱ assessȱ theȱ qualityȱ ofȱ
sedimentsȱ affectedȱ byȱ oilȱ spillsȱ inȱ differentȱ areasȱ ofȱ ȱ theȱ Spanishȱ Coast.ȱ Theȱ
organismsȱ wereȱ exposedȱ toȱ environmentalȱ samplesȱ ofȱ oilȬcontaminatedȱ
sedimentsȱduringȱfourȱweeks,ȱandȱafterȱtheȱexperimentȱaȱsuiteȱofȱbiomarkersȱofȱ
exposureȱ wasȱ measured:ȱ theȱ phaseȱ Iȱ detoxificationȱ systemȱ wasȱ assessedȱ byȱ
EthoxyresorufinȱOȬdeethylaseȱ(EROD)ȱactivity;ȱglutathioneȬSȬtransferaseȱ(GST)ȱ
isȱ aȱ phaseȱ IIȱ detoxificationȱ enzymeȱ butȱ isȱ alsoȱ implicatedȱ inȱ oxidativeȱ stressȱ
events;ȱglutathioneȱperoxidaseȱ(GPX),ȱglutathioneȱreductaseȱ(GR)ȱandȱtheȱferricȱ
reducingȱ abilityȱ ofȱ plasmaȱ (FRAP)ȱ assayȱ wereȱ analyzedȱ toȱ determineȱ theȱ
antioxidantȱ activityȱ ofȱ theȱ tissues.ȱ Theȱ biomarkerȱ resultsȱ wereȱ correlatedȱ withȱ
theȱchemicalȱcompoundsȱboundȱtoȱsedimentsȱ(PAHs,ȱPCBs,ȱZn,ȱCd,ȱPb,ȱCu,ȱNi,ȱ
Co,ȱV)ȱandȱaȱprincipalȱcomponentȱanalysisȱwasȱcarriedȱoutȱwithȱtheȱpurposeȱofȱ
linkingȱallȱtheȱvariables,ȱandȱtoȱdetectȱthoseȱcontaminatedȱsedimentsȱpotentiallyȱ
harmfulȱ toȱ theȱ biota.ȱ Resultsȱ showedȱ inductionȱ ofȱ biomarkersȱ inȱ bothȱ
invertebrateȱ speciesȱ andȱ significantȱ differencesȱ (pȱ <ȱ 0.05;ȱ pȱ <ȱ 0.01)ȱ wereȱ
ȱEnvironmentalȱToxicologyȱandȱChemistryȱ(aceptado)
- 117 -
establishedȱamongȱsedimentsȱaffectedȱbyȱdifferentȱspills.ȱTheȱuseȱofȱtheȱselectedȱ
biomarkersȱ togetherȱ withȱ theȱ sedimentȱ chemicalȱ analysisȱ assessesȱ theȱ
bioavailabilityȱofȱcontaminantsȱandȱhasȱprovenȱtoȱbeȱaȱsuitableȱtoolȱtoȱmonitorȱ
theȱenvironmentalȱqualityȱofȱsedimentsȱaffectedȱbyȱoilȱspills.ȱ
Keywords:ȱPAHs,ȱtoxicity,ȱbioassay,ȱoilȱspill,ȱWOEȱ
1.ȱIntroductionȱ
Theȱ presenceȱ ofȱ persistentȱ pollutantsȱ relatedȱ toȱ oilȱ spillsȱ suchȱ asȱ PAHsȱ
andȱ PCBsȱ andȱ toxicȱ metalsȱ (Cd,ȱ Pb,ȱ Zn,ȱ Cu,ȱ Ni,ȱ Co,ȱ V,ȱ etc)ȱ inȱ differentȱ
compartmentsȱ ofȱ theȱ marineȱ environmentȱ hasȱ becomeȱ aȱ majorȱ threatȱ toȱ theȱ
healthȱofȱmarineȱecosystemsȱdueȱtoȱaccumulationȱofȱtheirȱresiduesȱinȱtheȱtissuesȱ
ofȱ marineȱ organismsȱ [1].ȱ Biomarkersȱ haveȱ beenȱ shownȱ toȱ beȱ usefulȱ toolsȱ inȱ
characterizingȱtheȱhealthȱstatusȱofȱanimalsȱfromȱaffectedȱareas,ȱwhereȱcomplexȱ
mixturesȱ ofȱ pollutantsȱ areȱ usuallyȱ presentȱ [2,ȱ 3,ȱ 4].ȱ Biomarkersȱ presentȱ theȱ
inherentȱ capacityȱ toȱ detectȱ earlyȱ biologicalȱ effectsȱ withinȱ theȱ organismȱ andȱ toȱ
monitorȱtheȱtemporalȱprogressionȱ(orȱregression)ȱofȱtheȱdisturbanceȱofȱvariousȱ
levelsȱ ofȱ biologicalȱ organizationȱ [5].ȱ Underȱ controlledȱ conditionsȱ inȱ theȱ
laboratory,ȱitȱisȱrelativelyȱstraightforwardȱtoȱstandardiseȱbiomarkerȱassaysȱandȱ
toȱ regulateȱ theȱ chemicalȱ exposuresȱ thatȱ organismsȱ receive,ȱ soȱ thatȱ causeȬeffectȱ
andȱindeed,ȱexposureȬrelationships,ȱcanȱbeȱestablishedȱ[6].ȱ
Theȱfluctuationȱofȱdifferentȱbiomarkersȱinȱresponseȱtoȱdifferentȱtoxicantsȱ
providesȱaȱpatternȱofȱresultsȱwhichȱcanȱgiveȱcluesȱasȱtoȱtheȱtypeȱofȱpollutantȱthatȱ
isȱ causingȱ theȱ observedȱ effectȱ [6].ȱ Biomarkersȱ haveȱ beenȱ previouslyȱ usedȱ toȱ
assessȱ oilȱ spillȱ episodesȱ [5,ȱ 7,ȱ 8,ȱ 9].ȱ Inȱ theȱ presentȱ studyȱ aȱ suiteȱ ofȱ biomarkersȱ
wasȱchosenȱinȱorderȱtoȱinvestigateȱbiologicalȱresponsesȱofȱorganismsȱexposedȱtoȱ
oilȬcontaminatedȱ sedimentsȱ fromȱ theȱ Galicianȱ Coastȱ (NWȱ Spain),ȱ acutelyȱ
affectedȱ byȱ aȱ fuelȱ spillȱ (Prestige,ȱ 2002),ȱ andȱ theȱ Bayȱ ofȱ Algecirasȱ (Sȱ Spain),ȱ
chronicallyȱ affectedȱ byȱ differentȱ spills.ȱ Ethoxyresorufinȱ OȬdeethylaseȱ (EROD)ȱ
- 118 -
wasȱ selectedȱ asȱ theȱ phaseȱ Iȱ detoxificationȱ enzymeȱ implicatedȱ inȱ
monooxygenationȱ reactionsȱ ofȱ dioxinsȱ andȱ PAHs.ȱ GlutathioneȬSȬtransferaseȱ
(GST)ȱ isȱ aȱ phaseȱ IIȱ detoxificationȱ enzymeȱ butȱ isȱ alsoȱ implicatedȱ inȱ oxidativeȱ
stressȱ events,ȱ whileȱ glutathioneȱ peroxidaseȱ (GPX)ȱ andȱ glutathioneȱ reductaseȱ
(GR)ȱ wereȱ chosenȱ asȱ antioxidantȱ enzymesȱ togetherȱ withȱ theȱ ferricȱ reducingȱ
abilityȱ ofȱ plasmaȱ (FRAP)ȱ assay.ȱ Theȱ combinationȱ ofȱ biologicalȱ responsesȱ andȱ
chemicalȱdataȱofȱtheȱsedimentȱhelpsȱidentifyȱtheȱintegratedȱimpactȱofȱchemicalȱ
contaminationȱonȱorganisms.ȱManyȱauthorsȱagreeȱthatȱsedimentȱqualityȱisȱbestȱ
determinedȱbyȱintegratingȱtheȱinformationȱobtainedȱfromȱmeasuresȱofȱchemicalȱ
concentrationȱandȱfromȱspecificȱtestsȱtoȱdetermineȱsedimentȱtoxicityȱ[10,ȱ11].ȱȱ
Theȱ purposeȱ ofȱ thisȱ workȱ isȱ toȱ testȱ theȱ suitabilityȱ ofȱ usingȱ aȱ setȱ ofȱ
biomarkersȱinȱtwoȱinvertebrateȱspecies,ȱtheȱclamȱRuditapesȱphilippinarumȱandȱ
theȱ crabȱ Carcinusȱ maenas,ȱ inȱ orderȱ toȱ assessȱ theȱ environmentalȱ qualityȱ ofȱ
sedimentsȱ affectedȱ byȱ oilȱ spills.ȱ Toȱ achieveȱ thisȱ objectiveȱ theȱ selectedȱ
biomarkersȱ wereȱ linkedȱ withȱ theȱ concentrationȱ ofȱ contaminantsȱ inȱ theȱ
sedimentsȱandȱtheȱresultsȱareȱdiscussed.ȱȱ
2.ȱMaterialȱandȱmethodsȱ
2.1.ȱApproachȱ
Theȱsedimentsȱemployedȱinȱtheȱpresentȱstudyȱwereȱcollectedȱinȱtwoȱareasȱ
ofȱ theȱ Spanishȱ coastȱ affectedȱ byȱ oilȱ spills.ȱ Theȱ areaȱ ofȱ theȱ Galicianȱ coastȱ (NWȱ
Spain)ȱsufferedȱtheȱacuteȱimpactȱofȱtheȱPrestigeȱoilȱspillȱinȱ2002ȱwhereasȱtheȱBayȱ
ofȱAlgecirasȱ(SȱSpain)ȱisȱcontinuouslyȱaffectedȱbyȱminorȱspills,ȱincludingȱoilȱandȱ
otherȱ contaminantsȱ fromȱ industriesȱ andȱ dischargesȱ fromȱ commercialȱ shippingȱ
activitiesȱ[12].ȱAȱreferenceȱsiteȱwithȱnoȱorganicȱpollutionȱwasȱselectedȱinȱtheȱBayȱ
- 119 -
ofȱCádizȱ(SȱSpain).ȱThisȱsiteȱhasȱbeenȱwidelyȱvalidatedȱasȱaȱreferenceȱareaȱ[9,ȱ12,ȱ
13,ȱ14].ȱTheȱ10ȱselectedȱstudyȱsitesȱareȱshownȱinȱFigureȱ1.ȱ
ȱ
ȱ
ȱ
Atlantic Islands
National Park
• D72
•FIG
•D59
ƒA1ƒA2
ƒA3
Ría de CormeLaxe
ȱ
ȱ
Spain
ȱ
ȱ
•GR3’
•GR4
Bay of
Cádiz
•P1
ȱ
•CA1
ȱ
Bay of
Algeciras
N
E
W
S
ȱ
Figureȱ1.ȱMapȱofȱtheȱcoastalȱareaȱofȱGaliciaȱshowingȱtheȱlocationsȱofȱtheȱ
samplingȱ stations.ȱ FIG,ȱ D59ȱ andȱ D72ȱ refersȱ toȱ theȱ stationsȱ locatedȱ inȱ theȱ Ciesȱ
Islandȱ inȱ theȱ Atlanticȱ Islandȱ NationalȱParkȱ andȱA1,ȱA2ȱandȱA3ȱtoȱthoseȱinȱ theȱ
BayȱofȱCormeȬLaxe.ȱTheȱstationsȱlocatedȱinȱtheȱBayȱofȱAlgecirasȱareȱGR3’,ȱGR4ȱ
andȱP1.ȱTheȱstationȱCAȱlocatedȱinȱtheȱBayȱofȱCadizȱcorrespondsȱtoȱtheȱsedimentȱ
usedȱasȱreference.ȱ
2.2.ȱBioassaysȱ
Theȱ clamȱ Ruditapesȱ philippinarumȱ andȱ theȱ crabȱ Carcinusȱ maenasȱ wereȱ
obtainedȱfromȱanȱaquacultureȱfarmȱandȱwereȱkeptȱunderȱlaboratoryȱconditionsȱ
- 120 -
inȱtanksȱwithȱcontinuousȱwaterȱreplacementȱduringȱ10ȱdaysȱforȱacclimation.ȱ25Ȭ
Lȱ tanksȱ wereȱ employedȱ toȱ performȱ theȱ bioassayȱ withȱ crabs,ȱ whereasȱ 11ȬLȱ
aquariumsȱ wereȱ selectedȱ toȱ carryȱ outȱ theȱ experimentȱ withȱ clams.ȱ Sedimentȱ
collectedȱinȱtheȱstudyȱsitesȱwasȱplacedȱinȱreplicateȱinȱtheȱtanks:ȱ4ȱLȱofȱsedimentȱ
wasȱputȱinȱtheȱ25ȬLȱglassȱtanksȱandȱ2ȱLȱofȱsedimentȱsampleȱwasȱplacedȱinȱtheȱ
11ȬLȱ aquariums.ȱ Cleanȱ seaȱ waterȱ wasȱ thenȱ addedȱ andȱ afterȱ particleȱ settling,ȱ
aerationȱ wasȱ providedȱ toȱ maintainȱ adequateȱ oxygenȱ concentrationsȱ (greaterȱ
thanȱ 80%ȱ saturation).ȱ Subsequently,ȱ theȱ organismsȱ wereȱ transferredȱ toȱ theȱ
tanks,ȱ theȱ laboratoryȱ conditionsȱ wereȱ controlled,ȱ theȱ temperatureȱ wasȱ keptȱ atȱ
19±1ºCȱ andȱ theȱ naturalȱ photoperiodȱ wasȱ maintained.ȱ Theȱ bioassaysȱ wereȱ
performedȱinȱduplicateȱandȱlastedȱ28ȱdays;ȱoverȱthisȱtimeȱtheȱwaterȱinȱtheȱtanksȱ
wasȱreplacedȱandȱtheȱcrabsȱwereȱfedȱeveryȱweekȱwithȱaȱmixedȱdietȱofȱmusselsȱ
orȱfish,ȱwhileȱtheȱclamsȱwereȱfedȱȱwithȱanȱalgaeȱpreparation.ȱ
2.3.ȱBiochemicalȱanalysisȱ
Afterȱ 28ȱ daysȱ ofȱ theȱ exposureȱ periodȱ aȱ surveyȱ wasȱ carriedȱ outȱ andȱ theȱ
hepatopancreasȱ (inȱ crabs)ȱ andȱ digestiveȱ glandȱ (inȱ clams)ȱ wereȱ extractedȱ andȱ
keptȱ atȱ Ȭ80ºCȱ priorȱ toȱ homogenization.ȱ Theȱ samplesȱ wereȱ homogenizedȱ
accordingȱtoȱȱtheȱprocedureȱdevelopedȱbyȱLafontaineȱetȱal.ȱ[15].ȱ
Followingȱ homogenization,ȱ theȱ samplesȱ wereȱ centrifugedȱ atȱ 10,000gȱ forȱ
30ȱmin,ȱandȱtheȱsupernatantȱwasȱusedȱforȱtheȱbiomarkerȱdetermination.ȱMixedȱ
functionȱ oxygenaseȱ activity,ȱ whichȱ isȱ theȱ firstȱ modeȱ ofȱ detoxificationȱ ofȱ manyȱ
organicȱ pollutants,ȱ wasȱ measuredȱ usingȱ theȱ adaptedȱ ERODȱ assayȱ [16].ȱ Theȱ
FRAPȱassayȱallowsȱaȱmeasureȱofȱtheȱantioxidantȱcapacityȱandȱwasȱcarriedȱoutȱasȱ
describedȱbyȱBenzieȱandȱStrainȱ[17].ȱTheȱantioxidantȱGlutathioneȬSȬtransferaseȱ
(GST)ȱ activityȱ wasȱ determinedȱ byȱ monitoringȱ theȱ rateȱ ofȱ conjugationȱ ofȱ
glutathioneȱ (GSH)ȱ toȱ 1ȬchloroȬ2,4Ȭdinitrobenzeneȱ (CDNB)ȱ atȱ 340ȱ nmȱ [18].ȱ Theȱ
- 121 -
oxidationȱ ofȱ 1ȱ mMȱ NADPHȱ byȱ Glutathioneȱ Reductaseȱ activityȱ (GR)ȱ inȱ theȱ
presenceȱofȱ10ȱmMȱoxidizedȱglutathioneȱwasȱalsoȱmonitoredȱatȱ340ȱnmȱ[18].ȱTheȱ
phaseȱ IIȱ metabolizingȱ enzymeȱ Glutathioneȱ Peroxidaseȱ activityȱ (GPX)ȱ wasȱ
measuredȱaccordingȱtoȱMcFarlandȱetȱal.ȱ[18].ȱAllȱtheȱbiomarkerȱresponsesȱwereȱ
normalizedȱtoȱtheȱtotalȱproteinȱcontentȱ[19].ȱ
2.4.ȱChemicalȱanalysisȱ
Theȱ analysesȱ ofȱ PAHsȱ andȱ PCBsȱ wereȱ carriedȱ outȱ accordingȱ toȱ USEPAȱ
SWȬ846ȱ Methodȱ 827C78082.ȱ Briefly,ȱ followingȱ recommendationsȱ byȱ Ribaȱ etȱ al.ȱ
[20],ȱ driedȱ samplesȱ wereȱ Soxhletȱ extractedȱ withȱ nȬhexaneȱ forȱ 18ȱ h,ȱ andȱ theȱ
extractsȱ wereȱ isolatedȱ byȱ columnȱ chromatographyȱ onȱ Florisileȱ aluminoȬsilica.ȱ
Theȱ PCBsȱ andȱ PAHsȱ wereȱ elutedȱ andȱ theirȱ fractionsȱ wereȱ driedȱ inȱ aȱ rotatingȱ
evaporatorȱandȱreȬdissolvedȱinȱisooctane.ȱTheȱaromaticȱfractionsȱwereȱanalyzedȱ
usingȱȱaȱHewlettȱPackardȱ(HP)ȱ5890ȱSeriesȱIIȱgasȱchromatographerȱcoupledȱwithȱ
anȱ HPȱ 5970ȱ massȱ spectrometer.ȱ Theȱ PAHsȱ wereȱ analyzedȱ byȱ GCȬMSȱ usingȱ
selectedȱ ionȱ monitoringȱ (SIM).ȱ Analysisȱ ofȱ PCBsȱ suchȱ asȱ AROCLORȱ 1242ȱ andȱ
AROCLORȱ 1260ȱ wasȱ performedȱ usingȱ theȱ sameȱ instrumentȱ withȱ anȱ electronȱ
captureȱ detectorȱ (GC/ECD).ȱ Forȱ bothȱ setsȱ ofȱ organicȱ chemicals,ȱ PAHsȱ andȱ
AROCLOR,ȱ theȱ analyticalȱ procedureȱ showedȱ agreementȱ withȱ theȱ certifiedȱ
valuesȱofȱmoreȱthanȱ90%.ȱ
AȱtraceȱmetalȱanalysisȱwasȱcarriedȱoutȱasȱdescribedȱbyȱCasadoȬMartínezȱ
etȱ al.ȱ [21].ȱ Briefly,ȱ 2.5ȱ gȱ ofȱ sedimentsȱ (<0.065ȱ mm)ȱ wereȱ placedȱ inȱ Teflonȱ
containersȱandȱwereȱdigestedȱinȱaȱmicrowaveȱovenȱ(400W,ȱ15ȱmin,ȱtwice)ȱwithȱ
2NȱHNO3.ȱTheȱextractsȱwereȱpurifiedȱbyȱpassingȱthemȱthroughȱaȱCȬ18ȱcolumnȱ
andȱ metalȱ analysesȱ wereȱ performedȱ byȱ anodicȱ voltamperometryȱ (ȬZn,ȱ Cd,ȱ Pb,ȱ
Ni,ȱ Coȱ andȱ CuȬȱ Metrohmȱ Applicationȱ Bulletinȱ Nºȱ 147;ȱ Ȭȱ VȬȱ Metrohmȱ
Applicationȱ Noteȱ Nºȱ VȬ81).ȱ Theȱ coldȱ vapourȱ techniqueȱ wasȱ usedȱ forȱ Hgȱ andȱ
- 122 -
wasȱ quantifiedȱ usingȱ atomicȱ absorptionȱ spectrometry.ȱ Theȱ analyticalȱ
proceduresȱwereȱcheckedȱusingȱreferenceȱmaterialȱ(MESSȬ1ȱNRCȱandȱCRMȱ277ȱ
BCR)ȱandȱshowedȱaȱrecoveryȱgreaterȱthanȱ90%ȱofȱtheȱcertifiedȱconcentration.ȱ
2.5.ȱStatisticalȱanalysisȱ
Theȱ biomarkerȱ resultsȱ wereȱ analyzedȱ withȱ theȱ ANOVAȱ andȱ Tukeyȱ testȱ
withȱtheȱaimȱofȱdeterminingȱsignificantȱdifferencesȱ(pȱ<ȱ0.05;ȱpȱ<ȱ0.01)ȱbetweenȱ
theȱ resultsȱ obtainedȱ forȱ theȱ referenceȱ siteȱ andȱ theȱ otherȱ samplingȱ sitesȱ (SPSSȱ
11.5).ȱTheȱchemicalȱconcentrationsȱinȱsedimentsȱandȱbiomarkerȱresponsesȱwereȱ
correlatedȱ withȱ theȱ Pearsonȱ analysisȱ (pȱ <ȱ 0.05)ȱ inȱ orderȱ toȱ detectȱ relationshipsȱ
betweenȱ theȱ variablesȱ (STATISTICAȱ 6.0).ȱ Finallyȱ aȱ multivariateȱ analysisȱ wasȱ
carriedȱ outȱ withȱ theȱ purposeȱ ofȱ linkingȱ chemicalȱ andȱ biologicalȱ data;ȱ theȱ
principalȱ componentȱ analysisȱ (PCA)ȱ wasȱ usedȱ asȱ theȱ extractionȱ procedureȱ toȱ
deriveȱ aȱ reducedȱ numberȱ ofȱ newȱ variablesȱ (factors)ȱ asȱ linearȱ combinationsȱ ofȱ
theȱoriginalȱvariablesȱ(STATISTICAȱ6.0)ȱ[22].ȱ
3.ȱResultsȱ
3.1.ȱBiomarkerȱresponsesȱ
Theȱbiomarkerȱresponsesȱinȱcrabsȱandȱclamsȱafterȱ28ȱdaysȱofȱexposureȱtoȱ
theȱ sedimentȱ samplesȱ areȱ shownȱ inȱ Figureȱ 2.ȱ Theȱ GPXȱ activityȱ resultsȱ (Figureȱ
2.a)ȱshowedȱtheȱlowestȱvaluesȱinȱC.ȱmaenasȱexposedȱtoȱtheȱreferenceȱsediment.ȱ
Significantȱdifferencesȱ(pȱ<ȱ0.01)ȱinȱGPXȱinductionȱwereȱdetectedȱbetweenȱcrabsȱ
exposedȱtoȱtheȱreferenceȱsedimentȱandȱcrabsȱexposedȱtoȱtestȱsedimentsȱcollectedȱ
inȱA2ȱandȱA3ȱinȱCormeȬLaxeȱandȱGR3’ȱinȱtheȱBayȱofȱAlgeciras.ȱTheȱinductionȱofȱ
thisȱ biomarkerȱ inȱ clamsȱ didȱ notȱ presentȱ significantȱ differencesȱ betweenȱ
treatments.ȱTheȱinductionȱofȱtheȱantioxidantȱbiomarkerȱGRȱ(Figureȱ2.b)ȱinȱcrabsȱ
exposedȱtoȱsedimentȱfromȱA1ȱandȱclamsȱfromȱtheȱA2ȱtreatmentȱwasȱȱ
- 123 -
17.5
**
20
**
GR (n m o l/m g /m in )
GPX (nm ol/m g/m in)
30
**
10
**
10.5
7.0
3.5
*
0.0
0
Ca1 A1
A2 A3 FIG 59
Ca1 A1 A2 A3 FIG 59
72 GR3' GR4 P1
2500
72 GR3' GR4 P1
1.6
2000
1500
1000
*
500
*
0
Ca1 A1
A2 A3 FIG 59
EROD (pm ol/m g/m in)
GST (nm ol/m g/m in)
14.0
**
1.2
**
0.8
0.4
*
* *
0.0
72 GR3' GR4 P1
* *
Ca1 A1
A2 A3
**
*
* * ** **
FIG 59
72 GR3' GR4 P1
FRAP (uM /m g/m in)
20.0
**
16.0
Carcinusȱmaenas
12.0
Ruditapesȱphilippinarumȱ
8.0
* **
*
4.0
*
**
**
0.0
Ca1 A1
A2
A3 FIG 59
72 GR3' GR4 P1
Figureȱ 2.ȱ Generalȱ healthȱ biomarkersȱ forȱ bothȱ invertebrateȱ species,ȱ theȱ
clamȱ Ruditappesȱ philippinarumȱ andȱ theȱ crabȱ Carcinusȱ maenas:ȱ glutathioneȱ
peroxidaseȱ activityȱ GPXȱ (nmol/min/mgȱ prot),ȱ glutathioneȱ transferaseȱ GSTȱ
activityȱ (nmol/min/mgȱ prot),ȱ glutathioneȱ reductaseȱ GRȱ activityȱ (nmol/min/mgȱ
prot),ȱferricȱreducingȱabilityȱofȱplasmaȱFRAPȱactivityȱ(ΐM/mg/min)ȱandȱERODȱ
activityȱ (pmol/mg/min).ȱ Asterisksȱ indicateȱ significantȱ differencesȱ withȱ theȱ
referenceȱtreatmentȱCA1ȱ(*pȱ<ȱ0.05;ȱ**pȱ<ȱ0.01).ȱ
ȱ
ȱ
- 124 -
significantlyȱ differentȱ (pȱ <ȱ 0.05ȱ andȱ pȱ <ȱ 0.01ȱ respectively)ȱ fromȱ theȱ
referenceȱsite;ȱonȱtheȱotherȱhandȱcrabsȱexposedȱtoȱsedimentȱfromȱFIGȱandȱclamsȱ
exposedȱ toȱ GR3’ȱ presentedȱ significantȱ differencesȱ (pȱ <ȱ 0.05)ȱ andȱ lowerȱ valuesȱ
thanȱ theȱ referenceȱ siteȱ CA.ȱ Inȱ relationȱ toȱ ȱ theȱ phaseȱ Iȱ detoxificationȱ system,ȱ
clamsȱfromȱallȱtreatmentsȱshowedȱsignificantȱdifferencesȱwithȱtheȱreferenceȱsiteȱ
inȱERODȱactivityȱ(Figureȱ2.d);ȱtheseȱdifferencesȱwereȱgreaterȱ(p<0.01)ȱforȱthoseȱ
clamsȱthatȱhadȱbeenȱexposedȱtoȱtheȱsedimentsȱcollectedȱinȱtheȱBayȱofȱAlgecirasȱ
(GR3’,ȱ GR4ȱ andȱ P1).ȱ ȱ ERODȱ inductionȱ inȱ crabsȱ fromȱ A1,ȱ A2ȱ inȱ CormeȬLaxe,ȱ
GR3’ȱandȱGR4ȱinȱtheȱBayȱofȱAlgecirasȱwasȱalsoȱsignificantlyȱdifferentȱfromȱtheȱ
referenceȱ stationȱ (pȱ <ȱ 0.05).ȱ Theȱ antioxidantȱ activityȱ obtainedȱ fromȱ theȱ FRAPȱ
assayȱ(Figureȱ2.e)ȱshowedȱsignificantȱdifferencesȱbetweenȱȱcrabsȱexposedȱtoȱA2ȱ
(pȱ<ȱ0.05),ȱA3ȱ(pȱ<ȱ0.05),ȱFIGȱ(pȱ<ȱ0.05),ȱP1ȱ(pȱ<ȱ0.01)ȱandȱtheȱreferenceȱsite;ȱinȱtheȱ
caseȱ ofȱ clams,ȱ GR3’ȱ (pȱ <ȱ 0.01),ȱ GR4ȱ (pȱ <ȱ 0.01)ȱ andȱ D72ȱ (pȱ <ȱ 0.05)ȱ presentedȱ
significantȱ differencesȱ toȱ theȱ referenceȱ site,ȱ althoughȱ GR3’ȱ showedȱ higherȱ
valuesȱthanȱCAȱwhereasȱGR4ȱandȱD72ȱpresentedȱlowerȱvalues.ȱȱ
3.2.ȱChemicalȱanalysisȱ
ResultsȱofȱtheȱchemicalȱanalysisȱareȱshownȱinȱTable1.ȱSedimentsȱfromȱtheȱ
referenceȱ siteȱ didȱ notȱ presentȱ organicȱ contaminationȱ whereasȱ sedimentsȱ fromȱ
theȱ Bayȱ ofȱ Algecirasȱ (GR3’ȱ >ȱ GR4ȱ >ȱ P1),ȱ chronicallyȱ affectedȱ byȱ differentȱ spillȱ
andȱ CormeȬLaxeȱ (A1ȱ >ȱ A2ȱ >ȱ A3)ȱ presentedȱ higherȱ concentrationsȱ ofȱ PAHsȱ inȱ
theirȱ sedimentsȱ thanȱ sitesȱ fromȱ theȱ Ciesȱ Islandȱ (59ȱ >ȱ FIGȱ >72).ȱ ȱ ȱ Inȱ general,ȱ
chemicalȱanalysisȱdoesȱnotȱpresentȱaȱprevailingȱtendencyȱinȱtheȱconcentrationȱofȱ
metalsȱamongȱsedimentsȱfromȱtheȱdifferentȱareas.ȱSamplesȱcollectedȱinȱtheȱsiteȱ
GR3ȱfromȱtheȱBayȱofȱAlgecirasȱpresentedȱtheȱhighestȱvaluesȱofȱNiȱ(74.7ȱmgȱKgȬ1),ȱ
whereasȱ Zn,ȱ andȱ Pbȱ presentsȱ theirȱ maximumsȱ inȱ sedimentsȱ fromȱ theȱ areaȱ ofȱ
CormeȬLaxeȱ(A1ȱandȱA3).ȱȱȱ
- 125 -
Tableȱ 1.ȱ Concentrationȱ ofȱ PAHsȱ andȱ PCBsȱ (ΐgȱ kgȬ1ȱ dryȱ weight)ȱ andȱ
metalsȱ(mgȱkgȬ1ȱdryȱweight)ȱinȱtheȱsedimentȱsamplesȱusedȱinȱtheȱbioassays.ȱ
ȱȱ
Reference CormeȬLaxeȱ
CíesȱIslandȱ
BayȱofȱAlgecirasȱ
ȱȱ
Ca1ȱ
A1ȱ
A2ȱ
A3ȱ
FIGȱ
59ȱ
72ȱ
GR3ȇȱ GR4ȱ P1ȱ
PAHsȱ
n.d.ȱ
820ȱ
558ȱ
537ȱ
257ȱ
370
239ȱ
2961ȱ
802ȱ
PCBsȱȱ
n.d.ȱ
2.28ȱ 4.29ȱ 2.60ȱ
n.d.ȱ 6.52 4.76ȱ
22.0ȱ
1.75ȱ 0.84
Znȱȱ
21.3ȱ
244ȱ 31.8ȱ 65.7ȱ
76.2ȱ 43.4 37.5ȱ
138ȱ
35.3ȱ 56.7
Cdȱȱ
0.92ȱ
n.d.ȱ n.d.ȱ n.d.ȱ
n.d.ȱ n.d. n.d.ȱ
0.17ȱ
0.10ȱ 0.12
Pbȱ
2.28ȱ
14.3ȱ 4.25ȱ 44.0ȱ
26.6ȱ 9.13 6.54ȱ
21.6ȱ
6.21ȱ 12.3
Cuȱȱ
6.98ȱ
19.1ȱ n.d.ȱ 22.1ȱ
18.9ȱ n.d. 31.6ȱ
5.01ȱ
3.67ȱ 75.2
Niȱȱ
0.06ȱ
7.03ȱ 5.61ȱ 9.39ȱ
12.0ȱ 6.88 5.02ȱ
74.7ȱ
13.1ȱ 13.3
Coȱȱ
3.40ȱ
0.67ȱ 0.37ȱ 1.21ȱ
0.52ȱ n.d. 0.87ȱ
12.8ȱ
5.59ȱ n.d.
Vȱȱ
80.0ȱ
5.94ȱ 2.34ȱ 13.4ȱ
n.d.ȱ n.d. n.d.ȱ
26.1ȱ
n.d.ȱ 6.84
641ȱ
3.3.ȱCorrelationȱbetweenȱvariablesȱ
A correlation analysis was conducted in order to detect relationships between the
presence of contaminants in the sediments and the induction of biomarkers in the
invertebrates exposed to the same as biological mechanisms to defend against these
compounds. Significant correlations (p < 0.05 and p < 0.01) were observed between
sediment contamination and biological responses in the organisms exposed (table 2).
Similarly, significant associations (p < 0.05 and p < 0.01) were observed between
organic contaminants (PAHs and PCBs) bound to sediments, the metals Ni and Co and
the induction of EROD activity in both clams and crabs after the 28-dayexposure time. ȱ
- 126 -
ȱ
PAHs
PCBs
Zn
Cd
Pb
Cu
Ni
Co
V
GPX-crab
GPX-clam
GR-crab
GR-clam
GST-crab
GST-clam
EROD-crab
EROD-clam
FRAP-crab
FRAP-clam
.791**
Cu
Ni
Co
.754*
.851**
.648*
.727*
.709* .857**
.843** .725*
.882**
.882**
Pb
.969** .914**
.863** .799**
.970**
Cd
.969** .863**
.914** .799**
.843**
Zn
.905**
.905**
PAHs PCBs
.843**
.692*
.709*
.857**
.754*
.648*
.692*
.843**
.725*
.851**
.727*
.791**
GPX GPX GR GR GST GST EROD EROD FRAP FRAP
crab clam crab clam crab clam crab clam crab clam
- 127 -
.970**
V
Tableȱ 2.ȱ Pearsonȱ correlationȱ (*p<0.05,ȱ **p<0.01)ȱ resultsȱ amongȱ chemicalȱ compoundsȱ boundȱ toȱ sedimentsȱ andȱ
biomarkers:ȱ Ethoxyresorufinȱ OȬdeethylaseȱ (EROD)ȱ activity,ȱ glutathioneȬSȬtransferaseȱ (GST)ȱ activity,ȱ glutathioneȱ peroxidaseȱ
(GPX)ȱactivity,ȱglutathioneȱreductaseȱ(GR)ȱactivityȱandȱferricȱreducingȱabilityȱofȱplasmaȱ(FRAP)ȱactivity.ȱ
Meanwhile, the antioxidant GR and FRAP activity measured in both species was
significantly (p < 0.01) correlated with the presence of Zn and Cu in the sediment
respectively. A relationship has been shown between the metals V and Cd (p < 0.01)
although this association did not present any relationship with the biomarkers studied.
3.4.ȱPrincipalȱcomponentsȱanalysisȱ
Basedȱ onȱ correlationsȱ betweenȱ theȱ chemicalȱ andȱ biologicalȱ results,ȱ theȱ
principalȱcomponentsȱanalysisȱenablesȱgroupingȱofȱtheȱ19ȱoriginalȱvariablesȱintoȱ
4ȱ newȱ factorsȱ whichȱ accountȱ forȱ 63.3ȱ %ȱ ofȱ theȱ variance.ȱ Theȱ purposeȱ ofȱ thisȱ
analysisȱ isȱ ȱ toȱ reduceȱ theȱ numberȱ ofȱ variablesȱ withȱ theȱ minimumȱ lossȱ ofȱ
informationȱinȱorderȱtoȱsimplifyȱtheȱinterpretationȱofȱtheȱresults.ȱInȱtheȱpresentȱ
study,ȱitȱwasȱdecidedȱtoȱinterpretȱaȱgroupȱofȱvariablesȱasȱbeingȱassociatedȱwithȱ
aȱ particularȱ componentȱ whereȱ theȱ loadingȱ wasȱ 0.30ȱ orȱ higherȱ (Tableȱ 3),ȱ
approximatingȱ toȱ Comreys’ȱ cutȬoffȱofȱ0.55ȱ[23]ȱforȱaȱgoodȱassociationȱbetweenȱ
anȱ originalȱ variableȱ andȱ aȱ factor.ȱ Factorȱ 1ȱ (31.2ȱ %)ȱ linksȱ theȱ concentrationȱ ofȱ
PAHs,ȱ PCBs,ȱ Niȱ andȱ Coȱ inȱ sedimentsȱ withȱ theȱ inductionȱ ofȱ ERODȱ activityȱ inȱ
clamsȱ andȱ crabs,ȱ theȱ GPXȱ activityȱ inductionȱ inȱ crabsȱandȱ theȱ FRAPȱ activityȱ inȱ
clamsȱafterȱ28ȱdaysȱofȱexposure.ȱTheȱmetalsȱZnȱandȱPbȱinȱsedimentȱareȱrelatedȱ
withȱGPXȱandȱGRȱactivitiesȱinȱbothȱcrabsȱandȱclams,ȱGSTȱactivityȱinȱcrabsȱandȱ
FRAPȱinductionȱinȱclamsȱasȱdefinedȱbyȱFactorȱ2ȱ(17.3ȱ%).ȱFactorȱ3ȱ(14.9ȱ%),ȱwithȱ
negativeȱloading,ȱgroupsȱCuȱwithȱtheȱinductionȱofȱGPXȱandȱFRAPȱactivitiesȱinȱ
crabsȱafterȱ28ȱdaysȱofȱexposureȱtoȱtheȱsediments.ȱȱ
Afterȱdefiningȱtheȱmeaningȱofȱeachȱfactor,ȱtheȱPCAȱenablesȱȱidentificationȱ
ofȱ theȱ importanceȱ ofȱ eachȱ factorȱ atȱ eachȱ studyȱ siteȱ byȱ usingȱ theȱ factorȱ score.ȱ
Figureȱ3ȱshowsȱtheȱFactorȱscoreȱatȱeachȱofȱtheȱstations.ȱTheȱinfluenceȱofȱFactorȱ1,ȱ
relatedȱ withȱ theȱ biomarkerȱ responseȱ ofȱ theȱ organicȱ contaminantsȱ (PAHsȱ andȱ
PCHs),ȱandȱtheȱmetalsȱCoȱandȱNiȱinȱsediments,ȱisȱprevalentȱinȱtheȱstationsȱGR3’ȱ
(2.66)ȱandȱGR4ȱ(0.57)ȱfromȱtheȱBayȱofȱAlgeciras;ȱFactorȱ2,ȱwhichȱexplainsȱtheȱȱ
- 128 -
Tableȱ 3.ȱ Sortedȱ rotatedȱ factorȱ loadingsȱ ofȱ 19ȱ variablesȱ forȱ theȱ threeȱ
principalȱ factorsȱ resultingȱ fromȱ theȱ multivariateȱ analysisȱ ofȱ resultsȱ obtainedȱ
fromȱ theȱ chemicalȱ analysisȱ andȱ theȱ biomarkerȱ responsesinȱ crabsȱ andȱ clams:ȱ
EthoxyresorufinȱOȬdeethylaseȱ(EROD)ȱactivity,ȱglutathioneȬSȬtransferaseȱ(GST)ȱ
activity,ȱ glutathioneȱ peroxidaseȱ (GPX)ȱ activity,ȱ glutathioneȱ reductaseȱ (GR)ȱ
activityȱandȱferricȱreducingȱabilityȱofȱplasmaȱ(FRAP)ȱactivity.ȱ
ȱȱ
ȱȱ
PAHsȱ
PCBsȱ
Znȱ
Cdȱ
Pbȱ
Cuȱ
Niȱ
Coȱ
Vȱ
GPXȬcrabȬ28ȱ
GPXȬclamȬ28ȱ
GRȬcrabȬ28ȱ
GRȬclamȬ28ȱ
GSTȬcrabȬ28ȱ
GSTȬclamȬ28ȱ
ERODȬcrabȬ28ȱ
ERODȬclamȬ28ȱ
FRAPȬcrabȬ28ȱ
FRAPȬclamȬ28ȱ
FACTORȱ1ȱ FACTORȱ2ȱ FACTORȱ3ȱ
31.23%ȱ
17.25%ȱ
14.85%ȱ
ņȱ
ņȱ
0.80ȱ
ņȱ
0.34ȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.38ȱ
0.31ȱ
0.82ȱ
0.34ȱ
0.73ȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.44ȱ
0.95ȱ
0.91ȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.95ȱ
0.91ȱ
ņȱ
0.48ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.86ȱ
0.85ȱ
ņȱ
0.42ȱ
ȱ
ȱ
ȱ
- 129 -
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
Ȭ0.49ȱ
ņȱ
ņȱ
ņȱ
Ȭ0.30ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
Ȭ0.59ȱ
ņȱ
3.0
GR3'
ȱ
Factor 1
2.0
ȱ
1.0
GR4
ȱ
0.0
A2
-1.0
A3
A1
Ca1
FIG
59
P1
72
ȱ
ȱ
3.0
A1
ȱ
Factor 2
1.5
A2
GR3'
A3
0.0
P1
ȱ
FIG
Ca1
59
-1.5
72
ȱ
GR4
-3.0
ȱ
3.0
Ca1
ȱ
Factor 3
2.0
1.0
A3
A1
ȱ
GR3'
GR4
0.0
-1.0
-2.0
A2
ȱ
59
72
FIG
ȱ
P1
Figureȱ3.ȱFactorȱloadingsȱforȱtheȱthreeȱprincipalȱfactorsȱresultingȱfromȱtheȱ
multivariateȱanalysisȱofȱresultsȱobtainedȱfromȱtheȱchemicalȱanalysisȱandȱtheȱ
suiteȱofȱbiomarkers.ȱ
- 130 -
relationshipȱ betweenȱ theȱ contentȱ ofȱ Znȱ andȱ Pbȱ inȱ theȱ sedimentȱ andȱ theȱ
biomarkerȱ responsesȱ inȱ clamsȱ andȱ crabs,ȱ presentsȱ aȱ positiveȱ loadingȱ inȱ theȱ
stationsȱA1ȱ(2.02),ȱA2ȱ(0.36)ȱandȱA3ȱ(0.46)ȱfromȱCormeȬLaxeȱandȱGR3`ȱ(0.58)ȱandȱ
P1ȱ (0.07)ȱ fromȱ theȱ Bayȱ ofȱ Algeciras.ȱ Finally,ȱ Factorȱ 3,ȱ whichȱ withȱ negativeȱ
loadingȱ relatesȱ Cuȱ withȱ someȱ biomarkerȱ responsesȱ (GPXȱ andȱ FRAPȱ inductionȱ
inȱ crabs)ȱ showsȱ aȱ prevalenceȱ (negativeȱ scores)ȱ inȱ theȱ stationsȱ fromȱ theȱ Ciesȱ
IslandsȱFIGȱ(Ȭ0.88),ȱ59ȱ(Ȭ0.46),ȱ72ȱ(Ȭ0.16),ȱandȱA2ȱ(Ȭ0.38)ȱfromȱCormeȬLaxeȱandȱP1ȱ
(Ȭ1.40)ȱfromȱtheȱBayȱofȱAlgeciras.ȱ
4.ȱDiscussionȱȱ
Theȱ presentȱ studyȱ analysesȱ theȱ relationshipȱ betweenȱ biomarkerȱ
responsesȱinȱorganismsȱexposedȱtoȱsedimentsȱcontaminatedȱbyȱoilȱspillsȱȱinȱNWȱ
andȱSȱSpainȱandȱtheirȱchemicalȱcontent.ȱTwoȱinvertebrateȱspeciesȱwithȱdifferentȱ
feedingȱhabitsȱhaveȱbeenȱemployedȱinȱthisȱresearchȱasȱbioindicatorȱspecies,ȱtheȱ
shoreȱcrabȱCarcinusȱmaenasȱandȱtheȱclamȱRuditapesȱPhilippinarum.ȱȱ
Despiteȱ theȱ difficultyȱ ofȱ testingȱ complexȱ mixturesȱ ofȱ contaminants,ȱ theȱ
resultsȱ haveȱ shownȱ clearȱ relationshipsȱ betweenȱ theȱ differentȱ antioxidantȱ
enzymesȱ inȱ theȱ testedȱ organismsȱ inȱ theȱ presenceȱ ofȱ metals.ȱ Theȱ Phaseȱ Iȱ
detoxificationȱ systemȱ measuredȱ byȱ theȱ ERODȱ activityȱ wasȱ relatedȱ toȱ organicȱ
contaminantsȱ(PAHsȱandȱPCBs)ȱandȱmetalsȱboundȱtoȱcomplexȱorganicȱmixturesȱ
(Niȱ andȱ Co).ȱ Theseȱ contaminants,ȱ especiallyȱ PAHsȱ andȱ Ni,ȱ haveȱ ȱ oftenȱ beenȱ
linkedȱtoȱoilȱspills.ȱAntioxidantȱresponseȱ(GPXȱinȱcrabsȱandȱFRAPȱinȱclams)ȱwasȱ
alsoȱidentifiedȱforȱtheseȱcompounds.ȱERODȱactivityȱisȱoftenȱusedȱasȱaȱbiomarkerȱ
ofȱ exposureȱ toȱ lipophilicȱ organicȱ contaminantsȱ andȱ measuresȱ theȱ enzymaticȱ
activityȱ ofȱ ȱ phaseȱ Iȱ catalyzedȱ byȱ theȱ complexȱ CYP1A.ȱ Theȱ complexȱ CYP1Aȱ
transformsȱsomeȱlipophilicȱxenobioticsȱintoȱȱmoreȱwaterȬsolubleȱmetabolites,ȱsoȱ
thatȱtheyȱareȱeasierȱtoȱexcrete.ȱInductionȱofȱERODȱactivityȱhasȱbeenȱpreviouslyȱ
- 131 -
reportedȱ inȱ crabsȱ andȱ clamsȱ subsequentȱ toȱ organicȱ pollutantȱ exposureȱ [9,ȱ 15,ȱ
24].ȱ Theȱhighestȱ valuesȱ ofȱERODȱ activityȱ wereȱobtainedȱinȱorganismsȱexposedȱ
toȱsedimentsȱfromȱtheȱBayȱofȱAlgeciras,ȱandȱtheȱmultivariateȱanalysisȱlinkedȱthisȱ
inductionȱ withȱ organicȱ compoundsȱ (PAHsȱ andȱ PCBs).ȱ Inȱ addition,ȱ theȱ
inductionȱofȱantioxidantȱenzymesȱmeasuredȱbyȱtheȱGPXȱandȱFRAPȱanalysisȱwasȱ
linkedȱ withȱ organicȱ contaminantsȱ inȱ sedimentsȱ byȱ Factorȱ 1;ȱ Cheungȱ etȱ al.ȱ [25]ȱ
demonstratedȱ thatȱ someȱ PAHsȱ areȱ potentȱ oxidativeȱ stressȱ inducersȱ inȱ theȱ
marineȱ mussel,ȱ Pernaȱ viridis,ȱ andȱ obtainedȱ increasingȱ valuesȱ ofȱ antioxidantȱ
parameters,ȱ includingȱ GPX.ȱ Manyȱ pollutantsȱ (orȱ theirȱ metabolites)ȱ mayȱ elicitȱ
toxicityȱ relatedȱ toȱ oxidativeȱ stress.ȱ Oxygenȱ toxicityȱ canȱ beȱ aȱ potentȱ oxidantȱ
capableȱ ofȱ reactingȱ withȱ criticalȱ cellularȱ macromolecules,ȱ possiblyȱ leadingȱ toȱ
DNAȱ damageȱ andȱ cellȱ death.ȱ Defenseȱ systemsȱ thatȱ tendȱ toȱ inhibitȱ oxyradicalȱ
formationȱincludeȱantioxidantȱenzymesȱsuchȱasȱglutathioneȱreductaseȱ(GR)ȱandȱ
glutathioneȱ peroxidaseȱ (GPX)ȱ [9].ȱ Itȱ isȱ wellȱ knownȱ thatȱ GPXȱ transformsȱ
organohydroperoxideȱ toȱ alcoholȱ andȱ waterȱ atȱ theȱ expenseȱ ofȱ GSHȱ [26].ȱ Thus,ȱ
GPXȱactivityȱisȱlikelyȱtoȱbeȱinfluencedȱbyȱtheȱGSHȱlevelȱandȱGRȱactivity,ȱwhichȱ
regulatesȱtheȱlevelȱofȱGSHȱ[25].ȱTheȱresultsȱobtainedȱinȱtheȱmultivariateȱanalysisȱ
demonstratedȱaȱrelationshipȱofȱtheȱantioxidantȱbiomarkersȱGPXȱandȱGRȱinȱbothȱ
invertebrateȱspeciesȱdueȱtoȱtheȱpresenceȱofȱZnȱandȱPbȱinȱsediments.ȱThisȱmeansȱ
thatȱ theseȱ metalsȱ areȱ producingȱ someȱ stressȱ inȱ theȱ exposedȱ organisms,ȱ asȱ isȱ
reflectedȱinȱtheȱantioxidantȱresponses.ȱȱ
Theȱ inductionȱ ofȱ lipidȱ peroxidationȱ byȱ copperȱ isȱ wellȬknownȱ inȱ otherȱ
invertebratesȱ [27],ȱ whichȱ couldȱ explainȱ theȱ connectionȱ betweenȱ Cuȱ andȱ theȱ
inductionȱ ofȱ antioxidantȱ enzymesȱ explainedȱ byȱ Factorȱ 3ȱ inȱ theȱ MAA.ȱ Itȱ isȱ
importantȱtoȱnoteȱthatȱCuȱbelongsȱtoȱaȱgroupȱofȱmetalsȱthatȱareȱredoxȬactiveȱandȱ
areȱ capableȱ ofȱ directlyȱ generatingȱ freeȱ radicalsȱ [28]ȱ whichȱ mayȱ leadȱ toȱ
antioxidantȱ defenseȱ responses.ȱ Previousȱ studiesȱ alsoȱ reportedȱ anȱ inductionȱ ofȱ
- 132 -
GPXȱ activityȱ inȱ Mytilusȱ galloprovincialisȱ exposedȱ toȱ copperȱ inȱ controlledȱ
conditionsȱ orȱ toȱ complexȱ mixturesȱ ofȱ metalsȱ inȱ fieldȱ conditionsȱ [29].ȱ Theȱ
inductionȱ ofȱ FRAPȱ activityȱ inȱ crabsȱ andȱ clamsȱ seemsȱ toȱ beȱ ȱ lessȱ relevantȱ thanȱ
otherȱbiomarkersȱrelatedȱtoȱoxidativeȱstress,ȱalthoughȱtheȱcorrelationsȱobserved,ȱ
especiallyȱ withȱ metals,ȱ suggestȱ theȱ importanceȱ ofȱ analyzingȱ aȱ groupȱ ofȱ
biomarkersȱratherȱthanȱsingleȱuse.ȱȱȱ
GSTȱ activityȱ inductedȱ inȱ crabsȱ hasȱ beenȱ relatedȱ toȱ Znȱ andȱ Pbȱ
contaminationȱinȱtheȱmultivariateȱanalysisȱofȱFactorȱ2.ȱGlutathioneȱtransferasesȱ
phaseȱIIȱdetoxificationȱenzymesȱwhichȱutilizeȱglutathioneȱ(GSH)ȱasȱaȱsubstrateȱ
inȱ reactionsȱ whichȱ permitȱ theȱ biotransformationȱ andȱ disposalȱ ofȱ exogenousȱ
compoundsȱ [30];ȱ theȱ inductionȱ ofȱ GSTȱ activityȱ inȱ Carcinusȱ maenasȱ hasȱ beenȱ
previouslyȱrelatedȱwithȱmetalȱcontaminationȱ[9].ȱȱ
Theȱ resultsȱ obtainedȱ inȱ theȱ presentȱ studyȱ showedȱ theȱ activationȱ ofȱ
differentȱdefenceȱsystemsȱinȱbothȱtheȱorganismsȱtested.ȱThisȱwasȱmainlyȱrelatedȱ
withȱ anȱ inputȱ ofȱ chronicȱ fuelȱ oilȱ contaminationȱ intoȱ theȱ studiedȱ sediments,ȱ
predominantlyȱ inȱ theȱ Bayȱ ofȱ Algeciras,ȱ ȱ followedȱ byȱ CormeȬLaxe,ȱ whichȱ wasȱ
affectedȱbyȱanȱacuteȱoilȱimpact;ȱtheseȱresultsȱcorrespondȱwithȱhistopathologicalȱ
damageȱobservedȱinȱtheȱtissueȱofȱcrabsȱandȱclamsȱunderȱlaboratoryȱconditionsȱ
exposedȱ toȱ theseȱ sedimentsȱ (personalȱ observations).ȱ Theȱ Niȱ andȱ Coȱ sedimentȱ
contentȱcorrelatesȱwithȱtheȱorganicȱcontaminantsȱwhichȱareȱusualȱinȱtheȱcaseȱofȱ
hydrocarbonȱcontaminationȱepisodes.ȱȱZnȱandȱPbȱmetalȱcontaminationȱwasȱalsoȱ
detectedȱinȱtheȱareaȱofȱCormeȬLaxeȱandȱtheȱBayȱofȱAlgecirasȱȱ,ȱproducingȱstressȱ
inȱtheȱanimalsȱexposed;ȱbesides,ȱaȱsourceȱofȱCuȱinȱCíesȱandȱAlgecirasȱwasȱlinkedȱ
withȱ antioxidantȱ responsesȱ inȱ theȱ organismsȱ tested.ȱ Theȱ presenceȱ ofȱ metalsȱ inȱ
theseȱ areasȱ suggestsȱ ȱ thatȱ otherȱ alternativeȱ sourcesȱ ofȱ contaminantsȱ shouldȱ beȱ
investigatedȱapartȱfromȱtheȱPrestigeȱoilȱspill.ȱȱ
- 133 -
Bothȱ speciesȱ employedȱ inȱ theȱ presentȱ study,ȱ theȱ clamȱ Ruditapesȱ
philippinarumȱ andȱ theȱ crabȱ Carcinusȱ maenas,ȱ haveȱ biochemicallyȱ respondedȱ toȱ
theȱ contaminationȱ presentȱ inȱ theȱ sediments.ȱ Allȱ theȱ biomarkersȱ presentedȱ
higherȱ valuesȱ inȱ theȱ digestiveȱ glandȱ ofȱ clamsȱ thanȱ inȱ theȱ hepatopancreasȱ ofȱ
crabs,ȱexceptȱforȱGST.ȱTheȱuseȱofȱtwoȱinvertebrateȱspeciesȱwithȱdifferentȱfeedingȱ
habitsȱ allowsȱ aȱ moreȱ completeȱ studyȱ ofȱ theȱ biologicalȱ effectsȱ ofȱ contaminantsȱ
boundȱ toȱ sediments.ȱ Accordingȱ toȱ otherȱ authorsȱ [31,ȱ 32],ȱ theȱ informationȱ
providedȱ byȱ eachȱ biomarkerȱ individuallyȱ isȱ ofȱ limitedȱ relevance,ȱ asȱ thereȱ isȱ aȱ
considerableȱlikelihoodȱofȱmisinterpretation;ȱthus,ȱbiomarkersȱareȱbestȱusedȱasȱ
selectedȱ batteriesȱ ofȱ testsȱ ratherȱ thanȱ individually.ȱ Inȱ addition,ȱ combiningȱ
chemicalȱ analysisȱ withȱ suitesȱ ofȱ biomarkersȱ addressesȱ theȱ needȱ forȱ moreȱ
pragmaticȱ environmentalȱ assessmentȱ techniquesȱ linkingȱ environmentalȱ
degradationȱ withȱ itsȱ causesȱ [2].ȱ Theȱ higherȱ sensitivityȱ ofȱ sublethalȱ bioassaysȱ
comparedȱ toȱ acuteȱ toxicityȱ testsȱ indicatesȱ theȱ advantagesȱ ofȱ incorporatingȱ thisȱ
approachȱasȱpartȱofȱaȱmoreȱcompleteȱandȱintegratedȱstudyȱbasedȱonȱaȱweightȬ
ofȬevidenceȱapproach,ȱasȱpreviouslyȱrecommendedȱbyȱsomeȱauthorsȱ[8,ȱ9,ȱ33].ȱ
5.ȱConclusionsȱȱ
Inȱ theȱ presentȱ study,ȱ anȱ evaluationȱ hasȱ beenȱ carriedȱ outȱ ofȱ theȱ
environmentalȱqualityȱofȱcoastalȱareasȱaffectedȱbyȱdifferentȱcontaminantsȱ.ȱTheȱ
biomarkersȱ haveȱ demonstratedȱ thatȱ theyȱ areȱ activatedȱ dependingȱ onȱ theȱ kindȱ
andȱlevelȱofȱcontaminationȱandȱhaveȱprovenȱtoȱbeȱaȱsuitableȱtoolȱtoȱassessȱoilȬ
contaminatedȱsediments.ȱTheȱclamȱRuditapesȱphilippinarumȱandȱtheȱcrabȱCarcinusȱ
maenasȱ haveȱ demonstratedȱ theȱ importanceȱ ofȱ usingȱ differentȱ speciesȱ ofȱ
organismsȱ whenȱ assessingȱ environmentalȱ managementȱ andȱ haveȱ ȱ respondedȱ
satisfactorilyȱȱtoȱtheȱcontaminationȱpresentȱinȱtheȱsediments,ȱdemonstratingȱtheȱ
bioavailabilityȱ ofȱ organicȱ andȱ inorganicȱ contaminantsȱ relatedȱ toȱ oilȱ spills.ȱ Theȱ
- 134 -
applicationȱofȱtheȱmethodologyȱinȱtwoȱareasȱaffectedȱinȱdifferentȱmannersȱbyȱoilȱ
spillsȱ(acutely:ȱGalicianȱcoastȱandȱchronically:ȱBayȱofȱAlgeciras)ȱhasȱshownȱhowȱ
biomarkersȱ areȱ activatedȱ inȱ diverseȱ waysȱ dependingȱ onȱ theȱ sourceȱ ofȱ theȱ
pollutants.ȱ
Thisȱstudyȱhasȱdemonstratedȱtheȱimportantȱroleȱthatȱbiomarkersȱplayȱasȱ
partȱofȱtheȱWeightȱofȱEvidenceȱApproachȱ(WOE)ȱandȱitsȱuseȱinȱtheȱassessmentȱ
ofȱoilȱspillsȱisȱstronglyȱrecommended.ȱȱ
6.ȱAcknowledgementsȱ
Theȱ workȱ describedȱ wasȱ partlyȱ supportedȱ byȱ theȱ projectsȱ VEM2003Ȭ
20563/INTER,ȱ andȱ CTM2005Ȭ07282ȬC03Ȭ01/TECNOȱ financedȱ byȱ theȱ Spanishȱ
EducationȱandȱScienceȱ
Ministryȱ andȱ byȱ CISȱ fundedȱ byȱ theȱ Ministryȱ ofȱ Environment.ȱ Carmenȱ
MoralesȬCasellesȱthanksȱtheȱMinistryȱofȱEducationȱandȱScienceȱforȱfundingȱherȱ
researchȱ fellowshipȱ (FPU).ȱ Weȱ areȱ gratefulȱ forȱ theȱ supportȱ andȱ helpȱ ofȱ theȱ
membersȱofȱtheȱCISȱandȱtheȱICMANȬCSIC.ȱSpecialȱthanksȱareȱgivenȱtoȱAntonioȱ
Moreno,ȱJuditȱKalmanȱandȱtheȱErasmusȱstudentȱAurelian.ȱThanksȱareȱgivenȱtoȱ
theȱCSLMȱforȱtheȱEnglishȱrevisionȱandȱtoȱtheȱreviewersȱwhoȱhaveȱcontributedȱtoȱ
theȱimprovementȱofȱtheȱmanuscript.ȱȱ
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[25]ȱ Cheungȱ CCC,ȱ Siuȱ WHL,ȱ Richardsonȱ BJ,ȱ etȱ al.ȱ 2004.ȱ Antioxidantȱ responsesȱ toȱ
benzo[a]pyreneȱ andȱ Aeroclorȱ 1254ȱ exposureȱ inȱ theȱ greenȬlippedȱ mussel,ȱPernaȱ
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[28]ȱErcalȱN,ȱGurerȬorhanȱH,ȱAykinȬBurnsȱN.ȱ2001.ȱToxicȱmetalsȱandȱoxidativeȱstressȱ
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Toxicȱ18:ȱ245ȱ–ȱ252.ȱ
[31]ȱMooreȱMN,ȱSimpsonȱMG.ȱ1992.ȱMolecularȱandȱcellularȱpathologyȱinȱenvironmentȱ
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[32]ȱPachecoȱM,ȱȱSantosȱMA.ȱ2002.ȱBiotransformation,ȱgenotoxic,ȱandȱhistopathologicalȱ
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EcotoxicolȱEnvironȱSafȱ53:ȱ331–347.ȱ
[33]ȱCarballeiraȱA.ȱ2003.ȱConsiderationsȱinȱtheȱdesignȱofȱaȱmonitoringȱprogramȱofȱtheȱ
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- 139 -
- 140 -
VitellogeninȱvariationȱinȱtheȱcrabȱCarcinusȱmaenasȱexposedȱtoȱ
sedimentsȱaffectedȱbyȱoilȱspillsȱ(Spain)ȱ
C.ȱMoralesȬCaselles1,2*,ȱM.ȱL.ȱMartínȬDíaz1,2,ȱI.ȱRiba1,2,ȱT.ȱÁ.ȱDelValls1,2ȱ
§ȱ1ȱInstitutoȱdeȱCienciasȱMarinasȱdeȱAndalucía.ȱUnidadȱAsociadaȱdeȱCalidadȱAmbientalȱ
yȱPatologíaȱ(CSICȱ&ȱUCA);ȱȱPolígonoȱRíoȱSanȱPedroȱs/n.ȱ11510ȱPuertoȱRealȱ(Cádiz).ȱ
España.ȱ
CátedraȱUNESCO/UNITWIN/WiCop.ȱFacultadȱdeȱCienciasȱdelȱMarȱyȱAmbientales.ȱ
2ȱ
UniversidadȱdeȱCádiz.ȱȱPolígonoȱRíoȱSanȱPedroȱs/n.ȱ11510ȱPuertoȱRealȱ(Cádiz).ȱ
España.ȱ
Abstractȱ
Inȱtheȱpresentȱstudyȱtheȱinductionȱofȱvitellogeninȱhasȱbeenȱstudiedȱasȱaȱ
biomarkerȱ ofȱ exposureȱ inȱ crabsȱ inȱ orderȱ toȱ assessȱ itsȱ relationshipȱ withȱ
contaminantsȱ boundȱ toȱ sedimentsȱ [Zn,ȱ Pb,ȱ Cu,ȱ Ni,ȱ Co,ȱ Vȱ andȱ polyaromaticȱ
hydrocarbonsȱ(PAHs)]ȱaffectedȱbyȱdifferentȱoilȱspillsȱinȱSpain.ȱTwoȱdifferentȱ28Ȭ
daysȱ bioassaysȱ haveȱ beenȱ carriedȱ outȱ bothȱ underȱ fieldȱ andȱ laboratoryȱ
conditionsȱ byȱ exposingȱ theȱ crabȱ Carcinusȱ maenasȱ toȱ contaminatedȱ sedimentȱ
samples.ȱForȱtheȱfieldȱapproachȱtheȱorganismsȱwereȱlabelledȱandȱkeptȱinȱcagesȱ
locatedȱ inȱ theȱ studyȱ sitesȱ duringȱ theȱ exposureȱ period.ȱ Inȱ theȱ experimentȱ
conductedȱ underȱ laboratoryȱ conditionsȱ sedimentȱ fromȱ theȱ stationsȱ wasȱ
collectedȱandȱcarriedȱtoȱtheȱlaboratoryȱwhereȱlabelledȱcrabsȱwereȱplacedȱinȱ20ȱLȱ
tanksȱ withȱ theȱ sedimentȱ samples.ȱ Forȱ bothȱ bioassaysȱ haemolymphȱ wasȱ
extractedȱ fromȱ theȱ individualsȱ theȱ dayȱ 0ȱ andȱ 28ȱ ofȱ exposureȱ toȱ determineȱ theȱ
variationȱinȱtheȱlevelsȱofȱvitellogeninȱafterȱtheȱbioassay.ȱTheȱSpanishȱsedimentsȱ
selectedȱ forȱ thisȱ studyȱ hadȱ beenȱ affectedȱ inȱ aȱ differentȱ wayȱ byȱ oilȱ spills;ȱ theȱ
GalicianȱCoastȱ(NWȱSpain)ȱwasȱacutelyȱimpactedȱbyȱtheȱaccidentȱofȱtheȱtankerȱ
Prestigeȱ (2002)ȱ whereasȱ theȱ Bayȱ ofȱ Algecirasȱ (Southȱ Spain)ȱ suffersȱ chronicallyȱ
fromȱ continuousȱ inputȱ ofȱ differentȱ contaminantsȱ fromȱ shipsȱ andȱ industriesȱ
locatedȱinȱtheȱarea,ȱincludingȱoilȱspills.ȱResultsȱshowȱaȱrelationshipȱbetweenȱȱ
ȱPremioȱSecotoxȱ07ȱ“Bestȱoralȱpresentationȱofȱyoungȱscientist”ȱȱ
ȱJournalȱofȱEnvironmentalȱScienceȱandȱHealth:ȱPartȱAȱ(enviado)
- 141 -
vitellogeninȱ inductionȱ andȱ contaminants.ȱ Theȱ variationȱ ofȱ vitellogeninȱ
concentrationȱ wasȱ relatedȱ toȱ theȱ presenceȱ ofȱ PAHsȱ andȱ theȱ metalsȱ Pb,ȱ Niȱ andȱ
Cuȱ inȱ theȱ sediment,ȱ whichȱ occurredȱ mainlyȱ inȱ theȱ treatmentsȱ fromȱ theȱ Bayȱ ofȱ
Algeciras.ȱ Inȱ thisȱ sense,ȱ theȱ studyȱ showsȱ aȱ partialȱ recoveryȱ inȱ theȱ sedimentȱ
qualityȱ inȱ theȱ Galicianȱ Coastȱ threeȱ yearsȱ afterȱ theȱ spill,ȱ whereasȱ theȱ Bayȱ ofȱ
Algecirasȱisȱsignificantlyȱmoreȱpollutedȱthanȱtheȱsedimentsȱstudiedȱinȱtheȱareaȱ
ofȱGalicia.ȱȱȱȱȱȱȱȱȱ
Keywords:ȱȱvitellogenin,ȱoilȱspill,ȱecotoxicity,ȱinvertebrate,ȱPAHsȱ
1.ȱIntroductionȱ
Sediments,ȱ asȱ anȱ importantȱ partȱ ofȱ theȱ ecosystem,ȱ areȱ oftenȱ studiedȱ toȱ
assessȱenvironmentalȱquality.ȱComplexȱmixturesȱofȱcontaminantsȱhoweverȱcanȱ
beȱexaminedȱwithȱdifficulty,ȱthusȱtheȱstudyȱofȱbothȱchemicalȱandȱtoxicologicalȱ
effectsȱ turnsȱ outȱ toȱ beȱ aȱ suitableȱ toolȱ toȱ achieveȱ theȱ objectivesȱ proposedȱ inȱ
sedimentȱ qualityȱ assessment.ȱ Biomarkersȱ canȱ beȱ definedȱ asȱ measurementsȱ ofȱ
bodyȱ fluids,ȱ cellsȱ orȱ tissuesȱ thatȱ indicateȱ inȱ biochemicalȱ orȱ cellularȱ termsȱ theȱ
presenceȱ ofȱ contaminantsȱ orȱ theȱ magnitudeȱ ofȱ theȱ hostȱ responseȱ toȱ suchȱ
contaminantsȱ
[1]
.ȱ Combiningȱ chemicalȱ analysisȱ withȱ suitesȱ ofȱ biomarkersȱ
addressesȱ theȱ needȱ forȱ moreȱ pragmaticȱ environmentalȱ assessmentȱ techniquesȱ
linkingȱenvironmentalȱdegradationȱwithȱitsȱcausesȱ[2].ȱȱ
Theȱ chemicalsȱ introducedȱ intoȱ theȱ environmentȱ haveȱ theȱ potentialȱ toȱ
interactȱ withȱ neuroȬendocrineȱ signalingȱ cascades,ȱ resultingȱ inȱ signalȱ
perturbationsȱ
[3]
.ȱ Suchȱ alteredȱ signalingȱ canȱ resultȱ inȱ modificationsȱ toȱ
development,ȱ maturation,ȱ reproduction,ȱ andȱ otherȱ neuroȬendocrineȬregulatedȱ
processesȱ thatȱ hinderȱ populationȱ sustainabilityȱ
[4]
.ȱ Laboratoryȱ studiesȱ haveȱ
demonstratedȱ theȱ susceptibilityȱ ofȱ crustaceansȱ toȱ toxicantsȱ andȱ fieldȱ studiesȱ
haveȱ revealedȱ evidenceȱ ofȱ endocrineȱ disruptionȱ amongȱ variousȱ crustaceanȱ
populationsȱ[5].ȱ
- 142 -
Dueȱ toȱ theirȱ biologicalȱ andȱ ecologicalȱ characteristicsȱ crabsȱ areȱ suitableȱ
organismsȱ forȱ useȱ inȱ ecotoxicologicalȱ studiesȱ
[6]
.ȱ Inȱ theȱ presentȱ studyȱ
vitellogeninȱ (VTG)ȱ isȱ usedȱ asȱ aȱ biomarkerȱ toȱ assessȱ theȱ toxicityȱ causedȱ byȱ oilȱ
spillsȱ onȱ theȱ crabȱ Carcinusȱ maenas.ȱ Vitellogenesisȱ isȱ aȱ processȱ byȱwhichȱ femaleȱ
crustaceansȱ produceȱ andȱ sequesterȱ nutrientsȱ intoȱ developingȱ oocytesȱ
[7]
.ȱ
Vitellogeninȱ isȱ aȱ proteinȱ producedȱ inȱ theȱ hepatopancreasȱ andȱ transportedȱ
throughȱtheȱhaemolymphȱtoȱtheȱovary,ȱwhereȱitȱentersȱintoȱgrowingȱoocytesȱ [7].ȱ
Theȱ inhibitionȱ orȱ stimulationȱ ofȱ vitellogeninȱ levelsȱ inȱ haemolymphȱ couldȱ
provideȱaȱusefulȱindicatorȱofȱdirectȱrepercussionsȱonȱtheȱreproductiveȱcapacityȱ
inȱtheȱfemaleȱcrabsȱ[8].ȱ
Theȱ aimȱ ofȱ thisȱ studyȱ wasȱ toȱ investigateȱ theȱ relationshipȱ betweenȱ
contaminatedȱsedimentsȱwithȱtheȱinductionȱofȱvitellogeninȱinȱtheȱcrabȱCarcinusȱ
maenasȱ byȱ exposingȱ theȱ organismsȱ toȱ sedimentsȱ fromȱ theȱ Galicianȱ Coastȱ (NWȱ
Spain),ȱ threeȱ yearsȱ afterȱ anȱ oilȱ spillȱ (Prestige),ȱ andȱ sedimentsȱ fromȱ theȱ Bayȱ ofȱ
Algecirasȱ (Sȱ Spain),ȱ chronicallyȱ impactedȱ byȱ spillsȱ ofȱ differentȱ contaminantsȱ
includingȱ oilȱ spills.ȱ Exposuresȱ wereȱ performedȱ underȱ fieldȱ andȱ laboratoryȱ
conditionsȱ withȱ theȱ purposeȱ ofȱ studyȱ theȱ similaritiesȱ andȱ differencesȱ ofȱ bothȱ
methodologies.ȱȱ
2.ȱMaterialȱandȱmethodsȱ
Theȱsedimentsȱselectedȱforȱthisȱstudyȱwereȱaffectedȱinȱaȱdifferentȱwayȱbyȱ
oilȱspills;ȱtheȱGalicianȱCoastȱ(NWȱSpain)ȱwasȱacutelyȱimpactedȱbyȱtheȱaccidentȱ
ofȱtheȱtankerȱPrestigeȱ(2002)ȱwhereasȱtheȱBayȱofȱAlgecirasȱ(SȱSpain)ȱchronicallyȱ
suffersȱ continuousȱ inputȱ ofȱ differentȱ contaminantsȱ comingȱ fromȱ shipsȱ andȱ
industriesȱlocatedȱinȱtheȱarea,ȱincludingȱoilȱspills.ȱFigureȱ1ȱshowsȱtheȱlocationȱofȱ
theȱstudyȱsites.ȱ
ȱ
- 143 -
ȱ
ƒF ƒE
ƒD
Atlantic Islands
National Park
C
A
B
Ría de CormeLaxe
Spain
GR3
GR4
P1
Bay of
Algeciras
N
W
E
S
ȱ
Figureȱ 1.ȱ Mapȱ ofȱ ȱ studyȱ sites:ȱ theȱ coastalȱ areaȱ ofȱ Galiciaȱ showingȱ theȱ
locationsȱ ofȱ theȱ samplingȱ stations.ȱ A,ȱ Bȱ andȱ Cȱ refersȱ toȱ theȱ stationsȱ locatedȱ inȱ
theȱCiesȱIslandȱinȱtheȱAtlanticȱIslandȱNationalȱParkȱandȱD,ȱEȱandȱFȱtoȱthoseȱinȱ
theȱ Bayȱ ofȱ CormeȬLaxe.ȱ Theȱ stationsȱ locatedȱ inȱ theȱ Bayȱ ofȱ Algecirasȱ areȱ GR3,ȱ
GR4ȱandȱP1.ȱ
2.1.ȱSedimentȱsamplingȱandȱcharacterizationȱȱ
Sedimentsȱfromȱtheȱselectedȱsitesȱwereȱcarriedȱtoȱtheȱlaboratoryȱandȱwereȱ
sampledȱforȱphysicalȱcharacterizationȱandȱchemicalȱquantification.ȱTheȱanalysesȱ
ofȱ PAHsȱ wereȱ carriedȱ outȱ accordingȱ toȱ USEPAȱ SWȬ846ȱ Methodȱ 827C78082ȱ [9].ȱ
Forȱtraceȱmetalȱanalysesȱ(Zn,ȱPb,ȱCu,ȱNi,ȱCo,ȱV),ȱtheȱsedimentsȱwereȱdigestedȱasȱ
describedȱ byȱ Loringȱ andȱ Rantalaȱ [10]ȱ andȱ thenȱ measuredȱ byȱ atomicȱ absorptionȱ
spectrophotometryȱ (AAS).ȱ Organicȱ carbonȱ contentȱ wasȱ determinedȱ usingȱ theȱ
- 144 -
methodȱ ofȱ Gaudetteȱ etȱ al.ȱ [11]ȱ withȱ theȱ Elȱ Rayisȱ [12]ȱ modification.ȱ Forȱ sedimentȱ
grainȱ size,ȱ anȱ aliquotȱ ofȱ wetȱ sedimentȱ wasȱ analyzedȱ usingȱ aȱ Fristchȱ laserȱ
particleȱsizerȱ(modelȱAnalysetteȱ22)ȱfollowingȱtheȱmethodȱreportedȱbyȱDelVallsȱ
andȱChapmanȱ[13].ȱ
2.2.ȱToxicityȱtestsȱ
Intermoultȱ femalesȱ crabsȱ wereȱ collectedȱ fromȱ aȱ cleanȱ siteȱ inȱ theȱ Gulfȱ ofȱ
Cádizȱandȱwereȱacclimatizedȱforȱtwoȱweeksȱinȱtheȱlaboratory.ȱAfterȱthatȱperiodȱ
theȱsedimentȱsamplesȱwereȱplacedȱinȱ20ȬLȱaquariumsȱandȱseaȱwaterȱwasȱaddedȱ
(1:4).ȱAerationȱwasȱprovidedȱafterȱtheȱsedimentȱhadȱsettledȱdown.ȱCrabsȱwereȱ
labelledȱandȱaȱnumberȱofȱthemȱwereȱplacedȱinȱtheȱaquariumsȱinȱtheȱlaboratoryȱ
(8ȱperȱaquarium)ȱandȱinȱtheȱcagesȱwhichȱwereȱtransferredȱtoȱtheȱstudyȱsites.ȱTheȱ
bioassaysȱrunȱinȱreplicateȱandȱlastedȱ28ȱdays.ȱ
2.3.ȱVitellogeninȱdeterminationȱ
Haemolymphȱsamplesȱwereȱtakenȱfromȱtheȱbaseȱofȱaȱwalkingȱlegȱusingȱaȱ
syringeȱ theȱ daysȱ 0ȱ andȱ 28ȱ ofȱ theȱ bioassay.ȱ Theȱ samplesȱ wereȱ transferredȱ toȱ
microcentrifugeȱtubesȱandȱwereȱkeptȱintoȱliquidȱnitrogenȱbeforeȱstoringȱthemȱinȱ
theȱ Ȭ80ȱ ºCȱ freezer.ȱ Vitellogeninȱ determinationȱ wasȱ performedȱ usingȱ aȱ directȱ
EnzymeȬLinkedȱ Inmunosorbentȱ Assayȱ (ELISA)ȱ adaptedȱ fromȱ Paterakiȱ andȱ
Stratakisȱ
.ȱ Theȱ 96Ȭwellȱ microtiterȱ platesȱ wereȱ coatedȱ withȱ theȱ standardȱ
[14]
solutions,ȱ purifiedȱ VTGȱ (0,ȱ 22,ȱ 10,ȱ 20,ȱ 50,ȱ 75ȱ andȱ 100ȱ ngȱ 100ȱ ΐLȬ1)ȱ andȱ
haemolymphȱsamplesȱfromȱeachȱcrabȱ(200ȱΐL).ȱAȱpolyclonalȱantibodyȱraisedȱinȱ
rabbitsȱ againstȱ C.ȱ maenasȱ VTGȱ couldȱ identifyȱ vitellogeninȱ concentrations.ȱ Theȱ
plateȱwasȱreadȱatȱ405ȱnmȱandȱVTGȱstandardsȱwereȱfitȱtoȱaȱlinearȱregressionȱ(R2ȱ=ȱ
0.96;ȱ slopeȱ =ȱ 0.144).ȱ 28Ȭdayȱ VTGȱ resultsȱ wereȱ normalizedȱ withȱ theȱ 0Ȭdayȱ VTGȱ
concentrationsȱ([VTG*]ȱ=ȱ[28ȬdaysȱVTG]ȱ–ȱ[0ȬdaysȱVTG])ȱprovidingȱtheȱamountȱ
ofȱproteinsȱthatȱfluctuatesȱduringȱ28ȱdaysȱofȱexposure.ȱ
- 145 -
2.4.ȱStatisticalȱanalysisȱ
Contaminationȱ andȱ VTG*ȱ dataȱ wereȱ linkedȱ byȱ factorȱ analysis,ȱ usingȱ
principalȱ componentsȱ analysisȱ (PCA)ȱ asȱ theȱ extractionȱ procedureȱ
(STATISTICA®);ȱthisȱisȱaȱmultivariateȱstatisticalȱtechniqueȱforȱexploringȱvariableȱ
distributionsȱ ȱ Theȱ objectiveȱ ofȱ PCAȱ isȱ toȱ deriveȱ aȱ reducedȱ numberȱ ofȱ newȱ
variablesȱ asȱ linearȱ combinationsȱ ofȱ theȱ originalȱ variables.ȱ Thisȱ providesȱ aȱ
descriptionȱofȱtheȱstructureȱofȱtheȱdataȱwithȱtheȱminimumȱlossȱofȱinformationȱ[9].ȱ
3.ȱResultsȱandȱdiscussionȱ
3.1.ȱChemicalȱanalysisȱ
Sedimentsȱ wereȱ mainlyȱ contaminatedȱ byȱ PAHs,ȱ andȱ theȱ samplesȱ fromȱ
theȱ Bayȱ ofȱ Algeciras,ȱ continuouslyȱ affectedȱ byȱ oilȱ spills,ȱ turnedȱ outȱ toȱ beȱ theȱ
mostȱ contaminatedȱ byȱ theseȱ compoundsȱ (Tableȱ 1).ȱ Onȱ theȱ otherȱ hand,ȱ resultsȱ
doȱ notȱ showȱ aȱ prevailingȱ tendencyȱ inȱ theȱ concentrationȱ ofȱ metalsȱ amongȱ
sedimentsȱ fromȱ theȱ differentȱ areas.ȱ Highȱ levelsȱ ofȱ Znȱ wereȱ detectedȱ inȱ theȱ
stationsȱA,ȱCȱ(CiesȱIsland)ȱandȱFȱ(CormeȬLaxe)ȱfromȱGalicia,ȱwhereasȱCuȱlevelsȱ
wereȱhighȱP1ȱandȱNiȱinȱGR3,ȱbothȱstationsȱlocatedȱinȱtheȱBayȱofȱAlgeciras.ȱȱ
ȱ
ȱ
ȱ
ȱ
ȱ
- 146 -
Tableȱ 1.ȱ Valuesȱ ofȱ totalȱ organicȱ carbonȱ (%dryȱ weight),ȱ finesȱ (%ȱ ofȱ dryȱ
sedimentȱ<ȱ63ȱmm)ȱandȱtheȱconcentrationȱofȱcontaminantsȱ(metalsȱ(mgȱkg_1ȱdryȱ
weight);PAHsȱ andȱ PCBsȱ (mgȱ kg_1ȱ dryȱ weight))ȱ inȱ sedimentȱ samplesȱ fromȱ
Galiciaȱ(CíesȱIsland:ȱA,ȱB,ȱC;ȱandȱCormeȬLaxe:ȱD,ȱE,ȱF)ȱandȱAlgecirasȱBayȱ(GR3,ȱ
GR4ȱandȱP1).ȱNotȱdetectedȱisȱexpressedȱbyȱn.d.ȱ
Stationsȱ
Aȱ
Bȱ
Cȱ
Dȱ
Eȱ
Fȱ
GR3ȱ
GR4ȱ
P1ȱ
O.C.ȱ Finesȱ
0.28ȱ 4.32ȱ
0.26ȱ 2.81ȱ
0.30ȱ 2.76ȱ
0.31ȱ 3.79ȱ
0.37ȱ 5.50ȱ
0.65ȱ 5.95ȱ
2.15ȱ 69.4ȱ
3.19ȱ 59.3ȱ
3.86ȱ 35.4ȱ
Znȱȱ
377ȱ
91.0ȱ
164ȱ
25.0ȱ
19.9ȱ
271ȱ
138ȱ
35.3ȱ
56.7ȱ
Pbȱȱ
1.50ȱ
0.90ȱ
0.85ȱ
3.70ȱ
7.30ȱ
5.90ȱ
21.6ȱ
6.21ȱ
12.3ȱ
Cuȱȱ
5.20ȱ
1.40ȱ
1.40ȱ
0.70ȱ
0.43ȱ
4.20ȱ
5.01ȱ
3.67ȱ
75.2ȱ
Niȱȱ
13.3ȱ
2.40ȱ
4.50ȱ
1.70ȱ
1.50ȱ
5.70ȱ
74.7ȱ
13.1ȱ
13.3ȱ
Coȱȱ
0.30ȱ
0.20ȱ
0.10ȱ
0.34ȱ
0.35ȱ
0.36ȱ
12.8ȱ
5.59ȱ
n.d.ȱ
Vȱȱ
0.70ȱ
0.80ȱ
0.60ȱ
2.00ȱ
2.10ȱ
3.40ȱ
26.1ȱ
n.d.ȱ
6.84ȱ
PAHȱ
108ȱ
67.0ȱ
n.d.ȱ
38.0ȱ
52.0ȱ
323ȱ
3150ȱ
802ȱ
641ȱ
ȱ
3.2.ȱȱVitellogeneninȱanalysisȱ
Resultsȱ ofȱ vitellogeninȱ concentrationȱ inȱ haemolymphȱ decreasedȱ inȱ theȱ
majorityȱ ofȱ theȱ treatmentsȱ afterȱ 28ȱ daysȱ ofȱ exposureȱ (Fig.ȱ 2).ȱ Theȱ declineȱ wasȱ
detectedȱmainlyȱinȱtheȱlaboratoryȱtestsȱwhereasȱcagedȱcrabsȱpresentedȱaȱlowerȱ
variationȱinȱtheȱvitellogeninȱlevelsȱafterȱtheȱexposureȱperiod.ȱThisȱcouldȱbeȱdueȱ
toȱtheȱfactȱthatȱunderȱcagedȱcrabsȱinȱfieldȱwereȱsubjectedȱtoȱtheȱenvironmentalȱ
conditionsȱsuchȱasȱchangesȱinȱtheȱvariablesȱandȱcurrentsȱwhatȱmayȱdecreaseȱtheȱ
availabilityȱofȱtheȱcontaminants.ȱOrganismsȱexposedȱtoȱsedimentsȱfromȱtheȱBayȱ
ofȱ Algecirasȱ (GR3,ȱ GR4ȱ andȱ P1)ȱ sufferedȱ theȱ highestȱ variationsȱ inȱ vitellogeninȱ
levels.ȱ
ȱ
- 147 -
1.2
1.0
VTG
0.8
0.6
0.4
0.2
0.0
A
B
C
D
E
F
A-c
B-c
C-c
D-c
E-c
F-c
GR4
P1
GR3
GR4-c
P1-c
GR3-c
treatments
ȱ
Figureȱ 2.ȱ Levelsȱ ofȱ vitellogeninȱ (ngȱ 100mLȬ1)ȱ inȱ haemolymphȱ ofȱ crabsȱ
exposedȱtoȱsedimentsȱfromȱGaliciaȱ(CíesȱIsland:ȱA,ȱB,ȱC;ȱandȱCormeȬLaxe:ȱD,ȱE,ȱ
F)ȱ andȱ Algecirasȱ Bayȱ (GR3,ȱ GR4ȱ andȱ P1);ȱ samplesȱ fromȱ theȱ cagedȱ organismsȱ
haveȱtheȱsuffixȱ“–c”.ȱTheȱresultsȱshownȱmatchȱwithȱtheȱdayȱ0ȱ(dottedȱbar)ȱandȱ
dayȱ28ȱ(stripedȱbar)ȱofȱexposure.ȱ
3.3.ȱStatisticalȱanalysisȱ
Theȱvariablesȱ(O.C.,ȱfines,ȱZn,ȱPb,ȱCu,ȱNi,ȱCo,ȱV,ȱPAHs,ȱandȱVTG)ȱwereȱ
autoscaledȱ (standardized)ȱ soȱ asȱ toȱ beȱ treatedȱ withȱ equalȱ importanceȱ [15].ȱ Theȱ
applicationȱ ofȱ theȱ PCAȱ toȱ theȱ originalȱ 10ȱ variablesȱ indicatesȱ thatȱ theyȱ canȱ beȱ
groupedȱ inȱ twoȱ newȱ factorsȱ whichȱ explainȱ aȱ 72%ȱ ofȱ theȱ totalȱ varianceȱ inȱ theȱ
originalȱ dataȱ set.ȱ Aȱ groupȱ ofȱ variablesȱ asȱ thoseȱ associatedȱ withȱ aȱ particularȱ
componentȱwhereȱtheȱloadingȱwasȱ0.30ȱorȱhigherȱwasȱinterpretedȱ(Tableȱ2).ȱTheȱ
firstȱ principalȱ factor,ȱ #1ȱ isȱ predominantȱ (50%)ȱ andȱ itȱ groupsȱ theȱ variationȱ ofȱ
vitellogeninȱconcentrationȱinȱtheȱhaemolymphȱofȱtheȱcrabsȱwithȱtheȱpresenceȱofȱ
PAHsȱandȱtheȱmetalsȱPb,ȱNiȱandȱVȱinȱtheȱsedimentȱandȱitsȱassociationȱwithȱtheȱ
organicȱcarbonȱandȱgrainȱsize.ȱFactorȱ#2ȱ(22%),ȱshowsȱtheȱrelationshipȱbetweenȱ
- 148 -
theȱgrainȱsizeȱandȱtheȱtotalȱorganicȱcarbonȱinȱtheȱsedimentsȱwithȱtheȱpresenceȱofȱ
Pb,ȱCuȱandȱCoȱandȱtheȱlinkȱwithȱtheȱvariationȱinȱvitellogeninȱlevels.ȱ
Theȱ influenceȱ ofȱ theȱ twoȱ factorsȱ atȱ theȱ 18ȱ treatmentsȱ isȱ reflectedȱ byȱ theȱ
FactorȱscoreȱatȱtheseȱtreatmentsȱandȱisȱshownȱinȱFigureȱ3.ȱFactorȱ1ȱwhichȱshowsȱ
theȱ relationshipȱ ofȱ theȱ vitellogeninȱ variationȱ andȱ theȱ contaminationȱ byȱ PAHs,ȱ
Pb,ȱ Niȱ andȱ Vȱ hasȱ aȱ mainȱ prevalenceȱ inȱ theȱ stationsȱ fromȱ theȱ Bayȱ ofȱ Algecirasȱ
GR3ȱandȱGR4ȱbothȱinȱlaboratoryȱandȱfieldȱexposures.ȱFactorȱ#2,ȱwhichȱlinksȱtheȱ
variationȱ ofȱ vitellogeninȱ concentrationȱ withȱ theȱ metalsȱ Pb,ȱ Cuȱ andȱ Co,ȱ itȱ isȱ
mainlyȱprevalentȱinȱ theȱstationsȱGR4ȱandȱP1,ȱlaboratoryȱandȱcagedȱexposures,ȱ
inȱ theȱ Bayȱ ofȱ Algeciras.ȱ Treatmentsȱ fromȱ theȱ Galicianȱ Coastȱ didȱ notȱ presentȱ
positiveȱloadingȱinȱtheȱfactorȱscores.ȱ
Tableȱ 2.ȱ Sortedȱ rotatedȱ factorȱ loadingsȱ (pattern)ȱ ofȱ 10ȱ variablesȱ forȱ theȱ
twoȱprincipalȱfactorsȱresultingȱfromȱtheȱmultivariateȱanalysisȱofȱresultsȱobtainedȱ
fromȱtheȱchemicalȱanalysisȱandȱtheȱvitellogeninȱdetermination.ȱ
FACTOR1 FACTOR2
ȱȱ
ņȱ
ņȱ
Znȱȱ
Pbȱȱ
0.84ȱ
0.32ȱ
ņȱ
Cuȱȱ
0.95ȱ
ņȱ
Niȱȱ
0.93ȱ
ņȱ
Coȱȱ
0.95ȱ
ņȱ
Vȱȱ
0.54ȱ
ņȱ
PAHȱ
0.97ȱ
%C.O.ȱ
0.53ȱ
0.80ȱ
%finesȱ
0.91ȱ
0.33ȱ
VTGȱ
0.37ȱ
0.56ȱ
ȱ
ResultsȱobtainedȱinȱtheȱStatisticalȱanalysisȱhaveȱshownȱthatȱtheȱrangeȱofȱ
variationȱofȱtheȱvitellogeninȱconcentrationȱinȱtheȱhaemolymphȱofȱtheȱcrabsȱwasȱ
relatedȱtoȱtheȱPAHsȱandȱtheȱmetalsȱPb,ȱNi,ȱCu,ȱCoȱandȱV.ȱAlthoughȱVȱandȱCoȱȱ
- 149 -
3
GR3'
GR3'-c
Factor 1
2
GR4
1
GR4-c
0
-1
A
B
C
D
E
F
A-c
B-c
C-c
D-c
E-c
P1
F-c
P1-c
-2
4
P1
3
P1-c
Factor 2
2
1
GR4
GR4-c
0
-1
A
B
C
D
E
F
A-c
B-c
C-c
D-c
E-c
F-c
GR3'
GR3'-c
-2
Figureȱ 3.ȱ Estimatedȱ factorȱ scoresȱ forȱ theȱ twoȱ factorsȱ inȱ eachȱ ofȱ theȱ 18ȱ
cases.ȱTheȱfactorȱscoresȱquantifyȱtheȱprevalenceȱofȱeachȱfactorȱforȱeveryȱstationȱ
andȱ isȱ usedȱ toȱ establishȱ theȱ definitionȱ ofȱ eachȱ factor.ȱ Samplesȱ fromȱ theȱ cagedȱ
organismsȱhaveȱtheȱsuffixȱ“–c”.ȱ
areȱ includedȱ inȱ theȱ definitionsȱ ofȱ factorȱ #1ȱ andȱ 2ȱ respectively,ȱ theyȱ
appearȱ inȱ lowȱ concentrationsȱ inȱ theȱ sedimentsȱ whatȱ meansȱ thatȱ probablyȱ theȱ
correlationȱisȱdueȱtoȱbasalȱlevelsȱofȱtheseȱmetalsȱinȱtheȱenvironmentȱandȱdoȱnotȱ
supposeȱ contamination.ȱ Previousȱ studiesȱ showedȱ aȱ relationshipȱ betweenȱ
vitellogeninȱ andȱ metalsȱ
,ȱ althoughȱ inȱ thatȱ caseȱ thereȱ wasȱ aȱ vitellogeninȱ
[16]
inductionȱ alongȱ theȱ timeȱ ofȱ exposure,ȱ whereasȱ inȱ theȱ presentȱ caseȱ ofȱ studyȱ
vitellogeninȱ decreasedȱ inȱ theȱ majorityȱ ofȱ theȱ treatments.ȱ Otherȱ studiesȱ
ȱ
[17]
suggestedȱ thatȱ metalsȱ mayȱ interfereȱ withȱ theȱ ovarianȱ cycleȱ inȱ Carcinusȱ maenasȱ
and,ȱtherefore,ȱwithȱtheȱreproductionȱofȱthisȱspecies.ȱPrecedingȱinvestigationsȱinȱ
fishesȱ [18],ȱconsideredȱthatȱlowȱvitellogeninȱlevelsȱinȱfemalesȱcouldȱbeȱindicativeȱ
ofȱ pollutionȱ inducedȱ dysfunctionȱ atȱ theȱ reproductiveȱ endocrineȱ systemȱ level.ȱ
AlterationsȱinȱvitellogeninȬlikeȱproteinȱlevelsȱwereȱobservedȱinȱmusselsȱexposedȱ
- 150 -
toȱ organicȱ pollutantsȱ [19]ȱ whereasȱ studiesȱ withȱ femaleȱ clamsȱ exposedȱ toȱ PAHȱ
contaminationȱ presentedȱ lowȱ levelsȱ ofȱ alkaliȬlabileȱ phosphateȱ whichȱ positiveȱ
correlatesȱwithȱvitellogeninȱ[20].ȱȱ
4.ȱConclusionsȱ
Inȱ theȱ presentȱ studyȱ aȱ decreaseȱ ofȱ vitellogeninȱ concentrationȱ inȱ
haemolymphȱfromȱtheȱcrabȱCarcinusȱmaenasȱexposedȱtoȱcontaminatedȱsedimentsȱ
wasȱ detectedȱ afterȱ 28ȱ daysȱ ofȱ exposure.ȱ Theȱ variationȱ ofȱ vitellogeninȱ
concentrationȱ wasȱ relatedȱ toȱ theȱ presenceȱ ofȱ PAHsȱ andȱ theȱ metalsȱ Pb,ȱ Niȱ andȱ
Cuȱ inȱ theȱ sediment,ȱ whichȱ occurredȱ mainlyȱ inȱ theȱ treatmentsȱ fromȱ theȱ Bayȱ ofȱ
Algecirasȱ(chronicallyȱaffectedȱbyȱoilȱspills)ȱwhereasȱtheȱGalicianȱCoastȱ(acutelyȱ
impactedȱ byȱ anȱ oilȱ spill)ȱ didȱ notȱ presentȱ thisȱ association.ȱ Thisȱ pointsȱ toȱ aȱ
recoveryȱofȱtheȱareaȱaffectedȱbyȱtheȱoilȱspill.ȱAlthoughȱbothȱfieldȱandȱlaboratoryȱ
testsȱ presentedȱ theȱ sameȱ trendsȱ inȱ vitellogeninȱ variations,ȱ theȱ responseȱ wasȱ
lowerȱ underȱ fieldȱ conditionsȱ whichȱ meansȱ thatȱ laboratoryȱ testsȱ resultedȱ toȱ beȱ
moreȱsensitiveȱthanȱfieldȱstudiesȱinȱorderȱtoȱassessȱsedimentȱtoxicity.ȱ
5.ȱAcknowledgementsȱ
Theȱ workȱ describedȱ wasȱ partlyȱ supportedȱ byȱ theȱ projectsȱ VEM2003Ȭȱ
20563/INTER,ȱ andȱ CTM2005Ȭ07282ȬC03Ȭ01/TECNOȱ financedȱ byȱ theȱ Spanishȱ
Educationȱ andȱ Scienceȱ Ministryȱ andȱ byȱ CISȱ fundedȱ byȱ theȱ Ministryȱ ofȱ
Environment.ȱ Carmenȱ MoralesȬCasellesȱ thanksȱ theȱ Ministryȱ ofȱ Educationȱ andȱ
Scienceȱ forȱ fundingȱ herȱ researchȱ fellowshipȱ (FPU).ȱ Dr.ȱ Ribaȱ thanksȱ theȱ I3pȱ
programȱ forȱ supportingȱ herȱ participationȱ inȱ thisȱ research.ȱ Inmaculadaȱ Ribaȱ
thanksȱtheȱCSICȱforȱherȱI3Pȱcontract.ȱWeȱareȱgratefulȱforȱtheȱsupportȱandȱhelpȱofȱ
theȱ membersȱ ofȱ theȱ CISȱ andȱ theȱ ICMANȬCSIC.ȱ Specialȱ thanksȱ areȱ givenȱ toȱ
- 151 -
Antonioȱ Moreno,ȱ Pabloȱ Vidalȱ andȱ Trevorȱ Worsyȱ fromȱ theȱ Universityȱ ofȱ
Plymouth.ȱ
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Soil.ȱPollut.ȱ2005,ȱ160,ȱ245Ȭ258.ȱ
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[20]ȱ Gagnè,ȱ F.,ȱ Blaise,ȱ C.,ȱ Pellerin,ȱ J.,ȱ GauthierȬClerc,ȱ S.ȱ Alterationȱ ofȱ theȱ biochemicalȱ
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ȱ
ȱ
ȱ
ȱ
ȱ
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- 154 -
AȱmultibiomarkerȱapproachȱusingȱtheȱpolychaeteȱArenicolaȱ
marinaȱtoȱassessȱoilȱcontaminatedȱsedimentsȱȱ
CarmenȱMoralesȬCaselles1,ȱCeriȱLewis2,ȱTamaraȱGalloway2,ȱInmaculadaȱRiba1,ȱ
T.ȱÁngelȱDelVallsȱ1ȱ
1
ȱUnidadȱAsociadaȱdeȱCalidadȱAmbientalȱyȱPatologíaȱ(CSICȱ&ȱUCA)ȱinstitutoȱ
deȱCienciasȱMarinasȱdeȱAndalucíaȱ(ICMANȬCSIC),ȱUNESCOȱUNITWIN/WiCopȱ
Avda.ȱRepúblicaȱSaharauiȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱSpainȱ
2
SchoolȱȱofȱBiosciences,ȱHatherlyȱLaboratories,ȱUniversityȱofȱExeter,ȱPrinceȱofȱWalesȱ
Road,ȱExeter,ȱUK,ȱEX4ȱ4PS.telȱ(44)ȱ1392ȱ263436,ȱfaxȱ(44)ȱ1392ȱ263700ȱȱ
Abstractȱ
Marineȱandȱcoastalȱsedimentsȱcanȱaccumulateȱsubstantialȱconcentrationsȱ
ofȱ metalsȱ andȱ hydrocarbons,ȱ yetȱ theȱ consequencesȱ ofȱ thisȱ contaminationȱ forȱ
exposedȱ biotaȱ inȱ situȱ canȱ beȱ difficultȱ toȱ establish.ȱ Here,ȱ weȱ examineȱ theȱ
hypothesisȱ thatȱ exposureȱ toȱ contaminatedȱ sedimentsȱ canȱ leadȱ toȱ detrimentalȱ
effectsȱ toȱ sedimentȱ dwellingȱ species.ȱ Theȱ commonȱ lugwormȱ Arenicolaȱ marinaȱ
wasȱ exposedȱ inȱ theȱlaboratoryȱforȱ 14ȱdaysȱ toȱmarineȱ sedimentsȱcollectedȱfromȱ
sitesȱofȱcontaminationȱinȱSpainȱandȱEngland.ȱAȱsuiteȱofȱbiomarkersȱofȱsublethalȱ
toxicityȱ wasȱ combinedȱ withȱ analyticalȱ chemistryȱ toȱ testȱ forȱ relationshipsȱ
betweenȱ sedimentȱ contaminationȱ andȱ effect.ȱ ȱ Moderateȱ toȱ strongȱ correlationsȱ
betweenȱorganics,ȱmetalsȱandȱbiologicalȱresponsesȱwereȱobserved,ȱwithȱDNAȱȱ
ȱ
ȱEnvironementalȱScienceȱandȱTechnology
(enȱpreparación)
ȱ
- 155 -
damageȱ asȱ measuredȱ usingȱ theȱ Cometȱ assayȱ formingȱ theȱ largestȱ contributionȱ
towardsȱ theȱ observedȱ differences.ȱ Theȱ responseȱ ofȱ wormsȱ fromȱ sitesȱ
experiencesȱdifferentȱcontaminationȱloadsȱwereȱclearlyȱdistinguishable.ȱTheȱuseȱ
ofȱ A.ȱ marinaȱ inȱ thisȱ wayȱ providesȱ aȱ sensitive,ȱ holisticȱ approachȱ toȱ sedimentȱ
toxicityȱ assessment,ȱ enablingȱ comparisonsȱ betweenȱ chronically,ȱ andȱ acutelyȱ
pollutedȱsitesȱtoȱbeȱquantifiedȱandȱrecoveryȱofȱtheseȱsitesȱtoȱbeȱcharted.ȱȱ
1.ȱIntroductionȱ
Anȱ integratedȱ approachȱ toȱ marineȱ pollutionȱ monitoring,ȱ thatȱ combinesȱ
theȱ traditionalȱ chemicalȱ analysesȱ withȱ laboratoryȱ andȱ fieldȱ basedȱ toxicityȱ
testing,ȱisȱbecomingȱincreasinglyȱimportantȱinȱgainingȱbetterȱassessmentsȱofȱtheȱ
pollutionȱprocessȱinȱtheȱmarineȱandȱcoastalȱenvironmentȱ(Chapman.,ȱ2007).ȱTheȱ
waterȱ frameworkȱ directiveȱ (WFD)ȱ requiresȱ memberȱ statesȱ toȱ assessȱ theȱ
ecologicalȱ qualityȱ statusȱ (EQS)ȱ ofȱ waterȱ bodiesȱ andȱ toȱ achieveȱ “goodȱ waterȱ
status”ȱ forȱ allȱ Europeanȱ watersȱ byȱ 2015ȱ (EEC,ȱ 2000).ȱ Interestȱ isȱ thereforeȱ
focusedȱ onȱ developingȱ assessmentȱ toolsȱ toȱ monitorȱ littoralȱ ecosystemsȱ
followingȱ theȱ WFDȱ requirements.ȱ ȱ Biomarkersȱ haveȱ beenȱ shownȱ toȱ beȱ usefulȱ
toolsȱinȱcharacterizingȱtheȱhealthȱstatusȱofȱanimalsȱfromȱimpactedȱareas,ȱwhereȱ
complexȱ mixturesȱ ofȱ pollutantsȱ areȱ usuallyȱ presentȱ (Gallowayȱ etȱ al.,ȱ 2002;ȱ
Gallowayȱetȱal.,ȱ2004).ȱTheȱcombinationȱofȱchemicalsȱandȱbiomarkersȱasȱpartȱofȱ
aȱweightȱofȱevidenceȱ(WOE)ȱapproachȱallowsȱtheȱidentificationȱofȱtheȱimpactȱofȱ
chemicalȱ contaminationȱ onȱ differentȱ levelsȱ ofȱ biologicalȱ functionȱ andȱ couldȱ
makeȱ aȱ viableȱ additionȱ toȱ routineȱ managementȱ protocolsȱ forȱ protectingȱ theȱ
environmentȱ(Gallowayȱetȱal.,ȱ2004)ȱbutȱhasȱrarelyȱbeenȱachievedȱforȱsedimentȱ
dwellingȱspecies.ȱSinceȱmanyȱpersistentȱorganicsȱandȱmetalsȱareȱretainedȱwithinȱ
sediments,ȱ thisȱ representsȱ aȱ majorȱ knowledgeȱ gapȱ inȱ ecotoxicologicalȱ
monitoringȱprogrammesȱofȱtheȱmarineȱenvironment.ȱ
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ȱArenicolaȱ marinaȱ isȱ aȱ commonȱ intertidalȱ polychaeteȱ whichȱ isȱ highlyȱ
suitableȱforȱtheȱ biomonitoringȱ ofȱsedimentȬȱboundȱcontaminants:ȱ itȱlivesȱinȱ UȬ
shapedȱ burrowsȱ withinȱ theȱ sedimentȱ andȱ ingestsȱ largeȱ volumesȱ ofȱ sedimentȱ
whenȱfeeding,ȱthereforeȱisȱcontinuouslyȱexposedȱtoȱanyȱcontaminantsȱpresentȱinȱ
theȱ sediment.ȱ Itȱ isȱ availableȱ allȱ theȱ yearȱ round,ȱ oftenȱ inȱ reasonablyȱ highȱ
densities,ȱtoleratesȱaȱwideȱrangeȱofȱparticlesȱsizesȱandȱsalinitiesȱandȱhasȱaȱbroadȱ
geographicȱrangeȱ(Batȱetȱal.,ȱ1998).ȱArenicolaȱmarinaȱisȱalsoȱanȱimportantȱlinkȱinȱ
coastalȱ foodȱ chainȱ playingȱ anȱ importantȱ roleȱ inȱ sedimentȱ communityȱ
organizationȱ (Batȱ etȱ al.,ȱ 1998).ȱ Polychaeteȱ wormsȱ areȱ oftenȱ theȱ mostȱ abundantȱ
taxaȱ inȱ contaminatedȱ areasȱ andȱ theirȱ capacityȱ toȱ accumulateȱ andȱ metabolizeȱ
PAHsȱ mayȱ haveȱ importantȱ effectsȱ onȱ theȱ transportȱ andȱ fateȱ ofȱ PAHsȱ inȱ theȱ
marineȱenvironmentȱ(Selckȱetȱal.,ȱ2003).ȱTheȱstudyȱofȱwaterȬsolubleȱmetabolitesȱ
inȱ A.ȱ marinaȱ highlightsȱ theȱ presenceȱ ofȱ aȱ PAHȱ metabolisingȱ systemȱ inȱ theȱ
organismȱ(Christensenȱetȱal.,ȱ2002).ȱȱDuringȱtheȱpastȱfewȱyearsȱtheȱ10Ȭdayȱacuteȱ
sedimentȱassayȱusingȱA.ȱmarinaȱhasȱbeenȱwidelyȱadoptedȱforȱuseȱinȱevaluatingȱ
theȱ qualityȱ ofȱ sedimentsȱ (CEFAS,ȱ 1998;ȱ Thainȱ andȱ Bifieldȱ 2002).ȱAlthoughȱ thisȱ
bioassayȱ suppliesȱ informationȱ aboutȱ generalȱ health,ȱ itȱ doesȱ notȱ clarifyȱ howȱ orȱ
whyȱ theȱ organismsȱ areȱ affected.ȱ Biomarkersȱ studiesȱ inȱ Arenicolaȱ marinaȱ areȱ
few,ȱ (Hannamȱ etȱ al.,ȱ 2007;ȱ Lewisȱ etȱ alȱ (inȱ prep)),ȱ however,ȱ someȱ specificȱ andȱ
nonȬspecificȱbiomarkersȱhaveȱbeenȱinvestigatedȱinȱotherȱpolychaeteȱspeciesȱ(forȱ
example;ȱNereisȱdiversicolorȱ,ȱDurouȱetȱal.,ȱ2007);ȱCapitellaȱcapitataȱ,ȱBachȱetȱal.,ȱ
2005);ȱLaeonoreisȱacuta,ȱ(Montserratȱetȱal.,ȱ2006);ȱTubifexȱtubifexȱ,ȱMoslehȱetȱal.,ȱ
2007);ȱ Sipunclusȱ nudus,ȱ Matozzoȱ etȱ al.,ȱ 2002).ȱ Toȱ date,ȱ theseȱ techniquesȱ haveȱ
notȱpreviouslyȱbeenȱcombinedȱtoȱgiveȱaȱmultiȬbiomarkerȱapproachȱtoȱsedimentȱ
qualityȱmonitoringȱusingȱaȱsingleȱspecies.ȱ
Inȱ theȱ presentȱ study,ȱ weȱ useȱ aȱ novelȱ approachȱ toȱ sedimentȱ toxicityȱ
assessment,ȱ whichȱ combinesȱ aȱ multiȬȱ biomarkerȱ approachȱ usingȱ anȱ Arenicolaȱ
exposureȱ modelȱ withȱ analyticalȱ chemistryȱ toȱ addressȱ theȱ hypothesisȱ thatȱ
- 157 -
exposureȱ toȱ contaminatedȱ sedimentsȱ canȱ causeȱ detrimentalȱ biologicalȱ effects.ȱ
Marineȱ sedimentsȱwereȱ collectedȱ fromȱsitesȱaroundȱEuropeȱexhibitingȱvaryingȱ
degreesȱofȱanthropogenicȱimpactȱandȱincludedȱsitesȱrecoveringȱfromȱtheȱacuteȱ
impactsȱ provokedȱ byȱ theȱ tankerȱ Prestigeȱ (2002)ȱ inȱ theȱ Galicianȱ Coastȱ (NWȱ
Spain)ȱ(MoralesȬCasellesȱetȱal.,ȱaccepted)ȱandȱanȱareaȱchronicallyȱaffectedȱbyȱoilȱ
spillage.ȱSpecificȱquestionsȱincluded:ȱ(1)ȱcanȱaȱsignificantȱrelationshipȱbetweenȱ
contaminationȱandȱbiologicalȱresponseȱbeȱshown?ȱ(2)ȱcanȱweȱuseȱthisȱintegratedȱ
approachȱtoȱdistinguishȱbetweenȱacutelyȱandȱchronicallyȱimpactedȱsites?ȱ(3)ȱIsȱ
thisȱtechniqueȱsensitiveȱenoughȱtoȱchartȱrecovery?ȱ
2.ȱMaterialsȱandȱmethodsȱ
2.1.ȱStudyȱsiteȱ
Contaminatedȱ sedimentsȱ fromȱ twoȱ areasȱ ofȱ theȱ Spanishȱ coastȱ wereȱ
selected.ȱTheȱAtlanticȱIslandsȱNationalȱParkȱȱisȱlocatedȱinȱfrontȱofȱtheȱmouthȱofȱ
theȱ Riasȱ Baixasȱinȱtheȱ GalicianȱCoastȱ(NWȱSpain)ȱandȱ itȱitsȱconsideredȱaȱplaceȱ
withȱ highȱ ecologicalȱ revelance.ȱ Theseȱ islandsȱ playedȱ anȱ importantȱ roleȱ inȱ theȱ
Prestigeȱ oilȱ spillȱ (November,ȱ 2002)ȱ actingȱ asȱ aȱ barrierȱ thatȱ protectedȱ theȱ coastȱ
fromȱtheȱentranceȱofȱspilledȱoil.ȱSedimentsȱfromȱtheȱBayȱofȱAlgecirasȱ(SȱSpain)ȱ
haveȱsufferedȱaȱchronicȱimpactȱlastingȱseveralȱdecades,ȱcausedȱbyȱtheȱinputȱofȱ
oilȱandȱotherȱcontaminantsȱfromȱtheȱvariousȱindustriesȱȱlocatedȱinȱtheȱareaȱandȱ
fromȱ accidentalȱ spillsȱ andȱ deliberateȱ dischargesȱ fromȱ commercialȱ shippingȱ
activitiesȱ(MoralesȬCaselles,ȱetȱalȱ2007).ȱ
ȱToȱperformȱthisȱstudyȱ3ȱstationsȱwereȱselectedȱinȱtheȱNationalȱParkȱandȱ3ȱ
stationsȱinȱtheȱareaȱofȱtheȱBayȱofȱAlgecirasȱ(Figureȱ1).ȱTwoȱreferenceȱsitesȱwereȱ
selectedȱtoȱcarryȱoutȱthisȱstudy:ȱinȱtheȱEstuaryȱatȱExmouth,ȱSouthȱDevon,ȱU.K,ȱ
andȱ inȱ theȱ Bayȱ ofȱ Cádiz,ȱ Spain.ȱ Theȱ firstȱ wasȱ theȱ siteȱ ofȱ collectionȱ ofȱ Arenicolaȱ
marinaȱ specimensȱ whereasȱ theȱ secondȱ wasȱ chosenȱ becauseȱ itȱ hasȱ beenȱ widelyȱ
characterizedȱ inȱ previousȱ ecotoxicologicalȱ studiesȱ (DelVallsȱ etȱ al.,ȱ 1998,ȱ
- 158 -
MoralesȬCasellesȱetȱal.,ȱ2007);ȱinȱaddition,ȱorganicȱcontaminationȱwasȱbelowȱtheȱ
detectionȱlimitȱinȱbothȱplaces.ȱ
ȱ
ȱ
Atlantic Islands
National Park
(Galician Coast)
•AC1
Spain
•AC2
ȱ
N
•AC3
E
W
ȱ
United
Kingdom
S
ȱ Bay of Cádiz
•CR2
•CR1
•CR3
ȱ
Bay of Algeciras
•R2
R1•
ȱ
ȱ
Figureȱ 1.ȱ Mapȱ ofȱ generalȱ areasȱ sampledȱ andȱ locationsȱ ofȱ theȱ samplingȱ
stationsȱinȱGaliciaȱ(NWȱSpain)ȱandȱtheȱBayȱofȱAlgecirasȱ(SȱSpain)ȱ,ȱbothȱaffectedȱ
byȱ oilȱ spills,ȱ andȱ theȱ referenceȱ sitesȱ locatedȱ inȱ theȱ Bayȱ ofȱ Cádizȱ (Sȱ Spain)ȱ andȱ
Exmouthȱ(SȱUK).ȱ
2.2.ȱSampleȱcollectionȱandȱbioassayȱ
Sedimentȱ samplesȱwereȱ collectedȱandȱ transportedȱtoȱ theȱ laboratoryȱandȱ
subȬsampledȱ forȱ physicalȱ characterizationȱ andȱ chemicalȱ quantification.ȱ Afterȱ
that,ȱsedimentȱsamplesȱwereȱmaintainedȱinȱtheȱcoolerȱatȱ4ºȱCȱinȱtheȱdarkȱuntilȱ
theyȱ wereȱ usedȱ forȱ sedimentȱ toxicityȱ testing,ȱ butȱ forȱ noȱ longerȱ thanȱ 2ȱ weeks.ȱ
Arenicolaȱ marinaȱ specimensȱ wereȱ obtainedȱ fromȱ aȱ naturalȱ populationȱ fromȱ aȱ
‘clean’ȱ (http://www.environmentȬagency.gov.uk/)ȱ estuaryȱ atȱ Exmouth,ȱ Southȱ
Devon,ȱ U.Kȱ (50º36ȇ57ȇȇNȱ 3º26ȇ40ȇȇW).ȱ Organismsȱ wereȱ placedȱ inȱ 20ȱ Lȱ capacityȱ
aquariumsȱ withȱ clean,ȱ filteredȱ (0.5ΐm)ȱ seawaterȱ (FSW)ȱ andȱ sievedȱ sedimentȱ
- 159 -
(collectedȱinȱtheȱsameȱareaȱasȱtheȱorganisms)ȱandȱwereȱmaintainedȱinȱlaboratoryȱ
underȱ controlledȱ conditionsȱ forȱ acclimationȱ (7ȱ days)ȱ untilȱ theȱ startȱ ofȱ theȱ test.ȱ
Aerationȱwasȱprovidedȱwithȱaȱ12:12ȱlight:ȱdarkȱphotoperiod.ȱȱȱ
Theȱ toxicityȱ testȱ wasȱ conductedȱ inȱ replicateȱ (5)ȱ byȱ exposingȱ individualȱ
Arenicolaȱmarinaȱspecimensȱtoȱbulkȱsediment.ȱApproximately,ȱ250ȱgȱofȱsievedȱ(1ȱ
mm)ȱ sedimentȱ wasȱ placedȱ inȱ 2ȱ Lȱ beakersȱ withȱ 750ȱ mLȱ ofȱ wellȱ aeratedȱ FSW.ȱȱ
TwoȱA.ȱmarinaȱwereȱplacedȱinȱeachȱreplicateȱcontainerȱandȱmaintainedȱatȱ15ȱºCȱ
duringȱ theȱ 14ȱ dayȱ exposureȱ period.ȱ Theȱ behaviourȱ andȱ castsȱ assayȱ wasȱ
performedȱ afterȱ theȱ exposureȱ period.ȱ afterȱ whichȱ coelomicȱ fluidȱ wasȱ collectedȱ
forȱuseȱinȱtheȱcellularȱassays.ȱȱCoelomicȱfluidȱwasȱcarefullyȱwithdrawnȱfromȱtheȱ
posteriorȱpartȱofȱeachȱA.ȱmarinaȱspecimenȱusingȱaȱ21Gȱsyringe,ȱandȱstoredȱinȱiceȱ
priorȱ toȱ use.ȱ Theȱ wholeȱ bodyȱ wasȱ thenȱ frozenȱ atȱ Ȭ80ȱ ºCȱ forȱ subsequentȱ
biochemicalȱ biomarkersȱ analysis.ȱ Wormsȱ wereȱ homogenizedȱ withȱ PBSȱ pHȱ 7.5ȱ
andȱcentrifugedȱforȱ30ȱminutesȱatȱ10,000ȱgȱatȱ4ȱºC;ȱsupernatantȱwasȱemployedȱ
forȱ biochemicalȱ biomarkersȱ andȱ totalȱ proteinsȱ determinationȱ (Bradfordȱ etȱ al.,ȱ
1976).ȱ
Chemicalȱ analysis.ȱ Theȱ analysesȱ ofȱ PAHsȱ andȱ PCBsȱ boundȱ toȱ sedimentsȱ
wereȱcarriedȱoutȱaccordingȱtoȱUSEPAȱSWȬ846ȱMethodȱ827C78082ȱ(1994).ȱBrieflyȱ
driedȱ samplesȱ wereȱSoxhletȱ extractedȱ withȱ nȬhexaneȱ forȱ 18ȱ h,ȱ andȱ theȱ extractsȱ
wereȱisolatedȱbyȱcolumnȱchromatographyȱonȱFlorisileȱaluminoȬsilica.ȱPCBsȱandȱ
PAHsȱwereȱ elutedȱ andȱ theirȱ fractionsȱwereȱdriedȱinȱaȱ rotatingȱevaporatorȱandȱ
reȬdissolvedȱ inȱ isooctane.ȱ Aromaticȱ fractionsȱ wereȱ analyzedȱ onȱ aȱ
HewlettePackardȱ(HP)ȱ5890ȱSeriesȱIIȱgasȱchromatographerȱcoupledȱwithȱanȱHPȱ
5970ȱ massȱ spectrometer.ȱ PAHsȱ wereȱ analyzedȱ byȱ GCȬMSȱ usingȱ selectedȱ ionȱ
monitoringȱ (SIM).ȱ Analysisȱ ofȱ PCBsȱ asȱ AROCLORȱ 1242ȱ andȱ AROCLORȱ 1260ȱ
wasȱ performedȱ usingȱ theȱ sameȱ instrumentȱ withȱ anȱ electronȱ captureȱ detectorȱ
(GC/ECD).ȱ Forȱ bothȱ setȱ ofȱ organicȱ chemicals,ȱ PAHsȱ andȱ AROCLOR,ȱ theȱ
- 160 -
analyticalȱprocedureȱshowedȱagreementȱwithȱtheȱcertifiedȱvaluesȱofȱmoreȱthanȱ
90%.ȱ
TraceȱmetalȱanalysisȱwereȱanalyzedȱasȱdescribedȱbyȱCasadoȬMartínezȱetȱ
al.ȱ (2006);ȱ briefly,ȱ 2.5ȱ gȱ ofȱ sedimentsȱ (<0.065ȱ mm)ȱ wereȱ placedȱ inȱ Teflonȱ
containersȱ andȱ wereȱdigestedȱinȱmicrowaveȱ(400W,ȱ15ȱmin,ȱtwice)ȱwithȱHNO3ȱ
2N.ȱ Theȱ extractsȱ wereȱ purifiedȱ byȱ passingȱ throughȱ aȱ CȬ18ȱ columnȱ andȱ metalsȱ
analysesȱwereȱperformedȱbyȱanodicȱvoltamperimetryȱ(ȬZn,ȱPb,ȱNi,ȱCoȱandȱCuȬȱ
MetrohmȱApplicationȱBulletinȱNºȱ147;ȱȬȱVȬȱMetrohmȱApplicationȱNoteȱNºȱVȬ81).ȱ
Forȱ Hgȱ theȱ coldȱ vapourȱ techniqueȱ wasȱ usedȱ andȱ wasȱ quantifiedȱ usingȱ atomicȱ
absorptionȱ spectrometry.ȱ Theȱ analyticalȱ proceduresȱ wereȱ checkedȱ usingȱ
referenceȱ materialȱ (MESSȬ1ȱ NRCȱ andȱ CRMȱ 277ȱ BCR)ȱ andȱ showedȱ aȱ recoveryȱ
greaterȱthanȱ90%ȱofȱtheȱcertifiedȱconcentration.ȱ
Behaviourȱ andȱ Feedingȱ assay.ȱ Afterȱ 14ȱ daysȱ ofȱ exposureȱ theȱ individualȱ A.ȱ
marinaȱ specimensȱ wereȱ transferredȱ toȱ differentȱ beakersȱ containingȱ cleanȱ
sedimentȱ(fromȱtheȱreferenceȱsiteȱatȱExmouth)ȱandȱtheȱtimeȱtakenȱforȱthemȱtoȱreȬ
buryȱthemselvesȱcompletelyȱwasȱrecorded.ȱWormsȱwereȱtheȱreturnedȱtoȱtheȱtestȱ
sedimentȱ beakersȱ andȱ leftȱ forȱ 24ȱ hours;ȱ afterȱ whichȱ castsȱ wereȱ carefullyȱ
removed,ȱdriedȱandȱweighed.ȱȱ
Cometȱ assay.ȱ Theȱ Cometȱ assayȱ wasȱ performedȱ toȱ detectȱ singleȱ strandȱ
DNAȱbreaksȱinȱinvidicualȱcellsȱaccordingȱtoȱtheȱmethodsȱofȱSinghȱetȱal.ȱ(Singh,ȱ
1988)ȱ withȱ modificationsȱ specificȱ forȱ Arenicolaȱ marinaȱ (Lewisȱ etȱ al.,ȱ inȱ prep),ȱ
usingȱ alkalineȱconditions.ȱ ȱOneȱ hundredȱcellsȱperȱpreparationȱ wereȱquantifiedȱ
usingȱKineticȱCOMETȱSoftware.ȱȱ
Phagocytosis.ȱ Phagocytosisȱ activityȱ wasȱ determinedȱ byȱ measuringȱ theȱ
uptakeȱ ofȱ fluorescentȱ zymosanȱ particles,ȱ usingȱ trypanȱ blueȱ asȱ aȱ quenchingȱ
agentȱ Andersonȱ etȱ alȱ (1995).ȱ Inȱ brief,ȱ 50ȱ ΐLȱ ofȱ coelomicȱ fluidȱ wasȱ pipettedȱ inȱ
triplicateȱ wellsȱ ofȱ aȱ microtitreȱ plate;ȱ thenȱ 50ȱ ΐLȱ Flouresceinȱ isothiocyanateȱ
(FITC)ȱwasȱaddedȱtoȱtheȱwells.ȱIncubationȱwasȱperformedȱinȱdarkȱforȱ40ȱmin,ȱatȱ
- 161 -
21ȱºC.ȱ50ȱΐLȱofȱFluorescenceȱquenchingȱsolutionȱ(1.25ȱmLȱtrypanȱblueȱinȱ1ȱmMȱ
citrateȱbufferȱpHȱ4.5)ȱwasȱthenȱaddedȱtoȱtheȱwells,ȱandȱfluorescenceȱmeasuredȱ
usingȱ aȱ Hitachiȱ FȬ4500ȱ fluorescenceȱ spectrophotometerȱ Ώex/emȱ 485/535ȱ .ȱ Resultsȱ
wereȱcomparedȱtoȱaȱstandardȱcurveȱandȱnormalisedȱtoȱprotein.ȱ
Antioxidantȱ status.ȱ Antioxidantȱ statusȱ wasȱ measuredȱ suignȱ theȱ ferricȱ
reducingȱabilityȱofȱplasmaȱ(FRAP)ȱassayȱ(BenzieȱandȱStrain,ȱ1996)ȱasȱadaptedȱbyȱ
Haggerȱetȱal.,ȱ2005.ȱCoelomicȱfluid,ȱ50ȱΐl,ȱinȱduplicateȱwasȱincubatedȱforȱ10ȱminȱ
atȱ 25°Cȱ withȱ 200ȱ ΐLȱ ofȱ FRAPȱ reagentȱ (300ȱ mMȱ acetateȱ bufferȱ pHȱ 3.6,ȱ 2,4,6Ȭ
tripyridylȬ5Ȭtriazineȱ (TBTZ),ȱ 20ȱ mMȱ ironȱ chlorideȱ inȱ theȱ ratioȱ 10:1:1ȱ preparedȱ
immediatelyȱpriorȱtoȱanalysis)ȱinȱmicrotitreplatesȱandȱtheȱchangeȱinȱabsorbanceȱ
atȱ593nmȱnoted.ȱTheȱFRAPȱvalueȱwasȱcalculatedȱrelativeȱtoȱaȱstandardȱcurveȱofȱ
Fe(II)ȱ inȱ theȱ rangeȱ 100Ȭ500ȱ ΐmol/lȱ andȱ expressedȱ asȱ changeȱ inȱ absorbanceȱ perȱ
mgȱprotein.ȱȱ
Thiobarbituricȱacidȱreactiveȱsubstancesȱ(TBARS)ȱassay.ȱTheȱmeasurementȱofȱ
TBARSȱ wasȱ performedȱ toȱ evaluateȱ theȱ freeȱ radicalȬmediatedȱ oxidationȱ
(modifiedȱ byȱ Camejoȱ etȱ al.,ȱ 1999).ȱ ȱ Malondialdehydeȱ (MDA),ȱ aȱ secondaryȱ
productȱ inȱ lipidȱ peroxidationȱ bindsȱ toȱ thiobarbituricȱ acidȱ (TBA)ȱ whichȱ canȱ beȱ
measuredȱ spectrophotometrically.ȱ Briefly,ȱ 10ȱ ΐLȱ ofȱ freeȱ radicalȱ scavengerȱ 1ȱ
mmolȱ 1Ȭ1ȱ butylatedȱ hydroxytolueneȱ (2,6ȬDiȬ0ȱ tertȬbutylȬ4Ȭmethyphenol)ȱ
dissolvedȱ inȱ absoluteȱ ethanolȱ wasȱ addedȱ toȱ theȱ microplateȱ wellsȱ inȱ orderȱ toȱ
preventȱ furtherȱ oxidationȱ ofȱ theȱ samples;ȱ 40ȱ ΐLȱ ofȱ homogenateȱ andȱ 200ȱ ΐLȱ
phosphateȱbufferedȱsalineȱpHȱ7.4ȱwereȱaddedȱtoȱtheȱwells.ȱ50ȱΐLȱofȱ50ȱ%ȱ(w/v)ȱ
trichloroaceticȱ acidȱ andȱ 75ȱ ΐLȱ ofȱ 1.3ȱ %ȱ (w/v)ȱ thiobarbituricȱ acidȱ (TBA)ȱ
(dissolvedȱ inȱ 0.3%ȱ (w/v)ȱ NaOH)ȱ wereȱ includedȱ andȱ afterȱ 60ȱ minȱ atȱ 60ȱ ºCȱ
incubationȱ theȱ absorbanceȱ wasȱ readȱ atȱ 530ȱ nmȱ andȱ thenȱ againȱ atȱ 630ȱ nm.ȱ
Resultsȱ wereȱ comparedȱ toȱ aȱ standardȱ curveȱ preparedȱ usingȱ 1,1,3,3Ȭ
tetraethoxypropaneȱ(aȱstabilizedȱformȱofȱMDA)ȱandȱnormalisedȱtoȱprotein.ȱ
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Glutathioneȱ transferaseȱ (GST)ȱ assay.ȱ Theȱ phaseȱ IIȱ metabolizingȱ enzymeȱ
GlutathioneȬSȬtransferaseȱ (GST)ȱ wasȱ determinedȱ byȱ monitoringȱ theȱ rateȱ ofȱ
conjugationȱofȱglutathioneȱ(GSH)ȱtoȱ1ȬchloroȬ2,4Ȭdinitrobenzeneȱ(CDNB)ȱatȱ340ȱ
nmȱ (McFarlandȱ etȱ al.,1999).ȱ Supernatantsȱ wereȱ dilutedȱ (20ȱ ΐLȱ inȱ 1ȱ mLȱ
homogenizingȱ buffer)ȱ andȱ placedȱ inȱ theȱ 96ȱ wellsȱ plate.ȱ 0.5ȱ mLȱ 42ȱ mMȱ GSHȱ
wereȱ addedȱ toȱ aȱ mixtureȱ containingȱ 0.5ȱ mLȱ 42ȱ mMȱ CDNBȱ (inȱ ethanol)ȱ andȱ
CDNBȱassayȱbufferȱ(200ȱmMȱsodiumȱphosphate,ȱpHȱ6.5);ȱimmediatelyȱ200ȱΐLȱofȱ
theȱsolutionȱwereȱplacedȱintoȱtheȱwellsȱandȱtheȱplateȱwasȱreadȱatȱ340ȱnmȱeveryȱ
30ȱsecondsȱforȱ3ȱminutes.ȱȱ
GlutathioneȱReductaseȱ(GR)ȱassay.ȱTheȱoxidationȱofȱ1ȱmMȱNADPHȱbyȱGRȱ
inȱ theȱ presenceȱ ofȱ 10ȱ mMȱ oxidizedȱ glutathioneȱ wasȱ monitoredȱ atȱ 340ȱ nmȱ
(McFarlandȱetȱal.,1999).ȱInȱbrief,ȱ20ȱΐLȱofȱsupernatantȱwasȱpipettedȱinȱtriplicateȱ
wellsȱ ofȱ aȱ microtitreȱ plate.ȱ Aȱ solutionȱ containingȱ 2.5ȱ mLȱ 10ȱ mMȱ oxidizedȱ
glutathione,ȱ 2.5ȱ mLȱ 1mMȱ NADPHȱ andȱ 20ȱ mLȱ GRȱ bufferȱ (200ȱ mMȱ sodiumȱ
phosphate,ȱ pHȱ 7.6)ȱ wasȱ preparedȱ andȱ 200ȱ ΐLȱ wereȱ addedȱ toȱ theȱ wells.ȱ Plateȱ
wasȱreadȱatȱ340ȱnmȱeveryȱ2ȱminȱforȱ10ȱminȱ
Glutathioneȱ Peroxidaseȱ (GPX)ȱ assay.ȱ Theȱ antioxidantȱ GPXȱ activityȱ wasȱ
measuredȱ accordingȱ toȱ (McFarlandȱ etȱ al.,1999).ȱ Supernatantsȱ wereȱ dilutedȱ (10ȱ
ΐLȱ +ȱ 10ȱ ΐLȱ homogenizingȱ buffer)ȱ andȱ placedȱ inȱ theȱ 96ȱ wellsȱ plate.ȱ Aȱ solutionȱ
containingȱ inȱ excessȱ NADPH,ȱ reducedȱ gluthationeȱ andȱ glutathioneȱ reductaseȱ
wasȱpreparedȱandȱ200ȱΐLȱwereȱaddedȱtoȱtheȱwells.ȱAfterȱ2ȱminȱincubationȱ50ȱΐLȱ
ofȱ 1.25ȱ mMȱ hydrogenȱ peroxideȱ wasȱ pipettedȱ toȱ theȱ wells;ȱ NADPHȱ oxidationȱ
wasȱmeasuredȱatȱ340ȱnmȱatȱ10ȱsȱintervalsȱforȱ3ȱmin.ȱȱ
Statisticalȱanalysis.ȱDataȱforȱeachȱbiomarkerȱwereȱanalyzedȱusingȱANOVAȱ
inȱ orderȱ toȱ determineȱ significantȱ differencesȱ (p<0.05)ȱ amongȱ theȱ resultsȱ
obtainedȱ inȱ eachȱ collectionȱ siteȱ andȱ theȱ referenceȱ site.ȱ Correlationȱ betweenȱ
chemicalȱconcentrationsȱinȱsedimentsȱandȱbiomarkerȱresponsesȱwasȱcarriedȱoutȱ
usingȱaȱSpearmanȱcorrelationȱanalysisȱ(p<0.05).ȱMultivariateȱanalysesȱwereȱalsoȱ
- 163 -
performedȱ usingȱ theȱ MDS,ȱ SIMPER,ȱ ANOSIMȱ andȱ BIOENVȱ programsȱ ofȱ theȱ
PRIMERȱsoftwareȱpackageȱ(PlymouthȱMarineȱLaboratory,ȱUK).ȱȱAȱBrayȬCurtisȱ
dissimilarityȱ matrixȱ wasȱ producedȱ fromȱ fourthȱ rootȱ transformedȱ rawȱ
abundanceȱ data.ȱ ȱ NonȬmetricȱ MultiȬDimensionalȱ Scalingȱ (nMDS)ȱ wasȱ thenȱ
performedȱ toȱ produceȱ twoȬdimensionalȱ ordinationȱ plots.ȱ ȱ Inȱ ordinationȱ plots,ȱ
pointsȱ(sites)ȱcloseȱtoȱeachȱotherȱhaveȱsimilarȱbiomarkerȱresponses,ȱwhilstȱthoseȱ
farȱ apartȱ areȱ lessȱ similar.ȱ ȱ OneȬwayȱ ANOSIMȱ testsȱ wereȱ usedȱ toȱ testȱ forȱ
significantȱ differencesȱ betweenȱ biomarkerȱ responsesȱ inȱ reference,ȱ chronicallyȱ
pollutedȱ andȱ acutelyȱ pollutedȱ (i.e.ȱ Prestige)ȱ sites.ȱ ȱ Similarityȱ percentagesȱ
(SIMPER)ȱ wereȱ thenȱ usedȱ toȱ identifyȱ theȱ percentageȱ contributionȱ ofȱ eachȱ
biomarkerȱ toȱ theȱ multivariateȱ differencesȱ betweenȱ theȱ differentȱ sitesȱ (Clarkeȱ
andȱ Warwick,ȱ 1994)ȱ andȱ theȱ BESTȱ (BIOȬENV)ȱ programmeȱ wasȱ usedȱ toȱ
determineȱ whichȱ chemicalȱ parametersȱ measuredȱ ‘best’ȱ describeȱ theȱ patternȱ inȱ
biomarkerȱ responsesȱ observedȱ inȱ Arenicolaȱ marinaȱ specimensȱ exposedȱ toȱ theȱ
differentȱsediments.ȱ
3.ȱResultsȱ
3.1.ȱChemicalȱanalysisȱofȱsedimentsȱȱ
Theȱ concentrationȱ ofȱ organicȱ contaminantsȱ (PAHs)ȱ wasȱ muchȱ higherȱ inȱ
sedimentsȱcollectedȱinȱtheȱBayȱofȱAlgecirasȱ(CR2ȱ>ȱCR1ȱ>ȱCR3)ȱthanȱinȱtheȱareaȱ
ofȱ Galiciaȱ (AC3ȱ >ȱ AC2ȱ >AC1),ȱ whereasȱ theȱ concentrationȱ ofȱ metalsȱ didȱ notȱ
presentȱ aȱ clearȱ trendȱ amongȱ sedimentsȱ fromȱ theȱ differentȱ areasȱ (Tableȱ 1).ȱ Theȱ
referencesȱ (R1ȱ andȱ R2)ȱ presentedȱ theȱ lowestȱ levelsȱ ofȱ metals,ȱ andȱ noȱ organicȱ
contaminationȱwasȱdetected.ȱTheȱpredominantȱPAHȱinȱtheȱsedimentsȱcollectedȱ
inȱtheȱNationalȱParkȱwasȱtheȱnaphthaleneȱwhereasȱsedimentsȱlocatedȱinȱtheȱBayȱ
ofȱ Algecirasȱ mainlyȱ presentedȱ phenanthrene,ȱ fluorene,ȱ pyreneȱ andȱ
benzo[b]fluoranthene.ȱȱȱ
- 164 -
Tableȱ1.ȱTotalȱPAHs,ȱPCBsȱandȱmetalȱconcentrationȱ(Zn,ȱCd,ȱPb,ȱCu,ȱNi,ȱ
Co,ȱHgȱandȱV)ȱȬmgȱKgȬ1ȱdryȱsedimentȬ,ȱpercentageȱofȱfinesȱ(fines)ȱandȱorganicȱ
carbonȱ(O.C.)ȱmeasuredȱinȱtheȱsediments.ȱȱn.d:ȱnotȱdetected.ȱ
ȱȱ
Znȱ Cdȱ Pbȱ Cuȱ
C2ȱ
12.4ȱ 0.06 11.6ȱ 17.6ȱ 13.1ȱ 1.8ȱ 3.27 0.15ȱ n.d.ȱ
n.d.ȱ
1.22ȱ
22.7ȱ
Ca1ȱ
21.3ȱ 0.92 2.28ȱ 6.98ȱ 0.06ȱ 3.40 80.0 n.d.ȱ n.d.ȱ
n.d.ȱ
1.07ȱ
2.50ȱ
Niȱ
Coȱ
Vȱ
Hgȱ PAH PCBsȱ O.C.ȱ fines
GR4ȱ 35.3ȱ 0.10 6.21ȱ 3.67ȱ 13.1ȱ 5.59 n.d. 0.25a 802bȱ 1.75ȱ
3.19ȱ 59.3ȱ
GR3ȇȱ 138ȱ 0.17 21.6ȱ 5.01ȱ 74.7a 12.8 26.1 1.04a 3151b 22.0ȱ
2.15ȱ
69.4ȱ
P1ȱ
56.7ȱ 0.12 12.3ȱ 75.2aȱ 13.3ȱ n.d. 6.84 0.65a 641bȱ 0.84ȱ
3.86ȱ 35.4ȱ
D88ȱ
158aȱ n.d. 17.3ȱ 20.1ȱ 12.4ȱ n.d. n.d. 0.28a
13.0ȱ
n.d.ȱ
0.26ȱ
2.35ȱ
D79ȱ
107ȱ n.d. 21.0ȱ 39.1aȱ 21.1a 0.30 n.d. 0.09ȱ 80.0ȱ
n.d.ȱ
2.08ȱ
65.2ȱ
D60ȱ
161aȱ n.d. 43.4ȱ 16.7ȱ 14.7ȱ 0.20 n.d. 0.12ȱ
260ȱ
n.d.ȱ
2.07ȱ
50.0ȱ
0.15ȱ 624*ȱ
22.7ȱ
Ȭȱ
Ȭȱ
SQVsȱ 150ȱ 1.2ȱ 46.7ȱ 34ȱ
20.9ȱ
Ȭȱ
Ȭȱ
Chemicalȱ dataȱ wasȱ comparedȱ toȱ internationalȱ sedimentȱ qualityȱ
guidelinesȱ (SQGs)ȱ thatȱ specifyȱ theȱ levelsȱ ofȱ chemicalȱ contaminantsȱ associatedȱ
withȱbiologicalȱeffectsȱandȱthoseȱexceedingȱrecommendedȱlimitsȱareȱhighlightedȱ
inȱ Tableȱ 1.ȱ Followingȱ theȱ recommendationsȱ describedȱ byȱ MacDonaldȱ etȱ al.ȱ
(1996),ȱtheȱsedimentsȱfromȱtheȱBayȱofȱAlgecirasȱwouldȱbeȱconsideredȱasȱslightlyȱ
pollutedȱ byȱ PAHsȱ andȱ adverseȱ effectsȱ mightȱ beȱ predicted..ȱ Siteȱ AC3ȱ alsoȱ
presentȱ aȱ naphthaleneȱ contentȱ higherȱ thanȱ theȱ ERLȱ (160ȱ ΐgȱ kgȬ1)ȱ proposedȱ byȱ
NOAAȱ(1999)ȱ(ERL:ȱvaluesȱbelowȱwhichȱbiologicalȱeffectsȱareȱrare).ȱInȱtheȱBayȱ
ofȱAlgeciras,ȱsiteȱCR2ȱsurpassedȱtheȱERLȱdefinedȱforȱfluoreneȱ(19ȱΐgȱkgȬ1)ȱwithȱ
highȱvaluesȱofȱphenanthreneȱandȱfluoranthene.ȱCR3ȱfromȱtheȱBayȱofȱAlgecirasȱ
exceededȱ theȱ guidelineȱ forȱ Cu,ȱ asȱ didȱ AC2ȱ ȱ inȱ Galicia.ȱ Znȱ ERLȱ valueȱ isȱ
surpassedȱ byȱ theȱ sedimentsȱ AC1ȱ ȱ andȱ AC3;ȱ Niȱ sedimentȱ concentrationȱ goesȱ
aboveȱ theȱ ERLȱ definedȱ inȱ CR2ȱ andȱ AC2ȱ whereasȱ Hgȱ SQGȱ isȱ surpassedȱ byȱ
sedimentsȱ fromȱ theȱ Bayȱ ofȱ Algecirasȱ andȱ theȱ treatmentȱ AC1ȱ locatedȱ inȱ theȱ
- 165 -
Galicianȱ Coast.ȱ PCBsȱ wereȱ onlyȱ detectedȱ inȱ samplesȱ fromȱ theȱ chronicȱ sitesȱ
althoughȱlevelsȱwereȱbelowȱtheȱSQGs.ȱ
3.2.ȱBiomarkerȱresponsesȱ
AȱsummaryȱofȱallȱtheȱbiomarkersȱresultsȱmeasuredȱinȱArenicolaȱmarinaȱisȱ
shownȱ inȱ Figureȱ 2.ȱ Cometȱ resultsȱ obtainedȱ afterȱ 15ȱ daysȱ ofȱ exposureȱ confirmȱ
significantlyȱ differencesȱ (p<0.01)ȱ betweenȱ theȱ referenceȱ sitesȱ (R1ȱ andȱ R2)ȱ andȱ
theȱ stationsȱ fromȱ theȱ Bayȱ ofȱ Algecirasȱ CR1,ȱ CR2,ȱ CR3ȱ andȱ theȱ sitesȱ AC2ȱ andȱ
AC3ȱ locatedȱ inȱ theȱ AINP.ȱ Inȱ theȱ Behaviourȱ assayȱ CR2ȱ wasȱ significantlyȱ
differentȱ(p<0.05)ȱtoȱtheȱR1,ȱR2ȱandȱAC1ȱtreatmentsȱwhereasȱtheȱweightȱofȱcastsȱ
showedȱ thatȱ AC1ȱ wasȱ significantlyȱ differentȱ (p<0.01)ȱ toȱ allȱ theȱ stations,ȱ
includingȱtheȱreferenceȱtreatments.ȱDifferencesȱwereȱstatisticallyȱsignificantȱforȱ
theȱ phagocytosisȱ assayȱ althoughȱ responsesȱ presentedȱ aȱ highȱ increaseȱ inȱ
variation.ȱNoȱsignificantȱdifferencesȱwereȱdetectedȱinȱFRAP,ȱTBARS,ȱGPX,ȱGSTȱ
andȱ GRȱ byȱ usingȱ theȱ ANOVA.ȱ Inȱ general,ȱ treatmentȱ AC1ȱ fromȱ theȱ Galicianȱ
Coastȱandȱbothȱreferenceȱsitesȱpresentȱanalogousȱtrends.ȱInȱcontrast,ȱsitesȱfromȱ
theȱBayȱofȱAlgecirasȱgenerallyȱpresentȱmoreȱmarkedȱeffectsȱinȱtheȱinhibitionȱorȱ
increasingȱofȱtheȱanalyzedȱbiomarkers,ȱespeciallyȱsiteȱCR2.ȱ
- 166 -
40
4
3
TBARS
FRAP
30
20
10
2
1
0
0
C2
CA1
GR4
GR3
P1
D88
D79
C2
D60
1500
CA1
GR4
GR3
P1
D88
D79
D60
GR3
P1
D88
D79
D60
GR3
P1
D88
D79
D60
12
1200
9
900
GR
GST
6
600
3
300
0
0
C2
CA1
GR4
GR3
P1
D88
D79
C2
D60
-300
300
GR4
150
120
Phagocytosis
200
GPX
CA1
-3
100
90
60
30
0
C2
CA1
GR4
GR3
P1
D88
D79
D60
0
C2
-100
2500
GR4
5
*
*
2000
4
3
1500
casts
Time to bury
CA1
1000
2
1
500
0
0
C2
C2
CA1
GR4
GR3
P1
D88
D79
D60
*
*
D79
D60
CA1
GR4
GR3
P1
D88
D79
D60
-1
80
*
DNA tail 15-d
60
*
*
40
20
0
C2
CA1
GR4
GR3
P1
D88
Figureȱ 2.ȱ Generalȱ healthȱ biomarkers:ȱ glutathioneȱ peroxidaseȱ GPXȱ
(nmol/min/mgȱ prot),ȱ glutathioneȱ transferaseȱ GSTȱ (nmol/min/mgȱ prot),ȱ
glutathioneȱ reductaseȱ GRȱ (nmol/min/mgȱ prot),ȱ thiobarbituricȱ acidȱ reactiveȱ
substancesȱ TBARSȱ (nmol/mgȱ prot),ȱ ferricȱ reducingȱ abilityȱ ofȱ plasmaȱ FRAPȱ
- 167 -
(ΐM/mg),ȱ phagocytosisȱ (zymosanȱ perȱ mgȱ proteinȱ ȉȱ 106),ȱ behaviourȱ assayȱ (s),ȱ
castsȱ assayȱ (g)ȱ andȱ Cometȱ assayȱ (%ȱ DNAȱ inȱ tail)ȱ afterȱ 7ȱ andȱ 15ȱ daysȱ ofȱ
exposure.ȱ
Comparingȱtheȱbiomarkerȱresponsesȱbetweenȱtheȱdifferentȱsitesȱusingȱtheȱ
multivariateȱ ANOSIMȱ (Primerȱ 6ȱsoftware)ȱdemonstratedȱ aȱ significantȱ effectȱofȱ
chronicȱpollutionȱonȱtheȱbiomarkerȱresponsesȱofȱArenicolaȱmarinaȱ(Figureȱ3).ȱAnȱ
aȱ prioriȱ oneȬwayȱ ANOSIMȱ comparingȱ theȱ biomarkerȱ responsesȱ ofȱ Arenicolaȱ
marinaȱexposedȱtoȱ‘clean’ȱreferenceȱsedimentsȱwithȱthoseȱexposedȱtoȱsedimentsȱ
fromȱ chronicallyȱ pollutedȱ andȱ acutelyȱ pollutedȱ (Prestige)ȱ sites,ȱ revealsȱ aȱ
significantȱ differenceȱ inȱ biomarkerȱ responseȱ betweenȱ theȱ chronicallyȱ pollutedȱ
sitesȱ andȱ theȱ otherȱ sitesȱ (Rȱ =ȱ 0.281,ȱ Pȱ =ȱ 0.001).ȱ ȱ Noȱ significantȱ differenceȱ wasȱ
observedȱbetweenȱtheȱPrestigeȱaffectedȱsitesȱandȱtheȱtwoȱreferenceȱsitesȱ(Figureȱ
3).ȱ ȱ Similarityȱ percentagesȱ (SIMPER)ȱ wereȱ usedȱ toȱ identifyȱ theȱ percentageȱ
contributionȱofȱeachȱbiomarkerȱtoȱtheȱmultivariateȱdifferencesȱbetweenȱallȱsitesȱ
(Clarkeȱ andȱ Warwick,ȱ 1994).ȱ ȱ Theȱ cometȱ assayȱ wasȱ foundȱ toȱ makeȱ theȱ largestȱ
contributionȱ toȱ theȱ observedȱ differencesȱ betweenȱ theȱ referenceȱ sitesȱ andȱ theȱ
chronicallyȱ pollutedȱ sites,ȱ representingȱ 21.62%ȱ ofȱ theȱ dissimilarityȱ betweenȱ
sites,ȱwhilstȱtheȱburrowingȱassayȱmadeȱupȱ14.10%ȱofȱtheȱdissimilarityȱbetweenȱ
theȱPrestigeȱsitesȱandȱtheȱchronicallyȱpollutedȱsites.ȱ
ȱ
- 168 -
Transform: Fourth root
Normalise
Resemblance: D1 Euclidean distance
site health
control
chronic
Prestige
2D Stress: 0.16
Figureȱ 3.ȱ Twoȱ dimensionalȱ nonȬmetricȱ multidimensionalȱ scalingȱ plotȱ ofȱ
theȱ biomarkerȱ responsesȱ forȱ eachȱ Arenicolaȱ marinaȱ specimenȱ exposedȱ toȱ
sedimentsȱ fromȱ theȱ differentȱ experimentalȱ sites:ȱ representingȱ ‘clean’ȱ controlȱ
sites;ȱ sitesȱ affectedȱ byȱ chronicȱ pollutionȱ andȱ sitesȱ affectedȱ byȱ theȱ Prestigeȱ oilȱ
spillȱ(i.e.ȱanȱacuteȱpollutionȱincident).ȱ
3.3.ȱLinkingȱchemicalsȱandȱbiomarkersȱȱ
Theȱ chemicalȱ parametersȱ measuredȱ thatȱ bestȱ accountȱ forȱ theȱ patternȱ
observedȱ inȱ theȱ biomarkerȱ responsesȱ ofȱ Arenicolaȱ marinaȱ areȱ theȱ sumȱ ofȱ PAHsȱ
andȱtheȱPCBsȱ(Figureȱ4,ȱBESTȱanalysis,ȱSpearman’sȱRankȱcorrelationȱcoefficientȱ
0.361).ȱ Aȱ strongȱ positiveȱ relationshipȱ wasȱ observedȱ betweenȱ organicȱ
contaminantsȱ(PAHsȱandȱPCBs)ȱwithȱtheȱDNAȱdamageȱ(0.89,ȱ0.79,ȱrespectively),ȱ
theȱphagocyticȱresponseȱ(0.90,ȱ0.87,ȱrespectively)ȱandȱtheȱburialȱbehaviourȱ(0.90,ȱ
0.85,ȱrespectively).ȱAȱnegativeȱcorrespondenceȱwasȱdetectedȱwithȱtheȱweightȱofȱ
- 169 -
ȱ
casts.ȱ Onȱ theȱ otherȱ hand,ȱ theȱ phaseȱ IIȱ detoxificationȱ enzymeȱ GSTȱ presentedȱ aȱ
negativeȱ relationshipȱ withȱ theȱ organicȱ contaminants,ȱ whereasȱ noȱ correlationȱ
wasȱfoundȱwithȱtheȱantioxidantsȱenzymes.ȱȱ
4.ȱDiscussionȱ
Theȱ resultsȱ demonstrateȱ howȱ aȱ combinationȱ ofȱ multiȬbiomarkersȱ withȱ
analyticalȱchemistryȱcanȱbeȱusedȱtoȱinvestigateȱtheȱtoxicityȱofȱmarineȱsediments,ȱ
enablingȱ theȱ differentiationȱ ofȱ sitesȱ showingȱ differentȱ typesȱ ofȱ contamination.ȱ
Thereȱ areȱ clearȱ relationshipsȱ inȱ sublethalȱ assaysȱ thatȱ canȱ beȱ relatedȱ toȱ theȱ
putativeȱmodeȱofȱtoxicityȱofȱtheȱcontaminants.ȱȱ
Phenanthrene,ȱ fluorene,ȱ pyreneȱ andȱ benzo[b]fluorantheneȱ areȱ theȱ
predominantȱPAHsȱinȱsedimentsȱfromȱtheȱBayȱofȱAlgecirasȱwhichȱpresentedȱtheȱ
highestȱ impactȱ inȱ theȱ organismsȱ exposed.ȱ Theseȱ compoundsȱ areȱ consideredȱ
priorityȱ pollutantsȱ byȱ theȱ USEPAȱ onȱ theȱ basisȱ ofȱ theirȱ toxicity,ȱ frequencyȱ ofȱ
occurrenceȱandȱpotentialȱforȱhumanȱexposureȱ(MingȬHoȱYu,ȱ2005).ȱItȱisȱknownȱ
thatȱ PAHsȱ affectȱ organismsȱ throughȱ toxicȱ actionȱ byȱ theȱ interferenceȱ withȱ
cellularȱ membraneȱ functionȱ andȱ inductionȱ ofȱ enzymeȱ systemsȱ associatedȱ withȱ
theȱ membraneȱ (Albers,ȱ 2003).ȱ Althoughȱ unmetabolizedȱ PAHsȱ canȱ haveȱ toxicȱ
effects,ȱaȱmajorȱconcernȱinȱanimalsȱisȱtheȱabilityȱofȱtheȱreactiveȱmetabolites,ȱsuchȱ
asȱ epoxidesȱ andȱ dihydrodiols,ȱ ofȱ someȱ PAHsȱ toȱ bindȱ toȱ cellularȱ proteinsȱ andȱ
DNAȱ(Albers,ȱ2003).ȱThisȱcanȱexplainȱtheȱelevationȱinȱDNAȱdamageȱdetectedȱinȱ
thoseȱ organismsȱ exposedȱ toȱ sedimentsȱ fromȱ theȱ Bayȱ ofȱ Algeciras.ȱ ȱ Theȱ DNAȱ
damageȱobservedȱinȱtheȱlugwormsȱlinksȱtoȱtheȱresultsȱobtainedȱinȱtheȱbehaviourȱ
andȱcastsȱassaysȱconfirmingȱtheȱeffectsȱofȱtheȱtoxicantsȱonȱtheȱmetabolismȱandȱ
conduct.ȱ Theseȱ resultsȱ areȱ inȱ accordȱ withȱ previousȱ studiesȱ whichȱ usedȱ otherȱ
marineȱ speciesȱ andȱ demonstratedȱ thatȱ cometȱ assayȱ isȱ aȱ sensitiveȱ toolȱ forȱ
monitoringȱtheȱgenotoxicȱeffectsȱofȱPAHsȱ(PérezȬCadahíaȱetȱal.,ȱ2004).ȱ
- 170 -
(a)
Tra nsform: Fourth root
Norma lise
Resembla nce: D1 Euclidea n dista nce
PAH
400
ch
ch
1.6E3
ch
ch
P
ch ch
ch
ch ch
ch
ch
ch
cont
P
P
ch P
P
contcont
P
P
cont
controlcont cont
P
control
cont
P
P
P
P
P
cont
P
P
ch
2.8E3
ch
4E3
2D Stress: 0.16
(b)
PCBs
Tra nsform: Fourth root
Norma lise
Resembla nce: D1 Euclidea n dista nce
3
ch
ch
ch
ch
12
P
P
ch ch
ch
ch ch
ch
cont
P
ch
21
ch P
ch P
P
P
cont contcont
cont P
P
P
P
P
cont cont
P
cont
cont
P
ch
ch
30
cont
P
2D Stress: 0.16
Figureȱ 4.ȱ ȱ Twoȱ dimensionalȱ nonȬmetricȱ multidimensionalȱ scalingȱ plotsȱ
forȱ biomarkerȱ responsesȱ inȱ Arenicolaȱ marinaȱ forȱ theȱ controlȱ sitesȱ (cont);ȱ
chronicallyȱ pollutedȱ sitesȱ (ch)ȱ andȱ sitesȱ affectedȱ byȱ theȱ Prestigeȱ oilȱ spillȱ (P),ȱ
overlaidȱ withȱ circlesȱ proportionalȱ inȱdiameterȱ toȱtheȱconcentrationȱatȱeachȱsiteȱ
ofȱ(a)ȱPAH’sȱandȱ(b)ȱPCB’s.ȱ
- 171 -
ȱ
Exposureȱ toȱ PAHsȱ canȱ leadȱ toȱ theȱ formationȱ ofȱ reactiveȱ oxygenȱ speciesȱ
(ROS)ȱ whichȱ canȱ alsoȱ affectȱ immuneȱ functionȱ throughȱ lipidȱ peroxidationȱ andȱ
membraneȱ destabilisationȱ ofȱ haemocytesȱ (Diȱ Guilioȱ etȱ al.,ȱ 1989,ȱ Gallowayȱ andȱ
Goven,ȱ 2007).ȱ Thisȱ concursȱ withȱ observationsȱ ofȱ phagocyticȱ activityȱ fromȱ
previousȱ workȱ withȱ invertebrateȱ speciesȱ (Komiyamaȱ etȱ al.,ȱ 2003).ȱ Theȱ
correlationȱ betweenȱ organicȱ contaminantsȱ withȱ theȱ timeȱ ofȱ burrowingȱ inȱ theȱ
sedimentȱ suggestsȱ aȱ chemosensoryȱ response,ȱ whereasȱ theȱ weightȱ ofȱ castsȱ
decreaseȱseemsȱtoȱbeȱrelatedȱtoȱtheȱfeedingȱinhibitionȱbyȱtheȱpolychaete.ȱPCBsȱ
haveȱ beenȱ linkedȱ withȱ PAHsȱ andȱ similarȱ relationshipȱ withȱ biomarkersȱ haveȱ
beenȱ established.ȱ Thisȱ pointsȱ toȱ aȱ mixtureȱ ofȱ organicȱ pollutantsȱ whichȱ areȱ
affectingȱ theȱ qualityȱ ofȱ sedimentsȱ andȱ supposeȱ anȱ environmentalȱ riskȱ inȱ theȱ
areaȱofȱtheȱBayȱofȱAlgeciras.ȱ
Theȱ metalȱ contentȱ alsoȱ showedȱ correlationsȱ withȱ theȱ analyzedȱ
biomarkers.ȱ Niȱ showsȱ aȱ strongȱ relationshipȱ withȱ DNAȱ damageȱ (0.79),ȱ theȱ
phagocyticȱ response,ȱ theȱ burialȱ behaviourȱ andȱ casts.ȱ Copperȱ showedȱ aȱ
relationshipȱwithȱtheȱ antioxidantȱactivityȱanalyzedȱinȱtheȱTBARSȱassay,ȱwhichȱ
measuresȱoneȱofȱtheȱterminalȱproductsȱinȱtheȱperoxidativeȱbreakdownȱofȱlipids.ȱ
Theȱ inductionȱ ofȱ lipidȱ peroxidationȱ byȱ copperȱ isȱ wellȬknownȱ inȱ otherȱ
invertebratesȱ (Viarengo,ȱ 1989)ȱ andȱ theȱ TBARSȱ activityȱ hasȱ beenȱ previouslyȱ
associatedȱ withȱ Cuȱ (Quiniouȱ etȱ al.,ȱ 2007).ȱ Weakerȱ correlationsȱ wereȱ alsoȱ
detectedȱ amongȱ antioxidantȱ biomarkers:ȱ FRAPȱ associatesȱ withȱ GSTȱ andȱ GRȱ
whereasȱ TBARSȱ linksȱ withȱ GRȱ activity;ȱ howeverȱ noȱ relationshipsȱ wereȱ
observedȱforȱtheȱGPXȱantioxidantȱenzymeȱwithȱotherȱbiomarkersȱorȱpollutants.ȱ
Thisȱsuggestsȱcombinedȱregulationȱofȱtheseȱresponses.ȱ
AfterȱtheȱPrestigeȱoilȱspillȱinvestigationsȱhaveȱaddressedȱinȱdeterminingȱ
theȱ biologicalȱ effectsȱ andȱ environmentalȱ statusȱ afterȱ theȱ accidentȱ byȱ followingȱ
- 172 -
singleȱ linesȱ ofȱ evidence,ȱ suchȱ asȱ chemicalȱ analysesȱ [(CSIC,ȱ 2003;ȱ Francoȱ etȱ al.,ȱ
2006;ȱGonzálezȱetȱal.,ȱ2006)ȱandȱtoxicityȱincludingȱbiomarkersȱ(MariñoȬBalsaȱetȱ
al.,ȱ2003;ȱMartínezȬGómezȱetȱal.,ȱ2006;ȱMarigómezȱetȱal.,ȱ2006)ȱhoweverȱlittleȱhasȱ
beenȱdoneȱwithȱtheȱcombinationȱofȱbothȱfieldsȱ(MoralesȬCasellesȱ2006;ȱMoralesȬ
Caselles,ȱaccepted2).ȱInȱadditionȱmostȱofȱtheȱbiologicalȱassessmentsȱcarriedȱoutȱ
towardsȱtheȱPrestigeȱhaveȱbeenȱconductedȱinȱsitu,ȱunderȱfield;ȱunderȱcontrolledȱ
conditionsȱ inȱ theȱ laboratory,ȱ itȱ isȱ relativelyȱ straightforwardȱ toȱ standardiseȱ
biomarkerȱ assaysȱ andȱ toȱ regulateȱ theȱ chemicalȱ exposuresȱ thatȱ organismsȱ
receive,ȱ soȱ thatȱ causeȬeffectȱ andȱ indeed,ȱ exposureȬrelationships,ȱ canȱ beȱ
establishedȱ(Astleyȱetȱal.,ȱ1999).ȱȱ
LessȱattentionȱthanȱtheȱplayedȱinȱtheȱPrestigeȱaccidentȱhasȱbeenȱfocusedȱ
towardsȱ theȱ chronicȱ pollutionȱ ofȱ theȱ Bayȱ ofȱ Algecirasȱ inȱ recentȱ years.ȱ Theȱ
methodologyȱ employedȱ inȱ thisȱ studyȱ hasȱ shownȱ howȱ thisȱ areaȱ isȱ muchȱ moreȱ
pollutedȱandȱbiologicalȱeffectsȱonȱkeyȱinvertebratesȱexposedȱtoȱtheirȱsedimentsȱ
haveȱ beenȱ demonstrated.ȱ Otherȱ studiesȱ basedȱ onȱ theȱ Exxonȱ Valdezȱ oilȱ spillȱ
suggestedȱaȱrecoveryȱyearsȱafterȱtheȱepisodeȱandȱtheȱprevelanceȱofȱtheȱchronicȱ
pollutionȱdueȱtoȱtheȱhumanȱandȱindustrialȱactivitiesȱ(BoehmȱandȱPage,ȱ2007).ȱ
Researchȱ methodsȱ basedȱ inȱ biomarkersȱ haveȱ notȱ yetȱ beenȱ validatedȱ forȱ
applicationȱasȱaȱmonitoringȱmethodȱforȱoilȱspillsȱinȱaȱsystematicȱfashionȱ(Boehmȱ
andȱ Page,ȱ 2007).ȱ Theȱ selectedȱ biomarkersȱ analyzedȱ showsȱ importantȱ
relationshipsȱ withȱ pollutantsȱ andȱ theȱ proposedȱ methodologyȱ whichȱ integratesȱ
differentȱvariablesȱasȱpartȱofȱaȱweightȱofȱevidenceȱapproachȱhasȱdemonstratedȱ
toȱbeȱaȱsuitableȱtoolȱinȱoilȱspillȱassessmentsȱcomingȱfromȱdifferentȱsources.ȱ
Theȱ bioassayȱ performedȱ withȱ anȱ invertebrateȱ especieȱ thatȱ livesȱ inȱ theȱ
sediment,ȱ theȱ polychaeteȱ Arenicolaȱ marina,ȱ resultsȱ toȱ beȱ relativelyȱ simple,ȱ
rapid,ȱ economicȱ andȱ appropiateȱ toȱ testȱ theȱ environmentalȱ statusȱ ofȱ anȱ oilȬ
- 173 -
contamiantedȱ sediment.ȱ Thisȱ organismȱ hasȱ beenȱ oftenȱ usedȱ onȱ monitoringȱ
programs;ȱ however,ȱ theȱ applicationȱ ofȱ suitesȱ ofȱ assaysȱ andȱ chemistryȱ areȱ
illustratedȱhereȱforȱtheȱfirstȱtimeȱtoȱthisȱpolychaete.ȱTheseȱhaveȱtheȱpotentialȱtoȱ
chartȱrecoveryȱafterȱoilȱspillsȱandȱhaveȱallowedȱtheȱdifferentiationȱofȱsitesȱwithȱ
differentȱtypesȱofȱcontamination:ȱtheȱacutelyȱ(Prestige)ȱandȱchronicallyȱ(Bayȱofȱ
Algeciras)ȱ affectedȱ areasȱ andȱ theȱ referenceȱ sites.ȱ Theȱ Cometȱ assayȱ hasȱ
demonstratedȱ toȱ beȱ theȱ mostȱ sensitiveȱ ofȱ theȱ studiedȱ endpoints,ȱ showingȱ
importantȱ correlationsȱ withȱ theȱ mainȱ contaminants.ȱ Authorsȱ considerȱ theȱ
suitabilityȱ ofȱ theȱ usingȱ theseȱ toolsȱ inȱ assessingȱ environmentalȱ qualityȱ
assessmentȱandȱtoȱchartȱrecoveryȱinȱareasȱaffectedȱbyȱoilȱspills.ȱ
5.ȱAcknowledgmentsȱ
Supportedȱ byȱ Ministryȱ ofȱ Educationȱ andȱ Scienceȱ grantȱ VEM2003Ȭ20563,ȱ
EUȱ FP7ȱ FACEȬiTȱ grantȱ andȱ byȱ CISȱ fundedȱ byȱ theȱ Ministryȱ ofȱ Environment.ȱ
Carmenȱ MoralesȬCasellesȱ thanksȱ theȱ Ministryȱ ofȱ Educationȱ andȱ Scienceȱ forȱ
fundingȱherȱresearchȱfellowshipȱ(FPU).ȱWeȱareȱgratefulȱforȱtheȱsupportȱandȱhelpȱ
ofȱtheȱmembersȱofȱtheȱCISȱandȱtheȱSchoolȱofȱBiologicalȱSciencesȱ(Universityȱofȱ
Plymouth).ȱSpecialȱthanksȱareȱgivenȱtoȱChristopherȱPookȱandȱTrevorȱWorsey.ȱȱ
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ȱ
- 180 -
ȱ
Capítuloȱ4.ȱ
Evaluaciónȱdeȱefectosȱsubletalesȱinȱsituȱ
Laȱ calidadȱ deȱ losȱ ecosistemasȱ costerosȱ seȱ haȱ evaluadoȱ tradicionalmenteȱ
siguiendoȱ unaȱ metodologíaȱ clásicaȱ queȱ incluyeȱ elȱ muestreoȱ deȱ especiesȱ
autóctonas,ȱelȱestudioȱdeȱlasȱcomunidadesȱbentónicasȱoȱlaȱdeterminaciónȱdeȱlaȱ
toxicidadȱ bajoȱ condicionesȱ deȱ laboratorioȱ (Burtonȱ Jrȱ etȱ al.,ȱ 2005).ȱ Estaȱ serieȱ deȱ
estudiosȱresultanȱmuyȱútilesȱyȱenȱocasionesȱesenciales,ȱaunqueȱpresentanȱciertasȱ
limitacionesȱ(Tablaȱ1)ȱ(ej.ȱChapmanȱetȱal.,ȱ1992;ȱBurtonȱetȱal.,ȱ1996;ȱGrotheȱetȱal.,ȱ
1996).ȱ Elȱ estudioȱ deȱ laȱ toxicidadȱ inȱ situȱ medianteȱ organismosȱ enȱ jaulasȱ
proporcionaȱlaȱinformaciónȱqueȱfaltaȱenȱlosȱestudiosȱtradicionalesȱ(BurtonȱJrȱetȱ
al.,ȱ2005).
Laȱventajaȱprincipalȱdeȱlaȱexposiciónȱdeȱlosȱorganismosȱaȱlosȱsedimentosȱ
deȱestudioȱmedianteȱelȱusoȱdeȱjaulasȱradicaȱenȱlaȱobtenciónȱdeȱunaȱinformaciónȱ
sobreȱlaȱtoxicidadȱdeȱlosȱsedimentosȱevaluadaȱbajoȱcondicionesȱnoȱcontroladas,ȱ
queȱ noȱ sóloȱ permitenȱ estudiarȱ laȱ toxicidadȱ producidaȱ porȱ losȱ contaminantesȱ
presentesȱenȱelȱsedimento,ȱsinoȱqueȱpermiteȱevaluarȱelȱefectoȱproducidoȱporȱlasȱ
variacionesȱ fisicoquímicasȱ aȱ lasȱ queȱ seȱ veȱ sometidoȱ elȱ medioȱ yȱ queȱ puedenȱ
afectarȱaȱlaȱdisponibilidadȱdeȱlosȱcontaminantesȱ(MartínȬDíaz,ȱ2004).ȱAȱpesarȱdeȱ
lasȱdificultadesȱdeȱlaȱrealizaciónȱdeȱaproximacionesȱinȱsituȱestasȱpermitenȱllevarȱ
aȱ caboȱ unaȱ evaluaciónȱ másȱ realistaȱ deȱ losȱ efectosȱ biológicosȱ producidosȱ porȱ
contaminantesȱ delȱ medio.ȱ Ademásȱ esteȱ tipoȱ deȱ estudiosȱ sonȱ capacesȱ deȱ
ȱ
Ȭȱ181ȱȬȱ
Capítuloȱ4
identificarȱ fuentesȱ deȱ poluciónȱ ajenasȱ alȱ sedimentoȱ peroȱ queȱ puedenȱ serȱ laȱ
causaȱdeȱalteracionesȱbentónicasȱenȱelȱmedio,ȱyȱnoȱseȱpuedenȱdetectarȱmedianteȱ
laȱconsecuciónȱdeȱexperimentosȱdeȱlaboratorioȱaislados.
Tablaȱ 4.1.ȱ Ventajasȱ yȱ limitacionesȱ deȱ losȱ métodosȱ tradicionalesȱ deȱ
evaluaciónȱdeȱlaȱcalidadȱdeȱecosistemasȱcosterosȱ(segúnȱBurtonȱJrȱetȱal.,ȱ2005)ȱ
Metodologíaȱ
Ventajasȱ
Limitacionesȱprincipalesȱ
GuíasȱQuímicasȱ
Fácilesȱyȱestandarizadas.ȱ
Extrapolacionesȱaȱcampoȱyȱotrasȱ
especies.ȱEspecíficasȱdeȱlaȱzona.ȱ
Basadasȱenȱestudiosȱdeȱ
laboratorio.ȱȱ
Biotaȱautóctonaȱ
Ampliamenteȱutilizada.ȱ
Reflejaȱlosȱefectos.ȱInterésȱ
público.ȱ
Variabilidad.ȱEfectosȱindirectos.ȱ
Causasȱnaturalesȱdeȱestrésȱqueȱ
dificultanȱinterpretación.ȱ
Ensayosȱdeȱtoxicidadȱenȱ
laboratorioȱ
Ampliamenteȱutilizados.ȱ
Integraȱefectosȱdeȱ
contaminantesȱaȱcortoȱplazo.ȱ
Estandarizados.ȱȱ
Lasȱcondicionesȱnoȱsonȱigualesȱaȱ
lasȱnaturalesȱenȱȱcampo.ȱȱ
Bioacumulaciónȱ
Exposicionesȱrealistas.ȱÚtilesȱ
paraȱelaborarȱmodelosȱdeȱ
redesȱtróficas.ȱMedidasȱaȱ
largoȱplazo.ȱUtilizadasȱ
tradicionalmente.ȱ
Metabolismoȱyȱexcreciónȱdeȱ
algunosȱquímicos.ȱLaȱ
aclimatación,ȱȱadaptaciónȱyȱlosȱ
metalesȱesencialesȱpuedenȱ
confundirȱlaȱinterpretaciónȱdeȱlosȱ
efectosȱobservados.ȱȱȱ
Enȱelȱcapítuloȱ4ȱseȱpresentanȱcuatroȱartículos,ȱdeȱlosȱcualesȱelȱtrabajoȱX,ȱ
XIȱ yȱ XIIȱ muestranȱ losȱ resultadosȱ deȱ medidasȱ deȱ biomarcadoresȱ trasȱ
exposicionesȱenȱcampoȱdeȱdosȱespeciesȱdeȱinvertebrados.ȱEnȱprimerȱlugar,ȱenȱelȱ
trabajoȱ Xȱ seȱ recogenȱ losȱ resultadosȱ deȱ laȱ instalaciónȱ deȱ jaulasȱ enȱ puntosȱ deȱ
estudioȱ delȱ Golfoȱ deȱ Cádizȱ yȱ laȱ Costaȱ deȱ Galicia.ȱ Enȱ esteȱ estudioȱ seȱ
seleccionaronȱ dosȱ especiesȱ deȱ invertebradosȱ marinosȱ conȱ hábitosȱ distintosȱ deȱ
alimentación,ȱelȱcangrejoȱCarcinusȱmaenasȱyȱlaȱalmejaȱRuditapesȱPhilippinarum.ȱSeȱ
realizóȱunaȱexposiciónȱdeȱ28ȱdíasȱtrasȱlosȱcualesȱseȱllevaronȱaȱcaboȱmedidasȱdeȱ
biomarcadoresȱ deȱ exposiciónȱ (actividadȱ EROD,ȱ GPX,ȱ GSTȱ yȱ GR)ȱ yȱ unȱ
biomarcadorȱ deȱ efectoȱ (histopatología).ȱ Esteȱ experimentoȱ seȱ reprodujoȱ bajoȱ
- 182 -
Evaluaciónȱdeȱefectosȱsubletalesȱinȱsituȱ
condicionesȱ deȱ laboratorioȱ comoȱ seȱ explicaȱ enȱ elȱ Capítuloȱ 3.ȱ Alȱ compararȱ losȱ
resultadosȱ obtenidosȱ enȱ esteȱ experimentoȱ inȱ situȱ conȱ elȱ descritoȱ enȱ elȱ capítuloȱ
anteriorȱ desarrolladoȱ bajoȱ condicionesȱ deȱ laboratorio,ȱ comprobamosȱ comoȱ laȱ
inducciónȱ deȱ losȱ biomarcadoresȱ deȱ exposiciónȱ seȱ dabaȱ mayormenteȱ enȱ losȱ
organismosȱ expuestosȱ enȱ jaulasȱ ancladasȱ enȱ laȱ Bahíaȱ deȱ CormeȬLaxe,ȱ enȱ lugarȱ
deȱdarseȱenȱlosȱindividuosȱlocalizadosȱenȱlaȱzonaȱdeȱAlgeciras,ȱmientrasȱqueȱlosȱ
dañosȱ histopatológicosȱ “seȱ suavizaban”ȱ enȱ lasȱ exposicionesȱ enȱ campo.ȱ Esteȱ
hechoȱ puedeȱ significarȱ variasȱ cosas:ȱ a)ȱ que,ȱ enȱ general,ȱ losȱ efectosȱ biológicosȱ
bajoȱ condicionesȱ deȱ campoȱ sonȱ menoresȱ queȱ enȱ laboratorio,ȱ debidoȱ aȱ laȱ
renovaciónȱ continuaȱ deȱ aguaȱ queȱ disminuyeȱ laȱ biodisponibilidadȱ deȱ losȱ
contaminantes;ȱ b)ȱ queȱ lasȱ desembocadurasȱ deȱ losȱ ríosȱ Guadarranqueȱ yȱ
PalmonesȱenȱAlgeciras,ȱsujetosȱaȱunȱimportanteȱrégimenȱmareal,ȱsuponganȱunaȱ
renovaciónȱ mayorȱ deȱ agua;ȱ c)ȱ queȱ dadaȱ laȱ mezclaȱ complejaȱ deȱ contaminantesȱ
noȱmedidosȱenȱesteȱestudioȱpresentesȱenȱlosȱsedimentosȱyȱposiblementeȱtambiénȱ
enȱlasȱaguasȱdeȱlaȱBahíaȱdeȱAlgecirasȱseȱdenȱfenómenosȱdeȱsolapamientoȱentreȱ
laȱ inducción/inhibiciónȱ deȱ losȱ biomarcadores;ȱ d)ȱ queȱ losȱ factoresȱ abióticosȱ
afectenȱsignificativamenteȱaȱlaȱinducciónȱdeȱbiomarcadores,ȱprincipalmenteȱenȱ
laȱdesembocaduraȱdeȱlosȱríosȱenȱAlgeciras;ȱe)ȱqueȱlaȱaltaȱpresenciaȱdeȱbateasȱenȱ
laȱ Bahíaȱ deȱ CormeȬLaxeȱ supongaȱ unȱ estrésȱ aȱ laȱ biotaȱ debidoȱ aȱ posiblesȱ
sustanciasȱcontaminantesȱenȱpiensosȱoȱaȱlaȱaltaȱcargaȱorgánicaȱȱdelȱagua,ȱyȱqueȱ
expliquenȱ porȱ tanto,ȱ laȱ notableȱ inducciónȱ deȱ losȱ biomarcadoresȱ deȱ exposiciónȱ
observadosȱ bajoȱ condicionesȱ deȱ campoȱ yȱ queȱ noȱ fueronȱ vistosȱ trasȱ losȱ
experimentosȱ deȱ laboratorio.ȱ Paraȱ completarȱ esteȱ estudioȱ seȱ realizóȱ unaȱ
evaluaciónȱ deȱ laȱ cinéticaȱ deȱ variosȱ biomarcadoresȱ enȱ laȱ almejaȱ Ruditapesȱ
Philippinarum,ȱtalȱyȱcomoȱseȱmuestraȱenȱelȱtrabajoȱXI,ȱaclarandoȱdeȱmaneraȱmásȱ
efectivaȱ lasȱ posiblesȱ fuentesȱ deȱestrés;ȱasimismo,ȱseȱrealizóȱunȱestudioȱcinéticoȱ
deȱlasȱenzimasȱimplicadasȱenȱlaȱdetoxificaciónȱdeȱPAHȱenȱelȱcangrejoȱCarcinusȱ
maenasȱ(trabajoȱXII).ȱ
Ȭ 183ȱȬ
Capítuloȱ4
ȱ
ȱ
ȱ
ȱ
boya
ȱ
aguaȱ
ȱ
ȱ
ȱ
ȱ
ȱ
pesosȱ
ȱ
ȱ
sedimentoȱ
anclaje
ȱ
Figuraȱ 4.1.ȱ Esquemaȱ deȱ anclajeȱ yȱ utilizaciónȱ deȱ jaulasȱ bentónicasȱ
utilizadasȱenȱlosȱbioensayosȱenȱcampo.ȱ
Elȱúltimoȱtrabajoȱdeȱesteȱcapítulo,ȱXIII,ȱincluyeȱunaȱlíneaȱdeȱestudioȱajenaȱ
aȱ losȱ bioensayosȱ peroȱ deȱ granȱ importancia.ȱ Enȱ esteȱ trabajoȱ seȱ evalúaȱ laȱ
alteraciónȱdeȱlaȱfaunaȱbentónicaȱdeȱlasȱáreasȱdeȱestudioȱconȱelȱfinȱdeȱrelacionarȱ
losȱ efectosȱ deȱ laȱ biotaȱ autóctonaȱ conȱ losȱ contaminantesȱ presentesȱ enȱ losȱ
sedimentos.ȱ Deȱ estaȱ maneraȱ seȱ cubreȱ unaȱ deȱ lasȱ líneasȱ clásicasȱ dentroȱ deȱ losȱ
estudiosȱ deȱ calidadȱ ambientalȱ deȱ losȱ sedimentos.ȱ Enȱ esteȱ trabajoȱ seȱ observaȱ
comoȱ inicialmenteȱ laȱ macrofaunaȱ bentónicaȱ deȱ lasȱ costasȱ gallegasȱ seȱ vioȱ
afectadaȱ porȱ elȱ vertidoȱ delȱ petroleroȱ Prestige,ȱ aunqueȱ seȱ describeȱ unaȱ
recuperaciónȱimportanteȱqueȱhaȱsidoȱfinalmenteȱcomparadaȱconȱlaȱsituaciónȱdeȱ
laȱ biotaȱ deȱ laȱ zonasȱ deȱ estudioȱ localizadasȱ elȱ laȱ Bahíaȱ deȱ Algeciras,ȱ dondeȱ elȱ
- 184 -
Evaluaciónȱdeȱefectosȱsubletalesȱinȱsituȱ
impactoȱ delȱ conjuntoȱ deȱ fuentesȱ contaminantesȱ suponeȱ unȱ impactoȱ ambientalȱ
muchoȱmayor.ȱ
Bibliografíaȱ
BurtonȱJr.,ȱG.A.,ȱGreenberg,ȱM.S.,ȱRowland,ȱ
C.D.,ȱ Irvine,ȱ C.A.,ȱ Lavoie,ȱ D.R.,ȱ Brooker,ȱ
J.A.,ȱ Delia,ȱ L.M.,ȱ Raymer,ȱ F.N.,ȱ
McWilliam.ȱ2005.ȱInȱsituȱexposuresȱusingȱ
cagedȱ organisms:ȱ aȱ multiȬcompartmentȱ
approachȱ toȱ detectȱ aquaticȱ toxicityȱ andȱ
bioaccumulation.ȱ Environ.ȱ Pollut.ȱ 134,ȱ
133–144.ȱ
Burton,ȱ G.A.,ȱ Hickey,ȱ C.W.,ȱ DeWitt,ȱ T.H.,ȱ
Roper,ȱ D.S.,ȱ Morrisey,ȱ D.J.,ȱ Nipper,ȱ M.,ȱ
1996.ȱ Inȱ situȱ toxicityȱ testing:ȱ teasingȱ outȱ
environmentalȱstressors.ȱSETACȱNewsȱ16ȱ
(5),ȱ20–22.ȱ
Grothe,ȱ D.R.,ȱ Dickson,ȱ K.L.,ȱ ReedȬJudkins,ȱ
D.K.,ȱ 1996.ȱ Wholeȱ Effluentȱ Toxicityȱ
Testing:ȱ Anȱ Evaluationȱ ofȱ Methodsȱ andȱ
Predictionȱ ofȱ Receivingȱ Systemȱ Impacts.ȱ
SETACȱPress,ȱBocaȱRaton,ȱFL,ȱpp.ȱ346.ȱ
MartínȬDíaz,ȱ L.ȱ 2004.ȱ Determinaciónȱ deȱ laȱ
calidadȱ ambientalȱ deȱ sistemasȱ litoralesȱ yȱ
deȱ estuarioȱ deȱ laȱ penínsulaȱ ibéricaȱ
utilizandoȱ ensayosȱ deȱ campoȱ yȱ
laboratorio.ȱTesisȱDoctoral.ȱȱ
ȱ
ȱ
Chapman,ȱ P.M.,ȱ Power,ȱ E.A.,ȱ Burtonȱ Jr.,ȱ
G.A.,ȱ 1992.ȱ Integratedassessmentsȱ inȱ
aquaticȱ ecosystems.ȱ In:ȱ Burton,ȱ G.A.ȱ
(Ed.),Sedimentȱ Toxicityȱ Assessment.ȱ
LewisȱPublishers,ȱBocaȱRaton,ȱFL.ȱ
ȱ
Ȭ 185ȱȬ
ȱ
ȱ
ȱ
ȱ
Ȭȱ186ȱȬȱ
Sublethalȱresponsesȱinȱcagedȱorganismsȱexposedȱtoȱsedimentsȱ
affectedȱbyȱoilȱspillsȱ
CarmenȱMoralesȬCaselles1,2,*,ȱM.ȱLauraȱMartínȬDíaz1,2,ȱInmaculadaȱRiba1,2,ȱ
CarmenȱSarasquete1,ȱT.ȱÁngelȱDelVallsȱ1,2ȱ
1
IȱUnidadȱAsociadaȱdeȱCalidadȱAmbientalȱyȱPatologíaȱ(CSICȱ&ȱUCA)ȱinstitutoȱ
deȱCienciasȱMarinasȱdeȱAndalucíaȱ(ICMANȬCSIC),ȱAvda.ȱRepúblicaȱSaharauiȱs/n,ȱ
PuertoȱRealȱ11510,ȱCádiz,ȱSpainȱ
2
UNESCOȱUNITWIN/WiCop,ȱFacultadȱdeȱCienciasȱdelȱMarȱyȱAmbientales,ȱ
UniversidadȱdeȱCádiz,ȱPolígonoȱRíoȱSanȱPedroȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱSpain.ȱ
Abstractȱ
Theȱ currentȱ studyȱ wasȱ performedȱ toȱ determineȱ sublethalȱ responsesȱ inȱ
twoȱ invertebrateȱ speciesȱ byȱ usingȱ fieldȱ deploymentsȱ inȱ areasȱ affectedȱ byȱ oilȱ
spills,ȱ acuteȱ inȱ theȱ Galicianȱ Coastȱ (NNW,ȱ Spain)ȱ andȱ chronicȱ inȱ theȱ Bayȱ ofȱ
Algecirasȱ(SSW,ȱSpain).ȱTheȱorganismsȱemployedȱwereȱtheȱcrabȱCarcinusȱmaenasȱ
andȱ theȱ clamȱ Ruditapesȱ philippinarum.,ȱ andȱ duringȱ 28ȱ daysȱ theȱ animalsȱ wereȱ
exposedȱ inȱ cagesȱ underȱ fieldȱ conditionsȱ toȱ contaminatedȱ sediments.ȱ Differentȱ
biomarkersȱ ofȱ exposureȱ wereȱ determinedȱ afterȱ 28Ȭdayȱ exposure:ȱ
ethoxyresorufinȱ OȬdeethylaseȱ (EROD),ȱ phaseȱ Iȱ detoxificationȱ enzyme,ȱ
glutathioneȬSȬtransferaseȱ (GST)ȱ phaseȱ IIȱ detoxificationȱ enzymeȱ butȱ alsoȱ
implicatedȱ inȱ oxidativeȱ stressȱ events,ȱ glutathioneȱ peroxidaseȱ (GPX)ȱ andȱ
glutathioneȱ reductaseȱ (GR),ȱ bothȱ antioxidantȱ enzymes.ȱ Inȱ additionȱ
histopathologicalȱ effectsȱ inȱ targetȱ tissuesȱ ofȱ theȱ deployedȱ organismsȱ wereȱ
evaluated.ȱ Biomarkersȱ measurementsȱ wereȱ linkedȱ withȱ theȱ concentrationȱ ofȱ
chemicalsȱ inȱ theȱ sedimentsȱ inȱ orderȱ toȱ elucidateȱ theȱ type,ȱ sourceȱ andȱ
bioavailabilityȱ ofȱ contaminantsȱ producingȱ adverseȱ effectsȱ inȱ theȱ bioindicatorȱ
species.ȱResultsȱobtainedȱinȱtheȱpresentȱstudyȱhaveȱshownȱhowȱtheȱapplicationȱ
ofȱ theȱ selectedȱ batteryȱ ofȱ biomarkersȱ underȱ fieldȱ bioassaysȱ allowsȱ identifyingȱ
alternativeȱ sourcesȱ ofȱ stressȱ thatȱ areȱ notȱ possibleȱ toȱ observeȱ inȱ laboratoryȱ
experiments.ȱ
Keywords:ȱbiomarker,ȱhistopathology,ȱinvertebrate,ȱtoxicity,ȱcontaminantsȱȱ
ȱChemosphereȱ(enviado)
- 187 -
1.ȱIntroductionȱ
Measurementsȱ ofȱ anȱ organisms’ȱ responseȱ toȱ aȱ pollutantȱ atȱ theȱ
biochemicalȱorȱphysiologicalȱlevelȱcanȱdetectȱmoreȱquicklyȱandȱspecificallyȱtheȱ
presenceȱ ofȱ toxicȱ compounds,ȱ allowingȱ earlierȱ identificationȱ ofȱ change,ȱ beforeȱ
deleteriousȱ effectsȱ reachȱ higherȱ organizationȱ levelsȱ (Montserratȱ etȱ al.,ȱ 2003).ȱ
Overȱ theȱ pastȱ decade,ȱ biomarkersȱ haveȱ beenȱ usedȱ increasinglyȱ asȱ diagnosticȱ
toolsȱ toȱ investigateȱ sublethalȱ effectsȱ ofȱ toxicȱ exposureȱ andȱ toȱ elucidateȱ theȱ
variousȱmodesȱofȱactionȱofȱxenobioticsȱ(DeȱCoenȱetȱal.,ȱ2000).ȱTheȱapplicationȱofȱ
biomarkersȱunderȱfieldȱconditionsȱhasȱbeenȱproposedȱbyȱmanyȱauthorsȱinȱorderȱ
toȱ assessȱ chronicȱ responsesȱ inȱ aquaticȱ populationsȱ exposedȱ underȱ
environmentalȱ realisticȱ conditionsȱ (Suter,ȱ 1993;ȱ Depledgeȱ andȱ Fossi,ȱ 1994;ȱ Deȱ
Coenȱ etȱ al.,ȱ 2006;ȱ MartínȬDíazȱ etȱ al.,ȱ inȱ press).ȱ Fieldȱ studiesȱ poseȱ farȱ greaterȱ
difficultiesȱdueȱtoȱtheȱcomplexȱandȱfluctuatingȱnatureȱofȱtheȱenvironment,ȱandȱ
interactionsȱ amongȱ organismsȱ withinȱ ecologicalȱ communities.ȱ ȱ Theyȱ addressȱ
theȱ integratedȱ impactȱ ofȱ anthropogenicȱ andȱ environmentalȱ stressors.ȱ Dataȱ
collectedȱ inȱ fieldȱ studiesȱ mayȱ beȱ muchȱ harderȱ toȱ interpretȱ thanȱ dataȱ fromȱ
controlledȱ laboratoryȱ experimentsȱ (Astleyȱetȱalȱ1999).ȱ Itȱisȱalsoȱhighlightedȱtheȱ
potentialȱ useȱ ofȱ inȱ situȱ assaysȱ toȱ determineȱ theȱ toxicityȱ ofȱ sedimentsȱ usingȱ
differentȱ approachesȱ includingȱ cagingȱ animalsȱ (MartínȬDíaz,ȱ 2004).ȱ Sedimentȱ
toxicityȱ bioassaysȱ carriedȱ outȱ inȱ theȱ laboratoryȱ areȱ performedȱ underȱ strictlyȱ
controlledȱ parametersȱ andȱ thusȱ doȱ notȱ reflectȱ theȱ variabilityȱ inȱ exposureȱ thatȱ
mayȱoccurȱinȱnaturalȱsystems.ȱThisȱgivesȱriseȱtoȱuncertaintyȱinȱtheȱextrapolationȱ
ofȱ laboratoryȬbasedȱ testȱ resultsȱ toȱ naturalȱ environmentsȱ inȱ sedimentȱ riskȱ
assessmentȱ(Sibleyȱetȱal,ȱ1999).ȱ
Inȱorderȱtoȱevaluateȱtheȱexposureȱofȱcontaminantsȱrelatedȱtoȱoilȱspillsȱinȱ
theȱ organismsȱ usingȱ inȱ situȱ deploymentsȱ andȱ aȱ biomarkerȱ approach,ȱ theȱ
objectivesȱwereȱ asȱ follows:ȱ (1)ȱ toȱ testȱtheȱfeasibilityȱofȱaȱsuiteȱofȱbiomarkersȱtoȱ
assessȱ theȱ oilȬcontaminatedȱ sedimentȱ qualityȱ (2)ȱ toȱ identifyȱ theȱ contaminantsȱ
- 188 -
boundȱ toȱ sedimentsȱ whichȱ produceȱ theȱ sublethalȱ effectsȱ inȱ theȱ organismsȱ
exposedȱ (3)ȱ toȱ determineȱ theȱ differencesȱ betweenȱ theȱ biologicalȱ responsesȱ
associatedȱ withȱ aȱ deploymentȱ toȱ acutelyȱ andȱ chronicallyȱ oilȱ contaminatedȱ
sediments.ȱȱ
Aȱbatteryȱofȱbiomarkersȱofȱexposureȱofȱearlyȱbiologicalȱeffectsȱwasȱusedȱ
inȱorderȱtoȱassessȱsedimentȱtoxicityȱofȱtwoȱcoastalȱareasȱaffectedȱbyȱoilȱspills,ȱtheȱ
GalicianȱCoast,ȱacutelyȱimpactedȱbyȱtheȱsinkingȱofȱtheȱtankerȱPrestigeȱ(2002)ȱandȱ
theȱ Bayȱ ofȱ Algecirasȱ chronicallyȱ affectedȱ byȱ severalȱ spills.ȱ Twoȱ invertebrateȱ
speciesȱwithȱdifferentȱfeedingȱhabitsȱwereȱselectedȱtoȱcarryȱoutȱtheȱassessment,ȱ
theȱ crabȱ Carcinusȱ maenasȱ andȱ theȱ clamȱ Ruditapesȱ philippinarum.ȱ Theȱ suiteȱ ofȱ
biomarkersȱ employedȱ was:ȱ Ethoxyresorufinȱ OȬdeethylaseȱ (EROD),ȱ phaseȱ Iȱ
detoxificationȱenzymeȱimplicatedȱinȱmonooxygenationȱreactionsȱofȱdioxinsȱandȱ
PAHs;ȱ glutathioneȬSȬtransferaseȱ(GST)ȱphaseȱIIȱdetoxificationȱenzymeȱbutȱalsoȱ
implicatedȱ inȱ oxidativeȱ stressȱ events;ȱ glutathioneȱ peroxidaseȱ (GPX)ȱ andȱ
glutathioneȱreductaseȱ(GR),ȱantioxidantȱenzymesȱ(MartínȬDíazȱetȱal.,ȱinȱpress).ȱ
Histopathologicalȱalterationsȱinȱtargetȱtissuesȱwereȱalsoȱevaluatedȱbecauseȱitȱhasȱ
shownȱtoȱbeȱresponsiveȱandȱsensitiveȱtoȱaȱwideȱrangeȱofȱcontaminantsȱandȱhaveȱ
beenȱdevelopedȱandȱrecommendedȱasȱbiomarkersȱforȱmonitoringȱtheȱeffectsȱofȱ
pollutionȱ(Au,ȱ2004).ȱ
2.ȱMaterialsȱandȱmethodsȱ
2.1.ȱSitesȱdescriptionȱ
TheȱstudyȱwasȱperformedȱinȱtwoȱareasȱofȱtheȱSpanishȱCoast:ȱtheȱGalicianȱ
Coastȱ(NWȱSpain)ȱwasȱchosenȱasȱitȱwasȱaffectedȱbyȱtheȱPrestigeȱoilȱspillȱinȱ2002,ȱ
whatȱsupposedȱoneȱofȱtheȱmajorȱecologicalȱcatastrophesȱofȱtheȱIberianȱpeninsulaȱ
affectingȱ moreȱ thanȱ 1000ȱ kmȱ ofȱ coast;ȱ inȱ thisȱ sense,ȱ theȱ selectedȱ sitesȱ wereȱ
locatedȱinȱtheȱCiesȱIslandȱinȱtheȱAtlanticȱIslandȱNationalȱParkȱandȱinȱtheȱBayȱofȱ
CormeȬLaxe;ȱ theȱ secondȱ areaȱ ofȱ studyȱ wasȱ theȱ mouthȱ ofȱ theȱ Riverȱ Palmonesȱ
- 189 -
andȱ Guadarranqueȱ inȱ theȱ Bayȱ ofȱ Algecirasȱ (Sȱ Spain);ȱ thisȱ placeȱ wasȱ selectedȱ
becauseȱisȱhighlyȱindustrializedȱandȱthereȱareȱaȱlargeȱnumberȱofȱpetrochemicalȱ
activitiesȱ whichȱ compriseȱ severalȱ accidentalȱ oilȱ spills.ȱ Aȱ referenceȱ siteȱ wasȱ
selectedȱinȱaȱcleanȱareaȱinȱtheȱBayȱofȱCádizȱ(SȱSpain)ȱ(Ribaȱetȱal.,ȱ2004).ȱTheȱ10ȱ
selectedȱ studyȱ sitesȱ areȱ shownȱ inȱ Figureȱ 1:ȱ A,ȱ B,ȱ Cȱ locatedȱ inȱ Cíes,ȱ D,ȱ E,ȱ Fȱ inȱ
CormeȬLaxe,ȱ GR3,ȱ GR4ȱ andȱ P1ȱ inȱ theȱ Bayȱ ofȱ Algeciras,ȱ andȱ theȱ referenceȱ siteȱ
CAȱ inȱ theȱ Bayȱ ofȱ Cádizȱ widelyȱ characterizedȱ byȱ differentȱ ecotoxicologicalȱ
studiesȱ(DelVallsȱetȱal.,ȱ1998,ȱRibaȱetȱal.,ȱ2004,ȱMartínȬDíazȱetȱal.,ȱ2005)ȱ
ȱ
Atlantic Islands
National Park
ȱ
ƒF ƒE
ƒD
•C
ȱ
•A
•B
Ría de CormeLaxe
ȱ
Spain
ȱ
•GR3
•GR4
ȱ
•P1
Bay of
Algeciras
N
ȱ
E
W
ȱ
•CA
S
Bay of
Cádiz
ȱ
Figureȱ1.ȱMapȱofȱtheȱcoastalȱareaȱofȱGaliciaȱshowingȱtheȱlocationsȱofȱtheȱ
samplingȱstations.ȱA,ȱBȱandȱCȱrefersȱtoȱtheȱstationsȱlocatedȱinȱtheȱCiesȱIslandȱinȱ
theȱAtlanticȱIslandȱNationalȱParkȱandȱD,ȱEȱandȱFȱtoȱthoseȱinȱtheȱBayȱofȱCormeȬ
Laxe.ȱ Theȱ stationsȱ locatedȱ inȱ theȱ Bayȱ ofȱ Algecirasȱ areȱ GR3,ȱ GR4ȱ andȱ P1.ȱ Theȱ
stationȱ CAȱ locatedȱ inȱ theȱ Bayȱ ofȱ Cadizȱ correspondsȱ toȱ theȱ sedimentȱ usedȱ asȱ
reference.ȱ
- 190 -
2.2.ȱSamplingȱandȱdeploymentȱȱ
TheȱclamȱRuditapesȱphilippinarumȱwasȱobtainedȱfromȱanȱaquacultureȱfarmȱ
whereasȱtheȱcrabȱCarcinusȱmaenasȱwasȱcaughtȱinȱaȱcleanȱsiteȱlocatedȱinȱtheȱBayȱofȱ
Cádizȱ (SW,ȱ Spain)ȱ (Ribaȱ etȱ al.,ȱ 2003).ȱ Theȱ organismsȱ wereȱ transferredȱ toȱ theȱ
laboratoryȱ andȱ keptȱ inȱ tanksȱ withȱ continuousȱ waterȱ replacementȱ underȱ
controlledȱ conditionsȱ untilȱ theȱ beginningȱ ofȱ theȱ experiment.ȱ Theȱ testȱ animalsȱ
wereȱ carefullyȱ transportedȱ toȱ theȱ studyȱ sitesȱ andȱ placedȱ inȱ cagesȱ madeȱ withȱ
plasticȱmeshȱ(50cmȱxȱ25cmȱxȱ15cm)ȱdividedȱinȱtwoȱdifferentȱcompartments,ȱoneȱ
forȱcrabsȱ(n=20)ȱandȱoneȱforȱclamsȱ(n=40).ȱTheȱcagesȱwereȱpositionedȱwithȱlowȱ
tideȱ andȱ wereȱ wedgedȱ intoȱ theȱ sediment.ȱ Theȱ exposureȱ lastedȱ 28ȱ daysȱ duringȱ
whichȱ crabsȱ wereȱ fedȱ onceȱ perȱ weekȱ withȱ mixedȱ dietȱ ofȱ musselsȱ orȱ fish.ȱ
Sedimentȱ samplesȱ fromȱ theȱ studyȱ sitesȱ wereȱ collectedȱ andȱ transportedȱ toȱ theȱ
laboratoryȱwhereȱtheyȱwereȱkeptȱinȱdarkȱatȱ4ºCȱpriorȱtoȱchemicalȱanalysis.ȱ
ȱ2.3.ȱBiochemicalȱanalysisȱ
Deployedȱcrabsȱandȱclamsȱwereȱcollectedȱandȱdissectedȱafterȱ28ȱdaysȱofȱ
exposure;ȱ hepathopancreasȱ (inȱ crabs)ȱ andȱ digestiveȱ glandȱ (inȱ clams)ȱ wereȱ
extractedȱ andȱ keptȱ atȱ Ȭ80ºCȱ priorȱ homogenization.ȱ Theȱ samplesȱ wereȱ
homogenizedȱ withȱ TrisȬacetateȱ bufferȱ followingȱ theȱ procedureȱ developedȱ byȱ
Lafontaineȱetȱal.ȱ(2000).ȱSamplesȱwereȱcentrifugedȱatȱ10,000gȱforȱ30ȱmin,ȱandȱtheȱ
supernatantȱ wasȱ usedȱ forȱ theȱ biomarkersȱ determinationȱ andȱ theȱ totalȱ proteinȱ
contentȱdescribedȱbyȱBradfordȱ(1976).ȱTheȱphaseȱIIȱmetabolizingȱGlutathioneȬSȬ
transferaseȱ(GST)ȱactivityȱwasȱdeterminedȱbyȱmonitoringȱtheȱrateȱofȱconjugationȱ
ofȱ glutathioneȱ (GSH)ȱ toȱ 1ȬchloroȬ2,4Ȭdinitrobenzeneȱ (CDNB)ȱ atȱ 340ȱ nm,ȱ
methodologyȱ adaptedȱ fromȱ McFarlandȱ etȱ al.ȱ (1999).ȱ Theȱ oxidationȱ ofȱ 1ȱ mMȱ
NADPHȱ byȱ Glutathioneȱ Reductaseȱ (GR)ȱ inȱ theȱ presenceȱ ofȱ 10ȱ mMȱ oxidizedȱ
glutathioneȱ wasȱ monitoredȱ atȱ 340ȱ nm,ȱ andȱ theȱ methodȱ wasȱ similaryȱ adaptedȱ
fromȱ McFarlandȱ etȱ al.ȱ (1999).ȱ Mixedȱ functionȱ oxygenaseȱ activity,ȱ whichȱ isȱ theȱ
- 191 -
firstȱmodeȱofȱdetoxificationȱofȱmanyȱorganicȱpollutants,ȱwasȱmeasuredȱusingȱtheȱ
ERODȱ assayȱ (Gagnèȱ andȱ Blaiseȱ 1993).ȱ Theȱ antioxidantȱ enzymeȱ Glutathioneȱ
Peroxidaseȱ (GPX)ȱ wasȱ measuredȱ accordingȱ toȱ McFarlandȱ etȱ al.ȱ (1999).ȱ
Biomarkersȱresultsȱwereȱnormalizedȱwithȱtheȱproteinȱcontent.ȱ
2.4.ȱBiomarkerȱofȱeffect:ȱHistopathologyȱ
Gillsȱ andȱ digestiveȱ glandȱ tissuesȱ ofȱ theȱ organismsȱ wereȱ fixedȱ inȱ
phosphateȱ bufferedȱ 10%ȱ formaldehydeȱ (pHȱ 7.2)ȱ forȱ histopathologyȱ
determination.ȱ Afterȱ dehydrationȱ inȱ gradedȱ concentrationsȱ ofȱ ethanol,ȱ theȱ
samplesȱ wereȱ embeddedȱ inȱ paraffinȱ wax.ȱ Histologicalȱ sectionsȱ ofȱ 6ȱ toȱ 8ȱ ΐmȱ
thicknessȱwereȱstainedȱwithȱHaematoxylin–ȱEosinȱandȱHaematoxylin–VOFȱ[15].ȱ
Sectionsȱ wereȱ reviewedȱ byȱ lightȱ microscopyȱ Leitzȱ Laborluxȱ Sȱ andȱ
photographedȱ(SonyȱDKCȬCM30).ȱ
2.5.ȱChemicalȱanalysisȱ
Theȱ analysesȱ ofȱ PAHsȱ andȱ PCBsȱ boundȱ toȱ sedimentsȱ wereȱ carriedȱ outȱ
accordingȱ toȱ USEPAȱ SWȬ846ȱ Methodȱ 827C78082ȱ (USEPA,ȱ 1994).ȱ Brieflyȱ driedȱ
samplesȱ wereȱ Soxhletȱ extractedȱ withȱ nȬhexaneȱ forȱ 18ȱ h,ȱ andȱ theȱ extractsȱ wereȱ
isolatedȱ byȱ columnȱ chromatographyȱ onȱ Florisileȱ aluminoȬsilica.ȱ PCBsȱ andȱ
PAHsȱ wereȱelutedȱandȱtheirȱfractionsȱwereȱdriedȱinȱaȱ rotatingȱevaporatorȱandȱ
reȬdissolvedȱ inȱ isooctane.ȱ Aromaticȱ fractionsȱ wereȱ analyzedȱ onȱ aȱ
HewlettePackardȱ(HP)ȱ5890ȱSeriesȱIIȱgasȱchromatographerȱcoupledȱwithȱanȱHPȱ
5970ȱ massȱ spectrometer.ȱ PAHsȱ wereȱ analyzedȱ byȱ GCȬMSȱ usingȱ selectedȱ ionȱ
monitoringȱ (SIM).ȱ Analysisȱ ofȱ PCBsȱ asȱ AROCLORȱ 1242ȱ andȱ AROCLORȱ 1260ȱ
wasȱ performedȱ usingȱ theȱ sameȱ instrumentȱ withȱ anȱ electronȱ captureȱ detectorȱ
(GC/ECD).ȱ Forȱ bothȱ setȱ ofȱ organicȱ chemicals,ȱ PAHsȱ andȱ AROCLOR,ȱ theȱ
analyticalȱprocedureȱshowedȱagreementȱwithȱtheȱcertifiedȱvaluesȱofȱmoreȱthanȱ
90%.ȱ
- 192 -
TraceȱmetalȱanalysisȱwereȱanalyzedȱasȱdescribedȱbyȱCasadoȬMartínezȱetȱ
al.ȱ (2006c);ȱ briefly,ȱ 2.5ȱ gȱ ofȱ sedimentsȱ (<0.065ȱ mm)ȱ wereȱ placedȱ inȱ Teflonȱ
containersȱ andȱwereȱdigestedȱ inȱ microwaveȱ(400W,ȱ15ȱmin,ȱtwice)ȱwithȱHNO3ȱ
2N.ȱ Theȱ extractsȱ wereȱ purifiedȱ byȱ passingȱ throughȱ aȱ CȬ18ȱ columnȱ andȱ metalsȱ
analysesȱwereȱperformedȱbyȱanodicȱvoltamperimetryȱ(ȬZn,ȱCd,ȱPb,ȱNi,ȱCoȱandȱ
CuȬȱ Metrohmȱ Applicationȱ Bulletinȱ Nºȱ 147;ȱȬȱVȬȱMetrohmȱApplicationȱNoteȱNºȱ
VȬ81).ȱ Forȱ Hgȱ theȱ coldȱ vapourȱ techniqueȱ wasȱ usedȱ andȱ wasȱ quantifiedȱ usingȱ
atomicȱabsorptionȱspectrometry.ȱTheȱanalyticalȱproceduresȱwereȱcheckedȱusingȱ
referenceȱ materialȱ (MESSȬ1ȱ NRCȱ andȱ CRMȱ 277ȱ BCR)ȱ andȱ showedȱ aȱ recoveryȱ
greaterȱthanȱ90%ȱofȱtheȱcertifiedȱconcentration.ȱ
2.6.ȱStatisticalȱanalysisȱ
TheȱinductionȱofȱbiomarkersȱofȱresponseȱwasȱanalyzedȱwithȱtheȱANOVAȱ
andȱTukeyȱtestȱwithȱtheȱaimȱofȱdeterminingȱsignificantȱdifferencesȱ(pȱ<ȱ0.05;ȱpȱ<ȱ
0.01)ȱ amongȱ theȱ resultsȱ obtainedȱ forȱ theȱ referenceȱ (CA)ȱ siteȱ andȱ theȱ otherȱ
samplingȱ sites,ȱ usingȱ theȱ statisticalȱ packageȱ SPSSȱ 11.5.ȱ Multivariateȱ analysisȱ
wasȱ carriedȱ outȱ withȱ inȱ anȱ attemptȱ toȱ linkȱ contaminationȱ withȱ adverseȱ
biologicalȱmeasurements;ȱtheȱprincipalȱcomponentȱanalysisȱ(PCA)ȱwasȱusedȱasȱ
theȱextractionȱprocedureȱtoȱderiveȱaȱreducedȱnumberȱofȱnewȱvariablesȱ(factors)ȱ
asȱlinearȱcombinationsȱofȱtheȱoriginalȱvariablesȱ(STATISTICAȱ6.0).ȱ
3.ȱResultsȱandȱdiscussionȱ
3.1.ȱConcentrationȱofȱchemicalsȱinȱtheȱsedimentsȱ
Resultsȱ ofȱ theȱ concentrationȱ ofȱ chemicalsȱ inȱ theȱ studiedȱ sedimentsȱ areȱ
shownȱ inȱ tableȱ 1.ȱ Theȱ highestȱ concentrationȱ ofȱ PAHsȱ wasȱ foundȱ inȱ theȱ
sedimentsȱ fromȱ GR3ȱ (2961ȱ mgȱ KgȬ1ȱ dryȱ sediment)ȱ locatedȱ inȱ theȱ Bayȱ ofȱ
Algeciras,ȱfollowedȱbyȱsedimentsȱfromȱtheȱstationȱFȱ(820ȱmgȱKgȬ1ȱ dryȱsediment)ȱ
locatedȱinȱCormeȬLaxeȱandȱGR4ȱ(802ȱmgȱKgȬ1ȱdryȱsediment)ȱandȱP1ȱ(641ȱmgȱKgȬ1ȱȱ
- 193 -
Tableȱ1.ȱTotalȱPAHs,ȱPCBsȱandȱmetalȱconcentrationȱ(Zn,ȱCd,ȱPb,ȱNi,ȱCoȱandȱV)ȱȬ
mgȱKgȬ1ȱdryȱsedimentȬȱmeasuredȱinȱtheȱsedimentsȱfromȱGalicia:ȱAtlanticȱIslandsȱ
NationalȱParkȱ(A,ȱB,ȱC),ȱCormeȬLaxeȱ(D,ȱE,ȱF);ȱtheȱBayȱofȱAlgecirasȱ(GR3,ȱGR4ȱ
andȱ P1)ȱ andȱ theȱ Bayȱ ofȱ Cadizȱ (CA)ȱ usedȱ asȱ theȱ referenceȱ station.ȱ ȱ n.d:ȱ notȱ
detected.ȱ
ȱȱ
PAHsȱ PCBsȱ
Znȱ
Cdȱ
Pbȱ
Cuȱ
Niȱ
Coȱ
Vȱ
CAȱ
n.d.ȱ
n.d.ȱ
21.3ȱ
0.92ȱ 2.28ȱ
6.98ȱ
0.06ȱ
3.40ȱ 80.0ȱ
Aȱ
257ȱ
n.d.ȱ
76.2ȱ
n.d.ȱ 26.6ȱ
18.9dȱ
12.0ȱ
0.52ȱ n.d.ȱ
Bȱ
370ȱ
6.52ȱ
43.4ȱ
n.d.ȱ 9.13ȱ
n.d.ȱ
6.88ȱ
n.d.ȱ n.d.ȱ
Cȱ
239ȱ
4.76ȱ
37.5ȱ
n.d.ȱ 6.54ȱ
31.6dȱ
5.02ȱ
0.87ȱ n.d.ȱ
Dȱ
537ȱ
2.60ȱ
65.7ȱ
n.d.ȱ
44dȱ
22.1dȱ
9.39ȱ
1.21ȱ 13.4ȱ
Eȱ
558ȱ
4.29ȱ
31.8ȱ
n.d.ȱ 4.25ȱ
n.d.ȱ
5.61ȱ
0.37ȱ 2.34ȱ
Fȱ
820dȱ
2.28ȱ
243a,b,d n.d.ȱ 14.3ȱ
19.1dȱ
7.03ȱ
0.67ȱ 5.94ȱ
GR3ȱ
2961d,eȱ
22.0ȱ
138dȱ
0.17ȱ 21.6ȱ
5.01ȱ
GR4ȱ
802dȱ
1.75ȱ
35.3ȱ
0.10ȱ 6.21ȱ
3.67ȱ
P1ȱ
641dȱ
0.84ȱ
56.7ȱ
0.12ȱ 12.3ȱ 75.2a,c,d,eȱ
74.7a,d,eȱ 12.8ȱ 26.1ȱ
13.1ȱ
5.59ȱ n.d.ȱ
13.3ȱ
n.d.ȱ 6.84ȱ
Concentrationȱ thatȱ exceedsȱ theȱ ERLȱ (Effectsȱ RangeȬLow)ȱ definedȱ byȱ NOAAȱ (1999);ȱ bȱ
valueȱ thatȱ exceedsȱ theȱ sedimentȱ qualityȱ guidelineȱ suggestedȱ byȱ DelValls&Chapmanȱ
(1998);ȱ cȱ concentrationȱwhichȱsurpassȱtheȱguidelineȱdescribedȱbyȱRibaȱetȱal.ȱ(2004);ȱ dȱ valueȱ
thatȱexcedsȱtheȱguidelineȱproposedȱbyȱMcDonaldȱetȱal.ȱ(1996);ȱ eȱ concentrationȱthatȱsurpassȱ
theȱguidelinesȱdefinedȱbyȱDutchȱagencies,ȱTweedeȱKamer,ȱvergaderjaarȱ(1994–1995).ȱ
aȱ
ȱ
dryȱ sediment)ȱ inȱ theȱ Bayȱ ofȱ Algeciras;ȱ theseȱ couldȱ beȱ consideredȱ asȱ slightlyȱ
contaminatedȱ byȱ PAHsȱ andȱ adverseȱ effectsȱ couldȱ beȱ frequentȱ accordingȱ toȱ
McDonaldȱ etȱ al.ȱ (1996),ȱ andȱ inȱ theȱ caseȱ ofȱ GR3ȱ theȱ concentrationȱ ofȱ thisȱ
contaminantȱ alsoȱ exceedsȱ theȱ guidelineȱ proposedȱ byȱ theȱ Dutchȱ agenciesȱ
(Tweedeȱ Kamer,ȱ vergaderjaar,ȱ 1994Ȭ1995);ȱ onȱ theȱ otherȱ handȱ sedimentsȱ fromȱ
theȱ Ciesȱ Islandȱ presentȱ theȱ lowestȱ concentrationsȱ ofȱ PAHs,ȱ whereasȱ theseȱ
chemicalsȱwereȱnotȱdetectedȱinȱtheȱsamplingȱsiteȱlocatedȱinȱtheȱBayȱofȱCadiz.ȱNoȱ
specialȱ patternȱ wasȱ detectedȱ regardingȱ toȱ theȱ concentrationȱ ofȱ metalsȱ inȱ theȱ
- 194 -
differentȱ sitesȱ ofȱ study;ȱ GR3ȱ andȱ Fȱ exceedsȱ someȱ internationalȱ guidelinesȱ
definedȱ forȱ theȱ metalȱ Zn:ȱ GR3ȱ (MacDonaldȱ etȱ al.,ȱ 1996)ȱ andȱ Fȱ (NOAA,ȱ 1999;ȱ
DelVallsȱandȱChapman,ȱ1998;ȱMacDonaldȱetȱal.,ȱ1996).ȱAccordingȱtoȱMcDonaldȱ
etȱ al.ȱ (1996),ȱ sedimentsȱ fromȱ stationȱ Dȱ exceedȱ theȱ guidelineȱ proposedȱ forȱ Pb.ȱ
Sitesȱ Aȱ andȱ Cȱ fromȱ Ciesȱ andȱ D,ȱ Fȱ fromȱ CormeȬLaxeȱ surpassȱ theȱ proposedȱ
guidelineȱ describedȱ byȱ McDonaldȱ etȱ al.ȱ (1996)ȱ forȱ Cu,ȱ whereasȱ GR3ȱ exceedsȱ
variousȱ guidelinesȱ proposedȱ forȱ thisȱ metalȱ (NOAA,ȱ 1999;ȱ MacDonaldȱ etȱ al.,ȱ
1996;ȱ Tweedeȱ Kamer,ȱ vergaderjaar,ȱ 1994Ȭ1995;ȱ Ribaȱ etȱ al.,ȱ 2004).ȱ GR3ȱ alsoȱ
exceedsȱtheȱguidelinesȱforȱNiȱproposedȱbyȱdifferentȱinternationalȱagenciesȱandȱ
authorsȱ (NOAA,ȱ 1999;ȱ MacDonaldȱ etȱ al.,ȱ 1996;ȱ Tweedeȱ Kamer,ȱ vergaderjaar,ȱ
1994Ȭ1995).ȱȱ
3.2.ȱBiomarkersȱofȱexposureȱ
Meanȱ valuesȱ ofȱ theȱ biomarkersȱ ofȱ exposureȱ determinedȱ inȱ crabsȱ andȱ
clamsȱobtainedȱafterȱtheȱ28ȬdȱexposureȱareȱsummarizedȱinȱFigureȱ2.ȱInȱgeneral,ȱ
organismsȱdeployedȱinȱtheȱareaȱofȱCormeȬLaxeȱ(D,ȱE,ȱandȱF)ȱpresentȱtheȱhighestȱ
inductionȱ ofȱ theȱ biomarkersȱ ofȱ exposure.ȱ GPXȱ activitiesȱ forȱ crabsȱ showȱ
significantȱdifferencesȱ(p<0.01)ȱbetweenȱsitesȱD,ȱEȱandȱFȱ(CormeȬLaxe)ȱandȱtheȱ
referenceȱstationȱCA,ȱwhereasȱclamsȱexposedȱinȱsiteȱDȱpresentedȱalsoȱsignificantȱ
differencesȱ withȱ theȱ referenceȱ stationȱ regardingȱ toȱ thisȱ biomarker.ȱ Differencesȱ
obtainedȱ forȱ theȱ phaseȱ IIȱ enzymeȱ GSTȱ measuredȱ inȱ crabsȱ wereȱ significantlyȱ
amongȱD,ȱFȱandȱtheȱreferenceȱCA.ȱTheȱantioxidantȱenzymeȱGRȱactivityȱforȱboth,ȱ
crabsȱ andȱ clamsȱ resultedȱ significantlyȱ differentȱ fromȱ CAȱ forȱ theȱ threeȱ studyȱ
sitesȱlocatedȱinȱCormeȬLaxeȱ(D,ȱEȱandȱF).ȱInȱtheȱcaseȱofȱtheȱERODȱactivityȱwhichȱ
accountsȱ forȱ theȱ enzymaticȱ activityȱ occurringȱ inȱ theȱ phaseȱ Iȱ ofȱ detoxification,ȱ
significantȱ differencesȱ wereȱ detectedȱ forȱ thoseȱ crabsȱ thatȱ wereȱ placedȱ inȱ theȱ
locationsȱBȱ(AINP),ȱDȱ,ȱEȱandȱFȱ(CormeȬLaxe),ȱandȱclamsȱexposedȱtoȱsedimentsȱ
fromȱDȱ(CormeȬLaxe)ȱandȱGR3,ȱGR4,ȱP1ȱ(BayȱofȱAlgeciras).ȱȱ
- 195 -
GPX (nmol/mg/min)
200
ȱ
10000
*
ȱ
150
8000
GST (nmol/mg/min)
250
*
ȱ
50
*
*
ȱ
*
*
6000
4000
2000
ȱ
0
0
ȱ
25
CA
A
B
C
D
E
ȱ
F
CA
GR3 GR4 P1
A
B
C
D
E
F
GR3 GR4 P1
10.0
*
*
8.0
ȱ
*
15
10
EROD (pmol/mg/min)
GR (nmol/mg/min)
20
* *
*
ȱ
*
6.0
4.0
*
* * *
0.5
* * *
5
ȱ
0
0.0
CA
A
B
C
D
E
F
GR3 GR4 P1
CA
A
B
C
D
E
F
GR3 GR4 P1
ȱ
Figureȱ 2.ȱ Generalȱ healthȱ biomarkersȱ forȱ bothȱ invertebrateȱ species,ȱ theȱ
clamȱ Ruditappesȱ philippinarumȱ andȱ theȱ crabȱ Carcinusȱ maenas:ȱ glutathioneȱ
peroxidaseȱ activityȱ GPXȱ (nmol/min/mgȱ prot),ȱ glutathioneȱ transferaseȱ GSTȱ
activityȱ (nmol/min/mgȱ prot),ȱ glutathioneȱ reductaseȱ GRȱ activityȱ (nmol/min/mgȱ
prot)ȱ andȱ ERODȱ activityȱ (pmol/mg/min).ȱ Asterisksȱ indicateȱ significantȱ
differencesȱwithȱtheȱreferenceȱtreatmentȱCAȱ(*pȱ<ȱ0.05;ȱ**pȱ<ȱ0.01).ȱ
3.3.ȱBiomarkersȱofȱeffectȱ
Theȱrelationshipȱbetweenȱpollutantsȱandȱpathologiesȱinȱtargetȱtissuesȱhasȱ
beenȱ previouslyȱ reportedȱ (OrtizȬDelgadoȱ etȱ al.,ȱ 2007).ȱ Histopathologyȱ resultsȱ
showedȱnoȱalterationsȱinȱtheȱorganismsȱfromȱtheȱnegativeȱcontrolȱ(Figureȱ3ȱandȱ
Figureȱ4).ȱInȱgeneral,ȱdamageȱinȱcrabsȱandȱclamsȱtissuesȱwereȱlowerȱthanȱthoseȱ
detectedȱ inȱ laboratoryȱ deploymentsȱ (personalȱ observations).ȱ Mostȱ ofȱ theȱ
- 196 -
analyzedȱ organismsȱ showedȱ ȱ severalȱ ȱ histologicalȱ unȬspecificȱ lesionsȱ relatedȱ
withȱ symptomsȱ ofȱ generalȱ stress,ȱ includingȱ lossȱ ofȱ digestiveȱ epithelialȱ cells,ȱȱ
ruptureȱ ofȱ gillȱ epithelium,ȱ respiratoryȱ lamellarȱ fusionȱ ofȱ lifting,ȱ asȱ wellȱ asȱ
haemociticȱ infiltratesȱ orȱ lossȱ ofȱ ȱ connectiveȱ tissueȱ ofȱ theȱ gillsȱ andȱȱ
hepatopancreas,ȱ ȱ mostȱ whichȱ canȱ alsoȱ beȱ observedȱ inȱ differentȱ ȱ marineȱȱ
invertebrateȱ orȱ vertebrateȱ ȱ ȱ speciesȱ exposedȱ toȱ ȱ differentȱ ȱ inorganicȱ orȱ organicȱ
contaminants,ȱ parasiticȱ orȱ infectiousȱ diseases,ȱ nutritionalȱ stress,ȱ orȱ physicoȬ
chemicalȱdisordersȱ(RodriguezȱdeȱlaȱRuaȱetȱal.,ȱ2005;ȱOrtizȬDelgadoȱetȱal.,ȱ2007).ȱ
Organismsȱ exposedȱ toȱ sedimentsȱ fromȱ theȱ Bayȱ ofȱ Algecirasȱ wereȱ theȱ mostȱ
affectedȱfollowedȱbyȱclamsȱandȱcrabsȱexposedȱtoȱsedimentsȱfromȱCormeȬLaxe,ȱ
andȱfinallyȱorganismsȱfromȱtheȱCiesȱtreatmentȱwhichȱshowedȱalterationsȱdueȱtoȱ
generalȱ environmentalȱ stress.ȱ Theȱ lesionsȱ observedȱ inȱ clamsȱ thatȱ hadȱ beenȱ
exposedȱ toȱ sedimentsȱ fromȱ Algecirasȱ duringȱ 28ȱ daysȱ included:ȱ desquamationȱȱ
ofȱ ȱ digestiveȱ epithelium,ȱ occlusionȱ ofȱ theȱ ȱ digestiveȱ ȱ ducts,ȱ ȱ haemociticȱ
infiltrationsȱandȱweakȱalterationsȱorȱlossȱofȱtheȱȱsupportingȱdigestiveȱconnectiveȱ
tissueȱ (hepatopancreas),ȱ ciliarȱ alterations,ȱ lossȱ ofȱ ȱ supportȱ connectiveȱ tissue,ȱȱ
andȱ hypertrpophyȱ orȱ fusionȱ ofȱ lamellaeȱ (gills).ȱ Crabsȱ deployedȱ inȱ siteȱ GR3ȱ
presentedȱ disruptedȱ pillarȱ cells,ȱ epithelialȱ changes,ȱ desquamationȱ inȱ gillsȱ
presenceȱ ofȱ vacuolesȱ inȱ hepathopancreasȱ ofȱ cagedȱ organisms.ȱ Theȱ presenceȱ ofȱ
parasitesȱ inȱ someȱ ofȱ theȱ crabsȱ studiedȱ makeȱ moreȱ unclearȱ toȱ determineȱ theȱ
causeȱ ofȱ theȱ damages,ȱ inȱ thisȱ sense,ȱ aȱ betterȱ relationshipȱ withȱ pollutantsȱ wasȱ
shownȱ inȱ clamsȱ thanȱ crabs.ȱ Organismsȱ fromȱ theȱ referenceȱ siteȱ didȱ notȱ presentȱ
alterationsȱinȱtargetȱtissues.ȱȱ
ȱ
ȱ
ȱ
ȱ
- 197 -
ȱ
A
B
C
D
E
F
G
H
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
Figureȱ 3.ȱ Histologicalȱ sectionsȱ ofȱ gillsȱ andȱ digestiveȱ glandȱ ofȱ theȱ clamȱ
Ruditapesȱphilippinarumȱafterȱ28Ȭdȱexposureȱtoȱtheȱsediments:ȱ(A)ȱHistologicalȱ
sectionȱ ofȱ aȱ controlȱ gillȱ (dayȱ 0);ȱ (B)ȱ Histologicalȱ sectionȱ ofȱ aȱ controlȱ digestiveȱ
glandȱ(dayȱ0);ȱ(C)ȱHistologicalȱsectionȱofȱgillȱfromȱaȱclamȱexposedȱtoȱsedimentsȱ
fromȱAINP;ȱ(C)ȱHistologicalȱsectionȱofȱdigestiveȱglandȱfromȱaȱclamȱexposedȱtoȱ
sedimentsȱ fromȱ AINP;ȱ (D)ȱ Histologicalȱ sectionȱ ofȱ gillȱ fromȱ aȱ clamȱ exposedȱ toȱ
sedimentsȱfromȱCormeȬLaxe;ȱ(E)ȱHistologicalȱsectionȱofȱdigestiveȱglandȱfromȱaȱ
clamȱ exposedȱ toȱ sedimentsȱ fromȱ CormeȬLaxe;ȱ (F)ȱ Histologicalȱ sectionȱ ofȱ gillȱ
fromȱ aȱ clamȱ exposedȱ toȱ sedimentsȱ fromȱ Algeciras;ȱ (G)ȱ Histologicalȱ sectionȱ ofȱ
digestiveȱglandȱfromȱaȱclamȱexposedȱtoȱsedimentsȱfromȱAlgeciras.ȱ
ȱ
- 198 -
ȱ
A
B
C
D
E
F
G
H
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
Figureȱ 4.ȱ Histologicalȱ sectionsȱ ofȱ gillsȱ andȱ hepathopancreasȱ ofȱ theȱ crabȱ
Carcinusȱmaenasȱafterȱ28Ȭdȱexposureȱtoȱtheȱsediments:ȱ(A)ȱHistologicalȱsectionȱ
ofȱaȱcontrolȱgillȱ(dayȱ0);ȱ(B)ȱHistologicalȱsectionȱofȱaȱcontrolȱdigestiveȱglandȱ(dayȱ
0);ȱ(C)ȱHistologicalȱsectionȱofȱgillȱfromȱaȱclamȱexposedȱtoȱsedimentsȱfromȱAINP;ȱ
(C)ȱHistologicalȱsectionȱofȱhepathopancreasȱfromȱaȱclamȱexposedȱtoȱsedimentsȱ
fromȱ AINP;ȱ (D)ȱ Histologicalȱ sectionȱ ofȱ gillȱ fromȱ aȱ clamȱ exposedȱ toȱ sedimentsȱ
fromȱ CormeȬLaxe;ȱ (E)ȱ Histologicalȱ sectionȱ ofȱ hepathopancreasȱ fromȱ aȱ clamȱ
exposedȱ toȱ sedimentsȱ fromȱ CormeȬLaxe;ȱ (F)ȱ Histologicalȱ sectionȱ ofȱ gillȱ fromȱ aȱ
clamȱ exposedȱ toȱ sedimentsȱ fromȱ Algeciras;ȱ (G)ȱ Histologicalȱ sectionȱ ofȱ
hepathopancreasȱfromȱaȱclamȱexposedȱtoȱsedimentsȱfromȱAlgeciras.ȱ
ȱ
- 199 -
3.4.ȱLinkingȱchemicalsȱandȱbiomarkersȱȱ
Asȱitȱhasȱbeenȱshownȱabove,ȱinȱtheȱcurrentȱstudyȱtheȱhighestȱactivitiesȱofȱ
biomarkersȱofȱexposureȱwhereȱobservedȱinȱthoseȱindividualsȱdeployedȱ“inȱsitu”ȱ
inȱ theȱ Bayȱ ofȱ CormeȬLaxe.ȱ Studiesȱ carriedȱ outȱ withȱ theȱ sameȱ organismsȱ andȱ
similarȱ sedimentsȱ underȱ laboratoryȱ conditionsȱ (MoralesȬCasellesȱ etȱ al,ȱ
submitted)ȱ showedȱ higherȱ biomarkerȱ responsesȱ inȱ organismsȱ exposedȱ toȱ
sedimentsȱfromȱtheȱBayȱofȱAlgeciras,ȱmainlyȱdueȱtoȱtheȱconcentrationȱofȱPAHsȱ
inȱ theȱ sediments.ȱ Inȱ someȱ occasionsȱ inȱ situȱ exposuresȱ showedȱ greaterȱ toxicityȱ
thanȱlaboratoryȱexposuresȱtoȱsedimentsȱfromȱtheȱsameȱsitesȱ(Burtonȱetȱal.,ȱ2005).ȱ
Toȱ elucidateȱ theȱ sourceȱ andȱ typeȱ ofȱ contaminantȱ thatȱ isȱ producingȱ theȱ
stressȱ toȱ theȱ organismsȱ aȱ multivariateȱ analysisȱ wasȱ performedȱ toȱ linkȱ
biomarkersȱ ofȱ exposureȱ withȱ theȱ chemicalsȱ boundȱ toȱ sediments.ȱ Threeȱ newȱ
factorsȱwereȱdefinedȱtoȱdescribeȱtheȱ17ȱoriginalȱvariablesȱbyȱexplainingȱaȱ75ȱ%ȱ
ofȱ theȱ totalȱ varianceȱ (Tableȱ 2).ȱ ȱ Theȱ mainȱ Factorȱ (29.9ȱ %)ȱ linksȱ theȱ phaseȱ Iȱ
detoxificationȱ activityȱ determinedȱ byȱ ERODȱ inȱ clamsȱ andȱ crabs,ȱ theȱ GSTȱ andȱ
GPXȱactivityȱinȱclamsȱtoȱtheȱconcentrationȱofȱPbȱinȱtheȱsediment.ȱThisȱfactorȱhasȱ
aȱpositiveȱloadingȱprincipallyȱinȱsiteȱDȱfromȱCormeȬLaxeȱandȱfollowedȱbyȱsiteȱAȱ
inȱCiesȱ(Figureȱ5).ȱTheȱlowȱscoreȱinȱsiteȱAȱsuggestsȱthatȱPbȱboundȱtoȱsedimentsȱ
producedȱ someȱ stressȱ althoughȱ theȱ highȱ prevalenceȱ ofȱ thisȱ factorȱ inȱ siteȱ D,ȱ
whichȱ exceedsȱ theȱ sedimentȱ qualityȱ guidelineȱ proposedȱ byȱ McDonaldȱ etȱ alȱ
(1996)ȱ forȱ thisȱ contaminantȱ impliesȱ thatȱ thereȱ isȱ aȱ sourceȱ ofȱ Pbȱ whichȱ hasȱ
involvedȱtheȱactivationȱofȱ“earlyȱwarning”ȱbiomarkers.ȱPrecisely,ȱtheȱactivationȱ
ofȱ aȱ groupȱ ofȱ biomarkersȱ oftenȱ relatedȱ toȱ theȱ defenceȱ againstȱ organicȱ
compoundsȱ suggestsȱ thatȱ theȱ metalȱ Pbȱ comesȱ fromȱ anȱ organicȱ sourceȱ suchȱ asȱ
hydrocarbons.ȱȱ
Onȱ theȱ otherȱ handȱ theȱ factȱ thatȱ theȱ biomarkersȱ ofȱ exposureȱ haveȱ thisȱ
significantȱ inductionȱ inȱ theȱ organismsȱ exposedȱ toȱ sedimentsȱ fromȱ theȱ Bayȱ ofȱȱ
CormeȬLaxeȱ whichȱ wasȱ notȱ detectedȱ underȱ laboratoryȱ exposuresȱ (MoralesȬ
- 200 -
Casellesȱetȱal.,ȱsubmitted),ȱcouldȱbeȱrelatedȱtoȱtheȱexistenceȱofȱaȱnonȱmeasuredȱ
contaminantȱthatȱmightȱcomeȱfromȱtheȱseaȱwater.ȱInȱthisȱcaseȱaȱpossibleȱsourceȱ
couldȱbeȱtheȱpresenceȱofȱcagedȱmusselȱforȱaquacultureȱinȱtheȱproximitiesȱwhatȱ
mightȱ supposeȱ anȱ inputȱ ofȱ organicȱ matterȱ thereforeȱ aȱ causeȱ ofȱ stressȱ toȱ theȱ
deployedȱ organisms.ȱ Aȱ sourceȱ ofȱ organicȱ matterȱ toȱ theȱ surroundingȱ waterȱ
involvesȱ aȱ decreaseȱ inȱ theȱ dissolvedȱ oxygenȱ whichȱ isȱ usedȱ inȱ theȱ oxidationȱ
processes.ȱ Theȱ oxygenȱ contentȱ ofȱ waterȱ canȱ beȱ importantȱ inȱ determiningȱ theȱ
natureȱandȱtheȱrateȱofȱbothȱchemicalȱandȱbiochemicalȱtransformationsȱ(Walkerȱ
etȱal.,ȱ2006).ȱ
Tableȱ 2.ȱȱȱSortedȱ rotatedȱ factorȱ loadingsȱofȱ17ȱvariablesȱforȱtheȱthreeȱprincipalȱ
factorsȱ resultingȱ fromȱ theȱ multivariateȱ analysisȱ ofȱ resultsȱ obtainedȱ fromȱ theȱ
biomarkerȱresponsesȱinȱcrabsȱandȱclamsȱandȱtheȱchemicalȱanalysis.ȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱ
ȱ
ȱ
ȱȱ
ȱȱ
ȱ
GPXȬcrabȱ
GPXȬclamȱ
GRȬcrabȱ
GRȬclamȱ
GSTȬcrabȱ
GSTȬclamȱ
ERODȬcrabȱ
ERODȬclamȱ
PAHsȱ
PCBsȱ
Znȱ
Cdȱ
Pbȱ
Cuȱ
Niȱ
Coȱ
Vȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
FACTORȱ1ȱ FACTORȱ2ȱ FACTORȱ3ȱ
29.9ȱ
23.8ȱ
21.1ȱ
ņȱ
0.97ȱ
ņȱ
ņȱ
ņȱ
0.91ȱ
0.90ȱ
0.94ȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.93ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ȱ
ȱ
- 201 -
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
Ȭ0.98ȱ
Ȭ0.93ȱ
Ȭ0.46ȱ
ņȱ
ņȱ
ņȱ
Ȭ0.97ȱ
Ȭ0.88ȱ
ņȱ
0.93ȱ
ņȱ
0.85ȱ
0.82ȱ
0.94ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.51ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
Accordingȱ toȱ theȱ secondȱ factorȱ (23.8ȱ %)ȱ aȱ relationshipȱ isȱ observedȱ
betweenȱtheȱorganicȱcontaminantsȱPAHsȱandȱPCBsȱwithȱmetalsȱNi,ȱCoȱandȱZn.ȱ
Thisȱ factor,ȱ withȱ negativeȱ loading,ȱ doesȱ notȱ relateȱ theȱ associationȱ ofȱ theseȱ
contaminantsȱ withȱ biomarkersȱ andȱ accountsȱ forȱ aȱ contaminationȱ inȱ sedimentsȱ
fromȱsitesȱGR3ȱandȱGR4ȱfromȱtheȱBayȱofȱAlgecirasȱandȱFȱinȱCormeȬLaxeȱ(Figureȱ
5).ȱ Biomarkerȱ responsesȱ wereȱ expectedȱ inȱ organismsȱ exposedȱ toȱ theȱ
contaminationȱ boundȱ toȱ sedimentsȱ fromȱ Algeciras,ȱ asȱ itȱ wasȱ shownȱ inȱȱ
laboratoryȱ studiesȱ (MoralesȬCasellesȱ etȱ al.,ȱ submitted)ȱ howeverȱ biomarkersȱ ofȱ
exposureȱ wereȱ generallyȱ low,ȱ mainlyȱ inȱ crabs,ȱ inȱ comparisonȱ withȱ sedimentsȱ
fromȱCormeȬLaxe.ȱTheȱfactȱthatȱtheȱareaȱofȱtheȱdeployment,ȱinȱtheȱmouthȱofȱtheȱ
riverȱ Guadarranque,ȱ isȱ submittedȱ toȱ theȱ influenceȱ ofȱ naturalȱ tidesȱ couldȱ beȱ aȱ
reasonȱ ofȱ easingȱ theȱ bioavailabilityȱ ofȱ contaminantsȱ toȱ theȱ organisms.ȱ
Sedimentsȱofȱintertidalȱzonesȱalongȱtheȱseashoreȱexperienceȱfluctuatingȱoxygenȱ
levelsȱinȱaccordanceȱwithȱtidalȱmovementsȱ(Walkerȱetȱal.,ȱ2006).ȱAsȱtheȱoxygenȱ
contentȱ declines,ȱ thereȱ willȱ beȱ aȱ tendencyȱ forȱ oxidativeȱ transformationsȱ toȱ beȱ
replacedȱ byȱ reductiveȱ ones.ȱ Oxidationsȱ byȱ theȱ microsomalȱ monooxygenaseȱ
systemȱdependȱuponȱtheȱactivationȱofȱhemoproteinȱmolecularȱoxygenȱ(O2)ȱafterȱ
itȱ hasȱ beenȱ boundȱ toȱ anȱ associatedȱ hemoprotein,ȱ cytochromeȱ P450.ȱ (Walkerȱ etȱ
al.,ȱ2006).ȱȱ
Theȱthirdȱfactorȱ(21.1ȱ%)ȱconnectsȱtheȱconcentrationȱofȱZnȱinȱsedimentsȱtoȱ
theȱ biomarkersȱ ofȱ responseȱ relatedȱ toȱ antioxidantȱ activity:ȱ GRȱ inductionȱ inȱ
crabsȱandȱclamsȱandȱGSTȱandȱGPXȱactivityȱinȱcrabs.ȱSitesȱD,ȱEȱandȱFȱfromȱtheȱ
Bayȱ ofȱ CormeȬLaxeȱ presentȱ theȱ influenceȱ ofȱ thisȱ factorȱ (Figureȱ 5)ȱ whatȱ meansȱ
thatȱthisȱareaȱpresentsȱaȱstressȱdueȱtoȱtheȱpresenceȱofȱpollutionȱbyȱZn.ȱPreviousȱ
studiesȱhaveȱconsideredȱtheȱBayȱofȱCormeȬLaxeȱasȱnotȱcontaminatedȱandȱhaveȱ
attributedȱ theȱ presenceȱ ofȱ metalsȱ toȱ basalȱ levelsȱ (CobeloȬGarcíaȱ etȱ al.,ȱ 2005).ȱ
However,ȱ regardingȱ toȱ siteȱ Dȱ whichȱ levelsȱ ofȱ Znȱ surpassȱ severalȱ sedimentȱ
qualityȱ guidelinesȱ (tableȱ 1)ȱ anȱ anthropogenicȱ sourceȱ ofȱ thisȱ metalȱ isȱ probablyȱ
- 202 -
theȱ causeȱ ofȱ theȱ stressȱ shownȱ inȱ theȱ organisms,ȱ mainlyȱ inȱ crabs.ȱ Previousȱ
studiesȱ haveȱ shownȱ sublethalȱ responsesȱ inȱ Carcinusȱ maenasȱ exposedȱ toȱ
sedimentsȱcontaminatedȱbyȱZnȱ(MartínȬDíazȱetȱal.,ȱ2005).ȱȱ
Inȱgeneral,ȱhistopathologicalȱresponsesȱhaveȱshownȱmoderateȱdamageȱinȱ
theȱ studiedȱ organismsȱ deployedȱ inȱ CormeȬLaxeȱ whatȱ suggestsȱ aȱ successfulȱ
actionȱ ofȱ theȱ antioxidantȱ andȱ detoxificationȱ activities.ȱ However,ȱ defenceȱ mayȱ
involveȱaȱtradeȬoffȱbetweenȱproductionȱandȱsurvival:ȱincreasedȱsurvivalȱmayȱbeȱ
obtainedȱonlyȱatȱaȱcostȱofȱreducedȱgrowthȱofȱreproductionȱ(Walkerȱetȱal.,ȱ2006).ȱ
Inȱ thisȱ sense,ȱ moreȱ attentionȱ shouldȱ beȱ playedȱ toȱ thoseȱ areasȱ affectedȱ byȱ theȱ
inputȱ ofȱ contaminantsȱ whichȱ maybeȱ areȱ notȱ producingȱ lethalȱ responsesȱ
althoughȱsublethalȱeffectsȱareȱexpectedȱandȱcanȱleadȱtoȱsequentialȱchangesȱandȱ
reachȱecosystemȱlevels.ȱ
ȱ
- 203 -
D
3.0
Factor 1
2.0
1.0
A
0.0
-1.0
B
Ca1
F GR3'-J
P1-J
GR4-J
C
E
-2.0
1.5
Ca1
A
B
C
D
P1-J
E
Factor 2
0.0
GR4-J
F
-1.5
-3.0
GR3'-J
2.0
E
F
Factor 3
1.0
D
0.0
A
B
C
GR3'-J GR4-J
P1-J
-1.0
Ca1
-2.0
ȱ
Figureȱ5.ȱFactorȱloadingsȱforȱtheȱthreeȱprincipalȱfactorsȱresultingȱfromȱtheȱ
multivariateȱ analysisȱ ofȱ resultsȱ obtainedȱ fromȱ theȱ chemicalȱ analysisȱ andȱ theȱ
suiteȱofȱbiomarkers.ȱ
- 204 -
5.ȱConclusionsȱ
Inȱ theȱ presentȱ studyȱ sublethalȱ responsesȱ haveȱ beenȱ analyzedȱ inȱ
organismsȱdeployedȱinȱsitesȱaffectedȱbyȱdifferentȱspillsȱinȱtheȱareaȱofȱGaliciaȱandȱ
theȱ gulfȱ ofȱ Cádiz.ȱ Inȱ addition,ȱ biomarkersȱ resultsȱ haveȱ beenȱ linkedȱ withȱ
chemicalsȱ boundȱ toȱ sedimentsȱ inȱ orderȱ toȱ elucidateȱ theȱ cause,ȱ sourceȱ andȱ
bioavailabilityȱofȱadverseȱaffectsȱafterȱexposure.ȱAȱsetȱofȱbiomarkersȱincludingȱ
antioxidantȱ andȱ detoxificationȱ activitiesȱ haveȱ beenȱ evaluatedȱ inȱ additionȱ toȱ
histopathologicalȱ damagesȱ inȱ targetȱ tissues.ȱ Theȱ useȱ ofȱ twoȱ invertebrateȱ ofȱ
species,ȱ theȱ clamȱ Ruditapesȱ philippinarumȱ andȱ theȱ crabȱ Carcinusȱ maenasȱ withȱ
differenceȱfeedingȱhabitsȱprovidedȱaȱbetterȱassessmentȱofȱtheȱsubjectȱraised.ȱTheȱ
lowestȱ sublethalȱ responsesȱ wereȱ observedȱ inȱ organismsȱ exposedȱ toȱ sedimentsȱ
fromȱtheȱCíesȱIslandsȱinȱtheȱAINP,ȱalthoughȱtheȱpresenceȱofȱsomeȱmetalsȱcouldȱ
haveȱinducedȱsomeȱstressȱinȱtheȱdeployedȱanimals.ȱThisȱpointsȱtoȱaȱrecoveryȱofȱ
theȱareaȱfourȱyearsȱafterȱtheȱPrestigeȱoilȱspillȱalthoughȱtheȱinputsȱofȱsomeȱmetalsȱ
areȱ consideredȱ aȱ potentialȱ risk.ȱ Organismsȱ exposedȱ inȱ CormeȬLaxeȱ presentedȱ
highȱ levelsȱ ofȱ stressȱ thatȱ wereȱ notȱ observedȱ inȱ laboratoryȱ exposuresȱ (MoralesȬ
Casellesȱetȱal.,ȱsubmitted)ȱwhatȱsuggestsȱtheȱimpactȱofȱsourcesȱofȱcontaminants,ȱ
notȱonlyȱhydrocarbons,ȱsuchȱasȱtheȱmaterialȱfromȱtheȱaquacultureȱcages.ȱInȱtheȱ
caseȱ ofȱ theȱ bayȱ ofȱ Algecirasȱ theȱ toxicȱ effectsȱ ofȱ contaminantsȱ wereȱ probablyȱ
diminishȱ byȱ theȱ waterȱ removalȱ ofȱ theȱ tidalȱ fluctuationȱ althoughȱ targetȱ tissuesȱ
presentedȱtheȱhighestȱalterationsȱofȱallȱtheȱstudyȱsites.ȱȱ
Previousȱstudiesȱagreeȱwithȱtheȱfactȱthatȱinȱsituȱcagedȱorganismȱapproachȱ
shouldȱ beȱ usedȱ togetherȱ withȱ otherȱ assessmentȱ methodsȱ suchȱ asȱ laboratoryȱ
toxicityȱtestingȱ(Burtonȱetȱal.,ȱ2005).ȱInȱthisȱreportȱitȱhasȱbeenȱshownȱhowȱfieldȱ
studiesȱ haveȱ permittedȱ toȱ identifyȱ alternativeȱ sourcesȱ ofȱ stressȱ thatȱ areȱ notȱ
possibleȱtoȱobserveȱinȱlaboratoryȱexperiments.ȱTherefore,ȱauthorsȱconsiderȱthatȱ
bioassaysȱshouldȱnotȱlimitȱtoȱexperimentalȱdesignsȱunderȱlaboratoryȱconditionsȱ
andȱ proposeȱ thatȱ fieldȱ deploymentsȱ provideȱ theȱ lacksȱ regardingȱ toȱ theȱ
- 205 -
uncontrolledȱ circumstancesȱ ofȱ inȱ situȱ surveysȱ andȱ theȱ excessivelyȱ controlȱ ofȱ
laboratoryȱtests.ȱ
6.ȱAknowledgmentsȱ
Theȱ workȱ describedȱ wasȱ partlyȱ supportedȱ byȱ theȱ projectsȱ VEM2003Ȭ
20563/INTER,ȱ andȱ CTM2005Ȭ07282ȬC03Ȭ01/TECNOȱ financedȱ byȱ theȱ Spanishȱ
Educationȱ andȱ Scienceȱ Ministryȱ andȱ byȱ CISȱ fundedȱ byȱ theȱ Ministryȱ ofȱ
Environment.ȱ Carmenȱ MoralesȬCasellesȱ thanksȱ theȱ Ministryȱ ofȱ Educationȱ andȱ
Scienceȱ forȱ fundingȱ herȱ researchȱ fellowshipȱ (FPU).ȱ Dr.ȱ Ribaȱ thanksȱ theȱ I3pȱ
programȱ forȱ supportingȱ herȱ participationȱ inȱ thisȱ research.ȱ Weȱ areȱ gratefulȱ forȱ
theȱsupportȱandȱhelpȱofȱtheȱmembersȱofȱtheȱCISȱandȱtheȱICMANȬCSIC.ȱSpecialȱ
thanksȱareȱgivenȱtoȱAntonioȱMoreno,ȱPabloȱVidalȱandȱAugustoȱCésar.ȱ
7.ȱReferencesȱ
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Burton,ȱ G.A.,ȱ Greenberg,ȱ M.S.,ȱ Rowland,ȱ C.D.,ȱ Irvine,ȱ C.A.,ȱ Lavoie,ȱ D.R.,ȱ
Brooker,ȱ J.A.,ȱ Moore,ȱ L.,ȱ Raymer,ȱ D.F.N.,ȱ McWilliam,ȱ R.A.ȱ 2005.ȱ Inȱ situȱ
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CasadoȬMartínez,ȱ M.C.,ȱ Buceta,ȱ J.L.,ȱ Forja,ȱ J.M.,ȱ DelValls,ȱ A.ȱ 2006.ȱ
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ȱ
- 210 -
KineticȱofȱbiomarkersȱinȱtheȱclamȱRuditapesȱphilippinarumȱ
CarmenȱMoralesȬCaselles†ȱ‡ȱ*,ȱLauraȱMartínȬDíaz†ȱ‡,ȱInmaculadaȱRiba†ȱ‡,ȱ
CarmenȱSarasquete†,ȱT.ȱÁngelȱDelValls†ȱ‡ȱ
†ȱIȱUnidadȱAsociadaȱdeȱCalidadȱAmbientalȱyȱPatologíaȱ(CSICȱ&ȱUCA)ȱinstitutoȱ
deȱCienciasȱMarinasȱdeȱAndalucíaȱ(ICMANȬCSIC),ȱAvda.ȱRepúblicaȱSaharauiȱs/n,ȱ
PuertoȱRealȱ11510,ȱCádiz,ȱSpainȱ
‡ȱUNESCOȱUNITWIN/WiCop,ȱFacultadȱdeȱCienciasȱdelȱMarȱyȱAmbientales,ȱ
UniversidadȱdeȱCádiz,ȱPolígonoȱRíoȱSanȱPedroȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱ
Spain.ȱ
Abstractȱ
Theȱ detoxificationȱ andȱ antioxidantȱ systemsȱ inȱ theȱ clamȱ Ruditapesȱ
philippinarumȱ wasȱ studiedȱ byȱ analyzingȱ theȱ kineticsȱ ofȱ aȱ setȱ ofȱ biomarkers.ȱ
Phaseȱ Iȱ cytochromeȱ P450Aȱ (CYP1A)ȱ levelsȱ wereȱ measuredȱ asȱ digestiveȱ glandȱ
ethoxyresorufinȱOȬdeethylaseȱ(EROD)ȱactivityȱandȱbyȱgluthationeȬSȬtransferaseȱ
(Phaseȱ II),ȱ whereasȱ antioxidantȱ activityȱ wasȱ evaluatedȱ byȱ theȱ glutationeȱ
reductaseȱ (GR)ȱ andȱ glutationeȱ peroxidasaȱ (GPX)ȱ inductions.ȱ Analysesȱ wereȱ
performedȱ onȱ clamsȱ exposedȱ toȱ PAHsȱ contaminatedȱ sedimentsȱ underȱ fieldȱ
conditionsȱ afterȱ 7,ȱ 14,ȱ 21ȱ andȱ 28ȱ daysȱ ofȱ exposure.ȱ Histopathologicalȱ lesionsȱ
wereȱalsoȱdeterminedȱtoȱassessȱtheȱeffectsȱofȱpollutantsȱinȱtargetȱtissuesȱsuchȱasȱ
gillsȱ andȱ digestiveȱ glands.ȱ Bioassaysȱ wereȱ performedȱ underȱ fieldȱ conditionsȱ
usingȱcontaminatedȱsedimentsȱfromȱtwoȱareasȱofȱtheȱSpanishȱcoastȱaffectedȱbyȱ
oilȱ spills.ȱ Sedimentsȱ fromȱ theȱ selectedȱ sitesȱwereȱ chemicallyȱ characterizedȱ andȱ
theȱ dataȱ obtainedȱ wereȱ correlatedȱ withȱ theȱ kineticȱ approachȱ ofȱ biomarkers.ȱ
Resultsȱ showȱ anȱ importantȱ relationshipȱ betweenȱ theȱ phaseȱ Iȱ andȱ IIȱ
detoxificationȱ enzymesȱ inȱ theȱ clamȱ R.ȱ philippinarumȱ whereasȱ hitopathologicalȱ
lesionsȱ wereȱ mainlyȱ relatedȱ toȱ generalȱ stress.ȱ Inȱ addition,ȱ toxicityȱ testingȱ
followingȱaȱkineticȱapproachȱunderȱfieldȱconditionsȱcanȱbeȱconsideredȱaȱsuitableȱ
toolȱ toȱ monitoreȱ theȱ pollutantsȱ impact,ȱ asȱ wellȱ asȱ toȱ detectȱ otherȱ sourcesȱ ofȱ
contamination.ȱ
ȱȱȱEnvironementalȱToxicologyȱandȱChemistry (enviado)
- 211 -
Keywords:ȱ oilȱ spill,ȱ sedimentȱ quality,ȱ detoxification,ȱ histopathology,ȱ
contaminationȱ
1.ȱIntroductionȱ
Biomarkersȱ haveȱ beenȱ shownȱ toȱ beȱ usefulȱ toolsȱ inȱ characterizingȱ theȱ
healthȱ statusȱ ofȱ animalsȱ fromȱ impactedȱ areas,ȱ whereȱ complexȱ mixturesȱ ofȱ
pollutantsȱ areȱ usuallyȱ presentȱ [1,ȱ 2,ȱ 3,ȱ 4,ȱ 5].ȱ Ecotoxicityȱ studiesȱ basedȱ onȱ
biomarkersȱ allowȱ toȱ determineȱ theȱ impactȱ ofȱ environmentalȱ stressorsȱ andȱ toȱ
easilyȱfollowȱtheȱevolutionȱofȱtheȱecosystemȱtowardsȱdegradationȱorȱrestorationȱ
[6].ȱ
EthoxyresorufinȱOȬdeethylaseȱactivityȱ(EROD)ȱrepresentsȱaȱgoodȱmarkerȱ
inȱMFOȱ(mixedȬfunctionȱoxygenase),ȱwhichȱisȱtheȱfirstȱmodeȱofȱdetoxificationȱofȱ
manyȱ organicȱ pollutantsȱ (polycyclicȱ aromaticȱ hydrocarbonsȱ ȬPAHsȬ,ȱ
polychlorinatedȱ biphenylsȱ ȬPCBsȬ).ȱ Theȱ measurementȱ ofȱ ERODȱ activityȱ isȱ
successfullyȱ usedȱ asȱ aȱ potentialȱ biomarkerȱ ofȱ exposureȱ toȱ xenobioticȱ
contaminantsȱ inȱ marineȱ pollutionȱ monitoring.ȱ GlutathioneȬSȬtransferaseȱ (GST)ȱ
representsȱ aȱ phaseȱ IIȱ detoxificationȱ enzymeȱ butȱ alsoȱ implicatedȱ inȱ oxidativeȱ
stressȱ events;ȱ aȱ criticalȱ roleȱ forȱ GSTsȱ isȱ obviouslyȱ defenceȱ againstȱ oxidativeȱ
damageȱandȱperoxidativeȱproductsȱofȱDNAȱandȱlipidsȱ[7].ȱDueȱtoȱtheȱroleȱthatȱ
GSTsȱ playȱ inȱ conjugatingȱ reactiveȱ epoxideȱ speciesȱ andȱ otherȱ electrophiles,ȱ
inductionȱofȱtheseȱenzymesȱmustȱbeȱconsideredȱtoȱbeȱbeneficialȱ[7].ȱGlutathioneȱ
peroxidasesȱ (GPX)ȱ catalyseȱ mainlyȱ theȱ reductionȱ ofȱ organicȱ peroxidesȱ toȱ
alcoholsȱ usingȱ reducedȱ glutathioneȱ [8]ȱ whereasȱ glutathioneȱ reductaseȱ (GR)ȱ isȱ
alsoȱusedȱasȱantioxidantȱparameter.ȱInȱaddition,ȱhistopathologicalȱalterationsȱinȱ
gillsȱ andȱ digestiveȱ glandsȱ ofȱ bivalveȱ molluscsȱ tissuesȱ haveȱ beenȱ shownȱ toȱ beȱ
responsiveȱ andȱ sensitiveȱ toȱ aȱ wideȱ rangeȱ ofȱ contaminantsȱ andȱ haveȱ beenȱ
developedȱ andȱ recommendedȱ asȱ biomarkersȱ forȱ monitoringȱ theȱ effectsȱ ofȱ
pollutionȱ[9].ȱ
- 212 -
Theȱaimȱofȱthisȱstudyȱwasȱtoȱassessȱtheȱdetoxificationȱsystemȱofȱtheȱclamȱ
Ruditappesȱ philippinarumȱ exposedȱ toȱ oilȬcontaminatedȱ sedimentsȱ underȱ fieldȱ
conditionsȱ byȱ analyzingȱ theȱ kineticȱ ofȱ biomarkersȱ ofȱ exposureȱ relatedȱ toȱ theȱ
detoxificationȱsystemȱandȱantioxidantȱactivities.ȱ
2.ȱMethodologyȱȱ
Theȱ studyȱ sitesȱ chosenȱ inȱ theȱ presentȱ studyȱ haveȱ beenȱ affectedȱ byȱ oilȱ
spillsȱbyȱaȱdifferentȱway.ȱTheȱBayȱofȱAlgecirasȱ(SȱSpain)ȱsuffersȱchronicȱeffectsȱ
dueȱ toȱ theȱ severalȱ spillsȱ ofȱ oilȱ andȱ otherȱ compoundsȱ thatȱ supposeȱ anȱ inputȱ ofȱ
contaminantsȱ inȱ theȱ waterȱ andȱ sedimentȱ ofȱ theȱ zone.ȱ Onȱ theȱ otherȱ hand,ȱ theȱ
GalicianȱCoastȱ(NWȱSpain)ȱexperiencedȱoneȱofȱtheȱmajorȱaccidentalȱoilȱspillsȱinȱ
Europeȱ whenȱ inȱ Novemberȱ 2002,ȱ theȱ tankerȱ Prestigeȱ startedȱ droppingȱ heavyȱ
fuelȱoilȱbeyondȱ66,000ȱtons.ȱThreeȱsitesȱwereȱselectedȱinȱtheȱGalicianȱcoast,ȱtwoȱ
inȱ theȱ bayȱ ofȱ CormeȬLaxeȱ (CL1,ȱ CL2)ȱ andȱ oneȱ inȱ theȱ Atlanticȱ Islandȱ Nationalȱ
Parkȱ(AINP2);ȱbothȱareasȱwereȱimportantlyȱaffectedȱbyȱtheȱPrestigeȱoilȱspill,ȱandȱ
haveȱbeenȱrecoveredȱinȱlastȱfewȱyearsȱ[9].ȱTwoȱstationsȱwereȱalsoȱselectedȱinȱtheȱ
mouthȱofȱtheȱriverȱGuadarranqueȱinȱtheȱBayȱofȱAlgecirasȱ(ALG1ȱandȱALG2).ȱAȱ
referenceȱsiteȱwasȱselectedȱinȱaȱcleanȱareaȱinȱtheȱBayȱofȱCádizȱ(SȱSpain)ȱ[4]ȱȱ
Individualsȱ ofȱ Ruditapesȱ Philippinarumȱ wereȱ obtainedȱ fromȱ anȱ
aquacultureȱ farm,ȱ andȱ afterȱ oneȱ weekȱ ofȱ acclimation,ȱ clamsȱ wereȱ deployedȱ inȱ
cagesȱ (50cmȱ xȱ 25cmȱ xȱ 15cm)ȱ inȱ theȱ selectedȱ sitesȱ andȱ sedimentȱ samplesȱ wereȱ
carriedȱ toȱ theȱ laboratoryȱ toȱ performȱ theȱ chemicalȱ analysis.ȱ Theȱ experimentsȱ
lastedȱ28ȱdaysȱandȱsurveysȱwereȱperformedȱweekly.ȱȱ
Theȱ analysesȱ ofȱ PAHsȱ andȱ PCBsȱ boundȱ toȱ sedimentsȱ wereȱ carriedȱ outȱ
accordingȱ toȱ USEPAȱ SWȬ846ȱ Methodȱ 827C78082ȱ [11].ȱ Brieflyȱ driedȱ samplesȱ
wereȱSoxhletȱextractedȱwithȱnȬhexaneȱforȱ18ȱh,ȱandȱtheȱextractsȱwereȱisolatedȱbyȱ
columnȱ chromatographyȱ onȱ Florisileȱ aluminoȬsilica.ȱ PCBsȱ andȱ PAHsȱ wereȱ
elutedȱandȱtheirȱfractionsȱwereȱdriedȱinȱaȱrotatingȱevaporatorȱandȱreȬdissolvedȱ
- 213 -
inȱisooctane.ȱAromaticȱfractionsȱwereȱanalyzedȱonȱaȱHewlettePackardȱ(HP)ȱ5890ȱ
Seriesȱ IIȱ gasȱ chromatographerȱ coupledȱ withȱ anȱ HPȱ 5970ȱ massȱ spectrometer.ȱ
PAHsȱwereȱanalyzedȱbyȱGCȬMSȱusingȱselectedȱionȱmonitoringȱ(SIM).ȱAnalysisȱ
ofȱ PCBsȱ asȱ AROCLORȱ 1242ȱ andȱ AROCLORȱ 1260ȱ wasȱ performedȱ usingȱ theȱ
sameȱ instrumentȱ withȱ anȱ electronȱ captureȱ detectorȱ (GC/ECD).ȱ Forȱ bothȱ setȱ ofȱ
organicȱ chemicals,ȱ PAHsȱ andȱ AROCLOR,ȱ theȱ analyticalȱ procedureȱ showedȱ
agreementȱwithȱtheȱcertifiedȱvaluesȱofȱmoreȱthanȱ90%.ȱ
TraceȱmetalȱanalysisȱwereȱanalyzedȱasȱdescribedȱbyȱCasadoȬMartínezȱetȱ
al.[12];ȱbriefly,ȱ2.5ȱgȱofȱsedimentsȱ(<0.065ȱmm)ȱwereȱplacedȱinȱTeflonȱcontainersȱ
andȱ wereȱ digestedȱ inȱ microwaveȱ (400W,ȱ 15ȱ min,ȱ twice)ȱ withȱ HNO3ȱ 2N.ȱ Theȱ
extractsȱ wereȱ purifiedȱ byȱ passingȱ throughȱ aȱ CȬ18ȱ columnȱ andȱ metalsȱ analysesȱ
wereȱperformedȱbyȱanodicȱvoltamperimetryȱ(ȬZn,ȱPb,ȱNi,ȱCoȱandȱCuȬȱMetrohmȱ
ApplicationȱBulletinȱ Nºȱ147;ȱȬȱVȬȱMetrohmȱApplicationȱNoteȱNºȱVȬ81).ȱForȱHgȱ
theȱ coldȱ vapourȱ techniqueȱ wasȱ usedȱ andȱ wasȱ quantifiedȱ usingȱ atomicȱ
absorptionȱ spectrometry.ȱ Theȱ analyticalȱ proceduresȱ wereȱ checkedȱ usingȱ
referenceȱ materialȱ (MESSȬ1ȱ NRCȱ andȱ CRMȱ 277ȱ BCR)ȱ andȱ showedȱ aȱ recoveryȱ
greaterȱthanȱ90%ȱofȱtheȱcertifiedȱconcentration.ȱ
Testedȱorganismsȱwereȱcollectedȱfromȱcagesȱandȱwereȱdissectedȱtheȱdaysȱ
0,ȱ7,ȱ14,ȱ21ȱandȱ28ȱofȱexposure;ȱtheȱdigestiveȱglandȱwasȱwereȱextractedȱandȱkeptȱ
atȱȬ80ºCȱpriorȱhomogenizationȱȱ[1].ȱSamplesȱwereȱcentrifugedȱatȱ10,000gȱforȱ30ȱ
min,ȱandȱtheȱsupernatantȱwasȱusedȱforȱtheȱdetoxificationȱactivityȱdeterminationȱ
andȱ theȱ totalȱ proteinȱ contentȱ describedȱ byȱ Bradfordȱ [13].ȱ Mixedȱ functionȱ
oxygenaseȱ activity,ȱ whichȱ isȱ theȱ firstȱ modeȱ ofȱ detoxificationȱ ofȱ manyȱ organicȱ
pollutants,ȱ wasȱ measuredȱ usingȱ theȱ ERODȱ assayȱ [14].ȱ Theȱ oxidationȱ ofȱ 1ȱ mMȱ
NADPHȱ byȱ Glutathioneȱ Reductaseȱ activityȱ (GR)ȱ inȱ theȱ presenceȱ ofȱ 10ȱ mMȱ
oxidizedȱ glutathioneȱ wasȱ alsoȱ monitoredȱ atȱ 340ȱ nmȱ [15].ȱ Glutathioneȱ
Peroxidaseȱactivityȱ(GPX)ȱwasȱmeasuredȱaccordingȱtoȱMcFarlandȱetȱal.ȱ[14].ȱTheȱ
phaseȱ IIȱ metabolizingȱ enzymeȱ GlutathioneȬSȬtransferaseȱ (GST)ȱ activityȱ wasȱ
- 214 -
determinedȱ byȱ monitoringȱ theȱ rateȱ ofȱ conjugationȱ ofȱ glutathioneȱ (GSH)ȱ toȱ 1Ȭ
chloroȬ2,4Ȭdinitrobenzeneȱ (CDNB)ȱ atȱ 340ȱ nm,ȱ methodologyȱ adaptedȱ fromȱ
McFarlandȱ etȱ al.ȱ [15].ȱ Biomarkersȱ resultsȱ wereȱ normalizedȱ withȱ theȱ proteinȱ
content.ȱ Correlationȱ amongȱ chemicalsȱ andȱ biomarkersȱ wereȱ evaluatedȱ byȱ theȱ
Pearsonȱanalysisȱ(pȱ<ȱ0.05ȱandȱpȱ<ȱ0.01).ȱȱ
Gillsȱandȱliverȱtissuesȱofȱtheȱclamsȱwereȱalsoȱfixedȱinȱphosphateȱbufferedȱ
10%ȱ formaldehydeȱ (pHȱ 7.2)ȱ forȱ histopathologyȱ determinationȱ afterȱ 28ȱ daysȱ ofȱ
exposure.ȱ Afterȱ dehydrationȱ inȱ gradedȱ concentrationsȱ ofȱ ethanol,ȱ theȱ samplesȱ
wereȱ embeddedȱ inȱ paraffinȱ wax.ȱ Histologicalȱ sectionsȱ ofȱ 6ȱ toȱ 8ȱ ΐmȱ thicknessȱ
wereȱstainedȱwithȱHaematoxylin–ȱEosinȱandȱHaematoxylin–VOFȱ[16].ȱSectionsȱ
wereȱ reviewedȱ byȱ lightȱ microscopyȱLeitzȱ LaborluxȱSȱandȱ photographedȱ(Sonyȱ
DKCȬCM30).ȱ
3.ȱResultsȱ
3.1.ȱChemicalȱanalysisȱȱ
Chemicalȱ analysisȱ resultsȱ areȱ shownȱ inȱ tableȱ 1.ȱ Theȱ concentrationȱ ofȱ
PAHsȱinȱsedimentsȱisȱhigherȱinȱtheȱsedimentsȱfromȱtheȱBayȱofȱAlgecirasȱlocatedȱ
inȱtheȱsiteȱALG1ȱ(2961ȱΐgȱkgȬ1ȱdryȱweight),ȱfollowedȱbyȱthoseȱfromȱCL1ȱ(820ȱΐgȱ
kgȬ1ȱ dryȱ weight)ȱ inȱ CormeȬLaxeȱ andȱ ALG2ȱ (802ȱ ΐgȱ kgȬ1ȱ dryȱ weight)ȱ alsoȱ inȱ
Algeciras.ȱ Sedimentsȱ fromȱ theȱ AINPȱ presentȱ theȱ lowestȱ PAHsȱ contentȱ inȱ theirȱ
sediments.ȱ ALG2ȱ alsoȱ presentedȱ alsoȱ presentedȱ theȱ highestȱ concentrationȱ ofȱ
PCBsȱ(22ȱmgȱkgȬ1ȱdryȱweight),ȱCdȱ(0.17ȱmgȱkgȬ1ȱdryȱweight),ȱNiȱ(74.7ȱmgȱkgȬ1ȱdryȱ
weight),ȱ Coȱ (12.8ȱ mgȱ kgȬ1ȱ dryȱ weight)ȱ andȱ Vȱ (26.1ȱ mgȱ kgȬ1ȱ dryȱ weight).ȱ Theȱ
majorȱ amountȱ ofȱ Znȱ wasȱ foundȱ inȱ sedimentsȱ fromȱ CL1ȱ (244ȱ mgȱ kgȬ1ȱ dryȱ
weight),ȱ whereasȱ theȱ highestȱ concentrationȱ ofȱ Pbȱ wasȱ analyzedȱ inȱ sedimentsȱ
fromȱCL2ȱ(44ȱmgȱkgȬ1ȱdryȱweight)ȱbothȱsitesȱinȱCormeȬLaxe.ȱCuȱwasȱhigherȱinȱ
AINP2ȱ (31.6ȱ mgȱ kgȬ1ȱ dryȱ weight).ȱ ȱ Organicȱ contaminationȱ wasȱ notȱ detectedȱ inȱ
- 215 -
theȱ referenceȱ siteȱ (CA)ȱ whereasȱ theȱ metalȱ contentȱ inȱ sedimentsȱ wasȱ relativelyȱ
low.ȱ
Tableȱ 1.ȱ Concentrationȱ ofȱ PAHsȱ andȱ PCBsȱ (ΐgȱ kgȬ1ȱ dryȱ weight)ȱ andȱ
metalsȱ (mgȱ kgȬ1ȱ dryȱ weight)ȱ inȱ theȱ sedimentsȱ collectedȱ fromȱ theȱ studyȱ sitesȱ
(AINP:ȱ Atlanticȱ Nationalȱ Park,ȱ Galicia;ȱ CL:ȱ CormeȬLaxe,ȱ Galicia;ȱ ALG:ȱ Bayȱ ofȱ
Algeciras;ȱCA:ȱByaȱofȱCádiz).ȱn.d.:ȱnotȱdetected.ȱ
ȱ
ȱȱ
PAHsȱ PCBsȱ
Znȱ
Pbȱ
Cuȱ
Niȱ
Coȱ
ȱ
AINP2ȱ
239ȱ
4.76ȱ
37.5ȱ
6.54ȱ
31.6ȱ
5.02ȱ
0.87ȱ
CL1ȱ
820ȱ
2.28ȱ
244ȱ
14.3ȱ
19.1ȱ
7.03ȱ
0.67ȱ
ȱ CL2ȱ
537ȱ
2.60ȱ
65.7ȱ
44.0ȱ
22.1ȱ
9.39ȱ
1.21ȱ
ȱ ALG1ȱ
2961ȱ
22.0ȱ
138ȱ
21.6ȱ
5.01ȱ
74.7ȱ
12.8ȱ
ALG2ȱ
802ȱ
1.75ȱ
35.3ȱ
6.21ȱ
3.67ȱ
13.1ȱ
5.59ȱ
CAȱ
n.dȱ
n.d.ȱ
21.3ȱ
2.28ȱ
6.98ȱ
0.06ȱ
3.40ȱ
ȱ
3.2.ȱBiomarkersȱofȱexposureȱ
Biomarkerȱ responsesȱ ofȱ deployedȱ animalsȱ areȱ summarizedȱ inȱ Figureȱ 1ȱ
andȱ Figureȱ 2.ȱ Clamsȱ deployedȱ inȱ sitesȱ locatedȱ inȱ theȱ Galicianȱ Coastȱ presentedȱ
anȱ initialȱ increaseȱ ofȱ GPXȱ inductionȱ duringȱ theȱ firstȱ weekȱ ofȱ exposureȱ
(significantlyȱdifferentȱtoȱtheȱreferenceȱCA,ȱpȱ<ȱ0.01)ȱwhileȱtheȱfollowingȱweeksȱ
theȱ activityȱ reachedȱ basalȱ levelsȱ exceptȱ forȱ siteȱ CL2ȱ whichȱ continuedȱ risingȱ
showingȱaȱmaximumȱafterȱ28ȱdaysȱ(significantlyȱdifferentȱtoȱtheȱreferenceȱCA,ȱpȱ
<ȱ 0.01).ȱ Theȱ behaviourȱ ofȱ thisȱ biomarkerȱ inȱ organismsȱ collectedȱ fromȱ cagesȱ
placedȱ inȱ theȱ Bayȱ ofȱ Algecirasȱ wasȱ similarȱ toȱ thoseȱ fromȱ theȱ referenceȱ site,ȱ
althoughȱ differencesȱ wereȱ significantȱ (pȱ <ȱ 0.01).ȱ Theȱ GRȱ activityȱ showedȱ
significantȱ(pȱ<ȱ0.01)ȱdifferencesȱtoȱtheȱreferenceȱCAȱȱforȱtheȱsamplesȱcollectedȱinȱ
theȱ Galicianȱ Coast;ȱ thisȱ biomarkerȱ presentedȱ anȱ increaseȱ alongȱ theȱ exposureȱ
periodȱinȱclamsȱfromȱCormeȬLaxeȱwhereasȱtheȱotherȱstudyȱsitesȱpresentsȱsimilarȱ
- 216 -
inductionȱasȱtheȱreferenceȱsite,ȱexceptȱforȱaȱpeakȱdetectedȱinȱtheȱclamsȱfromȱtheȱ
AINPȱ whichȱ appearedȱ afterȱ 14ȱ daysȱ ofȱ deploymentȱ (significantlyȱ differentȱ toȱ
theȱreferenceȱCA,ȱpȱ<ȱ0.01).ȱALG2ȱalsoȱshowedȱsignificantȱdifferencesȱ(pȱ<ȱ0.05)ȱ
toȱ CAȱ theȱ lastȱ dayȱ ofȱ survey.ȱ Duringȱ theȱ firstȱ weekȱ ofȱ experimentȱ theȱ GSTȱ
activityȱ increasedȱ especiallyȱ inȱ clamsȱ collectedȱ inȱ theȱ Galicianȱ Coastȱ
(significantlyȱ differentȱ toȱ theȱ referenceȱ CA,ȱ pȱ <ȱ 0.01);ȱ aȱ secondȱ maximumȱ wasȱ
observedȱ inȱ theȱ lastȱ surveyȱ inȱ siteȱ CL2.ȱ Aȱ peakȱ wasȱ detectedȱ afterȱ 21ȱ daysȱ inȱ
clamsȱ collectedȱ fromȱ ALG1ȱ inȱ theȱ Bayȱ ofȱ Algeciras.ȱ ERODȱ activityȱ alsoȱ
presentedȱ anȱ intialȱ inductionȱ duringȱ theȱ firstȱ weekȱ ofȱ exposureȱ forȱ organismsȱ
deployedȱ inȱ sitesȱ fromȱ theȱ Galicianȱ Coastȱ althoughȱ theȱ generalȱ patternȱ wasȱ toȱ
estabilizeȱalongȱtheȱtime,ȱshowingȱsimilarȱresultsȱtheȱlastȱdayȱofȱtheȱexperiment.ȱ
Significantȱdifferencesȱ(pȱ<ȱ0.01)ȱwithȱtheȱreferenceȱsiteȱCAȱwhereȱobservedȱforȱ
allȱstudyȱsites.ȱȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
- 217 -
20
CL1
CL2
AINP2
ALG1
ALG2
CA
GR (nmol/min/mg)
15
10
5
0
0
5
10
15
20
25
30
20
25
30
time (d)
30
CL1
CL2
AINP2
ALG1
ALG2
CA
GPX (nmol/min/mg)
25
20
15
10
5
0
0
5
10
15
time (d)
Figureȱ1.ȱGPXȱandȱGRȱactivitiesȱ(nmol/min/mgȱprotein)ȱmeasuredȱinȱtheȱ
digestiveȱglandȱofȱR.ȱphilippinarumȱexposedȱalongȱ28ȱdaysȱtoȱoilȱcontaminatedȱ
sedimentsȱ (AINP:ȱ Atlanticȱ Nationalȱ Park,ȱ Galicia;ȱ CL:ȱ CormeȬLaxe,ȱ Galicia;ȱ
ALG:ȱBayȱofȱAlgeciras)ȱunderȱfieldȱconditionsȱ
- 218 -
7000
CL1
CL2
AINP2
ALG1
ALG2
CA
6000
GST (nmol/min/mg)
5000
4000
3000
2000
1000
0
0
5
10
15
20
25
30
time (d)
10
CL1
CL2
AINP2
ALG1
ALG2
CA
EROD (pmol/min/mg)
8
6
4
2
0
0
5
10
15
20
25
30
time (d)
Figureȱ 2.ȱ GSTȱ (nmol/min/mgȱ protein)ȱ andȱ ERODȱ (pmol/mg/min)ȱ
activitiesȱ inȱ theȱ digestiveȱ glandȱ ofȱ R.ȱ philippinarumȱ exposedȱ alongȱ 28ȱ daysȱ toȱ
oilȱcontaminatedȱsedimentsȱ(AINP:ȱAtlanticȱNationalȱPark,ȱGalicia;ȱCL:ȱCormeȬ
Laxe,ȱGalicia;ȱALG:ȱBayȱofȱAlgeciras)ȱunderȱfieldȱconditions.ȱ
ȱ
- 219 -
3.3.ȱBiomarkersȱofȱeffectȱȱ
Afterȱ 28ȱ daysȱ ofȱ exposureȱ toȱ theȱ sedimentsȱ clamsȱ presentedȱ differentȱ
alterationsȱ inȱ targetȱ tissuesȱ (gillsȱ andȱ gut).ȱ Mostȱ ofȱ theȱ analyzedȱ organismsȱ
showȱ lesionsȱ relatedȱ withȱ generalȱ stress,ȱ includingȱ lossȱ ofȱ epithelialȱ cells,ȱ
lamellaeȱ separationȱ andȱ haematiticȱ reaction.ȱ Organismsȱ exposedȱ toȱ sedimentsȱ
fromȱtheȱBayȱofȱAlgecirasȱwereȱtheȱmostȱaffectedȱfollowedȱbyȱclamsȱexposedȱtoȱ
sedimentsȱ fromȱ CormeȬLaxe,ȱ andȱ finallyȱclamsȱfromȱtheȱCiesȱtreatmentȱwhichȱ
showedȱalterationsȱdueȱtoȱgeneralȱenvironmentalȱstress.ȱȱ
4.ȱDiscussionȱ
Theȱfluctuationȱofȱdifferentȱbiomarkersȱinȱresponseȱtoȱdifferentȱtoxicantsȱ
providesȱaȱpatternȱofȱresultsȱwhichȱcanȱgiveȱcluesȱasȱtoȱtheȱtypeȱofȱpollutantȱthatȱ
isȱ causingȱ theȱ observedȱ effectȱ [17].ȱ Inȱ general,ȱ resultsȱ obtainedȱ inȱ thisȱ studyȱ
showedȱ anȱ inductionȱ ofȱ GPX,ȱ GSTȱ andȱ ERODȱ activityȱ presentedȱ aȱ maximumȱ
afterȱ theȱ firstȱ 7ȱ daysȱ ofȱ exposure,ȱ whereasȱ GRȱ showsȱ anȱ increaseȱ alongȱ theȱ
deploymentȱ period.ȱ Correlationsȱ amongȱ biomarkersȱ (tableȱ 2)ȱ wereȱ significantȱ
forȱtheȱinductionȱofȱtheȱdetoxificationȱsystemȱdeterminedȱbyȱERODȱactivityȱandȱ
theȱantioxidantȱenzymesȱGRȱandȱGPXȱȱtheȱdayȱ7ȱofȱexposure.ȱThisȱpointsȱtoȱtheȱ
factȱ thatȱ organismsȱ respondedȱ toȱ environmentalȱ stressorsȱ mainlyȱ duringȱ theȱ
firstȱ daysȱ ofȱ deployment.ȱ Theȱ correlationsȱ observedȱ indicatedȱ aȱ relationshipȱ
amongȱ organicȱ contaminantsȱ (PAHsȱ andȱ PCBs)ȱ andȱ metalsȱ Niȱ andȱ Coȱ inȱ theȱ
sediments,ȱalthoughȱnoȱlinksȱwereȱdetectedȱwithȱbiomarkers.ȱAȱconnectionȱwasȱ
detectedȱ betweenȱ Pbȱ andȱ theȱ inductionȱ ofȱ detoxificationȱ andȱ antioxidantȱ
enzymesȱ (GPX,ȱ GSTȱ andȱ ERODȱ activities)ȱ afterȱ 28ȱ daysȱ ofȱ exposure.ȱ Theȱ Pbȱ
originȱ inȱ pollutingȱ oilȱ wasȱ corroboratedȱ byȱ otherȱ authorsȱ [18];ȱ thereȱ isȱ oftenȱ aȱ
strongȱrelationshipȱbetweenȱleadȱconcentrationsȱinȱsoilȱandȱparentȱmaterialȱ[19],ȱ
soȱthisȱcompoundȱcouldȱbeȱrelatedȱtoȱanȱorganicȱcontaminationȱassociatedȱwithȱ
aȱsourceȱofȱhydrocarbonsȱnotȱlinkedȱtoȱtheȱPAHsȱboundȱsediment,ȱandȱwhichȱisȱȱ
- 220 -
0.118ȱ
Ȭ0.297ȱ
0.9141ȱ
0.7991ȱ
Ȭ0.109ȱ
Ȭ0.002ȱ
0.411ȱ
Ȭ0.238ȱ
Ȭ0.192ȱ
Ȭ0.044ȱ
0.129ȱ
Ȭ0.124ȱ
0.016ȱ
0.079ȱ
0.869ȱ
Ȭ0.255ȱ
Ȭ0.317ȱ
0.025ȱ
Ȭ0.117ȱ
0.458ȱ
0.214ȱ
Ȭ0.149ȱ
0.9691ȱ
0.8631ȱ
Ȭ0.209ȱ
Ȭ0.245ȱ
0.299ȱ
Ȭ0.185ȱ
Ȭ0.345ȱ
Ȭ0.269ȱ
Ȭ0.042ȱ
Ȭ0.133ȱ
Ȭ0.208ȱ
Ȭ0.117ȱ
0.758ȱ
Ȭ0.144ȱ
Ȭ0.479ȱ
Ȭ0.208ȱ
Ȭ0.313ȱ
0.272ȱ
Znȱ
Pbȱ
Cuȱ
Niȱ
Coȱ
GPXȱ7ȱ
GPXȱ14ȱ
GPXȱ21ȱ
GPXȱ28ȱ
GRȱ7ȱ
GR14ȱ
GRȱ21ȱ
GRȱ28ȱ
GSTȱ7ȱ
GSTȱ14ȱ
GSTȱ21ȱ
GSTȱ28ȱ
ERODȱ7ȱ
ERODȱ14ȱ
ERODȱ21ȱ
ERODȱ28ȱ
0.088ȱ
0.275ȱ
1ȱ
PCBsȱ
0.041ȱ
Ȭ0.198ȱ
Ȭ0.024ȱ
0.421ȱ
0.170ȱ
0.817ȱ
0.148ȱ
0.094ȱ
0.128ȱ
0.125ȱ
Ȭ0.035ȱ
0.414ȱ
0.068ȱ
0.485ȱ
0.294ȱ
0.492ȱ
0.187ȱ
0.335ȱ
0.020ȱ
0.266ȱ
1ȱ
0.275ȱ
0.458ȱ
0.9051ȱ
1ȱ
0.9051ȱ
PAHsȱ
Znȱ
PCBsȱ
Ȭ0.100ȱ
0.225ȱ
0.7931ȱ
Ȭ0.421ȱ
Ȭ0.304ȱ
0.047ȱ
Ȭ0.190ȱ
0.143ȱ
0.8111ȱ
0.085ȱ
0.817ȱ
0.257ȱ
Ȭ0.22ȱ
Ȭ0.206ȱ
Ȭ0.548ȱ
Ȭ0.095ȱ
Ȭ0.185ȱ
Ȭ0.522ȱ
Ȭ0.261ȱ
Ȭ0.258ȱ
0.056ȱ
Ȭ0.239ȱ
Ȭ0.406ȱ
Ȭ0.293ȱ
0.053ȱ
Ȭ0.236ȱ
0.121ȱ
0.138ȱ
Ȭ0.166ȱ
0.383ȱ
Ȭ0.277ȱ
Ȭ0.279ȱ
0.8821ȱ
1ȱ
Ȭ0.123ȱ
0.260ȱ
0.335ȱ
0.9141ȱ
0.9691ȱ
Niȱ
0.702ȱ
Ȭ0.640ȱ
Ȭ0.078ȱ
0.172ȱ
0.061ȱ
Ȭ0.203ȱ
Ȭ0.131ȱ
0.076ȱ
0.8741ȱ
0.171ȱ
0.310ȱ
Ȭ0.299ȱ
Ȭ0.052ȱ
Ȭ0.342ȱ
Ȭ0.123ȱ
1ȱ
0.129ȱ
0.020ȱ
Ȭ0.297ȱ
Ȭ0.149ȱ
Cuȱ
0.475ȱ
0.059ȱ
0.213ȱ
0.059ȱ
0.260ȱ
0.129ȱ
1ȱ
0.266ȱ
0.118ȱ
0.214ȱ
Pbȱ
0.215ȱ
Ȭ0.144ȱ
Ȭ0.323ȱ
Ȭ0.583ȱ
Ȭ0.073ȱ
0.746ȱ
Ȭ0.182ȱ
Ȭ0.307ȱ
Ȭ0.312ȱ
Ȭ0.044ȱ
Ȭ0.274ȱ
Ȭ0.566ȱ
Ȭ0.231ȱ
0.114ȱ
Ȭ0.423ȱ
Ȭ0.431ȱ
1ȱ
0.8821ȱ
Ȭ0.342ȱ
0.059ȱ
0.187ȱ
0.7991ȱ
0.8631ȱ
Coȱ
0.860 ȱ
0.949 ȱ
Ȭ0.152ȱ
Ȭ0.310ȱ
Ȭ0.321ȱ
0.641ȱ
0.455ȱ
0.816ȱ
0.8221ȱ
Ȭ0.611ȱ
0.176ȱ
Ȭ0.858ȱ
0.8372ȱ
0.9341ȱ
0.105ȱ
0.743ȱ
Ȭ0.400ȱ
Ȭ0.029ȱ
0.738ȱ
Ȭ0.176ȱ
0.392ȱ
0.894ȱ
Ȭ0.907ȱ
Ȭ0.028ȱ
0.388ȱ
1ȱ
0.581ȱ
Ȭ0.212ȱ
0.114ȱ
0.383ȱ
0.31ȱ
0.475ȱ
0.485ȱ
0.411ȱ
0.299ȱ
GPXȱ
21ȱ
0.26ȱ
0.416ȱ
0.559ȱ
0.496ȱ
0.7782ȱ
0.483ȱ
0.137ȱ
0.767ȱ
0.321ȱ
0.628ȱ
Ȭ0.482ȱ
0.699ȱ
1
0.124ȱ
1
0.27ȱ
1ȱ
0.581ȱ
0.675ȱ
0.675ȱ
Ȭ0.423ȱ
Ȭ0.277ȱ
Ȭ0.299ȱ
0.059ȱ
0.294ȱ
Ȭ0.002ȱ
Ȭ0.245ȱ
GPXȱ
14ȱ
Ȭ0.212ȱ
1ȱ
Ȭ0.431ȱ
Ȭ0.279ȱ
Ȭ0.052ȱ
0.213ȱ
0.492ȱ
Ȭ0.109ȱ
Ȭ0.209ȱ
GPXȱ
7ȱ
221
0.7401ȱ
0.919ȱ
0.095ȱ
0.305ȱ
0.9061ȱ
Ȭ0.013ȱ
Ȭ0.583ȱ
0.067ȱ
0.366ȱ
0.744ȱ
Ȭ0.115ȱ
0.274ȱ
1ȱ
0.388ȱ
0.124ȱ
0.27ȱ
Ȭ0.231ȱ
Ȭ0.166ȱ
0.076ȱ
0.125ȱ
0.138ȱ
0.321ȱ
0.861ȱ
Ȭ0.242ȱ
0.635ȱ
0.459ȱ
0.9881ȱ
0.199ȱ
0.221ȱ
0.568ȱ
0.6912ȱ
0.579ȱ
0.345ȱ
0.586ȱ
1ȱ
0.274ȱ
Ȭ0.323ȱ
Ȭ0.768ȱ
Ȭ0.146ȱ
0.473ȱ
Ȭ0.035ȱ
Ȭ0.417ȱ
0.347ȱ
0.9471ȱ
Ȭ0.003ȱ
Ȭ0.926ȱ
1ȱ
0.586ȱ
Ȭ0.115ȱ
Ȭ0.907ȱ
0.699ȱ
0.9491ȱ
Ȭ0.028ȱ
Ȭ0.274ȱ
Ȭ0.239ȱ
Ȭ0.712ȱ
0.917ȱ
0.757ȱ
Ȭ0.846ȱ
0.529ȱ
0.595ȱ
Ȭ0.192ȱ
Ȭ0.389ȱ
0.685ȱ
1ȱ
Ȭ0.926ȱ
0.345ȱ
0.744ȱ
0.894ȱ
0.767ȱ
Ȭ0.482ȱ
Ȭ0.044ȱ
0.056ȱ
0.121ȱ
0.061ȱ
Ȭ0.035ȱ
Ȭ0.203ȱ
0.129ȱ
Ȭ0.042ȱ
GRȱ
21ȱ
Ȭ0.044ȱ
Ȭ0.269ȱ
GRȱ
14ȱ
Ȭ0.566ȱ
Ȭ0.406ȱ
Ȭ0.131ȱ
0.414ȱ
0.171ȱ
0.068ȱ
Ȭ0.192ȱ
Ȭ0.345ȱ
GRȱ
7ȱ
0.8741ȱ
Ȭ0.238ȱ
Ȭ0.185ȱ
GPXȱ
28ȱ
0.081ȱ
0.843ȱ
0.06ȱ
0.662ȱ
0.114ȱ
0.528ȱ
0.223ȱ
0.403ȱ
1ȱ
0.685ȱ
Ȭ0.003ȱ
0.579ȱ
0.366ȱ
0.392ȱ
0.137ȱ
0.628ȱ
Ȭ0.312ȱ
Ȭ0.258ȱ
Ȭ0.236ȱ
0.172ȱ
0.128ȱ
Ȭ0.124ȱ
Ȭ0.133ȱ
GRȱ
28ȱ
Ȭ0.158ȱ
Ȭ0.479ȱ
0.014ȱ
0.638ȱ
0.035ȱ
0.495ȱ
0.412ȱ
1ȱ
0.403ȱ
Ȭ0.8242ȱ
0.056ȱ
0.416ȱ
0.523ȱ
Ȭ0.6792ȱ
0.193ȱ
1ȱ
0.412ȱ
0.223ȱ
Ȭ0.192ȱ
0.347ȱ
Ȭ0.389ȱ
0.568ȱ
0.9471ȱ
Ȭ0.583ȱ
Ȭ0.40ȱ
0.559ȱ
0.483ȱ
Ȭ0.182ȱ
Ȭ0.185ȱ
Ȭ0.522ȱ
Ȭ0.640ȱ
0.148ȱ
0.079ȱ
Ȭ0.117ȱ
GSTȱ
14ȱ
0.691 ȱ
2
0.067ȱ
Ȭ0.176ȱ
0.496ȱ
0.7782ȱ
Ȭ0.307ȱ
Ȭ0.261ȱ
0.053ȱ
Ȭ0.078ȱ
0.094ȱ
0.016ȱ
Ȭ0.208ȱ
GSTȱ
7ȱ
Ȭ0.087ȱ
0.382ȱ
0.5ȱ
Ȭ0.984ȱ
0.077ȱ
1ȱ
0.193ȱ
0.495ȱ
0.528ȱ
0.595ȱ
Ȭ0.417ȱ
0.221ȱ
Ȭ0.013ȱ
0.743ȱ
0.738ȱ
0.416ȱ
0.746ȱ
0.817ȱ
Ȭ0.293ȱ
0.702ȱ
0.817ȱ
0.869ȱ
0.758ȱ
GSTȱ
21ȱ
0.523ȱ
0.6772ȱ
0.818ȱ
Ȭ0.166ȱ
0.27ȱ
1ȱ
Ȭ0.314ȱ
Ȭ0.863ȱ
0.455ȱ
1ȱ
0.270ȱ
Ȭ0.984ȱ
Ȭ0.6792ȱ
0.077ȱ
0.638ȱ
0.662ȱ
Ȭ0.846ȱ
0.473ȱ
0.988 ȱ
1
0.305ȱ
Ȭ0.858ȱ
0.8372ȱ
0.9341ȱ
Ȭ0.583ȱ
Ȭ0.548ȱ
Ȭ0.19ȱ
0.085ȱ
0.421ȱ
Ȭ0.317ȱ
Ȭ0.479ȱ
EROD
7ȱ
0.035ȱ
0.114ȱ
0.529ȱ
Ȭ0.035ȱ
0.199ȱ
0.9061ȱ
0.105ȱ
Ȭ0.029ȱ
0.26ȱ
Ȭ0.073ȱ
Ȭ0.095ȱ
0.143ȱ
0.8111ȱ
0.17ȱ
Ȭ0.255ȱ
Ȭ0.144ȱ
GSTȱ
28ȱ
Ȭ0.211ȱ
0.904ȱ
1ȱ
0.455ȱ
Ȭ0.166ȱ
0.500ȱ
0.416ȱ
0.014ȱ
0.060ȱ
0.757ȱ
Ȭ0.146ȱ
0.459ȱ
0.095ȱ
0.455ȱ
0.8221ȱ
0.176ȱ
Ȭ0.323ȱ
Ȭ0.206ȱ
Ȭ0.421ȱ
0.047ȱ
Ȭ0.024ȱ
0.025ȱ
Ȭ0.208ȱ
Ȭ0.932ȱ
1ȱ
0.904ȱ
Ȭ0.863ȱ
0.818ȱ
0.382ȱ
0.056ȱ
Ȭ0.479ȱ
0.843ȱ
0.917ȱ
Ȭ0.768ȱ
0.635ȱ
0.919ȱ
0.641ȱ
0.816ȱ
Ȭ0.611ȱ
Ȭ0.144ȱ
Ȭ0.22ȱ
Ȭ0.10ȱ
Ȭ0.304ȱ
Ȭ0.198ȱ
Ȭ0.117ȱ
Ȭ0.313ȱ
1ȱ
Ȭ0.932ȱ
Ȭ0.211ȱ
Ȭ0.314ȱ
0.6771ȱ
Ȭ0.087ȱ
Ȭ0.8241ȱ
Ȭ0.158ȱ
0.081ȱ
Ȭ0.712ȱ
Ȭ0.323ȱ
Ȭ0.242ȱ
0.7402ȱ
Ȭ0.321ȱ
Ȭ0.31ȱ
Ȭ0.152ȱ
0.215ȱ
0.257ȱ
0.225ȱ
0.7931ȱ
0.041ȱ
0.088ȱ
0.272ȱ
ERODȱ ERODȱ ERODȱ
14ȱ
21ȱ
28ȱ
7,ȱ14,ȱ21ȱandȱ28ȱcorrespondȱtoȱtheȱsamplingȱdate
3
PAHsȱ
peroxidaseȱ(GPX)ȱactivity,ȱglutathioneȱreductaseȱ(GR),ȱglutathioneȬSȬtransferaseȱ(GST)ȱactivityȱandȱEthoxyresorufinȱOȬdeethylaseȱ(EROD)ȱactivity.ȱ
2
ȱ
Tableȱ2.ȱPearsonȱcorrelationȱ(*p<0.05,ȱ**p<0.01)ȱresultsȱamongȱchemicalȱcompoundsȱboundȱtoȱsedimentsȱandȱbiomarkers:ȱglutathioneȱ
1
producingȱ theȱ activiationȱ ofȱ theseȱ defenceȱ systems.ȱ Leadȱ isȱ notȱ essentialȱ toȱ
metabolismȱandȱitȱisȱ highlyȱ toxicȱ forȱbiota,ȱthisȱtoxicityȱisȱdeterminedȱbyȱtheirȱ
abilityȱ toȱ regulateȱ anomalousȱ concentrations,ȱ throughȱ variousȱ detoxificationȱ
mechanismsȱ[20].ȱTheȱcorrelationȱshownȱbetweenȱPbȱandȱbiomarkersȱoccursȱinȱ
theȱ dayȱ 28ȱ whatȱ suggestsȱ thatȱ itȱ isȱ afterȱ thisȱ periodȱ whenȱ theȱ metalȱ becameȱ
bioreactiveȱtoȱtheȱstudiedȱbiomarkers.ȱȱ
Inȱ general,ȱ theȱ behaviourȱ ofȱ GSTȱ wasȱ similarȱ toȱ ERODȱ inȱ mostȱ ofȱ theȱ
casesȱ whatȱ suggestsȱ aȱ relationshipȱ amongȱ theseȱ phaseȱ Iȱ andȱ IIȱ detoxificationȱ
biomarkers.ȱ Theȱ firstȱ increaseȱ ofȱ theȱ biomarkersȱ ofȱ effectȱ denotesȱ anȱ initialȱ
activationȱ ofȱ theȱ detoxificationȱ systemȱ whichȱ isȱ inhibitedȱ inȱ subsequentlyȱ
surveys.ȱWhenȱthereȱisȱanȱincreaseȱonȱtheȱCYPȱ450ȱcontent,ȱthereȱisȱnecessarilyȱ
anȱ incrementȱ ofȱ metabolitesȱ toȱ beȱ conjugatedȱ withȱ phaseȱ IIȱ enzymes,ȱ thusȱ
preventingȱ cellȱ damage;ȱ whenȱ theseȱ waterȱ solubleȱ compoundsȱ areȱ conjugatedȱ
withȱphaseȱIIȱenzymes,ȱGSTȱmayȱinterveneȱ[21].ȱȱ
Previousȱstudiesȱwithȱotherȱorganismsȱhaveȱobservedȱbothȱincreaseȱandȱ
decreaseȱ ofȱ GPXȱ activityȱ inȱ fieldȱ surveysȱ [7];ȱ resultsȱ obtainedȱ inȱ thisȱ
investigationȱ showȱ anȱ initialȱ fluctuationȱ ofȱ thisȱ biomarkerȱ (increaseȱ inȱ theȱ
GalicianȱCoastȱandȱdecreaseȱinȱtheȱBayȱofȱAlgeciras)ȱfollowedȱbyȱaȱestabilizationȱ
exceptȱforȱsiteȱCL2ȱinȱCormeȬLaxe.ȱȱ
Theȱ activationȱ ofȱ GRȱ playsȱ aȱ fundamentalȱ roleȱ inȱ theȱ faceȱ ofȱ oxidativeȱ
stressȱ maintainingȱ theȱ properȱ redoxȱ statusȱ ofȱ glutathione,ȱ whichȱ isȱ importantȱ
bothȱ asȱ cofactorȱ ofȱ severalȱ antioxidantȱ enzymesȱ andȱ asȱ indirectȱ scavengerȱ ofȱ
oxyradicalsȱ [22].ȱ Inȱ theȱ presentȱ studyȱ organismsȱ exposedȱ toȱ sedimentsȱ fromȱ
CormeȬLaxeȱ presentȱ aȱ continuousȱ inductionȱ ofȱ thisȱ biomarkerȱ suggestingȱ theȱ
presenceȱofȱaȱchronicȱsourceȱofȱstressȱwhichȱisȱnotȱrelatedȱtoȱtheȱcontaminantsȱ
measuredȱinȱtheȱsediments,ȱbutȱprobablyȱrelatedȱtoȱtheȱstressȱproducedȱbyȱtheȱ
presssureȱ ofȱ theȱ musselȱ raftsȱ [23]ȱ whichȱ mayȱ produceȱ negativeȱ impactsȱ toȱ theȱ
organismsȱexposed.ȱȱ
- 222 -
Biomarkerȱ fluctuationsȱ wereȱ relativelyȱ lowȱ inȱ siteȱ CAȱ inȱ comparissionȱ
withȱ theȱ otherȱ studyȱ sites,ȱ whatȱ confirmsȱ theȱ feasabilityȱ ofȱ thisȱ locationȱ asȱ
referenceȱ siteȱ inȱ ecotoxicologicalȱ studies.ȱ Higherȱ biomarkerȱ responsesȱ wereȱ
expectedȱinȱorganismsȱexposedȱtoȱtheȱcontaminationȱboundȱtoȱsedimentsȱfromȱ
theȱ Bayȱ ofȱ Algeciras,ȱ asȱ itȱ wasȱ shownȱ inȱ laboratoryȱ studiesȱ [24]ȱ howeverȱ
biomarkersȱ ofȱ exposureȱ wereȱ generallyȱ lowȱ inȱ comparisonȱ withȱ organismsȱ
exposedȱ toȱ sedimentsȱ fromȱ CormeȬLaxe.ȱ Inȱ theȱ caseȱ ofȱ theȱ cagedȱ organismsȱ
locatedȱinȱtheȱmouthȱofȱtheȱriverȱGuadarranqueȱtheȱvariationsȱofȱabioticȱfactorsȱ
dueȱtoȱtheȱinfluenceȱofȱtidesȱcouldȱaffectȱtheȱactivityȱofȱtheȱstudiedȱbiomarkers.ȱ
Previousȱstudiesȱhaveȱdemonstratedȱtheȱfluctuationsȱofȱdetoxificationȱenzymesȱ
inȱresponseȱtoȱchangesȱofȱtemperatureȱandȱsalinityȱ[21].ȱ
Theȱ histopahologicalȱ syntomsȱ ofȱ stressȱ agreeȱ withȱ theȱ presenceȱ ofȱ
contamiantsȱ notȱ onlyȱ inȱ theȱ sedimentȱ butȱ alsoȱ inȱ theȱ waterȱ inȱ theȱ areasȱ ofȱ
CormeȬLaxeȱ andȱ Algeciras.ȱ Organismsȱ deployedȱ inȱ theȱ AINPȱ presentedȱ slightȱ
reversibleȱ lesionsȱ relatedȱ withȱ generalȱ stressȱ butȱ notȱ withȱ contaminants.ȱ Theȱ
lesionsȱ wereȱ similarȱ toȱ theȱ tissuesȱ fromȱ organismsȱ exposedȱ toȱ sedimentsȱ fromȱ
theȱreferenceȱstationȱinȱtheȱBayȱofȱCádiz.ȱOnȱtheȱwhole,ȱorganisms’ȱlaboratoryȱ
deploymentsȱ (personalȱ observations)ȱ haveȱ concurredȱ inȱ moreȱ incidencesȱ ofȱ
lesionsȱ inȱ targetȱ tissuesȱ thanȱ inȱ theȱ fieldȱ exposuresȱ whatȱ suggestsȱ thatȱ theȱ
pollutantsȱcomeȱmainlyȱfromȱtheȱsediments;ȱunderȱfieldȱconditionsȱtheȱeffectsȱofȱ
theȱcontaminantsȱboundȱtoȱsedimentȱareȱrelievedȱdueȱtoȱtheȱopenȱwaterȱsystemȱ
whatȱ diminishȱ bioavailabilityȱ ofȱ contaminants.ȱ Theȱ applicationȱ ofȱ biomarkersȱ
underȱfieldȱconditionsȱinvolvesȱmoreȱrealisticȱconditionsȱforȱtheȱexperimentȱ[25,ȱ
26,ȱ27],ȱhoweverȱaȱlotȱofȱuncontrolledȱvariablesȱmayȱaffectȱbiomarkers;ȱpreviousȱ
studiesȱagreeȱwithȱtheȱfactȱthatȱinȱsituȱcagedȱorganismȱapproachȱshouldȱbeȱusedȱ
inȱ tandemȱ withȱ otherȱ assessmentȱ methodsȱ suchȱ asȱ laboratoryȱ toxicityȱ testingȱ
[28].ȱȱ
- 223 -
5.ȱConclusionsȱ
Inȱ theȱ presentȱ studyȱ aȱ setȱ ofȱ biomarkersȱ involvedȱ inȱ theȱ detoxificationȱ
system,ȱ antioxidantȱ ȱ activitiesȱ andȱ oneȱ biomarkerȱ ofȱ effectȱ (histopathology)ȱ
wereȱ assessedȱ inȱ theȱ clamȱ Ruditapesȱ philippinarumȱ exposedȱ underȱ fieldȱ
conditionsȱ toȱ sedimentȱ samplesȱ affectedȱ byȱ oilȱ spills.ȱ Theȱ setȱ ofȱ theȱ studiedȱ
biomarkersȱ presentedȱ anȱ importantȱ inductionȱ duringȱ theȱ firstȱ weekȱ ofȱ
deploymentȱ andȱ aȱ connectionȱ ofȱ theȱ phaseȱ Iȱ andȱ IIȱ enzymaticȱ activitiesȱ ofȱ theȱ
detoxificationȱ systemȱ inȱ theȱ clamȱ Ruditapesȱ philippinarumȱ wasȱ suggested.ȱ Theȱ
toxicityȱ ofȱ Pbȱ boundȱ toȱ sedimentsȱ wasȱ relatedȱ toȱ theȱ inductionȱ ofȱ biomarkersȱ
afterȱ 28ȱ daysȱ ofȱ deployment,ȱ whatȱ indicatesȱ theȱ importanceȱ ofȱ carryingȱ outȱ
kineticȱ studiesȱ inȱ fieldȱ studiesȱ whereȱ theȱ bioavailabilityȱ ofȱ contaminantsȱ
partiallyȱ dependsȱ onȱ abioticȱ parameters.ȱ Inȱ additionȱ theȱ evalluationȱ ofȱ
biomarkersȱ alongȱ theȱ timeȱ hasȱ allowedȱ toȱ distinguishȱ differentȱ sourcesȱ ofȱ
contaminantsȱnotȱrelatedȱtoȱsediments.ȱȱ
Authorsȱ considerȱ thatȱ Ruditapesȱ philippinarumȱ isȱ aȱ suitableȱ speciesȱ inȱ oilȱ
contaminatedȱ sedimentsȱ assessmentȱ byȱ includingȱ aȱ setȱ ofȱ antioxidant,ȱ phaseȱ Iȱ
andȱ IIȱ detoxificationȱ biomarkersȱ togetherȱ withȱ biomarkersȱ ofȱ effectȱ suchȱ asȱ
histopathology;ȱ moreoverȱ toxicityȱ testingȱ followingȱ aȱ kineticȱ approachȱ underȱ
fieldȱconditionsȱcontributesȱinȱaȱveryȱeffectiveȱwayȱtoȱmonitoreȱandȱdetermineȱ
theȱpollutantsȱeffects,ȱincludingȱthoseȱthatȱhaveȱnotȱbeenȱanalyzed.ȱ
6.ȱAcknowledgementsȱ
Theȱ workȱ describedȱ wasȱ partlyȱ supportedȱ byȱ theȱ projectsȱ VEM2003Ȭ
20563/INTER,ȱ andȱ CTM2005Ȭ07282ȬC03Ȭ01/TECNOȱ financedȱ byȱ theȱ Spanishȱ
Educationȱ andȱ Scienceȱ Ministryȱ andȱ byȱ CISȱ fundedȱ byȱ theȱ Ministryȱ ofȱ
Environment.ȱ Carmenȱ MoralesȬCasellesȱ thanksȱ theȱ Ministryȱ ofȱ Educationȱ andȱ
Scienceȱ forȱ fundingȱ herȱ researchȱ fellowshipȱ (FPU).ȱ Dr.ȱ Ribaȱ thanksȱ theȱ I3pȱ
programȱ forȱ supportingȱ herȱ participationȱ inȱ thisȱ research.ȱ ȱ Inmaculadaȱ Ribaȱ
- 224 -
thanksȱtheȱCSICȱforȱherȱI3Pȱcontract.ȱȱWeȱareȱgratefulȱforȱtheȱsupportȱandȱhelpȱ
ofȱ theȱ membersȱ ofȱ theȱ CISȱ andȱ theȱ ICMANȬCSIC.ȱ Specialȱ thanksȱ areȱ givenȱ toȱ
AntonioȱMorenoȱandȱPabloȱVidal.ȱȱ
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- 228 -
AȱkineticȱapproachȱinȱtheȱPAHsȱdetoxificationȱsystemȱinȱaȱmarineȱ
invertebrateȱspecie:ȱtheȱcrabȱCarcinusȱmaenasȱ
CarmenȱMoralesȬCaselles†ȱ‡ȱ*,ȱLauraȱMartínȬDíaz†ȱ‡,ȱInmaculadaȱRiba†ȱ‡,ȱ
CarmenȱSarasquete†,ȱT.ȱÁngelȱDelValls†ȱ‡ȱ
†ȱIȱUnidadȱAsociadaȱdeȱCalidadȱAmbientalȱyȱPatologíaȱ(CSICȱ&ȱUCA)ȱinstitutoȱ
deȱCienciasȱMarinasȱdeȱAndalucíaȱ(ICMANȬCSIC),ȱAvda.ȱRepúblicaȱSaharauiȱs/n,ȱ
PuertoȱRealȱ11510,ȱCádiz,ȱSpainȱ
‡ȱUNESCOȱUNITWIN/WiCop,ȱFacultadȱdeȱCienciasȱdelȱMarȱyȱAmbientales,ȱ
UniversidadȱdeȱCádiz,ȱPolígonoȱRíoȱSanȱPedroȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱ
Spain.ȱ
Abstractȱ
Toȱ testȱ theȱ hypothesisȱ thatȱ invertebratesȱ presentȱ aȱ significantȱ PAHsȬ
detoxificationȱ system,ȱ theȱ inductionȱ ofȱ ethoxyresorufinȱ OȬdeethylaseȱ (EROD)ȱ
activityȱ andȱ guthationeȬSȬtransferaseȱ (GST)ȱ wasȱ studiedȱ inȱ theȱ crabȱ Carcinusȱ
maenas.ȱAȱbioassayȱwasȱperformedȱbyȱexposingȱtheȱorganismsȱtoȱbulkȱsedimentȱ
contaminatedȱbyȱPAHsȱunderȱlaboratoryȱandȱfieldȱconditions.ȱSedimentsȱwereȱ
collectedȱ andȱ transferredȱ toȱ theȱ laboratoryȱ whereȱ theyȱ wereȱ subsampledȱ forȱ
chemicalȱanalysisȱandȱtoxicityȱtest;ȱcrabsȱwereȱkeptȱduringȱ28ȱdaysȱinȱtanksȱwithȱ
theȱ collectedȱ sedimentȱ samplesȱ whereasȱ cagedȱ crabsȱ wereȱ alsoȱ placedȱ inȱ theȱ
selectedȱ studyȱ sitesȱ duringȱ theȱ sameȱ period.ȱ Samplingȱ wasȱ performedȱ weeklyȱ
andȱ hepathopancreasȱ wereȱ homogenizedȱ andȱ centrifugedȱ forȱ theȱ biomarkersȱ
determination.ȱ Resultsȱ obtainedȱ showȱ theȱ relationshipȱ betweenȱ theȱ kineticȱ ofȱ
theȱ biomarkersȱ measuredȱ inȱ theȱ crabsȱ andȱ theȱ chemicalȱ characteristicsȱ ofȱ theȱ
sediment.ȱ Besides,ȱ itȱ demonstratesȱ theȱ capabilityȱ ofȱ theȱ mentionedȱ biologicalȱ
systemsȱinvolvedȱinȱtheȱdetoxificationȱofȱPAHsȱtoxicityȱinȱtheȱstudiedȱorganism.ȱȱȱ
Keywords:ȱbiomarker,ȱinvertebrate,ȱhistopathology,ȱERODȱactivity,ȱGST.ȱ
ȱ
ȱȱȱEnvironementalȱToxicologyȱ(enviado)
- 229 -
1.ȱIntroductionȱ
Theȱ presenceȱ ofȱ aȱ xenobioticȱ compoundȱ inȱ aȱ segmentȱ ofȱ anȱ aquaticȱ
ecosystemȱ doesȱ not,ȱ byȱ itself,ȱ indicateȱ injuriousȱ effects.ȱ Connectionsȱ mustȱ beȱ
establishedȱ betweenȱ externalȱ levelsȱ ofȱ exposure,ȱ internalȱ levelsȱ ofȱ tissueȱ
contaminationȱ andȱ earlyȱ adverseȱ effectsȱ (Vanȱ derȱ Oost,ȱ 2003).ȱ SedimentȬ
associatedȱchemicalsȱmayȱorȱmayȱnotȱbeȱbioavailable,ȱandȱthereȱisȱaȱpaucityȱofȱ
informationȱ onȱ theirȱ combinedȱ effectsȱ onȱ exposedȱ organismsȱ (Wernerȱ etȱ al.,ȱ
2004).ȱ
Aȱ
varietyȱ
ofȱ
molecular,ȱ
biochemical,ȱ
physiological,ȱ
histoȬ
cytopathological,ȱ organisimal,ȱ populationȱ andȱ communityȱ responsesȱ mayȱ beȱ
usedȱ toȱ identifyȱ exposureȱ toȱ certainȱ chemicals,ȱ provideȱ informationȱ onȱ spatialȱ
andȱ temporalȱ changesȱ inȱ theȱ concentrationȱ ofȱ contaminants,ȱ andȱ indicateȱ
environmentalȱ qualityȱ orȱ occurrenceȱ ofȱ adverseȱ ecologicalȱ consequences.ȱ (Au,ȱ
2004).ȱ Theȱ useȱ ofȱ biomarkersȱ whichȱ areȱ indicativeȱ ofȱ PAHsȱ exposureȱ mayȱ
provideȱ anȱ earlyȱ warningȱ ofȱ potentialȱ ecosystemȱ degradation,ȱ contaminantȱ
bioavailability,ȱandȱtheȱdefenceȱresponsesȱofȱexposedȱorganismsȱ(Goksøyrȱetȱal.,ȱ
1996;ȱGoksøyrȱ&ȱFörlin,ȱ1992;ȱReynoldsȱetȱal.,ȱ2003).ȱȱ
Theȱ ERODȱ activityȱ isȱ usedȱ asȱ aȱ biomarkerȱ ofȱ exposureȱ toȱ lipophilicȱ
organicȱcontaminantsȱsuchȱasȱPAHsȱandȱmeasuresȱtheȱenzymaticȱactivityȱofȱtheȱ
phaseȱ Iȱ catalyzedȱ byȱ theȱ complexȱ CYP1Aȱ whichȱ transformsȱ someȱ lipophilicȱ
xenobioticsȱinȱmetabolitesȱmoreȱwaterȱsolubleȱ(Bachȱetȱal.,ȱ2005).ȱGlutathioneȬSȬ
transferaseȱ (GST)ȱ representsȱ aȱ phaseȱ IIȱ detoxificationȱ enzymeȱ butȱ alsoȱ
implicatedȱ inȱ oxidativeȱ stressȱ events;ȱ aȱ criticalȱ roleȱ forȱ GSTsȱ isȱ obviouslyȱ
defenceȱ againstȱ oxidativeȱ damageȱ andȱ peroxidativeȱ productsȱ ofȱ DNAȱ andȱ
lipidsȱ(VanȱderȱOost,ȱ2003).ȱOnȱtheȱotherȱhandȱtheȱcapacityȱofȱmanyȱpollutantsȱ
toȱ alterȱ differentȱ cells,ȱ tissuesȱ orȱ organsȱ hasȱ ledȱ toȱ designȱ histopathologicalȱ
techniquesȱ inȱ orderȱ toȱ evaluateȱ theȱ effectsȱ ofȱ contaminantsȱ (Lowe,ȱ 1988;ȱ
Sarasqueteȱetȱal.,ȱ1997).ȱ
- 230 -
Inȱ theȱ presentȱ studyȱ bioassaysȱ underȱ fieldȱ andȱ laboratoryȱ conditionsȱ
haveȱbeenȱdevelopedȱtoȱelucidateȱtheȱdetoxificationȱsystemȱinȱtheȱcrabȱCarcinusȱ
maenasȱ exposedȱ toȱ sedimentsȱ affectedȱ byȱ oilȱ spills,ȱ byȱ analyzingȱ theȱ kineticȱ ofȱ
twoȱ biomarkersȱ ofȱ exposureȱ relatedȱ toȱ theȱ detoxificationȱ systemȱ (ERODȱ andȱ
GSTȱactivities)ȱandȱoneȱbiomarkerȱofȱeffectȱ(histopathology).ȱ
2.ȱMaterialȱandȱmethodsȱ
2.1.ȱStudyȱsitesȱ
Threeȱ differentȱ areasȱ wereȱ selectedȱ toȱ carryȱ outȱ theȱ bioassays,ȱ twoȱ ofȱ
themȱ inȱ theȱ Galicianȱ Coastȱ (NWȱ Spain)ȱ (theȱ Bayȱ ofȱ CormeȬLaxeȱ andȱ theȱ Ciesȱ
Island)ȱandȱtheȱotherȱoneȱinȱtheȱBayȱofȱAlgecirasȱ(SȱSpain).ȱInȱtotalȱ9ȱstudyȱsitesȱ
wereȱchosen,ȱ3ȱinȱtheȱCiesȱIslandsȱ(A,ȱB,ȱandȱC),ȱ3ȱinȱtheȱBayȱofȱCormeȬLaxeȱ(D,ȱ
EȱandȱF)ȱandȱ3ȱinȱtheȱBayȱofȱAlgecirasȱ(GR3,ȱGR4ȱandȱP1).ȱTheȱareaȱofȱGaliciaȱ
wasȱ affectedȱ inȱ 2002ȱ byȱ theȱ spillȱ ofȱ theȱ tankerȱ Prestigeȱ whereasȱ theȱ Bayȱ ofȱ
Algecirasȱ suffersȱ continuousȱ spillsȱ ofȱ oilȱ andȱ otherȱ compoundsȱ fromȱ theȱ
industriesȱandȱtheȱmaritimeȱactivitiesȱofȱtheȱarea.ȱȱ
2.2.ȱChemicalsȱinȱsedimentsȱ
ForȱPAHsȱandȱPCBsȱdeterminationȱdriedȱsamplesȱwereȱSoxhletȱextractedȱ
withȱ nȬhexaneȱ forȱ 18ȱ h,ȱ andȱ theȱ extractsȱ wereȱ isolatedȱ byȱ columnȱ
chromatographyȱ onȱ Florisileȱ aluminoȬsilica.ȱ PCBsȱ andȱ PAHsȱ wereȱ elutedȱ andȱ
theirȱfractionsȱwereȱdriedȱinȱaȱrotatingȱevaporatorȱandȱreȬdissolvedȱinȱisooctane.ȱ
PAHsȱwereȱanalyzedȱbyȱGCȬMSȱusingȱselectedȱionȱmonitoringȱ(SIM).ȱAnalysisȱ
ofȱ PCBsȱ asȱ AROCLORȱ 1242ȱ andȱ AROCLORȱ 1260ȱ wasȱ performedȱ usingȱ theȱ
sameȱ instrumentȱ withȱ anȱ electronȱ captureȱ detectorȱ (GC/ECD).ȱ Traceȱ metalȱ
analysisȱwereȱanalyzedȱasȱdescribedȱbyȱCasadoȬMartínezȱetȱal.ȱ(2006);ȱsedimentȱ
samplesȱwereȱdigestedȱinȱmicrowaveȱ(400W,ȱ15ȱmin,ȱtwice)ȱwithȱHNO3ȱ2N,ȱtheȱ
extractsȱ wereȱ purifiedȱ byȱ passingȱ throughȱ aȱ CȬ18ȱ columnȱ andȱ metalsȱ analysesȱ
- 231 -
wereȱ performedȱ byȱ anodicȱ voltamperimetryȱ (ȬZn,ȱ Cd,ȱ Pb,ȱ Ni,ȱ Coȱ andȱ CuȬȱ
MetrohmȱApplicationȱBulletinȱNºȱ147;ȱȬȱVȬȱMetrohmȱApplicationȱNoteȱNºȱVȬ81).ȱ
Forȱ Hgȱ theȱ coldȱ vapourȱ techniqueȱ wasȱ usedȱ andȱ wasȱ quantifiedȱ usingȱ atomicȱ
absorptionȱspectrometry.ȱȱ
2.3.ȱFieldȱandȱlaboratoryȱbioassaysȱ
IntermoultȱfemaleȱCarcinusȱmaenasȱwereȱcollectedȱfromȱaȱcleanȱsiteȱofȱtheȱ
BayȱofȱCádizȱ(Ribaȱetȱal.,ȱ2004)ȱfromȱanȱaquacultureȱfarmȱandȱwereȱkeptȱunderȱ
laboratoryȱconditionsȱduringȱthreeȱweeksȱforȱacclimatization.ȱAfterȱthatȱperiodȱ
theȱ crabsȱ wereȱ placedȱ inȱ cagesȱ whichȱ wereȱ deployedȱ inȱ theȱ studyȱ sitesȱ toȱ
conductȱ theȱ fieldȱ bioassay;ȱ simultaneously,ȱ aboutȱ 4ȱ Lȱ ofȱ sedimentȱ fromȱ theȱ
referenceȱ siteȱ andȱ theȱ otherȱ stationsȱ wereȱ placedȱ inȱ replicateȱ 25ȬLȱ glassȱ tanksȱ
withȱ cleanȱ seaȱ waterȱ beforeȱ theȱ beginningȱ ofȱ theȱ experiment.ȱ Afterȱ particleȱ
settling,ȱ aerationȱ wasȱ providedȱ toȱ maintainȱ adequateȱ oxygenȱ concentrationsȱ
(greaterȱthanȱ80%ȱsaturation).ȱCrabsȱwereȱalsoȱlocatedȱinȱtheseȱtanksȱcontainingȱ
sedimentȱfromȱtheȱdifferentȱstationsȱinȱorderȱtoȱperformȱtheȱtoxicityȱtestȱunderȱ
laboratoryȱ conditions.ȱ ȱ Bothȱ bioassaysȱ underȱ laboratoryȱ andȱ fieldȱ conditionsȱ
wereȱcarriedȱoutȱduringȱ28ȱdaysȱandȱoverȱthisȱtimeȱcrabsȱwereȱfedȱeveryȱweekȱ
withȱ aȱ mixedȱ dietȱ ofȱ musselsȱ orȱ fishȱ whereasȱ waterȱ fromȱ theȱ laboratoryȱ tanksȱ
wasȱreplacedȱeveryȱthreeȱdays.ȱ
2.4.ȱBiochemicalȱanalysisȱ
Samplingȱwasȱconductedȱeveryȱweekȱduringȱtheȱ28ȱdaysȱofȱtheȱexposureȱ
period;ȱ afterȱ dissection,ȱ hepathopancreasȱ wasȱ keptȱ atȱ Ȭ80ºCȱ priorȱ
homogenization.ȱ Theȱ samplesȱ wereȱ homogenizedȱ followingȱ theȱ procedureȱ
developedȱ byȱ Lafontaineȱ etȱ al.ȱ (2000).ȱ Mixedȱ functionȱ oxygenaseȱ activity,ȱ
implicatedȱinȱmonooxygenationȱreactionsȱofȱdioxinsȱandȱPAHs,ȱwasȱmeasuredȱ
usingȱ theȱ adaptedȱ Ethoxyresorufinȱ OȬdeethylaseȱ (EROD)ȱ (Gagnèȱ andȱ Blaise,ȱ
1993).ȱ Theȱ phaseȱ IIȱ metabolizingȱ enzymeȱ GlutathioneȬSȬtransferaseȱ (GST)ȱ
- 232 -
activityȱ wasȱ determinedȱ byȱ monitoringȱ theȱ rateȱ ofȱ conjugationȱ ofȱ glutathioneȱ
(GSH)ȱ toȱ 1ȬchloroȬ2,4Ȭdinitrobenzeneȱ (CDNB)ȱ atȱ 340ȱ nmȱ (McFarlandȱ etȱ al.,ȱ
1999).ȱ
Gillsȱandȱhepathopancreasȱtissuesȱwereȱfixedȱinȱphosphateȱbufferedȱ10%ȱ
formaldehydeȱ (pHȱ 7.2)ȱ forȱ histopathologyȱ determinationȱ afterȱ 28ȱ daysȱ ofȱ
exposure.ȱ Afterȱ dehydrationȱ inȱ gradedȱ concentrationsȱ ofȱ ethanol,ȱ theȱ samplesȱ
wereȱ embeddedȱ inȱ paraffinȱ wax.ȱ Histologicalȱ sectionsȱ ofȱ 6ȱ toȱ 8ȱ ΐmȱ thicknessȱ
wereȱ stainedȱ withȱ Haematoxylin–ȱ Eosinȱ andȱ Haematoxylin–VOFȱ (Gutiérrez,ȱ
1967).ȱ Sectionsȱ wereȱ reviewedȱ byȱ lightȱ microscopyȱ Leitzȱ Laborluxȱ Sȱ andȱ
photographedȱ(SonyȱDKCȬCM30).ȱ
3.ȱResultsȱ
3.1.ȱChemicalȱanalysisȱȱ
Sedimentsȱ fromȱ theȱ Ciesȱ Islandȱ presentedȱ theȱ lowestȱ concentrationsȱ ofȱ
PAHs,ȱwhereasȱtheȱhighestȱconcentrationȱofȱPAHsȱwasȱfoundȱinȱtheȱsedimentsȱ
fromȱGR3ȱ(2961ȱmgȱKgȬ1ȱdryȱsediment)ȱlocatedȱinȱtheȱBayȱofȱAlgeciras,ȱfollowedȱ
byȱsedimentsȱfromȱtheȱstationȱFȱ(820ȱmgȱKgȬ1ȱ dryȱsediment)ȱlocatedȱinȱCormeȬ
Laxe,ȱGR4ȱ(802ȱmgȱKgȬ1ȱ dryȱsediment)ȱandȱP1ȱ(641ȱmgȱKgȬ1ȱ dryȱsediment)ȱinȱtheȱ
BayȱofȱAlgeciras.ȱHighȱconcentrationsȱwereȱdetectedȱfor:ȱZnȱinȱGR3ȱ(138ȱmgȱKgȬ
1ȱ
dryȱsediment)ȱandȱFȱ(243ȱmgȱKgȬ1ȱ dryȱsediment);ȱPbȱinȱsiteȱDȱ(44ȱmgȱKgȬ1ȱ dryȱ
sediment);ȱCu:ȱAȱ(18.9ȱmgȱKgȬ1ȱdryȱsedimentȱ)ȱandȱCȱ(31.6ȱmgȱKgȬ1ȱdryȱsedimentȱ
)ȱ fromȱ Ciesȱ andȱ Dȱ (22.1ȱ mgȱ KgȬ1ȱ dryȱ sediment),ȱ Fȱ (19.1ȱ mgȱ KgȬ1ȱ dryȱ sediment)ȱ
fromȱCormeȬLaxeȱandȱP1ȱ(75.2ȱmgȱKgȬ1ȱdryȱsediment)ȱfromȱtheȱBayȱofȱAlgeciras;ȱ
NiȱinȱsiteȱGR3ȱ(74.7ȱmgȱKgȬ1ȱdryȱsediment).ȱ
3.2.ȱGSTȱandȱERODȱactivitiesȱ
AnalysisȱperformedȱinȱcrabsȱexposedȱtoȱsedimentsȱfromȱtheȱCiesȱIslandsȱ
showedȱthatȱduringȱ28ȱdaysȱofȱexposureȱERODȱactivityȱunderȱfieldȱconditionsȱ
- 233 -
keptȱlowȱandȱnoȱsignificantȱdifferencesȱwereȱdetectedȱinȱrelationȱwithȱtheȱdayȱ0.ȱ
UnderȱfieldȱconditionsȱcrabsȱdeployedȱinȱsitesȱA,ȱBȱandȱCȱpresentedȱaȱpeakȱinȱ
theȱinductionȱofȱthisȱsystemȱafterȱ7ȱȬ14ȱdaysȱofȱtheȱbeginingȱofȱtheȱbioassay;ȱtheȱ
activityȱ ofȱ theȱ GSTȱ enzymeȱ wasȱ alsoȱ higherȱ inȱ crabsȱ exposedȱ underȱ fieldȱ
conditions,ȱandȱtheȱmaximumȱwasȱobservedȱafterȱ14ȱ–ȱ21ȱdaysȱofȱexposure.ȱȱȱ
Theȱenzymesȱactivitiesȱdetectedȱinȱcrabsȱexposedȱtoȱsedimentsȱfromȱtheȱ
BayȱofȱCormeȬLaxeȱwasȱhigherȱforȱthoseȱorganismsȱdeployedȱinȱfieldȱthanȱcrabsȱ
fromȱ theȱ laboratoryȱ assays.ȱ Theȱ inductionȱ ofȱ ERODȱ andȱ GSTȱ activitiesȱ underȱ
controlledȱconditionsȱinȱlaboratoryȱwasȱnotȱsignificantȱcomparedȱwithȱtheȱfieldȱ
deploymentsȱwhereȱERODȱactivityȱshowedȱaȱmaximumȱtheȱdayȱ14ȱofȱexposureȱ
inȱcrabsȱfromȱsitesȱD,ȱEȱandȱF.ȱGSTȱactivityȱanalyzedȱinȱcrabsȱfromȱcagesȱlocatedȱ
inȱ stationȱ Dȱ presentedȱ similarȱ behaviourȱ thanȱ ERODȱ activity,ȱ withȱ aȱ peakȱ ofȱ
inductionȱ theȱ dayȱ 14.ȱ Inȱ contrast,ȱ crabsȱ collectedȱ inȱ sitesȱ Eȱ andȱ Fȱ showedȱ aȱ
maximumȱofȱinductionȱtheȱdayȱ28ȱofȱexposure,ȱwhichȱcorrespondȱtoȱtheȱlastȱdayȱ
ofȱtheȱexperiment.ȱ
Crabsȱ exposedȱ toȱ sedimentsȱ fromȱ theȱ Bayȱ ofȱ Algecirasȱ presentedȱ theȱ
highestȱvaluesȱofȱERODȱactivityȱinȱlaboratoryȱexposures.ȱInȱtheȱcaseȱofȱsiteȱGR3ȱ
whichȱ isȱ theȱ mostȱ contamiantedȱ siteȱ byȱ PAHs,ȱ theȱ maximumȱ inductionȱ wasȱ
observedȱ aȱ weekȱ afterȱ theȱ beginingȱ ofȱ theȱ assayȱ inȱ crabsȱ exposedȱ underȱ
laboratoryȱ conditions;ȱ aȱ similarȱ behaviourȱ wasȱ followedȱ byȱ theȱ inductionȱ ofȱ
GSTȱactivityȱanalyzedȱinȱtheȱsameȱcrabs.ȱAȱmaximumȱonȱofȱERODȱactivityȱwasȱ
alsoȱobservedȱtheȱdayȱ14ȱinȱcrabsȱexposedȱinȱlaboratoryȱtoȱsedimentsȱcololectedȱ
fromȱ siteȱ P1.ȱ Organismsȱ fromȱ GR4ȱ didȱ notȱ presentȱ significantȱ inductionsȱ ofȱ
ERODȱ activityȱ underȱ fieldȱ andȱ laboratoryȱ assaysȱ inȱ comparissionȱ withȱ otherȱ
sites.ȱInȱgeneral,ȱunderȱfieldȱdeploymentsȱtheȱinductionȱofȱGSTȱwasȱhigherȱthanȱ
inȱcrabsȱfromȱlaboratoryȱassays.ȱAȱpeakȱinȱGSTȱactivityȱwasȱobservedȱtheȱdayȱ21ȱ
inȱorganismsȱfromȱGR4ȱandȱdayȱ7ȱforȱcrabsȱcagedȱinȱsiteȱP1.ȱȱ
- 234 -
A
A
1800
12.0
1500
9.0
EROD
GST
1200
900
600
6.0
3.0
300
0
0.0
0
7
14
21
28
0
7
B
14
21
28
14
21
28
14
21
28
B
2100
12.0
1800
9.0
EROD
GST
1500
1200
900
600
6.0
3.0
300
0
0.0
0
7
14
21
28
0
7
C
4000
8.0
3000
6.0
EROD
GST
C
2000
4.0
2.0
1000
0.0
0
0
7
14
21
0
28
7
ȱ
Figureȱ 1.ȱ GSTȱ (nmol/min/mgȱ protein)ȱ andȱ ERODȱ (pmol/mg/min)ȱ
activitiesȱ inȱ theȱ hepatopancreasȱ ofȱ Carcinusȱ maenasȱ exposedȱ alongȱ 28ȱ daysȱ toȱ
sedimentsȱ fromȱ theȱ Ciesȱ Islands.ȱ Straigthȱ line:ȱ laboratoryȱ assays;ȱ dottedȱ line:ȱ
fieldȱassays.ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
- 235 -
ȱ
D
D
ȱ
2400
15.0
2000
12.0
ȱ
EROD
GST
1600
1200
800
9.0
6.0
3.0
400
ȱ0
0.0
0
7
ȱ
14
21
0
28
7
14
21
28
14
21
28
14
21
28
E
E
20.0
ȱ
7500
16.0
6000
12.0
EROD
GST
9000
4500
ȱ
3000
8.0
4.0
1500
0.0
0
ȱ
0
0
7
14
21
7
28
F
F
ȱ
7000
15.0
6000
12.0
ȱ
EROD
GST
5000
4000
3000
9.0
6.0
2000
3.0
1000
ȱ0
0.0
0
7
14
21
28
0
7
ȱ
Figureȱ 2.ȱ GSTȱ (nmol/min/mgȱ protein)ȱ andȱ ERODȱ (pmol/mg/min)ȱ
activitiesȱ inȱ theȱ hepatopancreasȱ ofȱ Carcinusȱ maenasȱ exposedȱ alongȱ 28ȱ daysȱ toȱ
sedimentsȱfromȱtheȱBayȱofȱCormeȬLaxe.ȱStraigthȱline:ȱlaboratoryȱassays;ȱdottedȱ
line:ȱfieldȱassays.ȱ
ȱ
ȱ
ȱ
ȱ
- 236 -
GR3
GR3
4.0
1800
1500
3.0
EROD
GST
1200
900
600
2.0
1.0
300
0.0
0
0
7
14
21
0
28
7
2000
1.0
1600
0.8
1200
0.6
800
400
28
14
21
28
14
21
28
0.4
0.2
0
0.0
0
7
14
21
28
0
7
P1
P1
2000
5.0
1600
4.0
1200
3.0
EROD
GST
21
GR4
EROD
GST
GR4
14
800
400
2.0
1.0
0
0.0
0
7
14
21
28
0
7
ȱ
Figureȱ 3.ȱ GSTȱ (nmol/min/mgȱ protein)ȱ andȱ ERODȱ (pmol/mg/min)ȱ
activitiesȱ inȱ theȱ hepatopancreasȱ ofȱ Carcinusȱ maenasȱ exposedȱ alongȱ 28ȱ daysȱ toȱ
sedimentsȱ fromȱ theȱ Bayȱ ofȱ Algeciras.ȱ Straigthȱ line:ȱ laboratoryȱ assays;ȱ dottedȱ
line:ȱfieldȱassays.ȱ
ȱ
ȱ
ȱ
ȱ
- 237 -
3.3.ȱHistologicalȱlesionsȱ
Asȱdesirableȱnoȱalterationsȱwereȱdetectedȱtheȱdayȱ0ȱofȱexposureȱpriorȱtoȱ
theȱbeginingȱofȱtheȱtests.ȱAllȱdeployedȱorganismsȱpresentedȱalterationsȱrelatedȱ
withȱ generalȱ stress,ȱ suchȱ asȱ ȱ lossȱ ofȱ ȱ connectiveȱ tissueȱ ofȱ theȱ gillsȱ andȱȱ
hepatopancreas,ȱ ruptureȱ ofȱ gillȱ epithelium,ȱ andȱ haemociticȱ infiltrates.ȱ Inȱ
general,ȱ damageȱ wasȱ higherȱ isȱ sitesȱ fromȱ Algecriasȱ followedȱ byȱ CormeȬLaxeȱ
andȱthenȱtheȱCiesȱIslandsȱwhichȱtissuesȱwereȱsimilarȱtoȱcontrols.ȱAlterationsȱinȱ
crabsȱ wereȱ lowerȱ underȱ fieldȱ exposuresȱ thanȱ thoseȱ detectedȱ inȱ laboratoryȱ
deployments;ȱ noȱ importantȱ lesionsȱ wereȱ detectedȱ exceptȱ forȱ siteȱ GR3ȱ whereȱ
diferentȱ alterationsȱ wereȱ observed,ȱ including:ȱ disruptedȱ pillarȱ cells,ȱ epithelialȱ
changes,ȱ desquamationȱ inȱ gills,ȱ presenceȱ ofȱ vacuolesȱ inȱ hepathopancreas,ȱ lossȱ
ofȱȱsupportȱconnectiveȱtissue,ȱnecrosis,ȱetc.ȱȱȱ
3.4.ȱCorrelationȱamongȱchemicalsȱandȱbiomarkersȱofȱresponseȱ
Theȱconcentrationȱofȱ PAHsȱinȱsedimentsȱhasȱbeenȱcorrelatedȱwithȱotherȱ
organicȱ chemicalsȱ suchȱ asȱ PCBsȱ andȱ theȱ metalsȱ Cd,ȱ Ni,ȱ Coȱ andȱ V.ȱ Thisȱ
substancesȱ hasȱ beenȱ alsoȱ correlatedȱ withȱ theȱ inductionȱ ofȱ ERODȱ activityȱ inȱ
crabsȱtheȱdayȱ7ȱandȱ28ȱofȱexposureȱunderȱlaboratoryȱconditions.ȱPbȱwasȱlinkedȱ
withȱtheȱinductionȱȱofȱERODȱactivityȱafterȱ28ȱdaysȱofȱfieldȱdeployment,ȱandȱCuȱ
wasȱalsoȱrelatedȱtoȱthisȱbiomarkerȱtheȱdayȱ14ȱofȱlaboratoryȱexposure.ȱȱȱ
AȱrelationshipȱhasȱbeenȱdetectedȱbetweenȱtheȱinductionȱofȱERODȱactivityȱ
inȱcrabsȱfromȱlaboratoryȱbioassaysȱtheȱdaysȱ14ȱandȱ21.ȱAȱsimilarȱcorrelationȱwasȱ
observedȱ inȱ ERODȱ activityȱ measuredȱ inȱ crabsȱ fromȱ organismsȱ collectedȱ fromȱ
cagesȱtheȱdaysȱ7ȱandȱ14.ȱTheȱinductionȱofȱtheȱphaseȱIIȱdetoxificationȱenzymesȱinȱ
crabsȱ afterȱ 28ȱ daysȱ ofȱ exposureȱ underȱ laboratoryȱ andȱ fieldȱ conditionsȱ wereȱ
linkedȱ inȱ additionȱ toȱ theȱ inductionȱ ofȱ thisȱ enzymeȱ theȱ dayȱ 21ȱ ofȱ fieldȱ
deployment.ȱ
- 238 -
ȱ
ȱ
A
B
ȱ
ȱ
ȱ
C
ȱ
ȱ
ȱ
ȱ
E
F
ȱ
ȱ
ȱ
Figureȱ 4.ȱ Histologicalȱ sectionsȱ ofȱ gillsȱ andȱ hepathopancreasȱ ofȱ theȱ crabȱ
Carcinusȱmaenasȱafterȱ28Ȭdȱexposureȱtoȱtheȱsediments:ȱ(A)ȱHistologicalȱsectionȱofȱ
aȱcontrolȱdigestiveȱglandȱ(dayȱ0);ȱ(B)ȱHistologicalȱsectionȱofȱaȱcontrolȱgillȱ(dayȱ
0);ȱ (C)ȱ Histologicalȱ sectionȱ ofȱ hepathopancreasȱ fromȱ aȱ cramȱ exposedȱ toȱ
sedimentsȱ fromȱ GR3’ȱ underȱ fieldȱ conditions;ȱ (D)ȱ Histologicalȱ sectionȱ ofȱ gillȱ
fromȱ aȱ crabȱ exposedȱ toȱ sedimentsȱ fromȱ GR3’ȱ underȱ fieldȱ conditions;ȱ (E)ȱ
Histologicalȱsectionȱofȱhepathopancreasȱfromȱaȱcramȱexposedȱtoȱsedimentsȱfromȱ
GR3’ȱ underȱ laboratoryȱ conditions;ȱ (F)ȱ Histologicalȱ sectionȱ ofȱ gillȱ fromȱ aȱ crabȱ
exposedȱtoȱsedimentsȱfromȱGR3’ȱunderȱlaboratoryȱconditions.ȱ
ȱ
- 239 -
Chemicals in sediment
Biomarkers (laboratory)
Biomarkers (field)
-0.26
0.13
-0.03
0.06
-0.14
.96(2)
-0.29
-0.11
0.63
0.22
-0.30
-0.17
-0.20
-0.05
-0.50
-0.14
-0.12
-0.27
-0.18
-0.04
.96(2)
-0.15
-0.13
.74(1)
0.25
-0.34
-0.18
-0.16
-0.35
-0.55
-0.30
-0.19
Gst 14
Gst 21
Gst 28
Erod 7
Erod 14
Erod 21
Erod 28
Gst 7
Gst 14
Gst 21
Gst 28
Erod 7
Erod 14
Erod 21
Erod 28
0.23
-0.17
0.34
0.31
-0.07
-0.02
-0.38
-0.12
0.23
-0.36
-0.39
-0.43
-0.52
-.7(1)
-0.46
-0.33
.74(1)
0.12
0.24
.89(2)
0.26
-0.18
-0.31
-0.15
-0.33
0.22
-0.04
0.08
.86(2)
.87(2)
.68(1)
Cd
0.66
-0.26
-0.13
-0.04
.74(1)
0.45
Gst 7
0.11
0.19
-0.03
0.29
0.22
0.41
0.22
.68(1)
0.04
-0.36
.91(2)
.84(2)
.78(1)
Zn
.90(2)
0.41
.86(2)
0.12
-0.22
.97(2)
.93(2)
.87(2)
PCBs
PCBs
Zn
Cd
Pb
Cu
Ni
Co
V
PAHs
-0.03
0.24
-0.58
-0.29
-0.19
0.17
0.20
.73(1)
-0.16
-0.22
-0.13
0.14
-0.13
-0.12
0.16
0.22
0.08
0.20
0.09
0.50
Pb
0.11
-0.06
-0.49
-0.33
-0.39
0.10
-0.11
-0.08
-0.52
0.66
.94(2)
-0.23
0.03
-0.26
-0.23
-0.23
-0.17
-0.34
-0.01
Cu
Chemicals in sediment
0.20
-0.16
-0.36
-0.34
-0.34
-0.59
-0.32
-0.19
.69(1)
0.02
-0.09
.99(2)
-0.24
-0.12
-0.13
0.52
.93(2)
.86(2)
Ni
biomarkersȱtheȱdaysȱ7,ȱ14,ȱ21ȱandȱ28ȱȱ
-0.01
-0.26
-0.20
-0.31
-0.41
-.7(1)
-0.29
-0.20
.90(2)
-0.23
-0.27
.92(2)
-0.25
-0.16
-0.06
0.46
.74(1)
Co
0.28
-0.33
-0.41
-0.21
-0.37
-0.29
-0.03
0.21
0.422
-0.17
-0.05
.87(2)
0.04
-0.24
-0.29
0.53
V
-0.12
-0.20
-0.29
-0.39
0.13
-0.33
0.27
0.26
0.21
-0.17
-0.40
0.63
-0.21
0.16
0.43
Gst 7
0.54
-0.08
-0.71
-0.29
.90(2)
-0.60
-0.33
0.23
-0.11
0.15
-0.13
-0.14
-0.43
- 240 -
-0.62
0.42
-0.13
-0.48
0.35
-0.34
-0.03
0.11
-0.04
-0.10
-0.61
-0.18
-0.41
0.32
0.10
-0.33
.73(1)
.80(2)
-0.02
0.52
0.23
-0.03
-0.22
-0.33
-0.12
-0.20
Gst 14 Gst 21 Gst 28
0.22
-0.23
-0.41
-0.34
-0.37
-0.57
-0.25
-0.20
0.69
0.10
0.22
Erod
7
0.42
-0.22
-0.50
-0.32
-0.31
0.25
-0.26
-0.26
-0.43
.90(1)
Erod
14
Biomarkers (laboratory assay)
0.25
0.43
-0.42
-0.30
-0.24
0.04
-0.31
-0.50
-0.40
Erod
21
-0.14
-0.28
0.09
-0.15
-0.34
-.8(1)
-0.32
-0.31
Erod
28
-0.36
-0.24
0.14
0.46
0.07
-0.49
0.09
Gst 7
-0.18
-0.08
-0.03
0.27
0.00
-0.15
.84(2)
0.56
0.32
0.12
-0.33
0.21
0.71
0.34
-0.10
Gst 14 Gst 21 Gst 28
.84(1)
-0.10
0.25
Erod
7
Biomarkers (field assay)
Table1.ȱ Spearmanȱ correlationȱ (1:ȱp<0.05;ȱ2:ȱp<0.01)ȱresultsȱamongȱchemicalȱcompoundsȱ boundȱ toȱ sedimentsȱ andȱ theȱ inductionȱ ofȱ
0.64
0.36
Erod
14
0.50
Erod
21
4.ȱDiscussionȱ
Crustaceansȱ haveȱ theȱ highestȱ totalȱ P450ȱ proteinȱ inȱ theȱ hepatopancreas,ȱ
butȱalsoȱsignificantȱactivityȱinȱgreenȱgland,ȱgonads,ȱandȱstomachȱ(James,ȱ1989),ȱ
howeverȱ Theȱ mechanismsȱ byȱ whichȱ xenobioticsȱ activateȱ geneȱ expressionȱ
leadingȱtoȱtheȱincreasedȱproductionȱofȱnewȱproteinsȱsuchȱasȱP450sȱareȱnotȱwellȱ
understoodȱ inȱ marineȱ organismsȱ (Snyder,ȱ 2000).ȱ Underȱ laboratoryȱ conditions,ȱ
theȱ concentrationȱ ofȱ PAHsȱ inȱ sedimentȱ hasȱ beenȱ positevelyȱ relatedȱ toȱ theȱ
inductionȱofȱERODȱactivityȱtheȱdayȱ7ȱandȱ28ȱofȱexposure.ȱThisȱcorrelationȱalsoȱ
includesȱ theȱ metalsȱ Ni,ȱ Co,Vȱ andȱ Cdȱ whichȱ couldȱ beȱ expectedȱ dueȱ toȱ theȱ
presenceȱofȱaȱcomplexȱmixtureȱofȱcontaminants,ȱspeciallyȱinȱsedimentsȱfromȱtheȱ
Bayȱ ofȱ Algeciras.ȱ Underȱ fieldȱ conditionsȱ thisȱ correlationdoesȱ notȱ occurȱ exceptȱ
forȱtheȱinductionȱofȱERODȱactivityȱtheȱdayȱ28ȱofȱdeploymentȱwhichȱisȱcorrelatedȱ
withȱPb.ȱThisȱmetalȱhasȱbeenȱoftenȱrelatedȱtoȱfuelȱusedȱbyȱoldȱcars;ȱinȱthisȱcaseȱ
thisȱ ERODȱ activityȱ couldȱ beȱ alsoȱ relatedȱ toȱ otherȱ organicȱ compoundsȱ notȱ
analyzedȱinȱtheȱtotalȱPAHsȱincludedȱinȱthisȱwork.ȱ
Relationshipsȱ betweenȱ theȱ sameȱ biomarkerȱ inȱ consequtiveȱ weeksȱ haveȱ
beenȱestablihedȱwhatȱindicatesȱthatȱbiomarkersȱfollowȱaȱmechanism.ȱInȱtheȱcaseȱ
ofȱ GSTȱ activityȱ correlationȱ haveȱ beenȱ observedȱ betweenȱ laboratoryȱ andȱ fieldȱ
assaysȱ showingȱ thatȱ theȱ stressȱ thatȱ causesȱ theȱ activationȱ ofȱ thisȱ enzymeȱ isȱ
presentȱinȱbothȱkindȱofȱexposures.ȱȱ
Inȱ general,ȱ theȱ fastestȱ inductionȱ ofȱ biomarkersȱ wasȱ obseredȱ inȱ crabsȱ
exposedȱ inȱ laboratoryȱ toȱ sedimentsȱ fromȱ siteȱ GR3ȱ withȱ theȱ highestȱ contentȱ ofȱ
PAHsȱinȱtheȱsediment.ȱInȱgeneral,ȱȱtheȱotherȱstudyȱsitesȱpresentȱaȱlaterȱinductionȱ
ofȱbiomarkersȱandȱnormalyȱERODȱactivityȱpresentȱpeaksȱaboutȱ14ȱdaysȱafterȱtheȱ
beginingȱofȱtheȱbioassays,ȱandȱtheȱsameȱhappendsȱtoȱGSTȱactivity;ȱinȱtheȱcaseȱofȱ
siteȱEȱandȱFȱtheȱGSTȱactivityȱkeepsȱgrowingȱfindingȱtheȱmaximumȱtheȱlastȱdayȱ
ofȱtheȱbioassay.ȱȱȱ
- 241 -
Differencesȱ hasȱ beenȱ detectedȱ amongȱ theȱ inductionȱ ofȱ biomarkersȱ ofȱ
exposureȱ inȱ crabsȱ exposedȱ toȱ theȱ areasȱ ofȱ study.ȱ Inȱ general,ȱ inȱ tehȱ caseȱ ofȱ theȱ
studyȱ sitesȱ locatedȱ inȱ theȱ Galiciaȱ Coast,ȱ theȱ inductionȱ ofȱ biomarkersȱ inȱ moreȱ
importantȱ inȱ cagedȱ organismsȱ underȱ fieldȱ conditions.ȱ Onȱ theȱ otherȱ handȱ
organismsȱ exposedȱ toȱ sedimentsȱ fromȱ theȱ Bayȱ ofȱ Algecirasȱ inȱ laboratoryȱ areȱ
highestȱ thanȱ thoseȱ deployedȱ inȱ field.ȱ Resultsȱ observedȱ inȱ histopathologicalȱ
analysis,ȱ useȱ asȱ biomarkerȱ ofȱ effect,ȱ indicateȱ thatȱ organismsȱ exposedȱ inȱ
laboratoryȱconditionsȱareȱmoreȱdamagedȱthanȱthoseȱinȱfield.ȱȱȱ
Previousȱ studiesȱ showedȱ howȱ theȱ diseaseȱ statusȱ inȱ theȱ crabȱ Carcinusȱ
maenasȱ mayȱ beȱ usedȱ asȱ aȱ highȬlevelȱ indicatorȱ ofȱ ecosystemȱ health.(Stentifordȱ
andȱ Feist,ȱ 2005).ȱ Histopathologicalȱ observationȱ showedȱ thatȱ crabsȱ exposedȱ toȱ
sedimentsȱfromȱtheȱBayȱofȱAlgecirasȱunderȱlaboratoryȱconditionsȱwereȱtheȱmostȱ
damagedȱfollowedȱbyȱandȱcrabsȱexposedȱtoȱsedimentsȱfromȱtheȱBayȱofȱCormeȬ
Laxe,ȱ andȱ finallyȱ organismsȱ fromȱ theȱ Ciesȱ treatmentȱ whichȱ showedȱ mainlyȱ
alterationsȱ dueȱ toȱ generalȱ environmentalȱ stress.ȱ Theȱ relationshipȱ betweenȱ
pollutantsȱandȱpathologiesȱinȱtargetȱtissuesȱhasȱbeenȱpreviouslyȱreportedȱ(OrtizȬ
Delgadoȱ etȱ al.,ȱ 2007).ȱ Inȱ thisȱ case,ȱ underȱ laboratoryȱ conditions,ȱ ȱ theȱ highestȱ
amountȱ ofȱ PAHsȱ inȱ theȱ sedimentȱ is,ȱ theȱ mostȱ histologicalȱ lesionsȱ inȱ crabsȱ areȱ
observed,ȱ asȱ inȱ theȱ caseȱ ofȱ siteȱGR3ȱfromȱtheȱ Bayȱ ofȱAlgeciras.ȱTheȱ alterationsȱ
shownȱinȱcrabsȱexposedȱtoȱsedimentȱfromȱGR3ȱhasȱbeenȱpreviouslyȱobservedȱinȱȱ
differentȱ ȱ marineȱ ȱ invertebrateȱ orȱ vertebrateȱ ȱ ȱ speciesȱ exposedȱ toȱ ȱ differentȱȱ
inorganicȱ orȱ organicȱ contaminants,ȱ parasiticȱ orȱ infectiousȱ diseases,ȱ nutritionalȱ
stress,ȱ orȱ physicoȬchemicalȱ disordersȱ (Rodriguezȱ deȱ laȱ Ruaȱ etȱ al.,ȱ 2005;ȱ OrtizȬ
Delgadoȱetȱal.,ȱ2007).ȱ
Althoughȱ aȱ causalȱ relationshipȱ mustȱ existȱ betweenȱ exposureȱ toȱ
contaminantsȱandȱbiologicalȱeffects,ȱsuchȱaȱcausalȱlinkȱdoesȱnotȱnecessarilyȱholdȱ
betweenȱtheȱtwoȱtypesȱofȱbiomarkersȱ(biomarkersȱofȱresponseȱandȱbiomarkersȱ
ofȱeffect),ȱexceptȱifȱbiomarkersȱshareȱaȱcommonȱmetabolicȱpathway.ȱ(Lafontaineȱ
- 242 -
etȱ al.,ȱ ȱ 2000).ȱ Inȱ thisȱ caseȱ histopathologicalȱ lesionsȱ areȱ higherȱ inȱ organismsȱ
exposedȱ toȱ theȱ sedimentȱ withȱ theȱ highestȱ contentȱ ofȱ PAHs;ȱ inȱ addition,ȱ theȱ
detoxificationȱ sytemȱ inductedȱ byȱ theȱ ERODȱ activityȱ theȱ dayȱ 7ȱ andȱ 28ȱ alsoȱ
correlatesȱ withȱ thisȱ contaminant.ȱ Previousȱ studiesȱ withȱ crabsȱ alsoȱ foundȱ
relationshipsȱ betweenȱ PAHsȱ andȱ theȱ inductionȱ ofȱ ERODȱ activityȱ (Fossiȱ etȱ al.,ȱ
2000;ȱMartínȬDíazȱetȱal.,ȱ2004)ȱ
5.ȱConclusionsȱ
Theȱ followingȱconclusionsȱ canȱbeȱdrawnȱfromȱtheȱresultsȱofȱtheȱpresentȱ
study:ȱ
1.ȱ Theȱ concentrationȱ ofȱ PAHsȱ inȱ sedimentsȱ hasȱ beenȱ correlatedȱ toȱ theȱ
inductionȱ ofȱ ERODȱ activityȱ inȱ theȱ hepatopancreasȱ ofȱ theȱ exposedȱ crabsȱ underȱ
laboratoryȱconditions.ȱȱ
2.ȱ Inȱ theȱ caseȱ ofȱ organismsȱ deployedȱ inȱ cagesȱ inȱ theȱ Bayȱ ofȱ Algeciras,ȱ
effectsȱofȱcontaminatnsȱboundȱtoȱsedimentsȱdecreaseȱconsiderablyȱasȱindicatedȱ
byȱbiomarkersȱofȱexposureȱandȱeffect.ȱThisȱcouldȱbeȱrelatedȱtoȱaȱdisminutionȱofȱ
bioavailabilityȱofȱcontaminantsȱdueȱtoȱtheȱwaterȱremovalȱmainlyȱproduceȱbyȱtheȱ
highȱinfluenceȱofȱtides.ȱ
3.ȱ Organismsȱ deployedȱ inȱ theȱ Galicianȱ Coastȱ probablyȱ presentȱ otherȱ
sourcesȱ ofȱ stressȱ notȱ relatedȱ toȱ sedimentȱ asȱ itȱ hasȱ beenȱ shownȱ inȱ theȱ lowȱ
biomarkerȱ activitiesȱ underȱ laboratoryȱ assaysȱ andȱ higherȱ activitiesȱ underȱ fieldȱ
conditions,ȱspeciallyȱinȱcrabsȱdeployedȱinȱtheȱBayȱofȱCormeȬLaxe.ȱHoweverȱtheȱ
sourcesȱofȱstressȱdidȱnotproduceȱsignificantȱhistpathologicalȱeffects.ȱ
4.ȱ Theȱ importanceȱ ofȱ carryingȱ outȱ kineticȱ approachesȱ ofȱ biomarkersȱ ofȱ
responseȱ inȱ marineȱ invertebrateȱ hasȱ beenȱ shown;ȱ theȱ incorporationȱ ofȱ
biomarkersȱ ofȱ effectȱ andȱ chemicalȱ dataȱ inȱ additionȱ toȱ theȱ combinationȱ ofȱ bothȱ
- 243 -
fieldȱ andȱ laboratoryȱ assaysȱ toȱ theȱ kineticȱ studyȱ helpsȱ toȱ elucidateȱ possibleȱ
sourcesȱofȱcontamination.ȱȱ
6.ȱAcknowledgementsȱ
Theȱ workȱ describedȱ wasȱ partlyȱ supportedȱ byȱ theȱ projectsȱ VEM2003Ȭ
20563/INTER,ȱ andȱ CTM2005Ȭ07282ȬC03Ȭ01/TECNOȱ financedȱ byȱ theȱ Spanishȱ
Educationȱ andȱ Scienceȱ Ministryȱ andȱ byȱ CISȱ fundedȱ byȱ theȱ Ministryȱ ofȱ
Environment.ȱ Carmenȱ MoralesȬCasellesȱ thanksȱ theȱ Ministryȱ ofȱ Educationȱ andȱ
Scienceȱ forȱ fundingȱ herȱ researchȱ fellowshipȱ (FPU).ȱ Dr.ȱ Ribaȱ thanksȱ theȱ I3pȱ
programȱ forȱ supportingȱ herȱ participationȱ inȱ thisȱ research.ȱ ȱ Inmaculadaȱ Ribaȱ
thanksȱtheȱCSICȱforȱherȱI3Pȱcontract.ȱWeȱareȱgratefulȱforȱtheȱsupportȱandȱhelpȱofȱ
theȱ membersȱ ofȱ theȱ CISȱ andȱ theȱ ICMANȬCSIC.ȱ Specialȱ thanksȱ areȱ givenȱ toȱ
AntonioȱMorenoȱandȱPabloȱVidal.ȱȱ
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perspectivesȱusingȱbiomarkersȱtoȱassessȱpollutionȱinȱaquaticȱecosystems.ȱ
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(1997).ȱHistofisiologíaȱeȱhistiopatologíaȱduranteȱelȱdesarrolloȱlarvarioȱdeȱ
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Vanȱ derȱ Oostȱ R,ȱ Beyerȱ J,ȱ Vermeulenȱ NPE.ȱ 2003.ȱ Fishȱ bioaccumulationȱ andȱ
biomarkersȱ inȱ environmentalȱ riskȱ assessment.ȱ Environȱ Toxicolȱ Pharmacolȱ
13:57Ȭ149ȱ
Werner,ȱ I.,ȱ Teh,ȱ S.J.,ȱ Datta,ȱ S.,ȱ Lu,ȱ X.,ȱ Young,ȱ T.M.ȱ Biomarkerȱ responsesȱ inȱ
Macomaȱ nasutaȱ (Bivalvia)ȱ exposedȱ toȱ sedimentsȱ fromȱ northernȱ Sanȱ
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ȱ
ȱȱ
ȱ
ȱ
- 247 -
ȱ
- 248 -
Aȱcomparativeȱanalysisȱofȱmacrobenthicȱcommunityȱstructureȱinȱ
relationȱtoȱdifferentȱoilȱcontaminatedȱsediments:ȱtheȱGalicianȱ
Coastȱ(acute,ȱPrestigeȱoilȱspill)andȱtheȱBayȱofȱAlgecirasȱ(chronicȱoilȱ
spills).ȱ
CarmenȱMoralesȬCaselles1,2,*,ȱAugustoȱCésar2,3,ȱRodrigoȱB.ȱChoueri2,ȱ
InmaculadaȱRiba1,2,ȱT.ȱÁngelȱDelValls1,2ȱ
1ȱUnidadȱAsociadaȱdeȱCalidadȱAmbientalȱyȱPatologíaȱ(CSICȱ&ȱUCA)ȱInstitutoȱ
deȱCienciasȱMarinasȱdeȱAndalucíaȱ(ICMANȬCSIC),ȱAvda.ȱRepúblicaȱSaharauiȱs/n,ȱ
PuertoȱRealȱ11510,ȱCádiz,ȱSpainȱ
2ȱUNESCOȱUNITWIN/WiCop,ȱFacultadȱdeȱCienciasȱdelȱMarȱyȱAmbientales,ȱ
UniversidadȱdeȱCádiz,ȱPolígonoȱRíoȱSanȱPedroȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱSpain.ȱ
3ȱDepartamentoȱdeȱEcotoxicologia,ȱUniversidadeȱSantaȱCecíliaȱ(UNISANTA),ȱRuaȱ
OswaldoȱCruz,ȱ266,ȱSantos,ȱSãoȱPaulo,ȱBrazil.ȱ
Abstractȱ
Theȱmacrobenthicȱfaunaȱandȱchemicalsȱconcentrationsȱinȱsedimentsȱwereȱ
examinedȱinȱtheȱGalicianȱCoastȱ(NWȱSpain)ȱalongȱthreeȱyearsȱafterȱtheȱPrestigeȱ
oilȱ spillȱ (November,ȱ 2002).ȱ Resultsȱ obtainedȱ pointsȱ toȱ anȱ initialȱ impactȱ toȱ theȱ
benthicȱcommunityȱdueȱtoȱtheȱfuelȱoilȱalthoughȱaȱrecoveryȱofȱtheȱenvironmentalȱ
qualityȱwasȱobservedȱfourȱyearsȱafterȱtheȱaccident.ȱSelectedȱsitesȱlocatedȱinȱtheȱ
GalicianȱCoastȱwereȱcomparedȱtoȱtheȱstatusȱofȱsedimentsȱchronicallyȱaffectedȱbyȱ
oilȱ spillsȱ inȱ theȱ Bayȱ ofȱ Algecirasȱ andȱ thoseȱ notȱ affectedȱ inȱ theȱ Bayȱ ofȱ Cadizȱ inȱ
orderȱtoȱassessȱtheȱrecuperationȱcapacityȱofȱtheȱecosystemȱafterȱanȱacuteȱimpact.ȱ
Theȱ methodologyȱ employedȱ includesȱ univariateȱ analysisȱ usingȱ conventionalȱ
communityȱ descriptiveȱ parametersȱ andȱ theȱ numericalȱ contributionȱ ofȱ majorȱ
taxonomicȱgroups.ȱResultsȱobtainedȱinȱtheȱareasȱofȱstudyȱhaveȱbeenȱlinkedȱwithȱ
theȱ physicochemicalȱ characterizationȱ ofȱ sedimentsȱ withȱ theȱ purposeȱ ofȱ
identifyingȱtheȱcauseȱandȱsourceȱofȱcontaminants.ȱ
ȱScienceȱofȱtheȱTotalȱEnvironmentȱ(enviado)
- 249 -
Keywords:ȱ sedimentȱ alteration,ȱ macrobenthicȱ populations,ȱ community,ȱ
environmentalȱdegradation,ȱPAHs.ȱȱȱȱ
1.ȱIntroductionȱ
TheȱoilȱtankerȱPrestigeȱbrokeȱdownȱinȱtheȱGalicianȱCoastȱ(NWȱSpain)ȱonȱ
Novemberȱ 2002ȱ andȱ approximatelyȱ 60ȱ000ȱ tonnesȱ ofȱ heavyȱ fuelȱ oilȱ wereȱ
releasedȱ intoȱ theȱ surroundingȱ waters,ȱ resultingȱ inȱ theȱ contaminationȱ ofȱ moreȱ
thanȱ 1000ȱ kmȱ ofȱ coastline;ȱ thisȱ accidentȱ supposedȱ oneȱ ofȱ theȱ majorȱ ecologicalȱ
catastrophesȱofȱtheȱIberianȱPeninsula.ȱResultsȱofȱworkȱinvestigatingȱtheȱimpactȱ
ofȱoilȱspillsȱonȱaȱvarietyȱofȱbiologicalȱcomponentsȱhaveȱconfirmedȱtheȱeffectsȱinȱ
aȱwideȱrangeȱofȱhabitatsȱandȱspeciesȱ(Petersonȱetȱal.,ȱ2001).ȱ
Theȱ assessmentȱ ofȱ inȱ situȱ alterationȱ ofȱ residentialȱ communityȱ structureȱ
hasȱ beenȱ oftenȱ performedȱ toȱ determineȱ theȱ effectsȱ ofȱ pollutantsȱ inȱ theȱ coastalȱ
environmentȱ (DelVallsȱ etȱ al.,ȱ 1998a.,ȱ GómezȬGesteiraȱ andȱ Dauvin,ȱ 2005).ȱ Fieldȱ
dataȱ onȱ theȱ communitiesȱ livingȱ inȱ theȱ sedimentsȱ allowȱ establishingȱ whetherȱ
thereȱisȱobservableȱpollutionȬinducedȱdegradationȱeffectȱinȱtheȱbiotaȱ(Chapmanȱ
etȱ al.,ȱ 1991,ȱ Chapman,ȱ 2007).ȱ Threeȱ symptomsȱ ofȱ stressȱ reducedȱ diversity,ȱ
retrogressionȱ toȱ opportunisticȱ species,ȱ andȱ reducedȱ sizeȱ ofȱ individualsȱ areȱ
documentedȱforȱaȱwideȱvarietyȱofȱnaturalȱandȱanthropogenicȱstressesȱinȱmarineȱ
environmentsȱ (Gray,ȱ 1989).ȱ Identificationȱ ofȱ theseȱ symptomsȱ inȱ aȱ benthicȱ
assemblageȱ mayȱ signalȱ aȱ changeȱ inȱ environmentalȱ conditionsȱ resultingȱ fromȱ
anthropogenicȱ influencesȱ (Newellȱ etȱ al.,ȱ 1999).ȱ Previousȱ studiesȱ (citedȱ inȱ
Blanchardȱ etȱ al.,ȱ 2002)ȱ indicatedȱ thatȱ someȱ benthicȱ organismsȱ canȱ respondȱ toȱ
nonȬtoxicȱ fractionsȱ ofȱ crudeȱ oilȱ asȱ theyȱ wouldȱ toȱ otherȱ formsȱ ofȱ organicȱ
enrichmentȱ (e.g.,ȱ Weston,ȱ 1990)ȱ andȱ thisȱ isȱ suggestedȱ byȱ theȱ responseȱ ofȱ anȱ
increaseȱinȱsomeȱpolychaeteȱspecies.ȱ
Multivariateȱ analysisȱ appearsȱ toȱ beȱ anȱ especiallyȱ sensitiveȱ toolȱ forȱ
detectingȱ changeȱ inȱ theȱ structureȱ ofȱ theȱ faunalȱ communityȱ (Warwickȱ andȱ
- 250 -
Clarke.,ȱ 1991).ȱ Theȱ integrationȱ ofȱ fieldȱ dataȱ withȱ chemicalsȱ analysisȱ permitsȱ
establishingȱtheȱpossibleȱcausesȱandȱsourcesȱofȱtheȱbenthicȱalteration.ȱ
Theȱscopeȱofȱthisȱstudyȱisȱtoȱexamineȱtheȱrecoveryȱofȱbenthicȱcommunityȱ
afterȱ theȱ Prestigeȱ oilȱ spillȱ andȱ toȱ compareȱ theȱ environmentalȱ statusȱ ofȱ theȱ
Galicianȱ Coastȱ afterȱ 4ȱ yearsȱ ofȱ theȱ spillȱ withȱ theȱ macrobenthicȱ structureȱ
analyzedȱinȱBayȱofȱAlgecirasȱthatȱisȱchronicallyȱimpactedȱbyȱdifferentȱoilȱspillsȱ
andȱwithȱthatȱanalyzedȱinȱtheȱBayȱofȱCadizȱconsideredȱnotȱcontaminatedȱneitherȱ
pollutedȱ byȱ thisȱ kindȱ ofȱ activitiesȱ orȱ contaminants..ȱ Theȱ comparativeȱ analysisȱ
willȱbeȱcarriedȱoutȱbyȱusingȱunivariateȱandȱmultivariateȱmethods.ȱ
2.ȱMaterialȱandȱmethodsȱ
2.1.ȱSitesȱdescriptionȱ
Figureȱ1ȱshowsȱtheȱselectedȱsitesȱinȱtheȱGalicianȱCoastȱ(NWȱSpain)ȱwereȱ
locatedȱ inȱ theȱ Ciesȱ Islandȱ (A,ȱ Bȱ andȱ C)ȱ inȱ theȱ Atlanticȱ Islandȱ Nationalȱ Parkȱ
(AINP)ȱandȱinȱtheȱBayȱofȱCormeȬLaxeȱ(D,ȱEȱandȱF).ȱBothȱareasȱwereȱimportantlyȱ
affectedȱ byȱ theȱ Prestigeȱ oilȱ spillȱ andȱ areȱ consideredȱ ofȱ highȱ ecologicalȱ
importance.ȱTheȱsecondȱareaȱofȱstudyȱwasȱtheȱmouthȱofȱtheȱRiverȱPalmonesȱ(P1)ȱ
andȱGuadarranqueȱ(GR3ȱandȱGR4)ȱinȱtheȱBayȱofȱAlgecirasȱ(SȱSpain);ȱthisȱplaceȱ
wasȱ selectedȱ becauseȱ isȱ highlyȱ industrializedȱ andȱ thereȱ areȱ aȱ largeȱ numberȱ ofȱ
petrochemicalȱactivitiesȱwhichȱcompriseȱseveralȱaccidentalȱoilȱspills.ȱAȱreferenceȱ
site,ȱCA,ȱwidelyȱcharacterizedȱbyȱdifferentȱecotoxicologicalȱstudiesȱ(DelȱVallsȱetȱ
al.,ȱ1998b,ȱRibaȱetȱal.,ȱ2004,ȱMartínȬDíazȱetȱal.,ȱ2005;ȱCesarȱetȱal.ȱ2007;ȱMoralesȬ
Casellesȱetȱal.,ȱ2007)ȱwasȱselectedȱinȱaȱcleanȱareaȱinȱtheȱBayȱofȱCádizȱ(SȱSpain).ȱȱ
2.2.ȱSampleȱcollectionȱ
Sedimentȱ samplesȱ wereȱ collectedȱ withȱ aȱ 0.025ȱ m2ȱ vanȱ Veenȱ grab.ȱ Onlyȱ
grabsȱ thatȱ achievedȱ adequateȱ penetrationȱ (2/3ȱ ofȱ totalȱ volume)ȱ toȱ collectȱ theȱ
superficialȱ 5ȱ cmȱ ofȱ theȱ sedimentȱ andȱ thatȱ showedȱ noȱ evidenceȱ ofȱ leakageȱ orȱ
- 251 -
surfaceȱ disturbanceȱ wereȱ retainedȱ forȱ theȱ study.ȱ Forȱ theȱ benthicȱ infaunalȱ
samples,ȱ theȱ entireȱ contentsȱ ofȱ theȱ grabȱ includingȱ overlyingȱ water,ȱ wereȱ wetȱ
sievedȱ atȱ theȱ studyȱ siteȱ withȱ aȱ 0.5ȱ mmȱ stainlessȱ steelȱ mesh.ȱ Residuesȱ wereȱ
gentlyȱ washed,ȱ placedȱ inȱ polyethyleneȱ bottles,ȱ preservedȱ withȱ 10ȱ %ȱ bufferedȱ
formalinȱ andȱ stainedȱ withȱ Roseȱ Bengal.ȱ Sedimentsȱ forȱ chemicalȱ analysesȱ wereȱ
collectedȱandȱtransportedȱtoȱaȱcooler.ȱSedimentȱsamplesȱwereȱkeptȱinȱdarkȱatȱ4ȱ
ºCȱpriorȱtoȱanalysis.ȱ
ȱȱ
ȱ
ȱ
ƒF ƒE
ƒD
Atlantic Islands
National Park
ȱ
Ría de CormeLaxe
•C
N
•A
•B
ȱ
E
W
S
ȱ
Spain
ȱ
ȱ
•GR3
•GR4
•P1
ȱ
Bay of
Algeciras
ȱ
•CA
ȱ
ȱ
Bay of
Cádiz
Figureȱ1.ȱMapȱofȱtheȱcoastalȱareaȱofȱGaliciaȱshowingȱtheȱlocationsȱofȱtheȱ
samplingȱstations.ȱA,ȱBȱandȱCȱrefersȱtoȱtheȱstationsȱlocatedȱinȱtheȱCiesȱIslandȱinȱ
theȱAtlanticȱIslandȱNationalȱParkȱandȱD,ȱEȱandȱFȱtoȱthoseȱinȱtheȱBayȱofȱCormeȬ
Laxe.ȱ Theȱ stationsȱ locatedȱ inȱ theȱ Bayȱ ofȱ Algecirasȱ areȱ GR3,ȱ GR4ȱ andȱ P1.ȱ Theȱ
stationȱ CAȱ locatedȱ inȱ theȱ Bayȱ ofȱ Cadizȱ correspondsȱ toȱ theȱ sedimentȱ usedȱ asȱ
reference.ȱ
- 252 -
2.3.ȱLaboratoryȱanalysisȱ
Theȱ organismsȱ collectedȱ inȱ theȱ studyȱ sitesȱ wereȱ separatedȱ fromȱ theȱ
remainingȱ sediment,ȱ sortedȱ andȱ identifiedȱ toȱ theȱ lowestȱ possibleȱ taxonȱ levelȱ
(speciesȱlevel,ȱorȱfamilyȱinȱcaseȱofȱPolychaeta).ȱIdentificationsȱtoȱtheȱfamilyȱlevelȱ
wereȱconsideredȱadequateȱmeasuresȱofȱfaunalȱcompositionȱforȱtheȱpurposesȱofȱ
thisȱstudy.ȱ
Polycyclicȱ aromaticȱ hydrocarbonsȱ (PAHs)ȱ boundȱ toȱ sedimentsȱ wereȱ
analyzedȱ byȱ usingȱ aȱ gasȱ chromatographȱ equippedȱ withȱ massȱ spectrometerȱ
(GC/MS)ȱ (USEPA,ȱ 1994).ȱ Brieflyȱ driedȱ samplesȱ wereȱ Soxhletȱ extractedȱ withȱ nȬ
hexaneȱforȱ18ȱh,ȱandȱtheȱextractsȱwereȱisolatedȱbyȱcolumnȱchromatographyȱonȱ
Florisil®.ȱ PAHsȱ wereȱ elutedȱ andȱ theirȱ fractionsȱ wereȱ driedȱ inȱ aȱ rotatingȱ
evaporatorȱandȱreȬdissolvedȱinȱisooctane.ȱAromaticȱfractionsȱwereȱanalyzedȱonȱ
aȱHewlettePackardȱ(HP)ȱ5890ȱSeriesȱIIȱgasȱchromatographȱcoupledȱwithȱanȱHPȱ
5970ȱ massȱ spectrometer.ȱ PAHsȱ wereȱ analyzedȱ byȱ GCȬMSȱ usingȱ selectedȱ ionȱ
monitoringȱ (SIM).ȱ Theȱ analyticalȱ procedureȱ showedȱ agreementȱ withȱ theȱ
certifiedȱvaluesȱofȱmoreȱthanȱ90%.ȱ
TraceȱmetalȱwereȱanalyzedȱasȱdescribedȱbyȱCasadoȬMartínezȱetȱal.ȱ(2006);ȱ
briefly,ȱ 2.5ȱ gȱ ofȱ sedimentsȱ (<0.065ȱ mm)ȱ wereȱ placedȱ inȱ Teflonȱ containersȱ andȱ
wereȱdigestedȱinȱmicrowaveȱ(400W,ȱ15ȱmin,ȱtwice)ȱwithȱHNO3ȱ2N.ȱTheȱextractsȱ
wereȱ purifiedȱ byȱ passingȱ throughȱ aȱ CȬ18ȱ columnȱ andȱ metalsȱ analysesȱ wereȱ
performedȱbyȱ anodicȱ voltamperimetryȱ(ȬZn,ȱCd,ȱPb,ȱNi,ȱCoȱandȱ CuȬȱMetrohmȱ
ApplicationȱBulletinȱ Nºȱ147;ȱȬȱVȬȱMetrohmȱApplicationȱNoteȱNºȱVȬ81).ȱForȱHgȱ
theȱ coldȱ vapourȱ techniqueȱ wasȱ usedȱ andȱ wasȱ quantifiedȱ usingȱ atomicȱ
absorptionȱ spectrometry.ȱ Theȱ analyticalȱ proceduresȱ wereȱ checkedȱ usingȱ
referenceȱ materialȱ (MESSȬ1ȱ NRCȱ andȱ CRMȱ 277ȱ BCR)ȱ andȱ showedȱ aȱ recoveryȱ
greaterȱthanȱ90%ȱofȱtheȱcertifiedȱconcentration.ȱ
- 253 -
OrganicȱcarbonȱcontentȱwasȱdeterminedȱusingȱtheȱmethodȱofȱGaudetteȱetȱ
al.ȱ (1974)ȱ withȱ theȱ Elȱ Rayisȱ (1985)ȱ modification.ȱ Forȱ sedimentȱ grainȱ size,ȱ anȱ
aliquotȱofȱwetȱsedimentȱwasȱanalyzedȱusingȱaȱFristchȱlaserȱparticleȱsizerȱ(modelȱ
Analysetteȱ 22)ȱ followingȱ theȱ methodȱ reportedȱ byȱ DelVallsȱ andȱ Chapmanȱ
(1998b).ȱ
2.4.ȱDataȱanalysisȱ
Descriptiveȱstatisticsȱwereȱusedȱinȱorderȱtoȱdescribeȱtheȱtheȱmacrobenthicȱ
communityȱ atȱ eachȱ site;ȱ univariateȱ methodsȱ includedȱ classicalȱ communityȱ
descriptiveȱ parameters,ȱ asȱ speciesȱ richnessȱ (Margaleff’sȱ R),ȱ ShannonȬWienerȱ
diversityȱ (H’),ȱ evennessȱ (Pielou’sȱ J),ȱ andȱ Simpson’sȱ dominanceȱ (Dȱ =ȱ 1ȬΏ’)ȱ
(GómezȬGesteiraȱandȱDauvin,ȱ2005;ȱChoueriȱetȱal.,ȱsubmitted).ȱSinceȱnumericalȱ
contributionȱ ofȱ majorȱ taxaȱ isȱ widelyȱ utilisedȱ toȱ evaluateȱ pollutionȱ effectsȱ
(DelValls,ȱ1998a;ȱChapmanȱetȱal.,ȱ1996),ȱanȱabundanceȱanalysisȱwasȱcarriedȱoutȱ
byȱ calculatingȱ theȱ proportionȱ ofȱ majorȱ taxa’sȱ (Polychaeta,ȱ Molluscaȱ andȱ
Crustacea)ȱabundanceȱtoȱtheȱtotalȱabundanceȱforȱeachȱsample.ȱȱ
Multivariateȱ analysisȱ wasȱ carriedȱ outȱ withȱ inȱ anȱ attemptȱ toȱ linkȱ
contaminationȱ withȱ benthicȱ alterationȱ parameters;ȱ theȱ principalȱ componentȱ
analysisȱ (PCA)ȱ wasȱ usedȱ asȱ theȱ extractionȱ procedureȱ whichȱ isȱ aȱ multivariateȱ
statisticalȱ techniqueȱ toȱ exploreȱ variableȱ distributionsȱ (Ribaȱ etȱ al.,ȱ 2003).ȱ Theȱ
objectiveȱ ofȱ PCAȱ isȱ toȱ deriveȱ aȱ reducedȱ numberȱ ofȱ newȱ variablesȱ asȱ linearȱ
combinationsȱ ofȱ theȱ originalȱ variables.ȱ Thisȱ providesȱ aȱ descriptionȱ ofȱ theȱ
structureȱ ofȱ theȱ dataȱ withȱ theȱ minimumȱ lossȱ ofȱ information.ȱ Aȱ hierarchicalȱ
classificationȱ techniqueȱ byȱ meansȱ ofȱ aȱ Clusterȱ analysisȱ wasȱ performedȱ toȱ
determineȱ theȱ percentageȱ disagreementȱ amongȱ theȱ studyȱ sitesȱ takingȱ intoȱ
accountȱtheȱphysicochemicalȱandȱbiologicalȱvariables,ȱresultsȱwereȱdisplayedȱinȱ
aȱdendrogram.ȱ
ȱ
- 254 -
3.ȱResultsȱandȱdiscussionȱ
3.1.ȱ Evolutionȱ ofȱ theȱ benthicȱ communityȱ andȱ chemicalȱ concentrationȱ inȱ
sedimentsȱfromȱtheȱGalicianȱcoastȱ(2004Ȭ2006)ȱ
Threeȱ surveysȱ wereȱ carriedȱ outȱ inȱ theȱ AINPȱ afterȱ theȱ Prestigeȱ oilȱ spillȱ
(2004,ȱ2005ȱandȱ2006)ȱwhereasȱtwoȱ(2005ȱandȱ2006)ȱwereȱperformedȱinȱtheȱBayȱ
ofȱ Cormeȱ Laxe.ȱ Theȱ decreaseȱ inȱ theȱ abundanceȱ ofȱ theȱ macroinfaunaȱ observedȱ
afterȱtheȱspillȱappearȱtoȱreflectȱtheȱlossesȱdueȱtoȱoilingȱtoxicityȱorȱindirectȱeffectsȱ
ofȱ oilingȱ andȱ cleanȬupȱ (Junoyȱ etȱ al.,ȱ 2004).ȱ Tableȱ 1ȱ showsȱ theȱ decreaseȱ inȱ theȱ
concentrationȱ ofȱ PAHsȱ inȱ sedimentsȱ 4ȱ yearsȱ afterȱ theȱ Prestigeȱ oilȱ spillȱ inȱ allȱ
studyȱ sitesȱ (MoralesȬCasellesȱ etȱ al.,ȱ accepted).ȱ Forȱ allȱ stationsȱ theȱ numberȱ ofȱ
speciesȱ increasedȱ fromȱ theȱ firstȱ surveyȱ tillȱ theȱ lastȱ one;ȱ thisȱ increaseȱ wasȱ
especiallyȱimportantȱinȱsitesȱBȱandȱCȱfromȱtheȱAINP.ȱTheȱspecificȱrichnessȱalsoȱ
increasedȱinȱsitesȱselectedȱinȱtheȱCíesȱIslandsȱwhereasȱitȱkeptȱsimilarȱinȱDȱandȱFȱ
andȱdecreasedȱinȱsiteȱEȱfromȱCormeȬLaxe.ȱDiversityȱpresentedȱaȱdiminutionȱinȱ
siteȱEȱwhileȱanȱincreaseȱofȱthisȱparameterȱwasȱobservedȱinȱtheȱotherȱstudyȱsites.ȱ
Inȱgeneral,ȱallȱstationsȱpresentedȱaȱhighȱpopulationȱofȱpolychaeteȱafterȱtheȱspillȱ
whichȱ decreasedȱ alongȱ theȱ timeȱ whereasȱ otherȱ taxonsȱ suchȱ asȱ molluscsȱ andȱ
crustaceanȱincreased.ȱItȱisȱknownȱthatȱtheȱabundanceȱofȱopportunisticȱtaxaȱsuchȱ
asȱ polychaeteȱ increaseȱ inȱ theȱ presenceȱ ofȱ petroleumȱ hydrocarbonsȱ (Federȱ andȱ
Blanchard,ȱ 1998)ȱ whereasȱ declinesȱ inȱ benthicȱ amphipodsȱ alsoȱ occurredȱ
followingȱtheȱAmocoȱCadizȱ(Dauvin,ȱ1982)ȱandȱtheȱAegeanȱSeaȱoilȱspillȱ(Parraȱ
andȱ LópezȬJamar,ȱ 1997).ȱ Univariateȱ analysesȱ ofȱ benthicȱ dataȱ fromȱ ourȱ studyȱ
showedȱ theȱ trendsȱ (i.e.ȱ lowȱ faunalȱ abundance,ȱ relativelyȱ highȱ dominanceȱ andȱ
lowȱ diversity)ȱ observedȱ followingȱ oilȱ spillsȱ (Federȱ etȱ al.,ȱ 1998),ȱ whatȱ indicatesȱ
thatȱ theȱ environmentȱ wasȱ negativelyȱ impactedȱ byȱ theȱ accidentȱ ofȱ theȱ tankerȱ
Prestige;ȱ however,ȱ theȱ variationȱ ofȱ theȱ benthicȱ parametersȱ alongȱ theȱ timeȱ andȱ
theȱ diminutionȱ ofȱ theȱ concentrationȱ ofȱ PAHsȱ boundȱ toȱ sedimentsȱ pointsȱ toȱ aȱ
- 255 -
recoveryȱofȱtheȱenvironmentalȱqualityȱinȱtheȱfollowingȱyearsȱafterȱtheȱoilȱspill,ȱ
whatȱhasȱbeenȱconfirmedȱbyȱotherȱauthorsȱ(Serranoȱetȱal.,ȱ2006).ȱȱ
Tableȱ1.ȱSummarizedȱresultsȱofȱtheȱconcentrationȱofȱPAHsȱinȱsedimentsȱ
theȱ andȱ benthicȱ alterationȱ parametersȱ measuredȱ forȱ theȱ studyȱ ofȱ theȱ
environmentalȱ qualityȱ inȱ theȱ Ciesȱ Islandȱ ȱ inȱ theȱ Atlanticȱ Islandȱ nationalȱ Parkȱ
2004Ȭ2006ȱ(firstȱsurvey:ȱAȬ1,ȱBȬ1,ȱCȬ1;ȱsecondȱsurvey:ȱAȬ2,ȱBȬ2,ȱCȬ2;ȱthirdȱsurvey:ȱ
AȬ3,ȱBȬ3,ȱCȬ3)ȱandȱtheȱBayȱofȱCormeȬLaxeȱ2005Ȭ2006ȱȱ(secondȱsurvey:ȱDȬ2,ȱEȬ2,ȱ
FȬ2;ȱthirdȱsurvey:ȱDȬ3,ȱEȬ3,ȱFȬ3).ȱȱ
Stationsȱ
AȬ1ȱ
AȬ2ȱ
AȬ3ȱ
BȬ1ȱ
BȬ2ȱ
BȬ3ȱ
CȬ1ȱ
CȬ2ȱ
CȬ3ȱ
DȬ2ȱ
DȬ3ȱ
EȬ2ȱ
EȬ3ȱ
FȬ2ȱ
FȬ3ȱ
Parametersȱmeasuredȱ
Moluscaȱ Polychaetaȱ Crustacea
PAHȱ Speciesȱȱ Specificȱ
Diversity
ΐgKgȬ1ȱ
Nºȱ
richnessȱ
%ȱ
ȱȱȱ%ȱ
%ȱ
390ȱ
3.0ȱ
1.8ȱ
1.5ȱ
0.1ȱ
53.0ȱ
33.3ȱ
119ȱ
5ȱ
12.0ȱ
4.3ȱ
2.4ȱ
21.0ȱ
34.5ȱ
108ȱ
7.09ȱ
28.5ȱ
5.1ȱ
15.3ȱ
20.0ȱ
37.0ȱ
2120ȱ
2ȱ
1.2ȱ
1.0ȱ
0.1ȱ
100.0ȱ
0.1ȱ
366ȱ
12ȱ
5.9ȱ
5.2ȱ
9.9ȱ
56.2ȱ
15.4ȱ
67ȱ
47ȱ
33.9ȱ
5.0ȱ
28.4ȱ
21.5ȱ
41.0ȱ
420ȱ
9.0ȱ
15.3ȱ
2.9ȱ
22.2ȱ
33.3ȱ
33.3ȱ
239ȱ
30.0ȱ
50.9ȱ
4.5ȱ
26.7ȱ
26.7ȱ
43.3ȱ
n.d.ȱ
25.0ȱ
42.4ȱ
4.3ȱ
39.1ȱ
21.7ȱ
39.1ȱ
537ȱ
9ȱ
25.7ȱ
2.9ȱ
33.3ȱ
33.3ȱ
33.3ȱ
38ȱ
10ȱ
28.6ȱ
3.0ȱ
30.0ȱ
20.0ȱ
50.0ȱ
558ȱ
12ȱ
66.7ȱ
5.0ȱ
2.0ȱ
30.6ȱ
100.0ȱ
52ȱ
12ȱ
32.1ȱ
3.0ȱ
40.1ȱ
22.2ȱ
51.4ȱ
820ȱ
15ȱ
55.6ȱ
2.3ȱ
40.0ȱ
26.7ȱ
33.3ȱ
323ȱ
13ȱ
48.2ȱ
2.9ȱ
15.4ȱ
23.1ȱ
61.5ȱ
Inȱorderȱtoȱelucidateȱifȱthereȱisȱstillȱdegradationȱofȱtheȱenvironmentȱinȱtheȱ
GalicianȱCoastȱfourȱyearsȱafterȱtheȱspill,ȱtheȱevaluationȱofȱtheȱsedimentȱquality,ȱ
includingȱ physicochemicalȱ andȱ benthicȱ parametersȱ wasȱ comparedȱ withȱ theȱ
situationȱofȱtheȱareaȱofȱtheȱbayȱofȱAlgeciras,ȱchronicallyȱaffectedȱbyȱoilȱspills.ȱ
ȱ
- 256 -
3.2.ȱ Physicochemicalȱ characterizationȱ ofȱ sedimentsȱ fromȱ theȱ Galicianȱ
CoastȱandȱtheȱBayȱofȱAlgecirasȱ(2006)ȱ
Summarisedȱ resultsȱ forȱ theȱ chemicalȱ dataȱ andȱ physicalȱ characterizationȱ
ofȱ theȱ sedimentsȱ areȱ shownȱ inȱ Tableȱ 2.ȱ ȱ Stationsȱ fromȱ theȱ Bayȱ ofȱ Algecirasȱ
presentedȱ theȱ highestȱ concentrationsȱ ofȱ organicȱ carbonȱ andȱ finesȱ inȱ theȱ
sediments.ȱ Theȱ highestȱ concentrationsȱ ofȱ PAHsȱ wereȱ observedȱ inȱ theȱ stationsȱ
locatedȱinȱtheȱBayȱofȱAlgecirasȱwhereasȱGR3ȱpresentedȱtheȱhighestȱvaluesȱofȱPbȱ
andȱ Niȱ oftenȱ relatedȱ toȱ hydrocarbons.ȱ Theȱ factȱ thatȱ thereȱ isȱ aȱ petrogenicȱ
industryȱclosedȱtoȱthisȱpointȱandȱtheȱpresenceȱofȱbunkeringȱactivitiesȱinȱtheȱBayȱ
couldȱexplainȱtheȱinputȱofȱhydrocarbonȱinȱtheȱarea.ȱOnȱtheȱotherȱhandȱneitherȱ
theȱreferenceȱstationȱnorȱtheȱsiteȱCȱshowedȱpresenceȱofȱPAHsȱinȱtheirȱsediments.ȱ
Noȱ generalȱ patternȱ wasȱ observedȱ forȱ otherȱ contaminantsȱ inȱ theȱ studiedȱ areasȱ
fromȱ CormeȬLaxeȱ andȱ Ciesȱ Island.ȱ Inȱ general,ȱ sitesȱ fromȱ theȱ Bayȱ ofȱ Algecirasȱ
presentȱtheȱhighestȱcontentȱofȱfinesȱandȱorganicȱcarbonȱinȱtheirȱsediments.ȱȱ
3.3.ȱ Theȱ benthicȱ communityȱ inȱ theȱ Galicianȱ Coastȱ andȱ theȱ Bayȱ ofȱ
Algecirasȱ(2006)ȱ
Theȱ descriptionȱ ofȱ theȱ benthicȱ communityȱ differsȱ dependingȱ onȱ theȱ
sampledȱ area.ȱ Forȱ sedimentsȱ collectedȱ inȱ theȱ referenceȱ stationȱ (CA)ȱ Molluscsȱ
wereȱ theȱ bestȱ representedȱ taxonȱ (78.5%)ȱ followedȱ byȱ Polychaeteȱ (12.7ȱ %)ȱ andȱ
Crustaceaȱ (8.8ȱ %)ȱ (Figureȱ 2).ȱ Surveysȱ inȱ theȱ Bayȱ ofȱ Algecirasȱ showedȱ aȱ
prevalenceȱ ofȱ polychaete,ȱ 45.3ȱ %ȱ inȱ GR4,ȱ 64.4ȱ %ȱ inȱ P1ȱ whereasȱ inȱ GR3ȱ allȱ theȱ
communityȱwasȱmadeȱupȱbyȱpolychaete.ȱTheȱdominanceȱdistributionȱofȱtaxaȱinȱ
BayȱofȱAlgecirasȱrevealsȱthatȱpollutantȱresistantȱgroups,ȱaccordingȱGrallȱandȱȱ
- 257 -
0.30ȱ
0.31ȱ
0.37ȱ
0.65ȱ
2.15ȱ
3.19ȱ
3.86ȱ
Cȱ
Dȱ
Eȱ
Fȱ
GR3ȱ
GR4ȱ
P1ȱ
35.4ȱ
59.3ȱ
69.4ȱ
5.95ȱ
5.50ȱ
3.79ȱ
2.76ȱ
2.81ȱ
56.7ȱ
35.3ȱ
138ȱ
271ȱ
19.9ȱ
25.0ȱ
164ȱ
91.0ȱ
12.3ȱ
6.21ȱ
21.6ȱ
5.90ȱ
7.30ȱ
3.70ȱ
0.85ȱ
0.90ȱ
1.50ȱ
75.2ȱ
3.67ȱ
5.01ȱ
4.20ȱ
0.43ȱ
0.70ȱ
1.40ȱ
1.40ȱ
5.20ȱ
13.3ȱ
13.1ȱ
74.7ȱ
5.70ȱ
1.50ȱ
1.70ȱ
4.50ȱ
2.40ȱ
13.3ȱ
- 258 -
0.65ȱ
0.25ȱ
1.04ȱ
3.40ȱ
2.10ȱ
2.00ȱ
0.60ȱ
0.80ȱ
0.70ȱ
641ȱ
802ȱ
2961ȱ
323ȱ
52.0ȱ
38.0ȱ
n.d.ȱ
67.0ȱ
108ȱ
n.d.ȱ
0.26ȱ
377ȱ
n.d.ȱ
Bȱ
4.32ȱ
0.06ȱ
0.28ȱ
6.98ȱ
Aȱ
2.28ȱ
2.50ȱ
1.07ȱ
CAȱ
21.3ȱ
Finesȱ
Znȱ
Pbȱ
Cuȱ
Niȱ
Hgȱ
PAHȱ
Ȭ1
Ȭ1
Ȭ1
Ȭ1
Ȭ1
%ȱ
mgKg mgKg mgKg mgKg mgKg ΐgKgȬ1ȱ
Physcochemicalȱanalysisȱ
studyȱ
O.C.ȱ%
sitesȱ
ȱȱ
ȱ
ȱ
ȱ
4.67ȱ
4.67ȱ
0.67ȱ
13ȱ
12ȱ
10ȱ
25ȱ
47ȱ
7.09ȱ
14ȱ
Speciesȱ
N.ȱ
1.3ȱ
1.2ȱ
0.0ȱ
48.2ȱ
32.1ȱ
28.6ȱ
42.4ȱ
33.9ȱ
28.5ȱ
2.6ȱ
1.24ȱ
1.29ȱ
0.0ȱ
2.9ȱ
3ȱ
3ȱ
4.3ȱ
5ȱ
5.1ȱ
1.64ȱ
0.68ȱ
0.72ȱ
0.0ȱ
0.20ȱ
0.19ȱ
0.15ȱ
0.06ȱ
0.10ȱ
0.50ȱ
0.66ȱ
Specificȱ
Diversity Dominanceȱ
richness
Benthicȱalterationsȱȱ
CormeȬLaxeȱ(D,ȱE,ȱF);ȱtheȱBayȱofȱAlgecirasȱ(GR3,ȱGR4ȱandȱP1)ȱandȱtheȱBayȱofȱCadizȱ(CA)ȱusedȱasȱtheȱreferenceȱstation.ȱȱȱ
specificȱ richness,ȱ diversityȱ andȱ dominance)ȱ measuredȱ inȱ theȱ sedimentsȱ fromȱ Galicia:ȱ Atlanticȱ Islandsȱ Nationalȱ Parkȱ (A,ȱ B,ȱ C),ȱ
Tableȱ2.ȱ TotalȱPAHs,ȱ PCBsȱandȱ metalȱconcentrationȱ(Zn,ȱPb,ȱCu,ȱNi,ȱandȱ Hg)ȱandȱ benthicȱ parametersȱ (numberȱ ofȱ species,ȱ
Glémarecȱ(1997)ȱclassification,ȱareȱmoreȱabundant.ȱPolychaetaȱ(Capitellidaeȱandȱ
Nereidae)ȱ wasȱ theȱ mostȱ commonȱ taxa,ȱ followedȱ byȱ Molluscaȱ (onlyȱ pollutionȬ
resistantȱ species,ȱ asȱ Cerastodermaȱ eduleȱ andȱ Abraȱ tenuis)ȱ (Choueriȱ etȱ al.,ȱ
submitted).ȱThisȱpatternȱofȱabundancyȱhasȱbeenȱshownȱinȱareasȱaffectedȱbyȱoilȱ
spillsȱ (Parraȱ andȱ LópezȬJamar,ȱ 1997;ȱ Serranoȱ etȱ al.,ȱ 2006).ȱ Otherȱ authorsȱ
considerȱ thatȱ theseȱ taxaȱ appearȱ toȱ beȱrespondingȱtoȱmoderateȱenhancementȱofȱ
theȱbenthosȱbyȱresidualȱhydrocarbonsȱinȱeffluentsȱasȱaȱfoodȱsourceȱ(Blanchardȱ
etȱal.,ȱ2002).ȱCrustaceaȱwasȱtheȱmostȱfrequentȱtaxaȱinȱallȱtheȱsitesȱlocatedȱinȱtheȱ
GalicianȱCoast,ȱ37.0ȱ%ȱinȱA,ȱ41.0ȱinȱB,ȱ39.1ȱ%ȱinȱC,ȱ50.0ȱ%ȱinȱD,ȱ51.4ȱ%ȱinȱEȱandȱ
61.5ȱ%ȱinȱF.ȱTheȱhighestȱnumberȱofȱspeciesȱwasȱdetectedȱinȱtheȱsitesȱBȱ(47)ȱandȱCȱ
(25)ȱfromȱtheȱCiesȱIslandsȱ(14),ȱfollowedȱbyȱtheȱreferenceȱstationȱ(CA)ȱwhereasȱ
theȱ lowestȱ numberȱ wasȱ observedȱ inȱ theȱ Bayȱ ofȱ Algeciras.ȱ Benthicȱ speciesȱ ofȱ
slowȱ growthȱ andȱ withȱ slowȱ recoveryȱ capability,ȱ mainlyȱ crustaceansȱ andȱ
echinoderms,ȱshowȱaȱhighȱsensitivityȱtoȱoilȱexposureȱ(Serranoȱetȱal.,ȱ2006).ȱWithȱ
regardȱtoȱotherȱpopulationȱparameters,ȱspeciesȱrichnessȱrangedȱfromȱ48.2ȱinȱFȱtoȱ
0ȱ inȱ GR3,ȱ whileȱ theȱ highestȱ diversityȱ wasȱ shownȱ inȱ theȱ AINP.ȱ Noȱ significantȱ
changesȱ inȱ benthicȱ communityȱ structure,ȱ characterizedȱ byȱ speciesȱ richness,ȱ
individualȱ abundance,ȱ andȱ diversityȱ wereȱ determinedȱ afterȱ theȱ Braerȱ oilȱ spillȱ
(Kingstonȱ etȱ al.,ȱ 1995)ȱ whatȱ agreesȱ withȱ theȱ affirmationȱ thatȱ fewȱ validȱ
generalizationsȱ aboutȱ ecologicalȱ effectsȱ canȱbeȱ appliedȱ toȱ mostȱ spillsȱ (Junoyȱetȱ
al.,ȱ2004).ȱNoȱdiversity,ȱthereforeȱnoȱdominanceȱwasȱfoundȱinȱtheȱstationȱGR3ȱinȱ
Algeciras.ȱ Changesȱ inȱ meanȱ abundance,ȱ biomass,ȱ orȱ diversityȱ atȱ aȱ stationȱ thatȱ
wereȱ unlike,ȱ orȱ outȱ ofȱ phaseȱ with,ȱ theȱ trendsȱ observedȱ forȱ otherȱ stationsȱ (anȱ
interactionȱ effect),ȱ indicateȱ possibleȱ influencesȱ byȱ sourcesȱ otherȱ thanȱ naturalȱ
factorsȱ (Jewettȱ etȱ al.,ȱ 1999;ȱ Blanchardȱ etȱ al.,ȱ 2002).ȱ However,ȱ lowȱ valuesȱ ofȱ
speciesȱ richnessȱ andȱ lowȱ diversityȱ andȱ highȱ dominanceȱ ofȱ fewȱ betterȱ adaptedȱ
speciesȱareȱexpectedȱforȱaquaticȱecosystemsȱlikeȱestuariesȱorȱaȱmouthȱofȱaȱriver,ȱ
whereȱ theȱ variationȱ ofȱ environmentalȱ conditionsȱ (salinity,ȱ pH,ȱ temperature)ȱ isȱ
stressingȱtoȱtheȱbiotaȱ(Choueriȱetȱal.,ȱsubmitted).ȱȱ
- 259 -
ȱ
CA
ȱ
GR3
8.8%
ȱ
12.7%
ȱ
ȱ
78.5%
100.0%
ȱ
GR4
P1
ȱ
10.2%
20.3%
25.4%
ȱ
34.4%
ȱ
ȱ
64.4%
45.3%
ȱ
% Molluscs
% Polychaete
% Crustacea
ȱ
Figureȱ 2.ȱ Distributionȱ ofȱ theȱ mainȱ taxaȱ inȱ sedimentsȱ fromȱ stationsȱ
selectedȱinȱtheȱGulfȱofȱCádiz.ȱȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
- 260 -
ȱ
A
ȱ
B
C
9.1%
15.3%
27.7%
28.4%
ȱ
20.0%
ȱ
41.0%
ȱ
21.5%
37.0%
ȱ
39.1%
39.1%
21.7%
E
D
F
ȱ
15.4%
30.0%
ȱ
35.3%
45.2%
50.0%
23.1%
ȱ
ȱ
ȱ
61.5%
20.0%
% Molluscs
19.5%
% Polychaete
% Crustacea
Other groups
ȱ
Figureȱ 3.ȱ Distributionȱ ofȱ theȱ mainȱ taxaȱ inȱ sedimentsȱ fromȱ stationsȱ
selectedȱinȱtheȱGalicianȱCoast.ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
- 261 -
3.4.ȱLinkingȱphysicochemicalȱcharacterizationȱwithȱbenthicȱalterationȱ
Aȱprincipalȱcomponentsȱanalysisȱwasȱperformedȱtowardsȱtwoȱobjectives:ȱ
toȱelucidateȱifȱtheȱbenthicȱalterationȱwasȱdueȱtoȱpollutantsȱboundȱtoȱsedimentsȱ
andȱ toȱ determineȱ whichȱ contaminantsȱ areȱ theȱ causeȱ ofȱ theȱ environmentalȱ
impact.ȱȱ
Theȱ multivariateȱ analysisȱ showsȱ thatȱ theȱ originalȱ variablesȱ canȱ beȱ
groupedȱinȱthreeȱnewȱfactorsȱthatȱexplainȱanȱ89ȱ%ȱofȱtheȱtotalȱvarianceȱ(Tableȱ3).ȱ
Theȱ firstȱ factorȱ (58.5ȱ %)ȱ linksȱ theȱ presenceȱ ofȱ Pb,ȱ Niȱ andȱ PAHsȱ boundȱ toȱ
sediment,ȱ concentrationȱ ofȱ organicȱ carbonȱ andȱ fines,ȱ withȱ allȱ theȱ parametersȱ
relatedȱwithȱtheȱbenthicȱalteration.ȱTheseȱcontaminantsȱareȱusuallyȱcomponentsȱ
ofȱ fuelȱ oilsȱ whatȱ suggestȱ thatȱ aȱ sourceȱ orȱ sourcesȱ ofȱ theseȱ compoundsȱ areȱ
producingȱ anȱ environmentalȱ impactȱ inȱ someȱ ofȱ theȱ studiedȱ areas.ȱ Thisȱ factorȱ
hasȱ mainlyȱ prevalenceȱinȱtheȱ stationȱGR3ȱfromȱtheȱBayȱofȱAlgecirasȱ(Figureȱ4)ȱ
meaningȱthatȱthereȱisȱenvironmentalȱdegradationȱinȱthisȱsiteȱdueȱtoȱtheȱinputȱofȱ
contaminantsȱ relatedȱ withȱ oilȱ spills.ȱ Theȱ stationȱ GR4ȱ presentsȱ theȱ influenceȱ ofȱ
thisȱ factorȱ (inȱ minorȱ degreeȱ thanȱ GR3),ȱ whatȱ alsoȱ meansȱ thatȱ theȱ alterationȱ ofȱ
theȱbenthicȱcommunityȱisȱdueȱtoȱtheȱspillsȱofȱoilȱthatȱoftenȱoccurȱinȱthisȱarea.ȱȱ
Bothȱ locationsȱ areȱ closeȱ toȱ aȱ petrochemicalȱ industryȱ whatȱ suggestȱ thatȱ
thereȱ isȱ aȱ chronicȱ inputȱ fromȱ thisȱ sourceȱ inȱ additionȱ toȱ accidentalȱ spillsȱ andȱ
otherȱ activitiesȱ inȱ theȱ Bayȱ ofȱ Algeciras.ȱ Theȱ secondȱ factorȱ (21.0ȱ %)ȱ correlates,ȱ
withȱnegativeȱloading,ȱtheȱmetalsȱZnȱandȱHgȱwithȱtheȱpopulationȱofȱmolluscs,ȱ
whereasȱ oppositeȱ relationshipȱ areȱ shownȱ amongȱ theseȱ contaminants,ȱ theȱ
percentageȱ ofȱ organicȱ carbonȱ inȱ theȱ sedimentȱ andȱ someȱ benthicȱ parametersȱ
suchȱasȱtheȱspecificȱrichnessȱandȱtheȱpopulationȱofȱcrustacean.ȱThisȱfactor,ȱcouldȱ
beȱ explainedȱ asȱ theȱ potentialȱ stressȱ thatȱ theseȱ contaminantsȱ mightȱ produceȱ toȱ
theȱ environmentȱ inȱ thoseȱ sitesȱ withȱ negativeȱ loadingȱ (Figureȱ 4)ȱ withȱ mainlyȱ
prevalenceȱofȱstationȱFȱandȱfollowedȱbyȱD,ȱEȱ(CormeȬLaxe)ȱandȱAȱ(CíesȱIsland).ȱ
Theȱpositiveȱloadingȱpresentedȱinȱtheȱreferenceȱsiteȱandȱrelatedȱtoȱtheȱalterationȱ
- 262 -
ofȱ theȱ populationȱ ofȱ crustaceanȱ itȱ isȱ probablyȱ relatedȱ toȱ theȱ highȱ activityȱ ofȱ
fishermenȱinȱtheȱareaȱwhoȱcollectȱdifferentȱspeciesȱofȱthisȱtaxon.ȱTheȱthirdȱfactorȱ
(9.4ȱ %)ȱ linksȱ theȱ concentrationȱ ofȱ Pbȱ andȱ Cuȱ boundȱ toȱ sedimentȱ withȱ theȱ
percentageȱ ofȱ finesȱ andȱ organicȱ carbonȱ inȱ theȱ sedimentȱ andȱ withȱ theȱ benthicȱ
alterationsȱ determinedȱ byȱ theȱ specificȱ richnessȱ andȱ theȱ disturbanceȱ ofȱ theȱ
populationsȱofȱpolychaetaȱandȱcrustacean.ȱThisȱrelationshipȱindicatesȱthatȱtheseȱ
metalsȱareȱproducingȱenvironmentalȱdegradationȱandȱcanȱbeȱconsideredȱaȱriskȱ
forȱ theȱ benthicȱ community.ȱ Factorȱ 3ȱ presentsȱ positiveȱ loadingȱ inȱ sitesȱ P1ȱ andȱ
GR4ȱfromȱtheȱBayȱofȱAlgecirasȱandȱstationȱFȱinȱCormeȬLaxe.ȱ
Tableȱ 3.ȱ Sortedȱ rotatedȱ factorȱ loadingsȱ ofȱ 15ȱ variablesȱ forȱ theȱ threeȱ
principalȱ factorsȱ resultingȱ fromȱ theȱ multivariateȱ analysisȱ ofȱ resultsȱ obtainedȱ
fromȱtheȱchemicalȱanalysisȱandȱtheȱbenthicȱalterationȱparameters.ȱ
ȱȱ
ȱȱ
Factorȱ1ȱ
58.5ȱ
Factorȱ2ȱ
21.0ȱ
Factorȱ3ȱ
9.4ȱ
Znȱ
ņȱ
Ȭ0.63ȱ
ņȱ
Pbȱ
0.86ȱ
ņȱ
0.42ȱ
Cuȱ
ņȱ
ņȱ
0.92ȱ
Niȱ
0.99ȱ
ņȱ
ņȱ
Hgȱ
ņȱ
Ȭ0.83ȱ
ņȱ
PAHȱ
0.98ȱ
ņȱ
ņȱ
O.C.ȱ
0.32ȱ
0.37ȱ
0.84ȱ
Finesȱ
0.77ȱ
ņȱ
0.44ȱ
SpeciesȱN.ȱ
0.99ȱ
ņȱ
ņȱ
Specificȱrichnessȱ
0.44ȱ
0.73ȱ
0.44ȱ
Diversityȱ
0.99ȱ
ņȱ
ņȱ
Dominanceȱ
0.99ȱ
ņȱ
ņȱ
Molluscsȱ
0.58ȱ
Ȭ0.69ȱ
ņȱ
Polychaeteȱ
Crustaceaȱ
0.87ȱ
ņȱ
0.47ȱ
0.55ȱ
0.72ȱ
0.34ȱ
- 263 -
GR3
3
Factor 1
2
1
GR4
0
A
B
CA
C
D
E
P1
F
-1
3
2
CA
GR4
Factor 2
1
B
P1
GR3
C
0
D
-1
E
A
-2
F
-3
3
P1
Factor 3
2
1
GR4
F
0
A
-1
CA
B
D
E
GR3
C
ȱ
Figureȱ4.ȱFactorȱloadingsȱforȱtheȱthreeȱprincipalȱfactorsȱresultingȱfromȱtheȱ
multivariateȱ analysisȱ ofȱ resultsȱ obtainedȱ fromȱ theȱ chemicalȱ analysisȱ andȱ theȱ
benthicȱalterationȱparameters.ȱ
- 264 -
ResultsȱobtainedȱwithȱtheȱMAAȱindicateȱthatȱfuelȱoilȱisȱnoȱmoreȱaffectingȱ
theȱbenthicȱcommunityȱofȱtheȱAINPȱandȱtheȱBayȱofȱCormeȬLaxeȱinȱtheȱGalicianȱ
Coast,ȱ whereasȱ sitesȱ evaluatedȱ inȱ theȱ Bayȱ ofȱ Algecirasȱ haveȱ shownȱ aȱ deepȱ
impactȱofȱchronicȱoilȱspillsȱwhichȱareȱtheȱcauseȱofȱtheȱimportantȱenvironmentalȱ
degradationȱofȱtheȱarea.ȱInȱaddition,ȱalthoughȱnoȱpollutionȱwasȱobservedȱinȱtheȱ
referenceȱsiteȱ(CA),ȱtheȱhighȱinfluenceȱofȱfishermenȱcouldȱbeȱconsideredȱaȱthreatȱ
forȱ someȱ taxonsȱ suchȱ asȱ crustaceans.ȱ Theȱ presenceȱ ofȱ metalsȱ boundȱ toȱ
sedimentsȱmainlyȱinȱAlgecirasȱandȱCormeȬLaxeȱandȱsomeȱsitesȱofȱtheȱAINPȱcanȱ
beȱ consideredȱ aȱ riskȱ forȱ theȱ benthicȱ communityȱ andȱ alterationȱ ofȱ theȱ
environmentȱcanȱbeȱexpected.ȱ
Figureȱ 5ȱ presentȱ theȱ resultsȱ ofȱ theȱ clusterȱ analysisȱ andȱ showsȱ theȱ
heterogeneityȱofȱtheȱareasȱofȱstudy.ȱSitesȱfromȱtheȱBaysȱofȱAlgecirasȱandȱCormeȬ
Laxeȱ appearȱ furtherȱ fromȱ theȱ referenceȱ siteȱ whereasȱ locationsȱ fromȱ theȱ AINPȱ
areȱ groupedȱ closeȱ toȱ theȱ referenceȱ station.ȱ Inȱ thisȱ sense,ȱ theȱ behaviourȱ ofȱ theȱ
CiesȱIslandsȱisȱquiteȱsimilarȱtoȱaȱreferenceȱsiteȱwhereasȱsitesȱfromȱCormeȬLaxe,ȱ
especiallyȱstationȱFȱpresentsȱanȱenvironmentalȱalterationȱlowerȱbutȱnearerȱtoȱtheȱ
degradationȱofȱtheȱBayȱofȱAlgeciras.ȱȱ
ȱ
Tree Diagram for 10 Cases
Single Linkage
Percent disagreement
1.01
ȱ
1.00
0.99
ȱ
ȱ
Linkage Distance
ȱ
0.98
0.97
0.96
0.95
0.94
ȱ
ȱ
0.93
0.92
GR3
F
GR4
P1
E
D
A
B
C
CA
Figureȱ 5.ȱ Classificationȱ treeȱ ofȱ theȱ studyȱ sitesȱ basedȱ onȱ theȱ clusterȱ
analysis.ȱTheȱstationȱCAȱcorrespondsȱtoȱtheȱreferenceȱsite.ȱA,ȱBȱandȱCȱrefersȱtoȱ
- 265 -
theȱstationsȱlocatedȱinȱtheȱCiesȱIslandȱinȱtheȱAtlanticȱIslandȱNationalȱParkȱandȱ
D,ȱEȱandȱFȱtoȱthoseȱinȱtheȱBayȱofȱCormeȬLaxe.ȱTheȱstationsȱlocatedȱinȱtheȱBayȱofȱ
AlgecirasȱareȱGR3,ȱGR4ȱandȱP1.ȱȱ
4.ȱConclusionsȱȱ
Thisȱ reportȱ showsȱ theȱ recoveryȱ ofȱ theȱ benthicȱ communityȱ fromȱ theȱ
Galicianȱ Coastȱ fourȱ yearsȱafterȱ theȱspillȱofȱtheȱtankerȱ Prestigeȱ(2002).ȱTheȱdataȱ
obtainedȱ wereȱ comparedȱ withȱ thoseȱ fromȱ anȱ areaȱ chronicallyȱ affectedȱ byȱ oilȱ
spills,ȱtheȱBayȱofȱAlgecirasȱandȱanȱareaȱnotȱcontaminatedȱinȱtheȱBayȱofȱCadiz.ȱAȱ
multivariateȱ analysisȱ wasȱ performedȱ toȱ determineȱ theȱ causeȱ ofȱ theȱ benthicȱ
alterations.ȱ Resultsȱ obtainedȱ showȱ aȱ highȱ environmentalȱ degradationȱ inȱ
sedimentsȱ fromȱ theȱ Bayȱ ofȱ Algecirasȱ whichȱ suffersȱ theȱ inputȱ ofȱ differentȱ
contaminantsȱ butȱ mainlyȱ fuelȱ oil.ȱ Biologicalȱ stressȱ wasȱ alsoȱ observedȱ dueȱ toȱ
sourcesȱofȱmetalȱcontaminationȱinȱallȱtheȱstudiedȱareasȱbutȱmainlyȱinȱAlgecirasȱ
andȱtheȱBayȱofȱCormeȬLaxe.ȱȱ
Theȱ presentȱ studyȱ showsȱ theȱ importanceȱ ofȱ theȱ combinationȱ ofȱ
physicochemicalȱ andȱ biologicalȱ dataȱ toȱ estimateȱ theȱ healthȱ statusȱ ofȱ theȱ
sediments.ȱBesides,ȱtheȱresultsȱachievedȱsuggestȱthatȱbenthicȱcommunityȱisȱableȱ
toȱrecuperateȱaȱfewȱyearsȱafterȱaȱmajorȱoilȱspillȱwhereasȱsedimentsȱaffectedȱbyȱ
lowȱalbeitȱcontinuousȱinputsȱpresentȱaȱchronicȱenvironmentalȱdegradation.ȱȱȱȱȱȱȱȱ
5.ȱAknowledgmentsȱ
Theȱ workȱ describedȱ wasȱ partlyȱ supportedȱ byȱ theȱ projectsȱ VEM2003Ȭ
20563/INTER,ȱ andȱ CTM2005Ȭ07282ȬC03Ȭ01/TECNOȱ financedȱ byȱ theȱ Spanishȱ
Educationȱ andȱ Scienceȱ Ministryȱ andȱ byȱ CISȱ fundedȱ byȱ theȱ Ministryȱ ofȱ
Environment.ȱ Carmenȱ MoralesȬCasellesȱ thanksȱ theȱ Ministryȱ ofȱ Educationȱ andȱ
Scienceȱ forȱ fundingȱ herȱ researchȱ fellowshipȱ (FPU).ȱ Dr.ȱ Ribaȱ thanksȱ theȱ I3pȱ
programȱ forȱ supportingȱ herȱ participationȱ inȱ thisȱ research.ȱ Cesar,ȱ A.ȱ thanksȱ
- 266 -
CAPES/MECȬBrazilȱ(BEXȬ3238/06Ȭ7)ȱforȱtheȱpostdoctoralȱscholarship.ȱTheȱworkȱ
wasȱ partiallyȱ fundedȱ byȱ theȱ Brazilian–Spanishȱ jointȱ projectȱ (CAPESȬBrazilȱ
#099/06ȱ andȱ MECȬSpainȱ PHBȱ 2005Ȭ0100ȬPC).ȱ Specialȱ thanksȱ areȱ givenȱ toȱ theȱ
membersȱofȱtheȱCIS.ȱȱ
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- 272 -
ȱ
Capítuloȱ5.ȱ
Aplicaciónȱdeȱunȱmétodoȱintegradoȱparaȱlaȱ
caracterizaciónȱdeȱsedimentosȱafectadosȱporȱ
vertidosȱdeȱpetróleoȱ
Enȱ laȱ actualidadȱ sabemosȱ queȱ paraȱ estudiarȱ laȱ calidadȱ ambientalȱ deȱ losȱ
sedimentosȱnoȱbastaȱconȱdeterminarȱlosȱnivelesȱdeȱcontaminantesȱpresentesȱenȱ
laȱ matriz,ȱ existenȱ aspectos,ȱ talesȱ comoȱ laȱ biodisponibilidadȱ deȱ losȱ
contaminantesȱ unaȱ vezȱ incorporadosȱ alȱ sedimento,ȱ laȱ acciónȱ concomitanteȱ deȱ
lasȱcondicionesȱfisicoquímicasȱdelȱmedioȱyȱlaȱposibilidadȱdeȱefectosȱsinérgicosȱoȱ
antagónicosȱ conȱ otrosȱ contaminantes,ȱ queȱ hacenȱ queȱ estasȱ medidasȱ distenȱ
muchoȱdeȱserȱunaȱvaloraciónȱobjetivaȱdeȱlaȱȈsaludȱambientalȈȱdeȱesosȱsistemasȱ
(DelValls,ȱ 2006).ȱ Asíȱ puesȱ elȱ riesgoȱ potencialȱ deȱ unaȱ sustanciaȱ químicaȱ
dependeráȱdeȱ(Chapman,ȱMasterȱlessons):ȱȱ
Bioaccessibilidad:ȱ laȱ fracciónȱ oȱ matrizȱ queȱ potencialmenteȱ puedeȱ
resultarȱȱdisponibleȱparaȱelȱorganismo.ȱ
Biodisponibilidad:ȱ laȱ sustanciaȱ queȱ inmediatamenteȱ esȱ disponibleȱ paraȱ
serȱincorporadaȱporȱlosȱorganismos.ȱ
Bioabsorción:ȱLoȱqueȱrealmenteȱesȱincorporadoȱporȱelȱorganismo.ȱ
ȱ
Ȭȱ273ȱȬȱ
Capítuloȱ5
Biorreactividad:ȱ Loȱ queȱ realmenteȱ esȱ capazȱ deȱ causarȱ toxicidadȱ (laȱ
fracciónȱbioabsorbidaȱmenosȱlaȱfracciónȱdepurada,ȱsecuestradaȱinternamenteȱoȱ
utilizadaȱporȱelȱorganismoȱparaȱcubrirȱsusȱnecesidades)ȱȱ
Seȱhanȱaplicadoȱdistintasȱmetodologíasȱparaȱevaluarȱlaȱcalidadȱambientalȱ
deȱ sedimentosȱ sinȱ limitarseȱ aȱ losȱ análisisȱ químicos.ȱ Estasȱ aproximacionesȱ
incluyenȱ medidasȱ deȱ laȱ bioacumulaciónȱ enȱ organismosȱ seleccionadosȱ comoȱ
indicadoresȱdeȱcontaminación,ȱlosȱefectosȱletalesȱy/oȱsubletalesȱobservadosȱtrasȱ
laȱ exposiciónȱ deȱ organismosȱ aȱ sedimentosȱ ‘supuestamente’ȱ contaminados,ȱ lasȱ
modificacionesȱ deȱ laȱ estructuraȱ deȱ lasȱ comunidadesȱ queȱ produceȱ laȱ
contaminación,ȱetc.ȱ(DelValls,ȱ2006).ȱSinȱembargo,ȱcadaȱunaȱdeȱestasȱpropuestasȱ
presentaȱsusȱlimitaciones.ȱȱ
Conȱelȱfinȱdeȱsuperarȱlasȱlimitacionesȱqueȱpresentanȱlasȱmetodologíasȱseȱ
propusoȱ laȱ realizaciónȱ deȱ unȱ estudioȱ integradoȱ enȱ elȱ queȱ seȱ incluyanȱ distintasȱ
tecnologíasȱ deȱ formaȱ queȱ hayaȱ unaȱ aproximaciónȱ loȱ másȱ certeraȱ aȱ laȱ realidad.ȱ
Paraȱelloȱseȱempleanȱlasȱllamadasȱlíneasȱdeȱevidenciaȱ(linesȱofȱevidence,ȱLOEs)ȱ
queȱincluyenȱcadaȱunaȱdeȱlasȱmetodologíasȱaplicadasȱdeȱformaȱindividualȱparaȱ
posteriormenteȱ realizarȱ unaȱ integraciónȱ globalȱ deȱ losȱ resultadosȱ dentroȱ delȱ
llamadoȱ “Weightȱ ofȱ Evidenceȱ (WOE)ȱ approach”ȱ oȱ “pesoȱ deȱ laȱ evidencia”,ȱ
contrarrestandoȱ asíȱ laȱ subjetividadȱ yȱ laȱ arbitrariedad.ȱ Lasȱ conclusionesȱ
proporcionadasȱporȱcadaȱmedidaȱindividual,ȱdentroȱdelȱmétodoȱintegrado,ȱsonȱ
consideradasȱenȱrelaciónȱconȱlasȱqueȱofrecenȱlosȱotrosȱcomponentesȱdelȱmétodo.ȱ
Deȱ estaȱ manera,ȱ seȱ evalúaȱ laȱ correlaciónȱ entreȱ losȱ resultadosȱ obtenidosȱ porȱ
técnicasȱ distintasȱ yȱ seȱ disponeȱ deȱ unosȱ resultadosȱ másȱ cercanosȱ aȱ laȱ realidadȱ
queȱcuandoȱseȱaplicanȱesasȱmismasȱtécnicasȱdeȱformaȱindividualizadaȱ(DelValls,ȱ
2006).ȱEsteȱtipoȱdeȱmetodologíaȱseȱhaȱaplicadoȱsatisfactoriamenteȱtrasȱepisodiosȱ
deȱ vertidosȱ contaminantesȱ (Ej.ȱ DelVallsȱ andȱ Chapman,ȱ 1998;ȱ Chapman,ȱ 2000;ȱ
Borgmannȱetȱal.,ȱ2001;ȱRibaȱetȱal.,ȱ2004;ȱLeeȱetȱal.,ȱ2006).ȱȱ
- 274 -
Métodosȱintegradosȱdeȱlaȱcalidadȱambientalȱȱ
Unaȱdeȱlasȱprincipalesȱventajasȱdeȱlaȱutilizaciónȱdeȱunȱmétodoȱintegradoȱ
seȱ encuentraȱ enȱ laȱ posibilidadȱ deȱ caracterizarȱ lasȱ llamadasȱ zonasȱ grisesȱ (Ȉgrayȱ
zoneȈ).ȱ Enȱ tornoȱ alȱ 70%ȱ deȱ lasȱ áreasȱ litoralesȱ yȱ deȱ estuarioȱ puedenȱ incluirseȱ
dentroȱdeȱestosȱecosistemas,ȱqueȱposeenȱunȱgradoȱdeȱpoluciónȱintermedioȱentreȱ
lasȱ zonasȱ claramenteȱ alteradasȱ yȱ lasȱ queȱ puedenȱ serȱ consideradasȱ comoȱ noȱ
estresadasȱ(DelValls,ȱ2006).ȱȱ
ȱ
1)ȱScreeningȱ
ȬȱAnálisisȱquímicos
Gradienteȱdeȱ
contaminación
ȬȱToxicidadȱagudaȱ
ȱ
2)ȱMétodoȱintegrado:ȱ
ȱ
Nivelesȱdeȱ
contaminantesȱenȱelȱ
sedimento
Contaminación
ȱ
Nivelesȱdeȱefectoȱ
biológicoȱmedidoȱ
ȱ
enȱlosȱorganismosȱ
autócotonos
Nivelesȱdeȱefectoȱ
Alteracionȱ
bentónicaȱ
WOEȱ
Toxicidadȱenȱ biológicoȱdeȱtipoȱagudoȱ
laboratorioȱ yȱcrónicoȱmedidosȱbajoȱ
condicionesȱcontroladasȱ
deȱlaboratorioȱ
ȱ
ȱ
ȱ
Efectosȱ
subletalesȱInȱsitu
Nivelesȱdeȱefectoȱbiológicoȱmedidoȱbajoȱ
condicionesȱdeȱcampo
ȱ
Figuraȱ 5.1.ȱ Representaciónȱ esquemáticaȱ delȱ desarrolloȱ delȱ modeloȱ
integradoȱ aplicadoȱ paraȱ elȱ seguimientoȱ delȱ impactoȱ delȱ vertidoȱ delȱ petroleroȱ
‘Prestige’ȱ yȱ suȱ comparaciónȱ conȱ laȱ calidadȱ ambientalȱ enȱ zonasȱ afectadasȱ porȱ
vertidosȱ deȱ petróleoȱ deȱ tipoȱ crónicoȱ (continuos)ȱ yȱ conȱ ausenciaȱ deȱ influenciaȱ
porȱ esteȱ tipoȱ deȱ vertidosȱ (Bahíaȱ deȱ Cádiz).ȱ Enȱ unaȱ primeraȱ faseȱ deȱ aplicaciónȱ
delȱ modeloȱseȱdesarrollaȱ laȱfaseȱ inicialȱdeȱ‘screening’ȱconȱlaȱaplicaciónȱdeȱsóloȱ
dosȱLOEsȱsobreȱunȱnúmeroȱextensoȱdeȱestaciones.ȱElȱresultadoȱdeȱlaȱaplicaciónȱ
deȱ estaȱ primeraȱ faseȱ vaȱ aȱ permitirȱ laȱ selecciónȱ deȱ unȱ númeroȱ menorȱ deȱ
estacionesȱ sobreȱ lasȱ queȱ seȱ vaȱ aȱ desarrollarȱ elȱ modeloȱ integradoȱ deȱ formaȱ
completa.ȱ Éstaȱ incluyeȱ cuatroȱ líneasȱ deȱ evidencia:ȱ contaminación,ȱ toxicidad,ȱ
alteraciónȱ‘inȱsitu’ȱyȱbioacumulaciónȱyȱbiomagnificación.ȱ
Ȭ 275ȱȬ
Capítuloȱ5
Enȱ esteȱ capítuloȱ seȱ presentanȱ tresȱ trabajosȱ realizadosȱ consecutivamente.ȱ
Enȱ elȱ trabajoȱ XIV,ȱ seȱ llevaȱ aȱ caboȱ unȱ estudioȱ integradoȱ deȱ laȱ calidadȱ deȱ losȱ
sedimentosȱ delȱ Parqueȱ Nacionalȱ deȱ lasȱ Islasȱ Atlánticasȱ yȱ laȱ Bahíaȱ deȱ CormeȬ
Laxeȱ aȱ loȱ largoȱ deȱ tresȱ años.ȱ Paraȱ realizarȱ esteȱ estudioȱ seȱ empleóȱ unaȱ
metodologíaȱclásicaȱbasadaȱenȱ3ȱlíneasȱdeȱevidencia:ȱa)ȱAnálisisȱquímicosȱdeȱlosȱ
sedimentos,ȱb)ȱToxicidadȱagudaȱbajoȱcondicionesȱdeȱlaboratorio,ȱyȱc)ȱalteraciónȱ
inȱ situȱ deȱ laȱ comunidadȱ bentónica.ȱ Esteȱ estudioȱ poneȱ deȱ manifiestoȱ laȱ
recuperaciónȱ deȱ laȱ “saludȱ ambiental”ȱ cuatroȱ añosȱ despuésȱ delȱ vertidoȱ delȱ
Prestige,ȱ principalmenteȱ enȱ lasȱ islasȱ Cíes,ȱ aunqueȱ detectaȱ ciertaȱ contaminaciónȱ
metálicaȱ potencialmenteȱ peligrosaȱ sobretodoȱ enȱ laȱ Bahíaȱ deȱ CormeȬLaxe.ȱ
Además,ȱlaȱ aplicaciónȱ deȱunȱ análisisȱdeȱlaȱ varianzaȱaȱ losȱresultadosȱobtenidosȱ
enȱlaȱintegraciónȱdeȱlosȱdatosȱpermitióȱestablecerȱdiferenciasȱsignificativasȱentreȱ
lasȱestacionesȱdeȱestudio.ȱ
Unaȱ vezȱ realizadoȱ elȱ estudioȱ presentadoȱ enȱ elȱ trabajoȱ XIVȱ seȱ decidióȱȱ
ampliarȱ elȱ númeroȱ deȱ líneasȱ deȱ evidencia,ȱ manteniendoȱ lasȱ anterioresȱ eȱ
incorporandoȱlosȱresultadosȱobtenidosȱenȱlosȱexperimentosȱsubletales,ȱtantoȱenȱ
campoȱ comoȱ enȱ laboratorio.ȱ Deȱ estaȱ formaȱ elȱ trabajoȱ XVȱ muestraȱ estaȱ
integraciónȱdondeȱseȱhaceȱhincapiéȱenȱlaȱimportanciaȱdelȱusoȱdeȱbiomarcadoresȱ
paraȱevaluarȱcalidadȱambientalȱdeȱlosȱsedimentosȱyȱlosȱriesgosȱpotenciales.ȱ
Elȱúltimoȱtrabajoȱdelȱcapítulo,ȱelȱXVI,ȱempleaȱlaȱinformaciónȱobtenidaȱaȱ
loȱ largoȱ deȱ laȱ tesisȱ doctoralȱ yȱ presentadaȱ enȱ losȱ anterioresȱ capítulosȱ paraȱ darȱ
respuestaȱalȱplanteamientoȱinicialȱdelȱestudio,ȱcompararȱlaȱcalidadȱambientalȱdeȱ
losȱ sedimentosȱ deȱ dosȱ áreasȱ afectadasȱ porȱ vertidosȱ deȱ petróleo.ȱ Paraȱ elloȱ seȱ
llevaȱ aȱ caboȱ unaȱ integraciónȱ completaȱ conȱ diversasȱ líneasȱ deȱ evidenciaȱ paraȱ
clarificarȱ elȱ estadoȱ deȱ losȱ sedimentosȱ deȱ laȱ costaȱ gallegaȱ yȱ deȱ laȱ Bahíaȱ deȱ
Algeciras.ȱȱȱ
- 276 -
Métodosȱintegradosȱdeȱlaȱcalidadȱambientalȱȱ
Bibliografíaȱ
Borgmann,ȱ
U.;ȱ
Norwood,ȱ
W.P.;ȱ
Reynoldson,ȱ T.B.;ȱ Rosa,ȱ F.ȱ 2001.ȱ
Identifyingȱ
causeȱ
inȱ
sedimentȱ
assessments:ȱ bioavailabilityȱ andȱ theȱ
Sedimentȱ Qualityȱ Triad.ȱ Can.ȱ J.ȱ Fish.ȱ
Aquat.ȱSci.ȱ58:ȱ950Ȭ969ȱ
Lee,ȱ M.R.;ȱ Correa,ȱ J.A.;ȱ Seed,ȱ R.ȱ 2006.ȱ Aȱ
sedimentȱ qualityȱ triadȱ assessmentȱ ofȱ theȱ
impactȱ ofȱ copperȱ mineȱ tailingsȱ disposalȱ
onȱ theȱ littoralȱ sedimentaryȱ environmentȱ
inȱ theȱ Atacamaȱ regionȱ ofȱ northernȱ Chile.ȱ
Mar.ȱPollut.ȱBull.ȱ52:ȱ1389Ȭ1395ȱ
Chapman,ȱP.M.ȱ2000.ȱTheȱSedimentȱQualityȱ
Triad:ȱ then,ȱ nowȱ andȱ tomorrow.ȱ Int.ȱ J.ȱ
Environ.ȱPollut.13:ȱ351Ȭ356ȱ
Riba,ȱ I.;ȱ Forja,ȱ J.M.;ȱ GómezȬParra,ȱ A.;ȱ
DelValls,ȱ T.A.ȱ 2004.ȱ Sedimentȱ qualityȱ inȱ
littoralȱ regionsȱ ofȱ theȱ Gulfȱ ofȱ Cádiz:ȱ aȱ
triadȱapproachȱtoȱaddressȱtheȱinfluenceȱofȱ
miningȱ activities.ȱ Environ.ȱ Pollut.ȱ 132:ȱ
341Ȭ353ȱ
DelValls,ȱT.A.;ȱChapman.ȱ1998.ȱSiteȬspecificȱ
sedimentȱ qualityȱ valuesȱ forȱ theȱ golfȱ ofȱ
Cádizȱ (Spain)ȱ andȱ Sanȱ Franciscoȱ Bayȱ
(USA),ȱ usingȱ theȱ sedimentȱ qualityȱ triadȱ
andȱ multivariateȱ análisis.ȱ Cienc.ȱ Mar.ȱ 24:ȱ
313Ȭ336ȱ
DelValls,ȱT.A.ȱ2006.ȱDiseñoȱyȱaplicaciónȱdeȱ
modelosȱ integradosȱ deȱ evaluaciónȱ deȱ laȱ
contaminaciónȱ yȱ susȱ efectosȱ sobreȱ losȱ
sistemasȱ marinosȱ yȱ litoralesȱ yȱ laȱ saludȱ
humana.ȱ Premioȱ CEPRECO.ȱ Ministerioȱ
deȱlaȱPresidencia.ȱEnȱprensa.ȱ
ȱ
Ȭ 277ȱȬ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
Ȭȱ278ȱȬȱ
UsingȱaȱclassicalȱWeightȬofȬEvidenceȱapproachȱforȱ4Ȭyears’ȱ
monitoringȱofȱtheȱimpactȱofȱanȱaccidentalȱoilȱspillȱonȱSedimentȱ
Qualityȱ
CarmenȱMoralesȬCaselles1,3,*,ȱInmaculadaȱRiba1,3,ȱCarmenȱSarasquete1,3,ȱT.ȱÁngelȱ
DelValls2,3ȱ
1
InstitutoȱdeȱCienciasȱMarinasȱdeȱAndalucíaȱ(ICMANȬCSIC),ȱAvda.ȱRepúblicaȱ
Saharauiȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱSpainȱ
2
UNESCOȱUNITWIN/WiCop,ȱFacultadȱdeȱCienciasȱdelȱMarȱyȱAmbientales,ȱ
UniversidadȱdeȱCádiz,ȱPolígonoȱRíoȱSanȱPedroȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱSpain.ȱ
3
ȱUnidadȱAsociadaȱdeȱCalidadȱAmbientalȱyȱPatologíaȱ(CSICȱ&ȱUCA),ȱAvda.ȱRepúblicaȱ
Saharauiȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱSpainȱ
Abstractȱ
ȱInȱtheȱpresentȱreport,ȱtheȱsuccessfulȱapplicationȱofȱaȱWeightȱofȱevidenceȱ
approachȱ (WOE)ȱ toȱ sedimentȱ qualityȱ assessmentȱ duringȱ aȱ fourȱ yearȱ impactȱ
periodȱ followingȱ anȱ oilȱ spillȱ isȱ discussed.ȱ Theȱ studyȱ assessesȱ theȱ sedimentȱ
qualityȱonȱtheȱGalicianȱCoastȱ(NWȱSpain)ȱwhichȱwasȱimpactedȱbyȱanȱaccidentalȱ
spillȱassociatedȱwithȱtheȱsinkingȱofȱtheȱtankerȱPrestigeȱ(2002).ȱTheȱassessmentȱisȱ
basedȱ onȱ threeȱ linesȱ ofȱ evidence:ȱ physicochemicalȱ characterizationȱ ofȱ theȱ
sediments;ȱdeterminationȱofȱacuteȱtoxicityȱbyȱconductingȱsedimentȱtoxicityȱtestsȱ
andȱ benthicȱ alterationȱ includingȱ taxonomicȱ identificationsȱ alongȱ withȱ
communityȱ descriptiveȱ statistics.ȱ Theȱ dataȱ obtainedȱ wereȱ integratedȱ usingȱ aȱ
WOEȱapproachȱbyȱmeansȱofȱtwoȱdifferentȱmethodologies:ȱȱmultivariateȱanalysisȱ
andȱ ANOVAȬbasedȱ pieȱ charts.ȱ Resultsȱ confirmȱ thatȱ PAHsȱ relatedȱ toȱ theȱ
Prestigeȱoilȱspillȱareȱtheȱmainȱcontaminantȱassociatedȱwithȱbiologicalȱeffectsȱinȱ
theȱareaȱwhichȱhasȱsinceȱrecoveredȱfromȱtheȱinitialȱacuteȱimpact.ȱAlso,ȱtheȱWOEȱ
allowedȱ theȱ identificationȱ ofȱ metalȱ contaminationȱ notȱ previouslyȱ describedȱ inȱ
theȱ areaȱ responsibleȱ forȱ toxicityȱ inȱ sedimentsȱ analyzed.ȱ Inȱ addition,ȱ theȱ
ȱEnvironmentȱInternationalȱ(aceptado)
- 279 -
methodologyȱproposedȱtoȱlinkȱtheȱ3ȱlinesȱofȱevidenceȱresultsȱshowsȱtheȱuseȱforȱ
theȱ firstȱ timeȱ ofȱ anȱ objectiveȱ indiceȱ basedȱ onȱ factorȱ analysisȱ whichȱ allowsȱ
pollutionȱ ofȱ theȱ sedimentsȱ studiedȱ toȱ beȱ qualitativelyȱ andȱ quantitativelyȱ
evaluatedȱ whileȱ demonstratingȱ theȱ WOEȱ approachȱ toȱ beȱ recommendableȱ inȱ
monitoringȱenvironmentalȱquality.ȱȱ
Keywords:ȱPAHs,ȱcontamination,ȱtoxicity,ȱWOE,ȱSedimentȱQualityȱTriadȱȱ
1.ȱIntroductionȱ
Chemicalȱ measurementsȱ inȱ theȱ environmentȱ provideȱ informationȱ onȱ
contaminationȱ (substancesȱ presentȱ whereȱ theyȱ wouldȱ notȱ normallyȱ occur,ȱ orȱ
aboveȱnaturalȱbackgroundȱconcentrations),ȱbutȱtheyȱdoȱnotȱprovideȱinformationȱ
onȱ pollutionȱ (contaminationȱ thatȱ causesȱ adverseȱ biologicalȱ effectsȱ inȱ theȱ
environment)ȱ (Chapman,ȱ 2007).ȱ Chemicalȱ analysesȱ areȱ anȱ importantȱ toolȱ inȱ
sedimentȱqualityȱassessment,ȱhoweverȱtheȱinformationȱobtainedȱdoesȱnotȱreportȱ
onȱ theȱ consequencesȱ thatȱ chemicalsȱ haveȱ onȱ theȱ organismsȱ exposedȱ toȱ them.ȱ
Biologicalȱ effectsȱ establishedȱ basedȱ onȱ laboratoryȱ testsȱ toȱ determineȱ toxicȱ
responsesȱ inȱ combinationȱ withȱ fieldȱ dataȱ onȱ theȱ communitiesȱ livingȱ inȱ theȱ
sedimentsȱ allowȱ itȱ toȱ beȱ establishedȱ whetherȱ thereȱ isȱ observableȱ pollutionȬ
inducedȱdegradationȱeffectȱaȱgivenȱsetȱofȱbiotaȱ(Chapmanȱetȱal.,ȱ1991).ȱȱ
Weightȱ ofȱ evidenceȱ (WOE)ȱ investigationsȱ determineȱ possibleȱ ecologicalȱ
impactsȱ owingȱ toȱ chemicalsȱ orȱ otherȱ stressorsȱ basedȱ onȱ multipleȱ linesȱ ofȱ
evidenceȱ(Chapman,ȱ2007).ȱTheȱclassicalȱSedimentȱQualityȱTriadȱ(SQT)ȱconsistsȱ
ofȱ sedimentȱ chemicalȱ analysis,ȱ examinationȱ ofȱ theȱ inȱ situȱ benthicȱ community,ȱ
andȱ measurementsȱ ofȱ sedimentȱ toxicityȱ (Borgmannȱ etȱ al.,ȱ 2001).ȱ Theȱ overallȱ
studyȱofȱtheseȱthreeȱcomponentsȱprovidesȱanȱassessmentȱofȱtheȱenvironmentalȱ
risk.ȱ Theȱ SQTȱ approach,ȱ acceptedȱ internationallyȱ asȱ theȱ mostȱ comprehensiveȱ
approachȱ availableȱ forȱ assessingȱ contaminatedȱ sedimentsȱ (Chapmanȱ andȱ
McDonald,ȱ 2005),ȱ formsȱ partȱ ofȱ theȱ WOEȱ frameworkȱandȱ isȱ expectedȱ toȱ beȱ anȱ
- 280 -
integralȱ componentȱ ofȱ largerȬscaleȱ assessmentsȱ (Chapmanȱ andȱ Hollert,ȱ 2006).ȱ
Thisȱ methodȱ hasȱ beenȱ successfullyȱ usedȱ toȱ assessȱ sedimentȱ qualityȱ followingȱ
contaminantȱ spillȱ episodesȱ (DelVallsȱ andȱ Chapman,ȱ 1998;ȱ Chapman,ȱ 2000;ȱ
Borgmannȱetȱal.,ȱ2001;ȱRibaȱetȱal.,ȱ2004;ȱLeeȱetȱal.,ȱ2006).ȱInȱtheȱpresentȱstudyȱthisȱ
methodologyȱwasȱperformedȱinȱorderȱtoȱdetermineȱwhetherȱtheȱWOEȱapproachȱ
isȱ ableȱ toȱ beȱ usedȱ asȱ aȱ goodȱ toolȱ inȱ assessingȱ sedimentȱ qualityȱ followingȱ theȱ
acuteȱ impactȱ ofȱ anȱ accidentalȱ oilȱ spill.ȱ Inȱ additionȱ theȱ suitabilityȱ ofȱ theȱ
applicationȱofȱtheȱWOEȱprocedureȱasȱaȱmonitoringȱinstrumentȱinȱenvironmentalȱ
riskȱassessmentȱisȱdemonstrated.ȱȱ
Theȱ broadȱ aimȱ ofȱ thisȱ studyȱ itȱ isȱ toȱ determineȱ theȱ qualityȱ ofȱ oilȱ spillȱ
affectedȱsedimentsȱbyȱapplyingȱaȱcompleteȱmethodology,ȱandȱmoreȱspecificallyȱ
toȱaddressȱtheȱfollowingȱ3ȱobjectives:ȱ(a)ȱtoȱmonitorȱtheȱimpactȱofȱanȱaccidentalȱ
oilȱ spillȱ usingȱ aȱ WOEȱ approachȱ duringȱ aȱ 4ȱ yearȱ period,ȱ (b)ȱ toȱ improveȱ
methodologicalȱaspectsȱinȱtheȱintegrationȱofȱtheȱ3ȬLOEȱresultsȱinȱorderȱtoȱavoidȱ
subjectivityȱ therebyȱ definingȱ aȱ newȱ andȱ moreȱ objectiveȱ processȱ ofȱ integration,ȱ
andȱ(c)ȱtheȱdeterminationȱofȱpollution,ȱcontaminationȱandȱnoȱimpactȱscenariosȱ
forȱtheȱstationsȱselectedȱandȱoverȱtheȱ4ȱyearȱperiod,ȱincludingȱtheȱidentificationȱ
ofȱtheȱcontaminantsȱresponsible.ȱȱ
2.ȱMaterialȱandȱmethodsȱ
2.1.ȱApproachȱ
Theȱ caseȱ studyȱ employedȱ forȱ theȱ improvementȱ ofȱ theȱ WOEȱ approach,ȱ
wasȱ thatȱ ofȱ theȱ impactȱ associatedȱ withȱ theȱ sinkingȱ ofȱ theȱ tankerȱ Prestigeȱ
(Novemberȱ2002)ȱwhichȱspiltȱaroundȱ60,000ȱtonsȱofȱheavyȱfuelȬoilȱwithȱtheȱmostȱ
affectedȱareaȱbeingȱtheȱGalicianȱCoastȱ(NWȱSpain).ȱAȱfirstȱstudyȱwasȱcarriedȱoutȱ
withȱ sedimentȱ samplesȱ collectedȱ inȱ theȱ Atlanticȱ Islandsȱ Nationalȱ Parkȱ (AINP)ȱ
- 281 -
duringȱ2004,ȱapproximatelyȱoneȱyearȱafterȱtheȱspill.ȱFigureȱ1ȱshowsȱtheȱareaȱofȱ
studyȱ andȱ theȱ 10ȱ stationsȱ selectedȱ forȱ theȱ firstȱ surveyȱdescribedȱ inȱ theȱ presentȱ
paper,ȱ3ȱstationsȱlocatedȱinȱtheȱOnsȱIslandȱ(D07,ȱD09ȱandȱD18)ȱandȱ7ȱinȱtheȱCiesȱ
Islandȱ(D60,ȱD66,ȱD69,ȱD79,ȱFIG,ȱGA1,ȱGA2).ȱ
ȱ
ȱ
Ons
•D18
•D07
•D09
ȱ
ȱ
Ría de
Pontevedra
ƒE
ƒF
ƒD
ȱ
Ría de
Corme-Laxe
ȱ
ȱ
Cíes
ȱ
N
ȱ
•C •A
•GA2/B
•FIG
•D66 •D79
•GA1 •D60
•D69
Ría de
Vigo
ȱ
ȱ
ȱ
ȱ
Galician
Coast
Spain
Atlantic Ocean
Mediterranean
Sea
ȱ
ȱ
Figureȱ 1.ȱ Mapȱ ofȱ theȱ coastalȱ areaȱ ofȱ Galiciaȱ (NWȱ Spain)ȱ showingȱ theȱ
samplingȱ sitesȱ inȱ theȱ areaȱ ofȱ CormeȬLaxeȱ (D,ȱ Eȱ andȱ F)ȱ andȱ theȱ Atlanticȱ Islandȱ
NationalȱParkȱ(Ons:ȱD07,ȱD09ȱandȱD18;ȱCíes:ȱGA1,ȱGA2/B,ȱFIG,ȱD60,ȱD66,ȱD69,ȱ
AȱandȱC).ȱȱ
- 282 -
Subsequentlyȱsurveysȱwereȱdesignedȱforȱmonitoringȱsedimentȱqualityȱatȱ
theȱ distinctȱ stationsȱ locatedȱ inȱ theȱ parkȱ andȱ theȱ surroundingȱ areaȱ withinȱ theȱ
fourȱyearȱperiod.ȱ4ȱstationsȱwereȱselectedȱinȱtheȱCiesȱIslandȱ(A,ȱB,ȱCȱandȱGA1),ȱ
locatedȱinȱtheȱAINP,ȱforȱtheȱperiodȱfromȱtheȱbeginningȱofȱ2004ȱthroughȱtoȱ2006ȱ
(3ȱ surveys).ȱ Theȱ resultsȱ obtainedȱ forȱ theȱ firstȱ studyȱ carriedȱ outȱ inȱ theȱ AINPȱ
(beginningȱ ofȱ 2004)ȱ suggestedȱ thatȱ theȱ areaȱ andȱ itsȱ surroundsȱ wereȱ probablyȱ
significantlyȱ affectedȱ byȱ theȱ spill.ȱ Thisȱ necessitatedȱ theȱ selectionȱ ofȱ aȱ
replacementȱ areaȱ onȱ theȱ Galicianȱ Coastȱ forȱ inclusionȱ inȱ theȱ sedimentȱ qualityȱ
monitoringȱ study.ȱ 3ȱ stationsȱ (D,ȱ E,ȱ F)ȱ inȱ CormeȬLaxeȱ (Figureȱ 1)ȱ wereȱ selectedȱ
andȱtheȱsameȱWOEȱapproachȱappliedȱoverȱ2ȱyears,ȱfromȱ2004/2005Ȭ2005/2006ȱ(2ȱ
surveys).ȱ
ȱ
Theȱ weightȬofȬevidenceȱ approachȱ (WOE)ȱ conductedȱ inȱ thisȱ studyȱ
includesȱ threeȱ linesȱ ofȱ evidenceȱ (LOEs)ȱ incorporatingȱ theȱ followingȱ samplingȱ
stationȱanalysesȱcarriedȱoutȱforȱeachȱofȱtheȱ3ȱdistinctȱsurveysȱdescribedȱabove:ȱ
(a)ȱsedimentȱcontamination:ȱphysicochemicalȱcharacterizationȱofȱtheȱsedimentsȱ
byȱ
analyzingȱ
PAHsȱ
benzo(a)anthracene,ȱ
perylene,ȱ
(acenaphtalene,ȱ
benzo(a)pyrene,ȱ
benzo(k)fluoranthene,ȱ
acenaphtylene,ȱ
anthracene,ȱ
benzo(b)fluoranthene,ȱ
benzo(g,h,i)ȱ
chrysene,ȱ
dibenzo(a,h)ȱ
anthracene,ȱ
fenanthrene,ȱfluoranthene,ȱfluorene,ȱindeneȱ(1,2,3,cdȱ)pyrene,ȱnaphthalene,ȱandȱ
pyrene),ȱtraceȱmetalsȱ(Zn,ȱPb,ȱCu,ȱNi,ȱCoȱandȱV),ȱgrainȱsizeȱandȱorganicȱcarbonȱ
(methodologiesȱdescribedȱinȱMoralesȬCasellesȱetȱal.,ȱ2006);ȱ(b)ȱsedimentȱtoxicity:ȱ
byȱ determinationȱ ofȱ acuteȱ toxicity,ȱ performingȱ bioassaysȱ withȱ bulkȱ sedimentȱ
suchȱasȱtheȱamphipodȱmortalityȱtestȱwithȱCorophiumȱvolutatorȱ(MoralesȬCasellesȱ
etȱ al.,ȱ 2007)ȱ andȱ theȱ polychaetaȱ mortalityȱ assayȱ (CasadoȬMartínezȱ etȱ al.,ȱ inȱ
press)ȱwithȱArenicolaȱmarinaȱasȱwellȱasȱtwoȱtestsȱusingȱsedimentȱelutriate,ȱtheseȱ
beingȱ theȱ commercialȱ assayȱ Microtox®ȱ (MoralesȬCasellesȱ etȱ al.,ȱ 2007)ȱ andȱ
- 283 -
embryo–larvalȱseaȱurchinȱbioassayȱ(methodologyȱdescribedȱbyȱFernándezȱetȱal,ȱ
2006;ȱdataȱobtainedȱfromȱFernándezȱetȱal.ȱnotȱpublished);ȱ(c)ȱ‘inȱsituȱalteration’:ȱ
Benthicȱ alterationȱ wasȱ selectedȱ andȱ determinedȱ byȱ measuringȱ parametersȱ inȱ
situȱ basedȱ onȱ taxonomicȱ identificationsȱ andȱ communityȱ descriptiveȱ statisticsȱ
(abundanceȬbiomassȱ analysis,ȱ speciesȱ richness,ȱ diversity,ȱ dominanceȱ andȱ
proportionsȱofȱtheȱmajorȱtaxonomicȱgroups)ȱ(DelVallsȱandȱChapman,ȱ1998).ȱ
2.2.ȱDataȱintegrationȱ
Theȱ integrationȱ ofȱ allȱ LOEȱ dataȱ obtainedȱ wasȱ performedȱ viaȱ theȱ twoȱ
followingȱmethodologies:ȱ(a)ȱaȱmultivariateȱanalysisȱapproachȱbasedȱonȱlinkingȱ
allȱ variablesȱ obtainedȱ inȱ determiningȱ theȱ environmentalȱ degradationȱ ofȱ theȱ
studiedȱecosystemsȱ(Ribaȱetȱal.,ȱ2004)ȱandȱ(b)ȱaȱrepresentationȱusingȱpieȱchartsȱ
usingȱ anȱ ANOVAȱ approachȱ andȱ byȱ meansȱ ofȱ theȱ determinationȱ ofȱ differentȱ
factorsȱ(Ribaȱetȱal.,ȱ2004).ȱȱ
Theȱ multivariateȱ analysisȱ wasȱ performedȱ usingȱ principalȱ componentsȱ
analysisȱ (PCA)ȱasȱtheȱextractionȱ procedure,ȱaȱmultivariateȱstatisticalȱtechniqueȱ
forȱexaminingȱvariableȱdistributionsȱ(Ribaȱetȱal.,ȱ2003).ȱTheȱobjectiveȱofȱPCAȱisȱ
toȱ deriveȱ aȱ reducedȱ numberȱ ofȱ newȱ variablesȱ asȱ linearȱ combinationsȱ ofȱ theȱ
originalȱvariables.ȱThisȱprovidesȱaȱdescriptionȱofȱtheȱstructureȱofȱtheȱdataȱwithȱ
minimumȱlossȱofȱinformation.ȱȱȱ
Forȱ theȱ representationȱ ofȱ theȱ pieȱ charts,ȱ theȱ factorsȱ obtainedȱ fromȱ theȱ
PCAȱ wereȱ subjectedȱ toȱ ANOVAȱ andȱ Tukeyȱ testsȱ whichȱ identifiedȱ significantȱ
differencesȱ inȱ sensitivityȱ amongȱ stationsȱ andȱ controlsȱ forȱ eachȱ factor.ȱ Inȱ thisȱ
sense,ȱthisȱnewȱmethodologyȱimprovesȱandȱupdatesȱthisȱkindȱofȱdataȱtreatmentȱ
previouslyȱreportedȱbyȱRibaȱetȱal.ȱ(2003).ȱ
ȱ
- 284 -
3.ȱResultsȱandȱdiscussionȱ
Summarizedȱ resultsȱ ofȱ theȱ differentȱ surveysȱ areȱ shownȱ inȱ tableȱ 1ȱ andȱ
tableȱ 2.ȱ Aȱ firstȱ approachȱ toȱ assessȱ theȱ impactȱ ofȱ theȱ oilȱ spillȱ onȱ theȱ Galicianȱ
CoastȱwasȱperformedȱinȱtheȱAINPȱaȱfewȱmonthsȱafterȱtheȱsinkingȱofȱtheȱtanker.ȱ
Theȱmultivariateȱanalysisȱwasȱusedȱinȱtheȱdataȱsetȱbyȱduplicateȱtoȱconnectȱandȱ
interpretȱ resultsȱ obtainedȱ fromȱ theȱ threeȱ linesȱ ofȱ evidenceȱ investigated.ȱ Theȱ
applicationȱ ofȱ theȱ MAAȱ allowsȱ theȱ averagedȱ variablesȱ (relatedȱ toȱ
contamination,ȱtoxicityȱandȱalteration)ȱtoȱbeȱgroupedȱinȱaȱnewȱsetȱofȱfactors.ȱInȱ
thisȱ studyȱ physicochemicalȱ dataȱ (metalsȱ –Zn,ȱ Pb,ȱ Cu,ȱ Ni,ȱ Hg,ȱ VȬȱ andȱ PAHs),ȱ
toxicityȱ(Microtox®ȱ test,ȱamphipodsȱassay,ȱpolychaetesȱtestȱandȱseaȱurchinȱtest)ȱ
andȱalterationȱ(numberȱofȱspecies,ȱspeciesȱrichness,ȱdiversityȱandȱproportionsȱofȱ
molluscs,ȱpolychaetesȱandȱcrustaceans)ȱwereȱincluded.ȱTheseȱoriginalȱvariablesȱ
canȱ beȱ groupedȱ inȱ fourȱ newȱ factorsȱ whichȱ explainȱ 84.0%ȱ ofȱ theȱ originalȱ dataȱ
varianceȱ(tableȱ3).ȱNegativeȱvaluesȱobtainedȱinȱtheȱanalysisȱareȱasȱimportantȱasȱ
theȱ positiveȱ values.ȱ Valuesȱ associatedȱ withȱ aȱ particularȱ componentȱ forȱ whichȱ
loadingȱwasȱ0.40ȱorȱhigherȱwereȱselectedȱtoȱinterpretȱaȱgroupȱofȱvariables.ȱThisȱ
approximatesȱComreys’ȱcutȬoffȱofȱ0.55ȱ(1973)ȱcorrespondingȱtoȱaȱgoodȱoriginalȱ
variableȱ factorȱ association,ȱ whileȱ takingȱ intoȱ accountȱ discontinuitiesȱ inȱ theȱ
magnitudesȱofȱloadingsȱapproximatingȱtheȱoriginalȱvariables.ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
- 285 -
2.9ȱ
14.78ȱ
85.3ȱ
106.9ȱ
Ga1ȱ
DȬ07ȱ
DȬ09ȱ
55.5ȱ
100.8ȱ
14ȱ
14.7ȱ
113.9ȱ
76.2ȱ
3.95ȱ
DȬ18ȱ
DȬ60ȱ
DȬ66ȱ
DȬ69ȱ
DȬ79ȱ
FIGȱ
Ga2ȱ
1.14ȱ
26.3ȱ
29.3ȱ
2.73ȱ
4.1ȱ
30.5ȱ
14ȱ
27.5ȱ
23.4ȱ
Pbȱ
mgKgȬ1ȱ
Znȱ
ȱ
Cuȱ
ȱ
ȱ
ȱ
ȱ
0.65ȱ
18.5ȱ
149.4ȱ
12.8ȱ
16.2ȱ
70.9ȱ
20.8ȱ
159.7ȱ
250.7ȱ
12.8ȱ
mgKgȬ1
Niȱ
Vȱ
Hgȱ
0.42ȱ
11.8ȱ
4.44ȱ
1.71ȱ
4.6ȱ
16.2ȱ
3.44ȱ
11.7ȱ
1.04ȱ
1.71ȱ
1.0ȱ
n.d.ȱ
13.7ȱ
n.d.ȱ
n.d.ȱ
125ȱ
54ȱ
116ȱ
81.2ȱ
1.0ȱ
0.01ȱ
0.04ȱ
0.09ȱ
0.05ȱ
0.06ȱ
0.12ȱ
0.04ȱ
0.07ȱ
0.08ȱ
0.01ȱ
mgKgȬ1ȱ mgKgȬ1ȱ mgKgȬ1
PAHȱ
2120ȱ
390ȱ
270ȱ
480ȱ
380ȱ
700ȱ
480ȱ
240ȱ
470ȱ
190ȱ
ΐgKgȬ1ȱ
15ȱ
50ȱ
25ȱ
20ȱ
30ȱ
60ȱ
20ȱ
45ȱ
25ȱ
25ȱ
5ȱ
50ȱ
5ȱ
15ȱ
10ȱ
15ȱ
10ȱ
20ȱ
15ȱ
10ȱ
1215ȱ
- 286 -
605ȱ
1006ȱ
364ȱ
450ȱ
486ȱ
358ȱ
390ȱ
1694ȱ
1367ȱ
80.0ȱ
4.8ȱ
8.7ȱ
29.8ȱ
5.0ȱ
100.0ȱ
15.8ȱ
100.0ȱ
3.3ȱ
15.0ȱ
2ȱ
3ȱ
8ȱ
7ȱ
6ȱ
12ȱ
24ȱ
12ȱ
17ȱ
17ȱ
1.2ȱ
1.8ȱ
18.6ȱ
4.3ȱ
15.4ȱ
21.1ȱ
18.6ȱ
9.3ȱ
20.5ȱ
14.3ȱ
1ȱ
1.5ȱ
2.552ȱ
2.574ȱ
1.231ȱ
2.386ȱ
4.089ȱ
3.036ȱ
3.76ȱ
4.574ȱ
0.1ȱ
0.1ȱ
50.0ȱ
42.9ȱ
33.3ȱ
25.0ȱ
25.0ȱ
33.3ȱ
23.5ȱ
22.9ȱ
100ȱ
66.66ȱ
37.5ȱ
42.86ȱ
50ȱ
58.33ȱ
62.5ȱ
66.66ȱ
41.17ȱ
42.86ȱ
0.1ȱ
33.3ȱ
12.5ȱ
14.3ȱ
16.7ȱ
0.1ȱ
8.3ȱ
0.1ȱ
17.7ȱ
34.3ȱ
ȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱBenthicȱalterationsȱȱȱ_________________ȱ
Molusc Polychaet Crustace
Corophiumȱ Arenicolaȱ Microtoxȱ Paracentrotusȱ speciesȱ specificȱ
Diversityȱ
%mortalityȱ
%mortalityȱ
IC50ȱ
%normalȱ
Nºȱ
richness
aȱ%ȱ
aȱ%ȱ
a%ȱ
ȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱChemicalȱanalysisȱ_________________ȱȱȱ ȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱToxicityȱtests_________________
mgKgȬ1ȱ
ȱ
ȱ
Ga1,ȱD60,ȱD66,ȱD69,ȱD79,ȱFIGȱandȱGa2ȱareȱplacedȱinȱtheȱCiesȱIsland).ȱn.d.:ȱnotȱdetected.ȱ
studyȱofȱtheȱsedimentsȱqualityȱinȱtheȱAtlanticȱIslandsȱNationalȱParkȱ(D07,ȱD09ȱandȱD18ȱareȱlocatedȱinȱtheȱOnsȱIslandȱwhereasȱ
Tableȱ 1.ȱ Summarizedȱ resultsȱ ofȱ chemicalȱ analysis,ȱ theȱ acuteȱ toxicityȱ testsȱ andȱ theȱ alterationȱ parametersȱ forȱ ourȱ firstȱ
76.2ȱ
123.8ȱ
377ȱ
3.95ȱ
12.7ȱ
91ȱ
41.1ȱ
37.5ȱ
164ȱ
65.7ȱ
25ȱ
34ȱ
19.9ȱ
214ȱ
271ȱ
AȬ1ȱ
AȬ2ȱ
AȬ3ȱ
BȬ1ȱ
BȬ2ȱ
BȬ3ȱ
CȬ1ȱ
CȬ2ȱ
CȬ3ȱ
DȬ2ȱ
DȬ3ȱ
EȬ2ȱ
EȬ3ȱ
FȬ2ȱ
FȬ3ȱ
21.2ȱ
13.8ȱ
GA1Ȭ2ȱ
GA1Ȭ3ȱ
14.78ȱ
5.9ȱ
14.6ȱ
7.3ȱ
4.31ȱ
3.7ȱ
42.5ȱ
0.85ȱ
6.54ȱ
7.13ȱ
0.9ȱ
0.72ȱ
1.14ȱ
1.5ȱ
10.1ȱ
26.3ȱ
2.1ȱ
1.6ȱ
2.9ȱ
Pbȱ
mgKgȬ1ȱ
Znȱ
mgKgȬ1ȱ
Cuȱ
4.2ȱ
20ȱ
0.43ȱ
n.d.ȱ
0.7ȱ
21.4ȱ
1.4ȱ
34.5ȱ
24.9ȱ
1.4ȱ
0.88ȱ
0.65ȱ
5.2ȱ
11.4ȱ
18.5ȱ
2.19ȱ
6.4ȱ
12.8ȱ
mgKgȬ1
Niȱ
Vȱ
Hgȱ
5.7ȱ
7.07ȱ
1.5ȱ
5.66ȱ
1.7ȱ
9.18ȱ
4.5ȱ
5.11ȱ
5.21ȱ
2.4ȱ
1.31ȱ
0.42ȱ
13.3ȱ
11.8ȱ
11.8ȱ
1.65ȱ
0.32ȱ
1.71ȱ
0.36ȱ
0.7ȱ
0.35ȱ
0.38ȱ
0.34ȱ
1.2ȱ
0.1ȱ
0.86ȱ
0.77ȱ
0.2ȱ
n.d.ȱ
n.d.ȱ
0.3ȱ
0.32ȱ
0.53ȱ
n.d.ȱ
n.d.ȱ
n.d.ȱ
3.4ȱ
5.81ȱ
2.1ȱ
2.33ȱ
2ȱ
13.2ȱ
0.6ȱ
n.d.ȱ
n.d.ȱ
0.8ȱ
1.01ȱ
n.d.ȱ
0.7ȱ
n.d.ȱ
n.d.ȱ
n.d.ȱ
n.d.ȱ
n.d.ȱ
mgKgȬ1ȱ mgKgȬ1ȱ mgKgȬ1
PAHȱ
323ȱ
820ȱ
52ȱ
558ȱ
38ȱ
537ȱ
n.d.ȱ
239ȱ
420ȱ
67ȱ
366ȱ
2120ȱ
108ȱ
119ȱ
390ȱ
n.d.ȱ
74ȱ
190ȱ
ΐgKgȬ1ȱ
20ȱ
40ȱ
17ȱ
36ȱ
10ȱ
30ȱ
17ȱ
28ȱ
33ȱ
20ȱ
23ȱ
50ȱ
23ȱ
22ȱ
25ȱ
10ȱ
15ȱ
15ȱ
17ȱ
40ȱ
17ȱ
35ȱ
39ȱ
40ȱ
22ȱ
28ȱ
36ȱ
28ȱ
35ȱ
50ȱ
28ȱ
10ȱ
5ȱ
0ȱ
5ȱ
5ȱ
- 287 -
4398ȱ
2185ȱ
21041ȱ
20827ȱ
3977ȱ
2436ȱ
1801ȱ
4651ȱ
723ȱ
9422ȱ
1523ȱ
605ȱ
5631ȱ
5231ȱ
1006ȱ
19762ȱ
3974ȱ
2486ȱ
76ȱ
30ȱ
85ȱ
35ȱ
55ȱ
67ȱ
85ȱ
68ȱ
46ȱ
88ȱ
79ȱ
20ȱ
79ȱ
87ȱ
95ȱ
97ȱ
89ȱ
85ȱ
48.2ȱ
55.6ȱ
32.1ȱ
66.7ȱ
28.6ȱ
25.7ȱ
42.4ȱ
50.9ȱ
15.3ȱ
33.9ȱ
5.9ȱ
1.2ȱ
28.5ȱ
12.0ȱ
1.8ȱ
39.1ȱ
43.0ȱ
14.3ȱ
2.9ȱ
2.3ȱ
3.0ȱ
5.0ȱ
3.0ȱ
2.9ȱ
4.3ȱ
4.5ȱ
2.9ȱ
5.0ȱ
5.2ȱ
1.0ȱ
5.1ȱ
4.3ȱ
1.5ȱ
6.1ȱ
2.0ȱ
4.6ȱ
15.4ȱ
40.0ȱ
40.1ȱ
2.0ȱ
30.0ȱ
33.3ȱ
39.1ȱ
26.7ȱ
22.2ȱ
28.4ȱ
9.9ȱ
0.1ȱ
15.3ȱ
2.4ȱ
0.1ȱ
34.7ȱ
23.5ȱ
22.9ȱ
23.1ȱ
26.7ȱ
22.2ȱ
30.6ȱ
20.0ȱ
33.3ȱ
21.7ȱ
26.7ȱ
33.3ȱ
21.5ȱ
56.2ȱ
100.0ȱ
20.0ȱ
21.0ȱ
53.0ȱ
18.0ȱ
32.1ȱ
42.9ȱ
61.5ȱ
33.3ȱ
51.4ȱ
100.0ȱ
50.0ȱ
33.3ȱ
39.1ȱ
43.3ȱ
33.3ȱ
41.0ȱ
15.4ȱ
0.1ȱ
37.0ȱ
34.5ȱ
33.3ȱ
40.5ȱ
41.0ȱ
37.1ȱ
48.2ȱ
55.6ȱ
32.1ȱ
66.7ȱ
28.6ȱ
25.7ȱ
42.4ȱ
50.9ȱ
15.3ȱ
33.9ȱ
5.9ȱ
1.2ȱ
28.5ȱ
12.0ȱ
1.8ȱ
39.1ȱ
43.0ȱ
14.3ȱ
ȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱBenthicȱalterationsȱȱȱ_________________ȱ
Molusc Polychaet Crustace
Corophiumȱ Arenicolaȱ Microtoxȱ Paracentrotusȱ speciesȱ specificȱ
Diversityȱ
%mortalityȱ
%mortalityȱ
IC50ȱ
%normalȱ
Nºȱ
richness
aȱ%ȱ
aȱ%ȱ
a%ȱ
ȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱChemicalȱanalysisȱ_________________ȱȱȱ ȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱȱToxicityȱtests_________________
GA1Ȭ1ȱ
ȱ
ȱ
Tableȱ2.ȱSummarizedȱresultsȱofȱchemicalȱanalysis,ȱtoxicityȱtestȱandȱbenthicȱalterationȱparametersȱmeasuredȱforȱtheȱstudyȱofȱtheȱ
sedimentsȱqualityȱinȱtheȱCiesȱIslandȱȱ2003Ȭ2006ȱ(firstȱsurvey:ȱGA1Ȭ1,ȱAȬ1,ȱBȬ1,ȱCȬ1;ȱsecondȱsurvey:ȱGA1Ȭ2,ȱAȬ2,ȱBȬ2,ȱCȬ2;ȱthirdȱ
survey:ȱGA1Ȭ3,ȱAȬ3,ȱBȬ3,ȱCȬ3)ȱandȱCormeȬLaxeȱ2004Ȭ2006ȱȱ(secondȱsurvey:ȱDȬ2,ȱEȬ2,ȱFȬ2;ȱthirdȱsurvey:ȱDȬ3,ȱEȬ3,ȱFȬ3).ȱn.d.:ȱnotȱ
detected.ȱ
ȱ
Tableȱ 3.ȱ Sortedȱ rotatedȱ factorȱ loadingsȱ (pattern)ȱ ofȱ 17ȱ variablesȱ forȱ theȱ fourȱ
principalȱ factorsȱ resultingȱ fromȱ theȱ multivariateȱ analysisȱ ofȱ theȱ singleȱ resultsȱ
obtainedȱ fromȱ theȱ chemicalȱ analysis,ȱ theȱ acuteȱ toxicityȱ testsȱ andȱ theȱ alterationȱ
parametersȱ forȱ theȱ studyȱ of the sediments quality in the Atlantic Islands National
Park.
ȱȱ
FACTORȱ1 FACTORȱ2 FACTORȱ3ȱ FACTORȱ4
ȱȱ
42.21ȱ
23.43ȱ
10.64ȱ
7.73ȱ
Znȱ
ņȱ
ņȱ
ņȱ
0.91ȱ
Pbȱ
ņȱ
ņȱ
ņȱ
0.81ȱ
Cuȱȱ
ņȱ
ņȱ
0.47ȱ
0.47ȱ
Niȱȱ
ņȱ
ņȱ
ņȱ
0.80ȱ
Vȱ
ņȱ
0.63ȱ
ņȱ
0.55ȱ
Hgȱ
ņȱ
0.32ȱ
ņȱ
0.73ȱ
PAHsȱ
0.65ȱ
0.54ȱ
ņȱ
ņȱ
Corophiumȱbioassayȱ
0.30ȱ
ņȱ
ņȱ
ņȱ
Arenicolaȱbioassayȱ
0.54ȱ
0.59ȱ
ņȱ
ņȱ
Microtoxȱtestȱ
ņȱ
ņȱ
ņȱ
ņȱ
Paracentrotusȱassayȱ
ņȱ
0.90ȱ
ņȱ
ņȱ
Numberȱofȱspeciesȱ
0.89ȱ
ņȱ
ņȱ
ņȱ
Specificȱrichnesȱ
0.94ȱ
ņȱ
ņȱ
ņȱ
Diversityȱ
0.79ȱ
ņȱ
ņȱ
ņȱ
%ȱMoluscaȱ
0.96ȱ
ņȱ
ņȱ
ņȱ
%ȱPolychaetaȱ
0.77ȱ
0.48ȱ
ņȱ
ņȱ
%ȱCrustaceaȱ
ņȱ
0.91ȱ
ņȱ
ņȱ
ȱ
- 288 -
Theȱfirstȱprincipalȱfactor,ȱ#1ȱisȱpredominantȱandȱaccountsȱforȱ42.2%ȱofȱtheȱ
variance.ȱThisȱfactorȱrepresentsȱtheȱdegradationȱofȱtheȱenvironmentȱassociatedȱ
withȱtheȱpresenceȱofȱPAHsȱinȱtheȱsedimentȱbyȱlinkingȱPAHsȱwithȱtheȱtoxicityȱofȱ
CorphiumȱandȱtheȱpolychaeteȱArenicolaȱmarinaȱasȱwellȱasȱinfaunaȱalterations.ȱTheȱ
secondȱfactorȱexplainsȱ23.4%ȱofȱtheȱvariance;ȱshowingȱtheȱrelationshipȱbetweenȱ
theȱ presenceȱ ofȱ PAHsȱ andȱ theȱ metalsȱ Hgȱ andȱ Vȱ inȱ theȱ sedimentȱ withȱ theȱ
adverseȱeffectsȱmeasuredȱinȱbothȱtheȱArenicolaȱandȱseaȱurchinȱtestsȱandȱaȱslightȱ
alterationȱinȱtheȱcrustaceansȱandȱpolychaeteȱcommunity.ȱTheseȱtwoȱfactorsȱ(#1ȱ
andȱ #2),ȱ certainlyȱ appearȱ asȱ aȱ consequenceȱ ofȱ theȱ tankerȱ Prestigeȱ whichȱ spiltȱ
hydrocarbonsȱ containingȱ PAHsȱ andȱ affectedȱ theȱ biotaȱ resultingȱ inȱ
environmentalȱ degradationȱ althoughȱ Factorȱ #2ȱ representsȱ moreȱ moderateȱ
effectsȱthanȱFactorȱ#1;ȱVȱisȱassociatedȱwithȱtheȱspillȱandȱhasȱanȱapparentlyȱminorȱ
effectȱonȱbiotaȱwhenȱcomparedȱtoȱPAHs.ȱFactorȱ#3ȱaccountsȱforȱaȱ10.6%ȱofȱtheȱ
varianceȱ andȱ isȱ relatedȱ toȱ Cuȱ contaminationȱ whichȱ isȱ notȱ responsibleȱ forȱ
biologicalȱ effectsȱ inȱ theȱ environment,ȱ whereasȱ factorȱ #4ȱ (7.7%)ȱ describesȱ theȱ
presenceȱ ofȱ metalȱ contaminationȱ (Zn,ȱ Pb,ȱ Cu,ȱ Niȱ ,ȱ Vȱ andȱ Hg)ȱ whichȱ doesȱ notȱ
produceȱtoxicȱeffectsȱandȱisȱnotȱproducingȱdegradationȱinȱtheȱenvironment.ȱThisȱ
metalȱcontaminationȱmayȱbeȱrelatedȱtoȱbasalȱlevelsȱofȱcontaminantsȱorȱowingȱtoȱ
sourcesȱotherȱthanȱtheȱPrestigeȱoilȱspillȱ
Figureȱ 2ȱ showsȱ theȱ factors’ȱ scoresȱ forȱ eachȱ ofȱ theȱ 10ȱ studiedȱ stations.ȱ
Factorȱ#1ȱdefinedȱasȱrepresentingȱtheȱenvironmentalȱdegradationȱcausedȱbyȱtheȱ
PAHsȱ boundȱ toȱ theȱ sedimentsȱ isȱ prevalentȱ inȱ theȱ stationsȱ FIGȱ (1.8)ȱ andȱ GA2ȱ
(2.0)ȱindicatingȱhighȱpollutionȱlevelsȱdueȱtoȱPAHsȱinȱtheseȱsites.ȱFactorȱ#2ȱwhichȱ
indicatesȱpollutionȱbyȱPAHsȱandȱVȱisȱprevalentȱinȱtheȱstationȱD09ȱ(1.4)ȱfromȱtheȱ
Onsȱ islandȱ andȱ D60ȱ (1.2)ȱ andȱ GA2ȱ (1.4)ȱ fromȱ Cies.ȱ Factorȱ #3,ȱ definedȱ asȱ
representingȱ Cuȱ contaminationȱ withȱ noȱ effectsȱ hasȱ aȱ positiveȱ loadingȱ inȱ theȱ
samplesȱfromȱOnsȱD07ȱ(1.5)ȱandȱD09ȱ(1.2)ȱandȱFIGȱ(0.4),ȱGA1ȱ(0.9)ȱinȱCies,ȱ
- 289 -
2.5
ȱ
FIG
2.0
GA2
1.5
FACTOR 1
ȱ
ȱ
1.0
0.5
0.0
D66
-0.5
ȱ
ȱ
ȱ
-1.0
D09
GA1
FACTOR 2
ȱ
D79
D07
2.0
GA2
D09
D60
1.0
0.5
0.0
D07
D18
-0.5
ȱ
D69
D60
-1.5
1.5
ȱ
D18
D66
-1.0
D69
GA1
D79
-1.5
ȱ
2.0
ȱ
D07
1.5
D09
GA1
FACTOR 3
ȱ
ȱ
1.0
FIG
0.5
GA2
0.0
-0.5
D69
D18
ȱ
D79
-1.0
D60
-1.5
D66
ȱ
2.0
ȱ
FACTOR 4
ȱ
ȱ
D60
1.5
FIG
D79
D09
1.0
0.5
D07
0.0
-0.5
D18
ȱ
-1.0
D66
D69
GA1
GA2
-1.5
ȱ
Figureȱ 2.ȱ Estimatedȱ factorȱ scoresȱ forȱ theȱ fourȱ factorsȱ inȱ eachȱ ofȱ theȱ 10ȱ
cases.ȱTheȱfactorȱscoresȱquantifyȱtheȱprevalenceȱofȱeachȱfactorȱforȱeveryȱstationȱ
andȱisȱusedȱtoȱestablishȱtheȱdefinitionȱofȱeachȱfactor.ȱ
- 290 -
whereasȱFactorȱ#4ȱdescribesȱtheȱpresenceȱofȱmetalsȱ(Zn,ȱPb,ȱCu,ȱNi,ȱVȱandȱHg)ȱ
inȱtheȱsedimentsȱfromȱOnsȱD07ȱ(0.1)ȱandȱD09ȱ(0.8),ȱCiesȱD60ȱ(1.4),ȱD79ȱ(0.8)ȱandȱ
FIGȱ(1.2).Theseȱresultsȱshowȱhowȱinȱ2004ȱtheȱAtlanticȱIslandsȱNationalȱParkȱwasȱ
significantlyȱ affectedȱ byȱ theȱ oilȱ spillȱ andȱ theȱ wayȱ inȱ whichȱ stationȱ GA1ȱ hasȱ
provedȱtoȱbeȱaȱsuitableȱsiteȱofȱreferenceȱforȱtheȱpresentȱresearch.ȱȱ
Factor 1
Factor 2
Factor 4
Factor 3
D07
Factor 1
Factor 2
Factor 4
Factor 3
Factor 2
Factor 4
Factor 3
D09
Factor 1
Factor 2
Factor 4
Factor 3
D60
D18
Factor 1
Factor 2
Factor 1
Factor 2
Factor 4
Factor 3
Factor 4
Factor 3
D66
Factor 1
Factor 2
Factor 1
Factor 2
Factor 4
Factor 3
Factor 4
Factor 3
D79
Factor 1
FIG
D69
Factor 1
Factor 2
Factor 4
Factor 3
GA2
ȱ
Figureȱ3.ȱPieȱchartsȱrepresentingȱtheȱsignificantȱdifferencesȱofȱtheȱfactorsȱ
scoreȱ inȱ everyȱ studyȱ siteȱ –Atlanticȱ Islandsȱ Nationalȱ Parkȱ (2003)Ȭȱ relatedȱ toȱ theȱ
referenceȱsiteȱGA1ȱ(black:ȱpȱ<ȱ0.01;ȱgrey:ȱ0.01<ȱpȱ>ȱ0.05;ȱwhite:ȱnotȱsignificantlyȱ
differences,ȱ p>0.05).ȱ Factorȱ #1:ȱ PAHsȬpollution;ȱ Factorȱ #2:ȱ PAHsȬHgȬVȬ
pollution;ȱ Factorȱ #3:ȱ CuȬcontamination;ȱ Factorȱ #4:ȱ ZnȬPbȬCuȬNiȬVȬHgȬ
contamination.ȱȱ
- 291 -
Withȱ theȱ aimȱ ofȱ identifyingȱ theȱ causeȱ ofȱ pollutionȱ (orȱ lackȱ thereof)ȱ inȱ
eachȱstudyȱsite,ȱanȱANOVAȱanalysisȱwasȱconductedȱbyȱusingȱtheȱfactorȱscoresȱ
obtainedȱ inȱ theȱ MAA.ȱ Figureȱ 3ȱ showsȱ theȱ pieȱ chartsȱ stemmingȱ fromȱ ANOVAȱ
results,ȱusingȱGA1ȱasȱtheȱnegativeȱcontrol.ȱ
Inȱ thisȱ firstȱ approachȱ inȱ whichȱ theȱ sedimentȱ statusȱ wasȱ establishedȱ (inȱ
2004)ȱ followingȱ theȱ spill,ȱ significantȱ pollutionȱ causedȱ byȱ PAHsȱ atȱ theȱ stationsȱ
FIGȱ andȱ GA2ȱ wasȱ detected.ȱ Pollutionȱ provokedȱ byȱ aȱ mixtureȱ ofȱ PAHsȱ andȱ Vȱ
affectingȱ bothȱ theȱ Ons:ȱ D09ȱ andȱ theȱ Ciesȱ Islands:ȱ D60,ȱ FIG,ȱ GA2ȱ wasȱ alsoȱ
detected.ȱHighȱlevelsȱofȱpollutionȱwereȱespeciallyȱidentifiedȱinȱtheȱstationȱGA2ȱ
onȱtheȱCiesȱisland.ȱAȱsourceȱofȱmetalsȱ(Zn,ȱPb,ȱCu,ȱNi,ȱHgȱandȱV)ȱwasȱidentifiedȱ
initially,ȱwhoseȱpresenceȱwasȱthoughtȱnotȱtoȱbeȱinȱconnectionȱwithȱtheȱoilȱspillsȱ
butȱ insteadȱ isȱ probablyȱ relatedȱ toȱ backgroundȱ levelsȱ orȱ contaminationȱ whichȱ
doesȱ notȱ causeȱ biologicalȱ effects.ȱ Suchȱ levelsȱ appearȱ atȱ D07,ȱ D09ȱ andȱ theȱ
stationsȱfromȱCiesȱIslandsȱD60,ȱD79ȱandȱFIG.ȱInȱFIGȱtheȱpresenceȱofȱmetalsȱhasȱ
beenȱ correlatedȱ withȱ alterationȱ inȱ oneȱ ofȱ theȱ analyses,ȱ butȱ noȱ toxicityȱ wasȱ
exhibited;ȱ perhapsȱ meaningȱ thatȱ alterationȱ ofȱ theȱ macrofaunaȱ wasȱ causedȱ byȱ
otherȱ sources,ȱ possiblyȱ physical,ȱ suchȱ asȱ theȱ assessedȱ beachȱ cleaningȱ afterȱ theȱ
spill.ȱ Previousȱ studiesȱ haveȱ demonstratedȱ thatȱ theȱ Prestigeȱ oilȱ spillȱ causedȱ Znȱ
contaminationȱ inȱ theȱ surroundingȱ waterȱ columnȱ (Pregoȱ andȱ CobeloȬGarcía,ȱ
2003).ȱContaminationȱbyȱcopperȱandȱleadȱwasȱalsoȱobservedȱinȱtheȱuppermostȱ
layerȱinȱtheȱshipwreckȱareaȱofȱtheȱNortheastȱAtlanticȱOceanȱ(PregoȱandȱCobeloȬ
García,ȱ2004;ȱCobeloȬGarciaȱetȱal.,ȱ2004),ȱhowever,ȱthisȱcontaminationȱwithȱCuȱisȱ
notȱlikelyȱtoȱbeȱrelatedȱbecauseȱlevelsȱofȱCuȱinȱtheȱfuelȱoilȱwereȱrelativelyȱlowȱ
(3.39ȱ mgȱ KgȬ1)ȱ suggestingȱ Cuȱ inputsȱ fromȱ theȱ nearbyȱ Riaȱ deȱ Vigo.ȱ Previousȱ
studiesȱhaveȱshownȱtheȱpresenceȱofȱtraceȱmetalȱcontaminationȱinȱtheȱRias,ȱcloseȱ
toȱtheȱAINPȱ(Carballeiraȱetȱal.,ȱ1997;ȱPérezȬLópezȱetȱal.,ȱ2003)ȱthisȱalsoȱpossiblyȱ
explainingȱtheȱpresenceȱofȱmetalsȱonȱtheȱCiesȱandȱOnsȱIslands.ȱȱ
- 292 -
TheȱapplicationȱofȱtheȱWOEȱapproachȱhasȱshownȱthatȱsomeȱmonthsȱafterȱ
theȱ spillȱ thereȱ wasȱ aȱ significantȱ impactȱ whichȱ provokedȱ degradationȱ ofȱ theȱ
ecosystemȱ inȱ partȱ ofȱ theȱ sedimentȱ fromȱ studyȱ sitesȱ locatedȱ inȱ theȱ Atlanticȱ
IslandsȱNationalȱPark.ȱAȱsourceȱofȱmetals,ȱwhichȱinȱsomeȱcasesȱareȱaffectingȱtheȱ
environmentȱ orȱ areȱ consideredȱ aȱ potentialȱ riskȱ hasȱ beenȱ detected.ȱ Resultsȱ
suggestedȱ thatȱ furtherȱ studiesȱ shouldȱ beȱ doneȱ inȱ orderȱ toȱ clarifyȱ whetherȱ theȱ
affectedȱAINPȱandȱsurroundingȱsitesȱhaveȱrecovered.ȱWithȱthisȱaimȱinȱmind,ȱtheȱ
WOEȱ investigationsȱ haveȱ beenȱ appliedȱ toȱ selectedȱ sitesȱ inȱ theȱ Ciesȱ Islandȱ
(AINP)ȱ andȱ CormeȬLaxe,ȱ withȱ theȱ freshȱ approachȱ ofȱ monitoringȱ overȱ aȱ fourȱ
yearȱperiodȱinȱorderȱtoȱassessȱtheȱrecoveryȱofȱanȱareaȱaffectedȱbyȱanȱoilȱspill.ȱȱ
Sedimentsȱ fromȱ GA1ȱ turnedȱ outȱ toȱ beȱ theȱ cleanestȱ givenȱ itȱ didȱ presentȱ
toxicityȱorȱinȱsituȱalterationȱmakingȱthisȱstationȱanȱappropriateȱselectionȱasȱtheȱ
referenceȱ siteȱ inȱ theȱ followingȱ assessments.ȱ Theȱ WOEȬmonitoringȱ focusedȱ onȱ
twoȱ areasȱ withȱ theȱ followingȱ proceduresȱ appliedȱ separately:ȱ (a)ȱ assessingȱ theȱ
Ciesȱ Islandȱ sedimentȱ qualityȱ monitoringȱ fromȱ theȱ beginningȱ ofȱ 2004ȱ toȱ 2006,ȱ
andȱ(b)ȱstudyingȱtheȱCormeȬLaxeȱsedimentȱstatusȱfromȱtheȱendȱofȱ2004ȱtoȱ2006.ȱ
Inȱthisȱsense,ȱthisȱstudyȱwasȱdesignedȱtoȱmonitorȱtheȱrecoveryȱorȱpersistenceȱofȱ
theȱ pollutionȱ causedȱ byȱ theȱ oilȱ spillȱ overȱ timeȱ usingȱ anȱ improvedȱ WOEȱ
approachȱbasedȱonȱtheȱclassicalȱSQT.ȱ
3.1.ȱCíesȱIslandȱ(2004Ȭ2006)ȱ
Theȱ sedimentȱ qualityȱ assessmentȱ atȱ theȱ Ciesȱ Islandȱ (AINP)ȱ hasȱ beenȱ
carriedȱ outȱ forȱ theȱ sameȱ 3ȱ sitesȱ inȱ distinctȱ samplingȱ campaignsȱ fromȱ 2003ȱ toȱ
2006.ȱResultsȱfromȱtheȱfirstȱsurveyȱ(2004)ȱcorrespondȱtoȱGA1Ȭ1,ȱAȬ1,ȱBȬ1ȱandȱCȬ
1;ȱ dataȱ fromȱ theȱ secondȱ surveyȱ (2004Ȭ2005)ȱ areȱ referredȱ toȱ asȱ GA1Ȭ2,ȱ AȬ2,ȱ BȬ2ȱ
andȱCȬ2,ȱwithȱtheȱresultsȱobtainedȱinȱtheȱthirdȱsurveyȱ(2005Ȭ2006)ȱcorrespondingȱ
toȱ Ga1Ȭ3,ȱ AȬ3,ȱ BȬ3ȱ andȱ CȬ3.ȱ Theȱ MAAȱ wasȱ carriedȱ outȱ byȱ treatingȱ eachȱ setȱ ofȱ
- 293 -
dataȱ asȱ anȱ independentȱ caseȱ inȱ orderȱ toȱ trackȱ theȱ monitoringȱ ofȱ theȱ sedimentȱ
qualityȱinȱeachȱstation.ȱȱ
Inȱ tableȱ 4ȱ theȱ MAAȱ carriedȱ outȱ onȱ originalȱ variableȱ data,ȱ includingȱ
replicatesȱisȱshown.ȱTheȱapplicationȱofȱtheȱstatisticalȱanalysisȱshowsȱthatȱtheȱ19ȱ
originalȱ variablesȱ canȱ beȱ groupedȱ inȱ fourȱ newȱ factors.ȱ Theseȱ factorsȱ explainȱ
88.5%ȱofȱtheȱoriginalȱdataȱvariance.ȱTheȱfirstȱFactorȱ(#1)ȱaccountsȱforȱ50.75ȱ%ȱofȱ
theȱvarianceȱandȱcorrespondsȱtoȱtheȱtoxicityȱandȱinȱsituȱalterationȱdueȱtoȱPAHsȱ
andȱ theȱ presenceȱ ofȱ Pbȱ responsibleȱ forȱ environmentalȱ degradationȱ (pollution);ȱ
Factorȱ#2ȱ(18.3%)ȱdepictsȱPb,ȱCu,ȱNiȱandȱVȱcontaminationȱforȱwhichȱnoȱtoxicityȱ
orȱ otherȱ effectsȱ onȱ biotaȱ isȱ appreciable;ȱ Factorȱ #3ȱ (10.9%)ȱ isȱ alsoȱ relatedȱ toȱ
contaminationȱ byȱ Zn,ȱ Pb,ȱ Niȱ andȱ Hgȱ havingȱ noȱ associatedȱ biologicalȱ effectsȱ
whileȱ Factorȱ #4ȱ (8.6%)ȱ showsȱ aȱ degreeȱ ofȱ toxicityȱ andȱ environmentalȱ
degradationȱdueȱtoȱPAHsȱcontamination.ȱȱ
Figureȱ4ȱshowsȱtheȱfactorȱscoresȱinȱtheȱ12ȱcases.ȱFactorȱ#1,ȱwhichȱdefinesȱ
pollutionȱdueȱtoȱPAHsȱandȱPb,ȱhasȱaȱpositiveȱloadingȱinȱAȬ1ȱ(2.0)ȱandȱBȬ1ȱ(2.2).ȱ
Theseȱ 2ȱ casesȱ correspondȱ toȱ firstȱ samplingȱ carriedȱ outȱ inȱ theȱ Ciesȱ Islands.ȱ Weȱ
canȱseeȱhowȱtheȱscoreȱforȱfactorȱ#1ȱatȱtheseȱ2ȱstationsȱ(AȱandȱB)ȱdecreasesȱwithȱ
timeȱinȱtheȱfollowingȱsurveys,ȱinȱresponseȱtoȱsedimentȱrecoveryȱfromȱtheȱeffectsȱ
ofȱ initialȱ pollutionȱ levelsȱ inȱ theȱ studiedȱ sitesȱ .ȱ Factorȱ #2ȱ andȱ #3ȱ demonstrateȱ
contaminationȱbyȱmetalsȱwhichȱwasȱnotȱassociatedȱwithȱdegradationȱinȱmostȱofȱ
theȱstations.ȱFactorȱ#4,ȱrelatedȱtoȱPAHȱtoxicity,ȱdecreasesȱinȱtheȱstationsȱBȱandȱCȱ
withȱtimeȱalthoughȱslightȱpersistenceȱisȱevidentȱinȱstationȱA.ȱȱ
ȱ
ȱ
ȱ
- 294 -
Tableȱ 4.ȱ Sortedȱ rotatedȱ factorȱ loadingsȱ (pattern)ȱ ofȱ 17ȱ variablesȱ forȱ theȱ
fourȱ principalȱ factorsȱ resultingȱ fromȱ theȱ multivariateȱ analysisȱ ofȱ theȱ singleȱ
resultsȱ obtainedȱ fromȱ theȱ chemicalȱ analysis,ȱ theȱ acuteȱ toxicityȱ testsȱ andȱ theȱ
alterationȱ parametersȱ forȱ theȱ studyȱ ofȱ theȱ sedimentsȱ qualityȱ inȱ theȱ Ciesȱ Islandȱ
2004Ȭ2006.ȱ
ȱȱ
ȱȱ
Znȱ
Pbȱ
Cuȱȱ
Niȱȱ
Vȱȱ
Hgȱ
PAHȱ
Corophiumȱbioassayȱ
Arenicolaȱbioassayȱ
Microtoxȱtestȱ
Paracentrotusȱassayȱ
Numberȱofȱspeciesȱ
Specificȱrichnesȱ
Diversityȱ
%ȱMoluscaȱ
%ȱPolychaetaȱ
%ȱCrustaceaȱ
Factorȱ1ȱ
50.73ȱ
Factorȱ2ȱ
18.26ȱ
Factorȱ3ȱ
10.93ȱ
Factorȱ4ȱ
8.62ȱ
ņȱ
0.41ȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.73ȱ
0.63ȱ
ņȱ
0.62ȱ
0.38ȱ
0.91ȱ
0.95ȱ
0.91ȱ
0.97ȱ
0.76ȱ
0.70ȱ
ņȱ
0.71ȱ
0.83ȱ
0.43ȱ
0.82ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.88ȱ
0.32ȱ
ņȱ
0.86ȱ
ņȱ
0.37ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.63ȱ
0.72ȱ
0.92ȱ
0.53ȱ
0.89ȱ
0.31ȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.57ȱ
Figureȱ5ȱshowsȱtheȱprevalenceȱofȱeachȱfactorȱinȱeveryȱstationȱaccordingȱtoȱ
theȱstatisticalȱdifferencesȱobtainedȱinȱtheȱANOVAȱanalysis,ȱinȱcomparisonȱwithȱ
theȱreferenceȱsite.ȱTheȱprevalenceȱofȱFactorȱ#1ȱandȱFactorȱ#4ȱwhichȱareȱrelatedȱtoȱ
PAHsȱ pollutionȱ decreasedȱ inȱ allȱ stationsȱ overȱ theȱ periodȱ 2004Ȭ2006ȱ givingȱ theȱ
impressionȱthatȱtheȱAINPȱhasȱbeenȱundergoingȱaȱprocessȱofȱrecoveryȱduringȱtheȱ
4ȱ yearsȱ followingȱ theȱ oilȱ spill.ȱ Onȱ theȱ otherȱ handȱ theȱ significantȱ differencesȱ
foundȱforȱFactorȱ#1ȱandȱFactorȱ#2ȱdidȱnotȱreflectȱsuchȱaȱrecoveryȱprocessȱduringȱ
thisȱperiod.ȱThisȱisȱperhapsȱrelatedȱtoȱtheȱpersistenceȱofȱmetalȱcontaminationȱinȱ
theȱ 3ȱ studyȱ sites,ȱ suggestingȱ thatȱ thisȱ contaminationȱ wasȱ presentȱ priorȱ toȱ theȱ
spillȱinȱtheȱstudiedȱarea.ȱȱ
- 295 -
3.5
B-1
FACTOR 1
2.5
A-1
1.5
0.5
A-2
GA1-2
-0.5
GA1-1
GA1-3
B-2
A-3
B-3
C-3
C-1
C-2
-1.5
3.0
2.0
A-2
1.0
C-1
C-2
A-1
GA1-1
0.0
GA1-2
-1.0
GA1-3
A-3
B-1
B-2
C-3
B-3
-2.0
3.5
A-3
FACTOR 3
2.5
1.5
A-1
A-2
-0.5
-1.5
C-3
B-3
0.5
B-2
GA1-1
C-1
B-1
GA1-2 GA1-3
C-2
-2.5
2.5
B-1
C-1
FACTOR 4
1.5
A-3
0.5
-0.5
-1.5
C-2
B-2
B-3
GA1-1
C-3
A-2
GA1-2
GA1-3
A-1
-2.5
Oȱ
Figureȱ4.ȱEstimatedȱfactorȱscoresȱforȱtheȱfourȱfactorsȱinȱeachȱofȱtheȱ12ȱcasesȱ
- 296 -
Factor 1
Factor 2
Factor 4
Factor 3
A-1
Factor 1
Factor 2
Factor 4
Factor 3
A-2
Factor 1
Factor 2
Factor 4
Factor 3
Factor 2
Factor 4
Factor 3
A-3
Factor 1
Factor 2
Factor 1
Factor 2
Factor 4
Factor 3
Factor 4
Factor 3
B-1
B-2
Factor 1
Factor 2
Factor 4
Factor 3
C-1
Factor 1
Factor 1
Factor 2
Factor 4
Factor 3
C-2
B-3
Factor 1
Factor 2
Factor 4
Factor 3
C-3
Figureȱ 5.ȱ Pieȱ chartsȱ showingȱ theȱ significantȱ differencesȱ ofȱ theȱ factorsȱ
scoreȱ inȱ everyȱ studyȱ siteȱ ȬCiesȱ (2003Ȭ2006)Ȭȱ relatedȱ toȱ theȱ referenceȱ siteȱ GA1ȱ
(black:ȱpȱ<ȱ0.01;ȱgrey:ȱ0.01<ȱpȱ>ȱ0.05;ȱwhite:ȱnotȱsignificantlyȱdifferences,ȱp>0.05).ȱ
Factorȱ#1:ȱPAHsȬPbȬpollution;ȱFactorȱ#2:ȱPbȬCuȬNiȬVȬcontamination;ȱFactorȱ#3:ȱ
ZnȬPbȬNiȬHgȬcontamination;ȱFactorȱ#4:ȱPAHsȬpollution.ȱȱ
ȱ
ȱ
ȱ
ȱ
- 297 -
Onȱ theȱ whole,ȱ theȱ analysisȱ performedȱ inȱ theȱ Ciesȱ Islandȱ forȱ theȱ periodȱ
2004Ȭ2006ȱhasȱshownȱanȱimportantȱdecreaseȱofȱtheȱinitialȱdegradationȱprovokedȱ
byȱ theȱ accidentalȱ oilȱ spill.ȱ Atȱ theȱ startȱ ofȱ 2004ȱ initialȱ pollutionȱ dueȱ toȱ PAHsȱ
boundȱ toȱ sedimentsȱ wasȱ detectedȱ whichȱ affectedȱ theȱ sedimentȱ qualityȱ inȱ
stationsȱ A,ȱ Bȱ andȱ C.ȱ Thisȱ contaminationȱ andȱ itsȱ biologicalȱ effectsȱ decreasedȱ inȱ
theȱfollowingȱsurveysȱandȱcurrentlyȱtheseȱsedimentsȱseemȱnotȱtoȱbeȱdegraded.ȱ
Theȱpresenceȱofȱmetalsȱcontaminationȱwasȱdetectedȱinȱtheȱstationsȱdespiteȱthisȱ
notȱhavingȱproducedȱenvironmentalȱbiologicalȱeffects.ȱItȱisȱpossibleȱtheseȱmetalsȱ
mayȱ notȱ beȱ availableȱ toȱ organismsȱ inȱ theirȱ presentȱ form,ȱ butȱ thatȱ ifȱ
environmentalȱconditionsȱeventuallyȱchanged,ȱtheyȱmayȱbecomeȱaȱthreatȱforȱtheȱ
environment.ȱȱ
3.2.ȱCormeȬLaxeȱ(endȱofȱ2004Ȭ2006)ȱ
Theȱsedimentȱqualityȱevaluationȱwasȱperformedȱatȱtheȱsameȱsitesȱduringȱ
distinctȱsurveysȱinȱtheȱperiodȱfromȱtheȱendȱofȱ2004ȱtoȱ2006.ȱResultsȱfromȱtheȱfirstȱ
surveyȱ(2004Ȭ2005)ȱcorrespondȱtoȱGA1Ȭ2,ȱDȬ2,ȱEȬ2ȱandȱFȬ2;ȱdataȱfromȱtheȱsecondȱ
campaignȱareȱreferredȱtoȱasȱGA1Ȭ3,ȱDȬ3,ȱEȬ3ȱandȱFȬ3.ȱȱ
Theȱ multivariateȱ approachȱ wasȱ conductedȱ asȱ describedȱ above.ȱ Afterȱ
applyingȱ theȱ principalȱ factorsȱ analysisȱ theȱ 17ȱ variablesȱ wereȱ groupedȱ inȱ twoȱ
newȱfactorsȱ(tableȱ5).ȱTheseȱfactorsȱexplainȱ62.96ȱ%ȱofȱtheȱoriginalȱdataȱvariance.ȱ
Theȱfirstȱfactor,ȱ#1ȱisȱpredominantȱandȱexplainsȱaȱ41.3ȱ%ȱofȱtheȱvariance.ȱItȱlinksȱ
theȱpollutionȱcausedȱbyȱPAHsȱandȱmetalsȱ(Zn,ȱPb,ȱCu,ȱNi,ȱVȱandȱHg)ȱboundȱtoȱ
sedimentȱ byȱ relatingȱ theseȱ contaminantsȱ withȱ theȱ toxicityȱ (amphipods,ȱ
polychaete,ȱ seaȱ urchin)ȱ andȱ alterationȱ (numberȱ ofȱ speciesȱ andȱ percentageȱ ofȱ
crustacea).ȱTheȱsecondȱfactor,ȱ#2,ȱexplainsȱ21.6ȱ%ȱofȱtheȱvarianceȱandȱdepictsȱtheȱ
relationshipȱ betweenȱ certainȱ metalsȱ (Cuȱ andȱ V)ȱ withȱ alterationȱ (specificȱ
richness,ȱ%ȱofȱpolychaete)ȱandȱpotentialȱtoxicityȱ(Microtox®ȱtest).ȱȱ
- 298 -
Tableȱ 5.ȱ Sortedȱ rotatedȱ factorȱ loadingsȱ (pattern)ȱ ofȱ 17ȱ variablesȱ forȱ theȱ
fourȱ principalȱ factorsȱ resultingȱ fromȱ theȱ multivariateȱ analysisȱ ofȱ theȱ singleȱ
resultsȱ obtainedȱ fromȱ theȱ chemicalȱ analysis,ȱ theȱ acuteȱ toxicityȱ testsȱ andȱ theȱ
alterationȱ parametersȱ forȱ theȱ studyȱ ofȱ theȱ sedimentsȱ qualityȱ inȱ CormeȬLaxeȱ
2004Ȭ2006.ȱ
ȱȱ
FACTORȱ1ȱ
FACTORȱ2ȱ
ȱȱ
41.32ȱ
Znȱ
0.55ȱ
21.64ȱ
ņȱ
Pbȱ
0.79ȱ
Cuȱȱ
0.70ȱ
0.59ȱ
Niȱȱ
0.94ȱ
ņȱ
Vȱȱ
0.64ȱ
Hgȱ
0.88ȱ
0.72ȱ
ņȱ
PAHȱ
0.89ȱ
Corophiumȱbioassayȱ
0.88ȱ
Arenicolaȱbioassayȱ
0.83ȱ
Microtoxȱtestȱ
ņȱ
Paracentrotusȱassayȱ
0.72ȱ
Numberȱofȱspeciesȱ
0.50ȱ
ņȱ
Specificȱrichnesȱ
Diversityȱ
%ȱMoluscaȱ
%ȱPolychaetaȱ
%ȱCrustaceaȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.87ȱ
ņȱ
ņȱ
ņȱ
0.92ȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.49ȱ
0.78ȱ
ņȱ
ȱ
Figureȱ 6ȱ showsȱ theȱ factorȱ scoresȱ forȱ theȱ 8ȱ cases.ȱ Factorȱ #1,ȱ whichȱ isȱ
definedȱasȱtheȱpollutionȱcausedȱbyȱPAHsȱandȱtheȱmetalsȱZn,ȱPb,ȱCu,ȱNi,ȱVȱandȱ
Hg,ȱhasȱaȱpositiveȱloadingȱinȱDȬ2ȱ(1.7),ȱEȬ2ȱ(0.3),ȱFȬ2ȱ(1.5)ȱandȱFȬ3ȱ(0.1).ȱTheȱthreeȱ
firstȱcasesȱcorrespondȱtoȱtheȱfirstȱCormeȬLaxeȱsurveyȱ(2004Ȭ2005)ȱwhereasȱFȬ3ȱȱ
- 299 -
2.5
D-2
F-2
FACTOR 1
1.5
E-2
0.5
F-3
D-3
-0.5
E-3
GA1-2
GA1-3
-1.5
1.5
D-2
FACTOR 2
0.5
GA1-2
F-2
D-3
-0.5
E-3
F-3
GA1-3
E-2
-1.5
ȱ
Figureȱ6.ȱEstimatedȱfactorȱscoresȱforȱtheȱtwoȱfactorsȱinȱeachȱofȱtheȱ8ȱcases.ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
- 300 -
correspondsȱ toȱ theȱ secondȱ stationȱ Fȱ surveyȱ (2005Ȭ2006).ȱ Weȱ canȱ seeȱ howȱ theȱ
scoreȱ forȱ factorȱ 1ȱ inȱ D,ȱ E,ȱ andȱ Fȱ decreasedȱ asȱ timeȱ wentȱ on,ȱ whileȱ remainingȱ
positiveȱ forȱ stationȱ F,ȱ thisȱ indicatingȱ thatȱ sedimentsȱ fromȱ theseȱ stationsȱ haveȱ
recoveredȱ butȱ thatȱ degradationȱ persistsȱ inȱ stationȱ F.ȱ Factorȱ #2ȱ showsȱ metalȱ
contaminationȱbyȱCuȱandȱVȱlinkedȱtoȱalterationȱandȱpotentialȱtoxicityȱhavingȱaȱ
positiveȱloadingȱforȱDȬ2ȱ(0.8).ȱTheȱfactorȱscoreȱdecreasedȱwithȱtimeȱinȱstationsȱDȱ
andȱFȱwhereasȱstationȱEȱpresentedȱanȱincreaseȱdespiteȱitȱnotȱhavingȱaȱpositiveȱ
loading.ȱFigureȱ7ȱshowsȱtheȱprevalenceȱofȱeachȱfactorȱinȱeveryȱstationȱaccordingȱ
toȱ theȱ statisticalȱ differencesȱ obtainedȱ inȱ theȱ ANOVAȱ analysesȱ inȱ comparisonȱ
withȱtheȱreferenceȱsiteȱ(GA1).ȱTheȱsignificantȱdifferencesȱinȱFactorȱ#1ȱcomparedȱ
withȱ theȱ referenceȱ stationȱ didȱ notȱ decreaseȱ duringȱ theȱ 2004Ȭ2006ȱ periodȱ withȱ
respectȱtoȱtheȱstationsȱD,ȱEȱandȱF,ȱmeaningȱthatȱalthoughȱrecoveryȱwasȱdetectedȱ
inȱtheȱMAAȱanalysis,ȱDȱandȱEȱareȱnotȱasȱcleanȱasȱtheȱreferenceȱstation,ȱwhereasȱ
siteȱ Fȱ continuedȱ toȱ presentȱ degradationȱ dueȱ toȱ aȱ mixtureȱ ofȱ variousȱ
contaminantsȱ includingȱ PAHsȱ andȱ metals.ȱ Onȱ theȱ otherȱ handȱ theȱ potentialȱ
pollutionȱ dueȱ toȱ theȱ metalsȱ Cuȱ andȱ Vȱ (Factorȱ #2)ȱ presentȱ inȱ Dȱ andȱ E,ȱ wasȱ notȱ
apparentȱ forȱ theȱ finalȱ survey,ȱ insofarȱ asȱ significantȱ differencesȱ comparedȱ withȱ
theȱcontrolȱGA1ȱwereȱconcerned.ȱȱ
Theȱ CormeȬLaxeȱ studyȱ showedȱ thatȱ theȱ presenceȱ ofȱ bothȱ PAHsȱ andȱ aȱ
mixtureȱ ofȱ metalsȱ Zn,ȱ Pb,ȱ Cu,ȱ Ni,ȱ Vȱ andȱ Hgȱ initiallyȱ causedȱ environmentalȱ
degradationȱatȱtheȱstationsȱD,ȱEȱandȱF.ȱTheȱrecoveryȱofȱtheȱstationsȱinȱDȱandȱEȱ
(MAA)ȱshowȱthatȱtheȱsourceȱofȱtheȱPAHsȱpollutionȱisȱrelatedȱtoȱtheȱPrestigeȱoilȱ
spill.ȱHowever,ȱtheȱpresenceȱofȱmetalsȱwithȱdifferentȱcharacteristicsȱfromȱthoseȱ
boundȱtoȱtheȱoriginalȱfuelȱoilȱfromȱtheȱPrestige,ȱsuggestȱtheȱpossibleȱexistenceȱofȱ
anotherȱ sourceȱ orȱ sourcesȱ ofȱ contaminationȱ inȱ theȱ area.ȱ Previousȱ studiesȱ haveȱ
shownȱ thatȱ theȱ neighbouringȱ Riasȱ andȱ coastalȱ watersȱ actȱ asȱ aȱ sourceȱ ofȱ
dissolvedȱ andȱ particulateȱ traceȱ metalsȱ (CobeloȬGarcíaȱ etȱ al.,ȱ 2005).ȱ Researchȱ
- 301 -
resultsȱ obtainedȱ showȱ sedimentsȱ fromȱ theȱ stationsȱ Dȱ andȱ Eȱ toȱ haveȱ recoveredȱ
whereasȱdegradationȱremainsȱatȱtheȱFȱsiteȱnearestȱtoȱtheȱcoast,ȱhighlightingȱtheȱ
influenceȱ ofȱ theȱ mentionedȱ causesȱ differentȱ thanȱ theȱ Prestige.ȱ Initialȱ potentialȱ
degradationȱ causedȱ byȱ Cuȱ andȱ Vȱ wasȱ alsoȱ detected,ȱ althoughȱ notȱ presentȱ forȱ
theȱfinalȱsurvey.ȱ
ȱ
F2
F1
ȱ
F2
ȱ
D-2
F1
D-3
ȱ
F2
F1
F2
ȱ
E-2
F1
ȱ
E-3
ȱ
F2
F2
F1
F1
ȱ
F-2
ȱ
F-3
Figureȱ 6.ȱ Pieȱ chartsȱ showingȱ theȱ significantȱ differencesȱ ofȱ theȱ factorsȱ
scoreȱinȱeveryȱstudyȱsiteȱ–CormeȬLaxeȱ(2004Ȭ2006)Ȭȱrelatedȱtoȱtheȱreferenceȱsiteȱ
GA1ȱ (black:ȱ pȱ <ȱ 0.01;ȱ grey:ȱ 0.01<ȱ pȱ >ȱ 0.05;ȱ white:ȱ notȱ significantlyȱ differencesȱ
p>0.05).ȱ Factorȱ #1:ȱ PAHsȬZnȬȱ PbȬNiȬCuȬVȬHgȬpollution;ȱ Factorȱ #2:ȱ CuȬVȬ
pollution.ȱȱ
- 302 -
4.ȱConclusionsȱ
TheȱWOEȱapproachȱemployedȱinȱthisȱstudyȱhasȱbeenȱappliedȱtoȱ3ȱlinesȱofȱ
evidenceȱ (contamination,ȱ toxicityȱ andȱ alteration)ȱ inȱ addressingȱ 3ȱ distinctȱ
objectives.ȱFirstȱofȱallȱaȱrevisionȱofȱtheȱsedimentȱqualityȱfollowingȱtheȱPrestigeȱoilȱ
spillȱhasȱbeenȱcarriedȱoutȱinȱtheȱAtlanticȱIslands,ȱanȱareaȱwithȱaȱhighȱecologicalȱ
relevance.ȱThisȱwasȱachievedȱbyȱapplyingȱaȱmultivariateȱandȱANOVAȱanalyses.ȱ
Inȱ orderȱ toȱ assessȱ theȱdevelopmentȱofȱtheȱqualityȱofȱtheȱsedimentsȱaffectedȱbyȱ
theȱ spillȱ aȱ setȱ ofȱ stationsȱ wasȱ studiedȱ usingȱ theȱ sameȱ timeȬdependentȱ
methodologyȱ forȱ theȱ Ciesȱ Islandȱ (2004Ȭ2006)ȱ andȱ inȱ CormeȬLaxeȱ (2004Ȭ2006).ȱ
Resultsȱ obtainedȱ haveȱ identifiedȱ PAHsȱ relatedȱ toȱ theȱ Prestigeȱ oilȱ spillȱ asȱ theȱ
mainȱcontaminantȱinȱtheȱsitesȱstudiedȱonȱtheȱGalicianȱCoast.ȱAȱsourceȱofȱmetalsȱ
hasȱbeenȱidentifiedȱinȱtheȱAtlanticȱIslandsȱNationalȱParkȱwhichȱseemsȱnotȱtoȱbeȱ
producingȱ biologicalȱ effectsȱ althoughȱ furtherȱ researchȱ ofȱ thisȱ inputȱ ofȱ metalsȱ
shouldȱ beȱ carriedȱ outȱ especiallyȱ forȱ theȱ Onsȱ Island.ȱ Inȱ CormeȬLaxeȱ anȱ
additionalȱ sourceȱ orȱ sourcesȱ ofȱ aȱ mixtureȱ ofȱ contaminantsȱ wasȱ alsoȱ detected.ȱ
Pollutionȱ hasȱ decreasedȱ inȱ recentȱ yearsȱ inȱ bothȱ theȱ Atlanticȱ Islandsȱ Nationalȱ
ParkȱandȱCormeȬLaxeȱareasȱ,ȱalthoughȱthereȱisȱstillȱsomeȱdegradationȱpresentȱinȱ
someȱareas,ȱparticularlyȱȱinȱCormeȬLaxe.ȱȱȱ
TheȱinformationȱobtainedȱinȱthisȱstudyȱhasȱdemonstratedȱthatȱWOEȱisȱaȱ
suitableȱ toolȱ forȱ monitoringȱ environmentalȱ riskȱ assessmentȱ allowingȱ sourcesȱ
andȱfateȱofȱcontaminantsȱtoȱbeȱdifferentiatedȱinȱadditionȱtoȱtheirȱpotentialȱrisk.ȱ
Theȱ innovativeȱ applicationȱ ofȱ theȱ classicalȱ WOEȱ methodologyȱ hasȱ provedȱ
usefulȱ inȱ obtainingȱ moreȱ objectiveȱ resultsȱ andȱ itsȱ useȱ isȱ recommendedȱ inȱ theȱ
designȱandȱimplementationȱofȱmonitoringȱprogramsȱinȱareasȱthatȱhaveȱsufferedȱ
contaminationȱ episodesȱ throughȱ theȱ selectingȱ ofȱ appropriateȱ linesȱ ofȱ evidenceȱ
onȱaȱcaseȱbyȱcaseȱbasisȱȱ
- 303 -
5.ȱAcknowledgementsȱ
Theȱ workȱ describedȱ wasȱ partlyȱ supportedȱ byȱ theȱ projectsȱ VEM2003Ȭ
20563/INTER,ȱ andȱ CTM2005Ȭ07282ȬC03Ȭ01/TECNOȱ financedȱ byȱ theȱ Spanishȱ
Educationȱ andȱ Scienceȱ Ministryȱ andȱ byȱ theȱ CISȱ fundedȱ byȱ theȱ Ministryȱ ofȱ
Environment.ȱ Carmenȱ MoralesȬCasellesȱ thanksȱ theȱ Ministryȱ ofȱ Educationȱ andȱ
Scienceȱforȱ fundingȱherȱ researchȱ fellowshipȱ(FPU).ȱ Specialȱthanksȱareȱgivenȱ toȱ
theȱ CISȱ membersȱ forȱ theirȱ supportȱ andȱ helpȱ inȱ theȱ chemicalȱ analysisȱ andȱ theȱ
benthicȱ communityȱ information;ȱ authorsȱ thankȱ Nuriaȱ Fernándezȱ forȱ theȱ
ParacentrotusȱdataȱandȱherȱhelpȱinȱtheȱfirstȱbioassayȱcarriedȱoutȱwithȱArenicola.ȱ
Weȱ appreciateȱ theȱ contributionȱ ofȱ theȱ reviewersȱ andȱ theȱ helpȱ ofȱ Thomasȱ
Ransome.ȱ
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- 306 -
Aȱweightȱofȱevidenceȱapproachȱforȱqualityȱassessmentȱinȱ
sedimentsȱimpactedȱbyȱanȱoilȱspill:ȱtheȱroleȱofȱaȱnewȱlineȱofȱ
evidenceȱusingȱaȱsetȱofȱbiomarkersȱ
CarmenȱMoralesȬCaselles1,2,*,ȱInmaculadaȱRiba1,2,ȱCarmenȱSarasquete1,ȱT.ȱÁngelȱ
DelValls1,2ȱ
1
ȱUnidadȱAsociadaȱdeȱCalidadȱAmbientalȱyȱPatologíaȱ(CSICȱ&ȱUCA)ȱinstitutoȱdeȱ
CienciasȱMarinasȱdeȱAndalucíaȱ(ICMANȬCSIC),ȱAvda.ȱRepúblicaȱSaharauiȱs/n,ȱPuertoȱ
Realȱ11510,ȱCádiz,ȱSpainȱ
UNESCOȱUNITWIN/WiCop,ȱFacultadȱdeȱCienciasȱdelȱMarȱyȱAmbientales,ȱ
2ȱ
UniversidadȱdeȱCádiz,ȱPolígonoȱRíoȱSanȱPedroȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱSpain.ȱ
Abstractȱ
Aȱ setȱ ofȱ biomarkersȱ hasȱ beenȱ chosenȱ andȱ analyzedȱ inȱ targetȱ tissuesȱ ofȱ
twoȱ invertebrateȱ speciesȱ afterȱ aȱ 28Ȭdȱ exposureȱ toȱ sedimentsȱ fromȱ theȱ Galicianȱ
Coastȱ inȱ anȱ attemptȱ toȱ incorporateȱ aȱ newȱ lineȱ ofȱ evidenceȱ (LOE)ȱ toȱ aȱ classicalȱ
weightȱ ofȱ evidenceȱ (WOE),ȱ anȱ approachȱ designedȱ toȱ assessȱ sedimentȱ qualityȱ
fourȱ yearsȱ afterȱ theȱ oilȱ spillȱ ofȱ theȱ tankerȱ Prestigeȱ (2002).ȱ ȱ Sublethalȱ bioassaysȱ
withȱ crabsȱ andȱ clamsȱ wereȱ carriedȱ outȱ underȱ laboratoryȱ andȱ fieldȱ conditionsȱ
andȱ includedȱ theȱ determinationȱ of:ȱ Ethoxyresorufinȱ OȬdeethylaseȱ (EROD)ȱ
activityȱ toȱ assessȱ theȱ phaseȱ Iȱ detoxificationȱ system;ȱ glutathioneȬSȬtransferaseȱ
(GST)ȱasȱaȱphaseȱIIȱdetoxificationȱenzymeȱbutȱalsoȱimplicatedȱinȱoxidativeȱstressȱ
events;ȱglutathioneȱperoxidaseȱ(GPX),ȱglutathioneȱreductaseȱ(GR)ȱandȱtheȱferricȱ
reducingȱ abilityȱ ofȱ plasmaȱ (FRAP)ȱ assayȱ wereȱ analyzedȱ toȱ determineȱ theȱ
tissuesȇȱ antioxidantȱ activity.ȱ Theȱ integrationȱ ofȱ biomarkersȱ withȱ sedimentsȱ
contamination,ȱ acuteȱ toxicityȱ andȱ benthicȱ alterationȱ parametersȱ provideȱ anȱ
“earlyȱwarning”ȱtoolȱwhichȱnotȱonlyȱindicatesȱtheȱenvironmentalȱqualityȱofȱanȱ
area,ȱ itȱ alsoȱ constitutesȱ anȱ advisoryȱ toolȱ forȱ ȱ potentialȱ ecologicalȱ risks.ȱ Theȱ
presentȱ studyȱ demonstratesȱ thatȱ theȱ useȱ ofȱ theȱ setȱ ofȱ biomarkersȱ asȱ partȱ ofȱ aȱ
WOEȱ approachȱ designedȱ toȱ assessȱ contaminatedȱ sedimentsȱ contributesȱ addedȱ
ȱEnvironmentȱToxicologyȱandȱChemistryȱ(enviado)
- 307 -
valueȱtoȱtheȱclassicalȱLOEsȱandȱallowsȱcharacterizingȱtheȱenvironmentalȱstatusȱ
ofȱtheȱstudiedȱareaȱinȱaȱmoreȱpreciseȱandȱaccurateȱway.ȱ
Keywords:ȱȱPAHs,ȱcontamination,ȱtoxicity,ȱsublethal,ȱWOE.ȱ
1.ȱIntroductionȱ
Chemicalȱ analysisȱ normallyȱ isȱ theȱ mainȱ toolȱ inȱ sedimentȱ qualityȱ
assessmentȱ evenȱ thoughȱ chemicalȱ concentrationsȱ aloneȱ areȱ inadequateȱ forȱ
predictionȱofȱbiologicalȱconsequences.ȱTheȱbiologicalȱeffectsȱcanȱbeȱestablishedȱ
basedȱonȱlaboratoryȱtestsȱthatȱdetermineȱtoxicȱresponses,ȱasȱwellȱasȱfieldȱdataȱonȱ
theȱ communitiesȱ livingȱ inȱ theȱ sedimentsȱ allowȱ toȱ establishȱ whetherȱ thereȱ isȱ
observableȱ pollutionȬinducedȱ degradationȱ effectȱ inȱ theȱ biotaȱ [1].ȱ Weightȱ ofȱ
evidenceȱ (WOE)ȱ investigationsȱ determinesȱ possibleȱ ecologicalȱ impactsȱ fromȱ
chemicalsȱorȱotherȱstressorsȱbasedȱonȱmultipleȱlinesȱofȱevidenceȱ(LOEs)ȱ[2]ȱandȱ
haveȱ beenȱ widelyȱ usedȱ inȱ recentȱ yearsȱ toȱ assessȱ sedimentȱ qualityȱ aroundȱ theȱ
worldȱ[3,4]ȱincludingȱdifferentȱareasȱinȱtheȱIberianȱPeninsulaȱ[5,6,7,8,9].ȱȱ
Sinceȱ theȱ sinkingȱ ofȱ theȱ tankerȱ Prestigeȱ (2002),ȱ whichȱ spiltȱ aboutȱ 63,000ȱ
tonnesȱ ofȱ heavyȱ fuelȱ oilȱ (aȱ mixtureȱ ofȱ saturatedȱ hydrocarbons,ȱ aromaticȱ
hydrocarbons,ȱresins,ȱandȱasphaltenes,ȱwithȱmostȱofȱtheȱPAHsȱbeingȱofȱmediumȱ
toȱ highȱ molecularȱ weight)ȱ andȱ mainlyȱ affectedȱ theȱ Galicianȱ Coast,ȱ severalȱ
investigationsȱ haveȱ focusedȱ onȱ determiningȱ theȱ biologicalȱ effectsȱ andȱ
environmentalȱ statusȱ afterȱ thisȱ dramaticȱ episodeȱ byȱ followingȱ singleȱ linesȱ ofȱ
evidence,ȱ suchȱ asȱ chemicalȱ analysesȱ [10,11,12],ȱ toxicityȱ [13,14,15,16]ȱ orȱ benthicȱ
alterationȱ [17,18].ȱ Recently,ȱ authorsȱ presentedȱ aȱ reportȱ [9]ȱ whereȱ aȱ classicalȱ
WOEȱ approachȱ basedȱ onȱ threeȱ linesȱ ofȱ evidenceȱ (physicochemicalȱ
characterizationȱ ofȱ theȱ sediments,ȱ determinationȱ ofȱ acuteȱ toxicityȱ andȱ benthicȱ
alteration)ȱ wasȱ carriedȱ outȱ inȱ theȱ Galicianȱ Coast;ȱ theȱ sedimentȱ qualityȱ ofȱ theȱ
areaȱ wasȱ monitoredȱ duringȱ theȱ timeȱ andȱ aȱ generalȱ recoveryȱ ofȱ theȱ
- 308 -
environmentalȱ healthȱ wasȱ observed.ȱ Howeverȱ thereȱ wereȱ signsȱ thatȱ otherȱ
sourcesȱ ofȱ contaminantsȱ apartȱ fromȱ theȱ Prestigeȱ oilȱ spillȱ couldȱ beȱ producingȱ
someȱ environmentalȱ stressȱ toȱ theȱ exposedȱ organisms.ȱ Theȱ aimȱ ofȱ theȱ presentȱ
studyȱisȱtoȱuseȱaȱnewȱlineȱofȱevidenceȱ(LOE)ȱwithȱtheȱweightȱofȱevidenceȱ(WOE)ȱ
approachȱ toȱ improveȱ theȱ sedimentȱ qualityȱ assessmentȱ conductedȱ byȱ previousȱ
studiesȱ inȱ theȱ Galicianȱ Coast.ȱ Theȱ linesȱ ofȱ evidenceȱ selectedȱ constituteȱ anȱ
improvementȱofȱtheȱclassicalȱWOEȱincludingȱlaboratoryȱandȱfieldȱstudiesȱbasedȱ
onȱ biomarkersȱ determinations.ȱ Biomarkersȱ canȱ actȱ asȱ anȱ importantȱ earlyȱ
warningȱ systemȱ byȱ tellingȱ whetherȱ environmentalȱ pollutantsȱ areȱ presentȱ atȱ
sufficientlyȱ highȱ concentrationsȱ toȱ causeȱ anȱ effectȱ [19].ȱ Chemicalsȱ suchȱ asȱ theȱ
PolyciclicȱAromaticȱHydrocarbonsȱ(PAHs)ȱhaveȱveryȱshortȱbiologicalȱhalfȬlivesȱ
inȱmostȱspeciesȱbutȱmayȱneverthelessȱhaveȱlongȬtermȱeffectsȱ[19].ȱInȱthisȱsense,ȱ
someȱcompoundsȱmightȱnotȱproduceȱacuteȱtoxicȱeffectsȱbutȱsublethalȱeffectsȱcanȱ
beȱexpected.ȱTheȱmainȱaimsȱofȱthisȱresearchȱare:ȱ(a)ȱtoȱproveȱtheȱfeasibilityȱandȱ
viabilityȱ ofȱ incorporatingȱ newȱ linesȱ ofȱ evidenceȱ toȱ theȱ classicalȱ methodologyȱ
employedȱinȱtheȱWOEȱandȱitsȱapplicationȱtoȱassessȱtheȱenvironmentalȱqualityȱofȱ
oilȱ contaminatedȱ sedimentsȱ ,ȱ (b)ȱ toȱ monitorȱ theȱ sedimentȱ qualityȱ 4ȱ yearsȱ afterȱ
theȱ impactȱ ofȱ anȱ accidentalȱ oilȱ spillȱ byȱ usingȱ aȱ newlyȱ WOEȱ approach,ȱ (c)ȱ toȱ
determineȱtheȱextentȱofȱtheȱimpactȱfromȱtheȱspillȱaddressingȱtheȱcontamination,ȱ
pollutionȱ andȱ noȱ effectsȱ inȱ theȱ stationsȱselected,ȱincludingȱtheȱidentificationȱofȱ
theȱcontaminantsȱresponsibleȱforȱtheȱdamage.ȱȱȱȱ
2.ȱMaterialȱandȱmethodsȱ
2.1.ȱApproachȱ
TheȱstudyȱwasȱperformedȱonȱtwoȱareasȱofȱtheȱGalicianȱCoastȱ(NWȱSpain)ȱ
importantlyȱaffectedȱbyȱtheȱPrestigeȱoilȱspillȱinȱ2002ȱ(Figureȱ1):ȱCiesȱIslandȱ(A,ȱB,ȱ
C)ȱ inȱ theȱ Atlanticȱ Islandȱ Nationalȱ Parkȱ andȱ theȱ Bayȱ ofȱ CormeȬLaxeȱ (D,ȱ E,ȱ F).ȱ
- 309 -
Ciesȱ Island,ȱ locatedȱ inȱ theȱ Atlanticȱ Islandȱ Nationalȱ Parkȱ actedȱ asȱ aȱ naturalȱ
barrierȱprotectingȱtheȱriasȱfromȱtheȱentranceȱofȱtheȱfuel.ȱTheȱBayȱofȱCormeȬLaxeȱ
isȱ alsoȱ consideredȱ aȱ placeȱ withȱ highȱ ecologicalȱ relevanceȱ withȱ aȱ lowȱ
anthropogenicȱ andȱ industrialȱ influenceȱ withȱ fishingȱ andȱ farmingȱ beingȱ theȱ
mainȱeconomicȱactivities.ȱ
ȱ
ȱ
ȱ
ƒE
ƒF
Ría de ƒD
Corme-Laxe
ȱ
ȱ
Spain
ȱ
ȱ
•C •A
•B
ȱ
ȱ
N
Cíes
Islands
E
W
S
ȱ
Figureȱ 1.ȱ Mapȱ ofȱ theȱ coastalȱ areaȱ ofȱ Galiciaȱ (NWȱ Spain)ȱ showingȱ theȱ
samplingȱ sitesȱ inȱ theȱ Atlanticȱ Islandȱ Nationalȱ Parkȱ (A,ȱ B,ȱ C)ȱ andȱ theȱ areaȱ ofȱ
CormeȬLaxeȱ(D,ȱEȱandȱF).ȱ
Theȱ4ȱlinesȱofȱevidenceȱemployedȱinȱtheȱWOEȱapproachȱincluded:ȱȱ
(a)ȱ sedimentȱ contamination:ȱ physicochemicalȱ characterizationȱ ofȱ
sedimentsȱ byȱ analyzingȱ PAHsȱ (acenaphtalene,ȱ acenaphtylene,ȱ anthracene,ȱ
benzo(a)anthracene,ȱ
perylene,ȱ
benzo(a)pyrene,ȱ
benzo(k)fluoranthene,ȱ
benzo(b)fluoranthene,ȱ
chrysene,ȱ
dibenzo(a,h)ȱ
benzo(g,h,i)ȱ
anthracene,ȱ
fenanthrene,ȱfluoranthene,ȱfluorene,ȱindeneȱ(1,2,3,cdȱ)pyrene,ȱnaphthalene,ȱandȱ
- 310 -
pyrene)ȱ usingȱ GCȬMSȱ withȱ selectedȱ ionȱ monitoringȱ andȱ traceȱ metalsȱ (Zn,ȱ Pb,ȱ
Cu,ȱNiȱandȱHg)ȱwithȱanodicȱvoltamperimetryȱ[16];ȱȱ
(b)ȱ acuteȱ toxicityȱ andȱ bioaccumulation:ȱ ȱ byȱ performingȱ sedimentȱ
bioassaysȱsuchȱasȱtheȱcommercialȱassayȱMicrotox®ȱ[20],ȱtheȱamphipodȱmortalityȱ
testȱ withȱ Corophiumȱ volutatorȱ [20],ȱ theȱ ȱ polychaetaȱ mortalityȱ assayȱ [21]ȱ andȱ
bioaccumulationȱexperimentȱwithȱArenicolaȱmarinaȱ[22];ȱȱ
(c)ȱ‘inȱsituȱalteration’:ȱBenthicȱalterationȱwasȱselectedȱandȱdeterminedȱbyȱ
measuringȱ parametersȱ inȱ situȱ basedȱ inȱ taxonomicȱ identificationsȱ andȱ
communityȱdescriptiveȱstatisticsȱ(abundanceȬbiomassȱanalysis,ȱspeciesȱrichness,ȱ
diversity,ȱdominanceȱandȱproportionsȱofȱtheȱmajorȱtaxonomicȱgroups)ȱ[5];ȱȱ
(d)ȱ laboratoryȱ andȱ fieldȱ studiesȱ basedȱ onȱ biomarkersȱ byȱ usingȱ twoȱ
invertebrateȱ species,ȱ theȱ crabȱ Carcinusȱ maenasȱ andȱ theȱ clamȱ Ruditapesȱ
philippinarum,ȱ andȱ aȱ setȱ ofȱ biomarkersȱ [23,24]:ȱ mixedȱ functionȱ oxygenaseȱ
activity,ȱ whichȱ isȱ theȱ firstȱ modeȱ ofȱ detoxificationȱ inȱ manyȱ organicȱ pollutants,ȱ
wasȱ measuredȱ usingȱ theȱ adaptedȱ ERODȱ assay;ȱ theȱ phaseȱ IIȱ metabolizingȱ
enzymeȱGlutathioneȬSȬtransferaseȱ(GST)ȱactivityȱwasȱdeterminedȱbyȱmonitoringȱ
theȱ rateȱ ofȱ conjugationȱ ofȱ glutathioneȱ (GSH)ȱ toȱ 1ȬchloroȬ2,4Ȭdinitrobenzeneȱ
(CDNB)ȱ atȱ 340ȱ nm;ȱ theȱ oxidationȱ ofȱ 1ȱ mMȱ NADPHȱ byȱ Glutathioneȱ Reductaseȱ
activityȱ(GR)ȱinȱtheȱpresenceȱofȱ10ȱmMȱoxidizedȱglutathioneȱwasȱalsoȱmonitoredȱ
atȱ340ȱnm;ȱtheȱantioxidantȱGlutathioneȱPeroxidaseȱactivityȱ(GPX)ȱwasȱanalyzedȱ
byȱ determiningȱ theȱ oxidationȱ ofȱ NADPHȱ withȱ theȱ presenceȱ ofȱ 1.25ȱ mMȱ
hydrogenȱperoxide;ȱtheȱFRAPȱassay,ȱferricȱreducingȱabilityȱofȱplasma,ȱallowsȱaȱ
measureȱ ofȱ theȱ antioxidantȱ capacity;ȱ allȱ theȱ biomarkersȱ responsesȱ wereȱ
normalizedȱwithȱtheȱtotalȱproteinȱcontent.ȱ
2.2.ȱDataȱintegrationȱȱ
- 311 -
Theȱ dataȱ obtainedȱ fromȱ theȱ differentȱ LOEsȱ wereȱ integratedȱ throughȱ aȱ
multivariateȱ analysisȱ approachȱ basedȱ onȱ linkingȱ allȱ theȱ variablesȱ obtainedȱ [7]ȱ
andȱ aȱ pieȱ chartȱ representationȱ ofȱ comparisonsȱ betweenȱ sitesȱ ofȱ multivariateȱ
factorsȱ [7,9].ȱ Theȱ multivariateȱ analysisȱ wasȱ performedȱ usingȱ principalȱ
componentȱanalysisȱ(PCA)ȱasȱtheȱextractionȱprocedure,ȱwhichȱisȱaȱmultivariateȱ
statisticalȱtechniqueȱtoȱexploreȱvariableȱdistributionsȱ[25].ȱTheȱoriginalȱdataȱsetȱ
usedȱ inȱ theȱ analysisȱ includedȱ theȱ variablesȱ obtainedȱ fromȱ theȱ 4ȬLOEsȱ andȱ itsȱ
objectiveȱ wasȱ toȱ deriveȱ aȱ reducedȱ numberȱ ofȱ newȱ variablesȱ asȱ linearȱ
combinationsȱ ofȱ theȱ originalȱ variables.ȱ Thisȱ providesȱ aȱ descriptionȱ ofȱ theȱ
structureȱ ofȱ theȱ dataȱ withȱ theȱ minimumȱ lossȱ ofȱ information.ȱ Forȱ theȱ
representationȱ ofȱ theȱ pieȱ charts,ȱ theȱ newȱ factorsȱ obtainedȱ fromȱ theȱ PCAȱ wereȱ
submittedȱ toȱ ANOVAȱ andȱ Tukeyȱ testsȱ whichȱ identifiedȱ significantȱ differencesȱ
inȱ sensitivityȱ amongȱ stationsȱ andȱ controlsȱ forȱ eachȱ factorȱ [9];ȱ everyȱ studyȱ siteȱ
hasȱ aȱ pieȱ chartȱ dividedȱ intoȱ theȱ obtainedȱ factorsȱ whichȱ useȱ differentȱ coloursȱ
dependingȱonȱtheȱlevelȱofȱsignificantȱdifferencesȱinȱrelationȱwithȱtheȱreference.ȱ
3.ȱResultsȱandȱdiscussionȱ
Tableȱ 1ȱ showsȱ theȱ summarizedȱ resultsȱ ofȱ theȱ differentȱ parametersȱ
analyzed.ȱ Inȱ general,ȱ theȱ concentrationȱ ofȱ chemicalsȱ variesȱ amongȱ theȱ stationsȱ
fromȱtheȱAINPȱ(A,ȱBȱandȱC)ȱandȱthoseȱfromȱCormeȬLaxeȱ(D,ȱE,ȱandȱF)ȱalthoughȱ
noȱgeneralȱpatternȱwasȱobserved,ȱexceptȱforȱHgȱwhichȱwasȱhigherȱinȱallȱCormeȬ
Laxeȱ sites.ȱ Stationȱ Aȱ andȱ Fȱ presentedȱ theȱ highestȱ contentsȱ inȱ metals.ȱ Mainly,ȱ
acuteȱtoxicityȱandȱPAHsȱbioaccumulationȱwasȱhigherȱinȱorganismsȱexposedȱtoȱ
sedimentsȱ fromȱ theȱ Bayȱ ofȱ CormeȬLaxeȱ whereasȱ biomarkersȱ responsesȱ whereȱ
alsoȱ higherȱ inȱ theȱ areaȱ ofȱ CormeȬLaxe,ȱ bothȱ underȱ fieldȱ andȱ laboratoryȱ
deployments.ȱ Itȱ wasȱ notȱ observedȱ aȱ generalȱ patternȱ inȱ benthicȱ parametersȱ
betweenȱtheȱsamplingȱsites.ȱ
- 312 -
ȱ
ȱ
ȱ
Tableȱ 1.ȱ Summarizedȱ resultsȱ ofȱ chemicalȱ analysisȱ (mgKgȬ1ȱ forȱ metals,ȱ
ΐgKgȬ1ȱ forȱ PAHs)ȱ theȱ acuteȱ toxicityȱ testsȱ (Corophiumȱ andȱ Arenicola:ȱ %ȱ
mortality;ȱ Microtox:ȱ IC50;ȱ bioaccumulationȱ ofȱ PAHs:ȱ ΐgKgȬ1),ȱ biomarkerȱ
responsesȱ underȱ fieldȱ andȱ laboratoryȱ conditionsȱ (glutathioneȱ peroxidaseȱ
activityȱ GPX:ȱ nmol/min/mgȱ prot,ȱ glutathioneȱ transferaseȱ GSTȱ activityȱ
nmol/min/mgȱprot,ȱglutathioneȱreductaseȱGRȱactivityȱnmol/min/mgȱprot,ȱferricȱ
reducingȱ abilityȱ ofȱ plasmaȱ FRAPȱ activityȱ ΐM/mg/minȱ andȱ ERODȱ activityȱ
pmol/mg/min)ȱandȱtheȱalterationȱparametersȱforȱsedimentsȱfromȱtheȱAINPȱ(A,ȱ
B,ȱC)ȱandȱCormeȬLaxeȱ(D,ȱE,ȱF).ȱn.d.:ȱnotȱdetected;ȱn.a.:ȱnotȱavailable.ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
- 313 -
Benthicȱalterationsȱȱ
Biomarkersȱ(field)ȱ
Biomarkersȱ(laboratory)ȱ
Toxicityȱtestsȱ
Chemicalȱ
analysesȱ
ȱȱ
ȱȱ
Znȱȱ
Pbȱ
Cuȱ
Niȱ
Hgȱ
PAHȱ
Corophiumȱ
Arenicolaȱ
Microtoxȱ
Bioaccumulationȱ
PAHȱ
GPXȬcrabȬlabȱ
GPXȬclamȬlabȱ
GRȬcrabȬlabȱ
GRȬclamȬlabȱ
GSTȬcrabȬlabȱ
GSTȬclamȬlabȱ
ERODȬcrabȬlabȱ
ERODȬclamȬlabȱ
FRAPȬcrabȬlabȱ
FRAPȬclamȬlabȱ
GPXȬcrabȬfieldȱ
GPXȬclamȬfieldȱ
GRȬcrabȬfieldȱ
GRȬclamȬfieldȱ
GSTȬcrabȬfieldȱ
GSTȬclamȬfieldȱ
ERODȬcrabȬfieldȱ
ERODȬclamȬfieldȱ
FRAPȬcrabȬfieldȱ
FRAPȬclamȬfieldȱ
Numberȱofȱspeciesȱ
specificȱrichnessȱ
Diversityȱ
Dominanceȱ
%ȱMolluscaȱ
%ȱPolychaeteȱ
%ȱCrustaceaȱ
Aȱ
377ȱ
1.5ȱ
5.2ȱ
13.3ȱ
0.7ȱ
108ȱ
23ȱ
28ȱ
5631ȱ
Bȱ
91ȱ
0.9ȱ
1.4ȱ
2.4ȱ
0.8ȱ
67ȱ
20ȱ
28ȱ
9422ȱ
Cȱ
164ȱ
0.85ȱ
1.4ȱ
4.5ȱ
0.6ȱ
n.d.ȱ
17ȱ
22ȱ
1801ȱ
Dȱ
25ȱ
3.7ȱ
0.7ȱ
1.7ȱ
2ȱ
38ȱ
10ȱ
39ȱ
3977ȱ
Eȱ
19.9ȱ
7.3ȱ
0.43ȱ
1.5ȱ
2.1ȱ
52ȱ
17ȱ
17ȱ
21041ȱ
Fȱ
271ȱ
5.9ȱ
4.2ȱ
5.7ȱ
3.4ȱ
323ȱ
20ȱ
17ȱ
4398ȱ
2927ȱ
11.6ȱ
2.1ȱ
1.1ȱ
2.1ȱ
140ȱ
1293ȱ
0.1ȱ
0.3ȱ
3.9ȱ
10.6ȱ
17.8ȱ
10.5ȱ
0.7ȱ
2.9ȱ
1098ȱ
2061ȱ
0.1ȱ
0.2ȱ
2.7ȱ
10.4ȱ
28.5ȱ
5.1ȱ
15.3ȱ
0.50ȱ
15.3ȱ
20.0ȱ
37.0ȱ
2573ȱ
9.7ȱ
2.9ȱ
0.7ȱ
1.6ȱ
218ȱ
839ȱ
0.1ȱ
0.3ȱ
2.1ȱ
7.8ȱ
23.1ȱ
3.6ȱ
1.4ȱ
1.3ȱ
1564ȱ
372ȱ
3.0ȱ
0.1ȱ
n.a.ȱ
3.1ȱ
33.9ȱ
5ȱ
28.4ȱ
0.10ȱ
28.4ȱ
21.5ȱ
41.0ȱ
2666ȱ
8.2ȱ
4.5ȱ
0.9ȱ
2.3ȱ
407ȱ
1624ȱ
0.1ȱ
0.4ȱ
2.6ȱ
4.0ȱ
15.9ȱ
4.0ȱ
1.4ȱ
3.8ȱ
690ȱ
1199ȱ
0.0ȱ
0.1ȱ
n.a.ȱ
2.6ȱ
42.4ȱ
4.3ȱ
39.1ȱ
0.06ȱ
39.1ȱ
21.7ȱ
39.1ȱ
2616ȱ
19.3ȱ
6.1ȱ
0.9ȱ
3.4ȱ
430ȱ
1199ȱ
0.0ȱ
0.4ȱ
2.9ȱ
13.7ȱ
41.4ȱ
25.5ȱ
9.9ȱ
9.7ȱ
1489ȱ
3366ȱ
8.5ȱ
0.6ȱ
2.4ȱ
23.6ȱ
28.6ȱ
3ȱ
30ȱ
0.15ȱ
30.0ȱ
20.0ȱ
50.0ȱ
3912ȱ
19.5ȱ
3.1ȱ
0.6ȱ
11.7ȱ
684ȱ
910ȱ
0.1ȱ
0.4ȱ
2.9ȱ
12.1ȱ
193.1ȱ
3.2ȱ
9.5ȱ
14.7ȱ
7523ȱ
131ȱ
0.4ȱ
0.1ȱ
n.a.ȱ
2.0ȱ
32.1ȱ
3ȱ
40.1ȱ
0.19ȱ
32.4ȱ
20.4ȱ
47.2ȱ
3285ȱ
15.9ȱ
4.2ȱ
1.5ȱ
4.0ȱ
1071ȱ
848ȱ
0.1ȱ
0.2ȱ
1.6ȱ
6.4ȱ
125.7ȱ
7.0ȱ
23.4ȱ
8.0ȱ
6073ȱ
1558ȱ
0.5ȱ
0.1ȱ
n.a.ȱ
6.6ȱ
48.2ȱ
2.9ȱ
15.4ȱ
0.20ȱ
15.4ȱ
23.1ȱ
61.5ȱ
- 314 -
Theȱ multivariateȱ analysisȱ wasȱ usedȱ inȱ theȱ originalȱ dataȱ setȱ byȱ usingȱ
replicatesȱ(notȱaverages)ȱinȱorderȱtoȱlinkȱtheȱresultsȱobtainedȱfromȱtheȱdifferentȱ
linesȱ ofȱ evidenceȱ investigated.ȱ Theȱ factorȱ analysisȱ revealsȱ thatȱ theȱ originalȱ
variablesȱ canȱ beȱ groupedȱ intoȱ threeȱ newȱ factorsȱ whichȱ explainȱ aȱ 79ȱ %ȱ ofȱ theȱ
totalȱ varianceȱ (Tableȱ 2).ȱ Theȱ multivariateȱ analysisȱ isȱ aȱ toolȱ thatȱ allowsȱ usȱ toȱ
interpretȱ aȱ largeȱ groupȱ ofȱ differentȱ variablesȱ byȱ groupingȱ themȱ usingȱ
correlations;ȱ inȱ additionȱ itȱ indicatesȱ theȱ importanceȱ ofȱ eachȱ factorȱ inȱ everyȱ
singleȱstudyȱsite.ȱ
Tableȱ 2.ȱ Sortedȱ rotatedȱ factorȱ loadingsȱ ofȱ 36ȱ variablesȱ forȱ theȱ threeȱ
principalȱ factorsȱ resultingȱ fromȱ theȱ multivariateȱ analysisȱ ofȱ theȱ singleȱ resultsȱ
obtainedȱ fromȱ theȱ chemicalȱ analysis,ȱ theȱ acuteȱ toxicityȱ tests,ȱ theȱ suiteȱ ofȱ
biomarkersȱandȱtheȱalterationȱparametersȱforȱtheȱstudyȱofȱtheȱsedimentsȱqualityȱ
inȱ theȱ Galicianȱ Coast.ȱ Chemicals:ȱ loadingsȱ areȱ relatedȱ toȱ theȱ concentrationȱ ofȱ
contaminantsȱinȱsediments;ȱAcuteȱeffects:ȱloadingsȱexplainȱtheȱtoxicityȱdetectedȱ
byȱ theȱ acuteȱ assaysȱ andȱ theȱ bioaccumulationȱ ofȱ PAHsȱ inȱ Arenicola.ȱ Sublethalȱ
effects:ȱ loadingsȱ areȱ relatedȱ toȱ theȱ inductionȱ ofȱ biomarkers.ȱ Benthicȱ alteration:ȱ
loadingsȱ areȱ relatedȱ toȱ alterationȱ ofȱ theȱ biotaȱ (decreaseȱ ofȱ numberȱ ofȱ species,ȱ
specificȱ richness,ȱ diversity,ȱ diminuitionȱ inȱ theȱ percentageȱ ofȱ molluscsȱ andȱ
crustaceaȱ andȱ increaseȱ onȱ theȱ polychaeteȱ population.ȱ Theȱ groupȱ ofȱ variablesȱ
selectedȱ forȱ theȱ interpretationȱ presentedȱ aȱ loadingȱ 0.40ȱ orȱ higherȱ forȱ aȱ goodȱ
associationȱbetweenȱanȱoriginalȱvariableȱandȱaȱfactor.ȱ
ȱ
ȱ
ȱ
ȱ
- 315 -
ȱ
ȱȱ
ȱȱ
Benthicȱalterationȱ
Sublethalȱeffectsȱ(laboratoryȱ&ȱfield)ȱ
Acuteȱ
effectsȱ
Chemicalsȱ
ȱ
ȱ
Znȱȱ
Pbȱȱ
Cuȱȱ
Niȱȱ
Hgȱ
PAHȱȱ
Corophiumȱ
Arenicolaȱȱ
Microtoxȱ
Bioaccumulationȱ
GPXȬcrabȬlabȱ
GPXȬclamȬlabȱ
GRȬcrabȬlabȱ
GRȬclamȬlabȱ
GSTȬcrabȬlabȱ
GSTȬclamȬlabȱ
ERODȬcrabȬlabȱ
ERODȬclamȬlabȱ
FRAPȬcrabȬlabȱ
FRAPȬclamȬlabȱ
GPXȬcrabȬfieldȱ
GPXȬclamȬfieldȱ
GRȬcrabȬfieldȱ
GRȬclamȬfieldȱ
GSTȬcrabȬfieldȱ
GSTȬclamȬfieldȱ
ERODȬcrabȬfieldȱ
ERODȬclamȬfieldȱ
FRAPȬcrabȬfieldȱ
FRAPȬclamȬfieldȱ
Numberȱofȱspeciesȱ
specificȱrichnessȱ
Diversityȱ
%ȱMoluscaȱ
%ȱPolychaetaȱ
%ȱCrustaceaȱ
Factorȱ1ȱ
35.2ȱ
Factorȱ2ȱ
24.5ȱ
Factorȱ3ȱ
19.2ȱ
ņȱ
0.95ȱ
ņȱ
ņȱ
0.95ȱ
0.59ȱ
ņȱ
Ȭ0.41ȱ
ņȱ
0.73ȱ
0.48ȱ
ņȱ
0.41ȱ
0.65ȱ
0.85ȱ
Ȭ0.53ȱ
0.62ȱ
ņȱ
Ȭ0.46ȱ
ņȱ
0.88ȱ
ņȱ
0.91ȱ
0.77ȱ
0.89ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.91ȱ
ņȱ
ņȱ
0.62ȱ
Ȭ0.96ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.90ȱ
ņȱ
0.93ȱ
0.76ȱ
ņȱ
0.76ȱ
0.75ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.78ȱ
0.49ȱ
ņȱ
ņȱ
ņȱ
Ȭ0.89ȱ
ņȱ
ņȱ
ņȱ
- 316 -
Ȭ0.52ȱ
0.87ȱ
ņȱ
ņȱ
Ȭ0.77ȱ
0.53ȱ
ņȱ
ņȱ
ņȱ
ņȱ
Ȭ0.78ȱ
ņȱ
ņȱ
0.64ȱ
ņȱ
0.99ȱ
ņȱ
ņȱ
ņȱ
0.75ȱ
0.88ȱ
0.98ȱ
0.76ȱ
0.99ȱ
0.56ȱ
ņȱ
ņȱ
ņȱ
Ȭ0.68ȱ
ņȱ
ņȱ
ņȱ
Ȭ0.46ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.97ȱ
0.96ȱ
ņȱ
ņȱ
Factorȱ#1ȱ
Theȱ mainȱ factor,ȱ Factorȱ 1,ȱ accountsȱ forȱ aȱ 35.2ȱ %ȱ ofȱ theȱ varianceȱ andȱ
showsȱ theȱ relationshipȱ betweenȱ differentȱ variablesȱ relatedȱ withȱ chemicals,ȱ
sublethalȱresponses,ȱbioaccumulationȱandȱbenthicȱalteration.ȱTheȱconcentrationȱ
ofȱPAHs,ȱPbȱandȱHgȱinȱsedimentȱisȱrelatedȱtoȱtheȱbioaccumulationȱofȱPAHsȱinȱ
Arenicolaȱ marinaȱ exposedȱ underȱ laboratoryȱ conditions,ȱ butȱ oppositeȱ toȱ theirȱ
mortalityȱinȱtheȱacuteȱassays.ȱAȱsetȱofȱantioxidantȱandȱdetoxificationȱbiomarkersȱ
includingȱ GPX,ȱ GR,ȱ GST,ȱ ERODȱ andȱ FRAPȱ activitiesȱ analyzedȱ inȱ crabsȱ underȱ
laboratoryȱconditionsȱareȱcorrelatedȱinȱtheȱFactorȱ#1,ȱinȱadditionȱtoȱGRȱandȱGSTȱ
activityȱinȱtheȱdigestiveȱglandȱofȱclams.ȱERODȱandȱFRAPȱinȱtheȱfieldȱexposuresȱ
wereȱ notȱ correlatedȱ toȱ otherȱ variables.ȱ Theȱ aforementionedȱ contaminantsȱ andȱ
theȱtoxicityȱvariablesȱareȱslightlyȱconnectedȱtoȱtheȱbenthicȱalterationȱexplainedȱ
byȱ alterationȱ ofȱ theȱ specificȱ richnessȱ andȱ anȱ increaseȱ ofȱ theȱ polychaeteȱ
population,ȱ whileȱ aȱ positiveȱ developmentȱ ofȱ crustaceansȱ wasȱ detected.ȱ Theȱ
combinationȱ ofȱ thisȱ largeȱ groupȱ ofȱ variablesȱ inȱ Factorȱ #1ȱ isȱ interpretedȱ asȱ aȱ
contaminationȱ byȱ PAHsȱ mixedȱ withȱ theȱ metalsȱ Hgȱ andȱ Pbȱ whichȱ areȱ notȱ
producingȱ lethalȱ effects,ȱ althoughȱ PAHsȱ bioaccumulationȱ andȱ subȬlethalȱ
responsesȱ inȱ organismsȱ areȱ generatedȱ resultingȱ inȱ aȱ slightȱ alterationȱ ofȱ theȱ inȱ
situȱ benthicȱ community.ȱ Environmentalȱ alterationsȱ dueȱ toȱ theseȱ contaminantsȱ
haveȱbeenȱreportedȱbyȱotherȱauthorsȱ[26].ȱThisȱfactorȱhasȱaȱpositiveȱeffectȱinȱtheȱ
stationsȱ Eȱ (1.0)ȱ andȱ Fȱ (1.4)ȱ locatedȱ inȱ CormeȬLaxeȱ (Figureȱ 2).ȱ Theȱ
hydrodynamicsȱ ofȱ theȱ Bayȱ ofȱ CormeȬLaxeȱ suggestȱ anȱ accumulationȱ ofȱ
contaminantsȱincludingȱfuelȱoilȱfromȱtheȱPrestigeȱ[27,ȱ28]ȱwhatȱcouldȱexplainȱtheȱ
sedimentȱcontaminationȱandȱeffectsȱofȱtheȱstudyȱsites.ȱPreviousȱstudiesȱdidȱnotȱ
detectȱ theȱ presenceȱ ofȱ Pbȱ andȱ Hgȱ concentrationsȱ inȱ emulsifiedȱ samplesȱ ofȱ theȱ
Prestigeȱfuelȱ(withȱ54–59%ȱwater)ȱ[29]ȱalthoughȱtheȱPbȱoriginȱinȱtheȱpollutingȱoilȱ
wasȱcorroboratedȱbyȱotherȱauthorsȱ[30].ȱTakingȱthisȱfactorȱintoȱaccountȱseemsȱtoȱ
- 317 -
describeȱtheȱpollutionȱcausedȱbyȱtheȱremainingȱcontaminantsȱfromȱtheȱfuelȱspillȱ
byȱtheȱtankerȱPrestige,ȱasȱwasȱshownȱinȱtheȱpreviousȱstudyȱ[22].ȱ
3
2
1
0
-1
-2
A
B
Factor 1
C
Factor 2
D
E
F
Factor 3
ȱ
Figureȱ 2.ȱ Estimatedȱ factorȱ scoresȱ forȱ theȱ threeȱ factorsȱ inȱ eachȱ ofȱ theȱ
6cases.ȱTheȱfactorȱscoreȱquantifyȱtheȱprevalenceȱofȱeachȱfactorȱforȱeveryȱstationȱ
andȱisȱusedȱtoȱestablishȱtheȱdefinitionȱofȱeachȱfactor.ȱ
Despiteȱ toxicityȱ testsȱ didȱ notȱ demonstrateȱ theȱ acuteȱ effectsȱ ofȱ theseȱ
contaminantsȱ(PAHs,ȱPbȱandȱHg),ȱtheȱbioaccumulationȱofȱPAHsȱexperiencedȱbyȱ
A.ȱ marinaȱ inȱ theȱ conductedȱ bioassaysȱ demonstrateȱ theȱ bioavailabilityȱ ofȱ theseȱ
substances.ȱ Theȱ inductionȱ ofȱ differentȱ biomarkersȱ inȱ theȱ hepathopancreasȱ ofȱ
crabsȱandȱinȱtheȱdigestiveȱglandȱofȱclamsȱhaveȱbeenȱrelatedȱtoȱtheȱpresenceȱofȱ
theseȱcontaminantsȱwhatȱsuggestsȱthatȱtheȱdeployedȱorganismsȱsufferedȱstressȱ
dueȱtoȱtheȱpresenceȱofȱthisȱsubstancesȱinȱtheȱsediments;ȱtheȱcorrelationȱobservedȱ
amongȱ theȱ biomarkersȱ andȱ theȱ differentȱ variablesȱ definedȱ byȱ Factorȱ #1ȱ isȱ
strongerȱ forȱ thoseȱ enzymeȱ activitiesȱ measuredȱ inȱ organismsȱ exposedȱ underȱ
laboratoryȱ conditionsȱ whatȱ implyȱ thatȱ fieldȱ deploymentsȱ resultȱ inȱ lessȱ
sensitivity.ȱ Mostȱ likely,ȱ theȱ effectsȱ ofȱ theȱ contaminantsȱ withinȱ sedimentȱ areȱ
reducedȱ becauseȱ ofȱ theȱ flushingȱ actionȱ ofȱ theȱ openȱ waterȱ environment.ȱ Inȱ
- 318 -
addition,ȱ theȱ crabȱ Carcinusȱ maenasȱ hasȱ shownȱ toȱ beȱ moreȱ perceptiveȱ thanȱ theȱ
clamȱ Ruditappesȱ philippinarumȱ toȱ assessȱ thisȱ kindȱ ofȱ pollution,ȱ althoughȱ inȱ
general,ȱaȱgoodȱcorrelationȱwasȱdetectedȱamongȱtheȱbiomarkersȱinducedȱinȱbothȱ
invertebrateȱ species.ȱ Onȱ theȱ otherȱ handȱ someȱ ofȱ theȱ variablesȱ relatedȱ toȱ theȱ
benthicȱ alterationȱ presentȱ inȱ Factorȱ #1ȱ corroborateȱ theȱ effectsȱ observedȱ inȱ theȱ
sublethalȱexperiments.ȱ
Factorȱ#2ȱ
Theȱsecondȱfactor,ȱ Factorȱ #2ȱ(24.5ȱ%ȱofȱtheȱvariance)ȱ connectsȱtheȱ setȱofȱ
biomarkersȱmeasuredȱunderȱfieldȱconditionsȱ(ERODȱandȱFRAPȱactivityȱinȱcrabsȱ
andȱclams,ȱandȱGPXȱandȱGSTȱinȱclams),ȱtheȱmortalityȱofȱArenicolaȱinȱtheȱacuteȱ
experiment,ȱnoȱtoxicityȱforȱamphipods,ȱtheȱalterationȱinȱtheȱnumberȱofȱspeciesȱ
andȱ theȱ decreaseȱ ofȱ theȱ polychaeteȱ population.ȱ Positiveȱ andȱ negativeȱ
correlationsȱ forȱ aȱ fewȱ biomarkersȱ wereȱ alsoȱ detectedȱ underȱ laboratoryȱ
conditions,ȱ andȱ noȱ accordanceȱ withȱ theȱ amphipodȱ toxicityȱ testȱ wasȱ observed.ȱ
Theȱ relationshipsȱ betweenȱ theȱ biologicalȱ responsesȱ identifiedȱ inȱ Factorȱ #2ȱ areȱ
notȱ correlatedȱ withȱ anyȱ ofȱ theȱ chemicalsȱ analyzedȱ whatȱ suggestȱ thatȱ aȱ
contaminantȱorȱgroupȱofȱcompoundsȱboundȱorȱnotȱtoȱtheȱsedimentȱwhichȱwereȱ
notȱanalyzedȱareȱtheȱcauseȱofȱtheȱbiologicalȱeffects.ȱTakingȱintoȱaccountȱthatȱtheȱ
acuteȱ toxicityȱ observedȱ byȱ theȱ Arenicolaȱ andȱ theȱ restȱ ofȱ theȱ bioassaysȱ wasȱ
relativelyȱ lowȱ (lessȱ thanȱ 30%ȱ mortalityȱ inȱ mostȱ ofȱ theȱ cases)ȱ [22],ȱ aȱ sourceȱ ofȱ
contaminantȱnotȱrelatedȱtoȱsedimentȱisȱtheȱmostȱprobableȱcauseȱofȱtheseȱeffects.ȱ
StationȱAȱ(0.1)ȱlocatedȱonȱCiesȱIslandȱandȱmainlyȱtheȱsiteȱDȱ(2.0)ȱinȱtheȱbayȱofȱ
CormeȬLaxeȱ presentȱ positiveȱ loadingȱ ofȱ Factorȱ #2ȱ (Figureȱ 2).ȱ Theȱ goodȱ
correlationȱ experiencedȱ byȱ theȱ biomarkersȱ measuredȱ inȱ bothȱ crabsȱ andȱ clamsȱ
underȱ fieldȱ conditionsȱ suggestȱ thatȱ theseȱ locationsȱ sufferȱ theȱ stressȱ ofȱ nonȬ
measuredȱ variable/sȱ whichȱ inȱ theȱ caseȱ ofȱ siteȱ Dȱ couldȱ beȱ relatedȱ toȱ theȱ
proximityȱofȱaquacultureȱinfrastructuresȱforȱmusselȱgrowth.ȱOtherȱauthorsȱ[31],ȱ
- 319 -
haveȱ describedȱ theȱ negativeȱ impactsȱ ofȱ theseȱ raftsȱ forȱ musselȱ aquaculture,ȱ
including:ȱ theȱ dischargeȱ ofȱ aȱ largeȱ volumeȱ ofȱ bioȬdepositsȱ containingȱ highȱ
concentrationsȱ ofȱ nutrients;ȱ theȱ releaseȱ ofȱ drugsȱ andȱ pesticidesȱ intoȱ theȱ
environment;ȱanȱincreaseȱinȱsedimentationȱandȱaccumulationȱofȱorganicȱmatter;ȱ
anȱincreaseȱinȱtheȱconcentrationȱofȱnutrientsȱinȱsedimentsȱandȱwatersȱ(mainlyȱNȱ
andȱ P).ȱ Negativeȱ effectsȱ onȱ wildȱ populationsȱ ofȱ animalsȱ haveȱ alsoȱ beenȱ
reflected,ȱrangingȱfromȱgeneticȱinteractionȱandȱdiseaseȱtransmission,ȱtoȱchangesȱ
inȱtheȱcompositionȱofȱtheȱstructureȱofȱbenthicȱfaunaȱdueȱtoȱaȱchangeȱfromȱoxicȱ
toȱanoxicȱconditionsȱ[31].ȱ
Factorȱ#3ȱ
Theȱ thirdȱ factor,ȱ Factorȱ #3ȱ accountsȱ forȱ aȱ 19.2ȱ %ȱ ofȱ theȱ varianceȱ andȱ
showsȱ theȱ sedimentsȱ contaminationȱ byȱ theȱ metalsȱ Zn,ȱ Cuȱ andȱ Ni,ȱ andȱ theȱ
PAHs;ȱ thisȱ contaminantsȱ areȱ relatedȱ toȱ acuteȱ toxicityȱ determinedȱ byȱ theȱ
amphipodsȱassay,ȱwhichȱisȱnotȱsignificantȱ(theȱtoxicityȱdetectedȱwasȱnotȱenoughȱ
toȱ considerȱ sedimentȱ samplesȱ asȱ toxicȱ accordingȱ toȱ thisȱ acuteȱ assay:ȱ samplesȱ
whereȱ theȱ mortalityȱ rateȱ ofȱ theȱ amphipodsȱ isȱ 20%ȱ higherȱ thanȱ theȱ mortalityȱ
recordedȱ inȱ theȱ referenceȱ andȱ showȱ significantlyȱ differentȱ (*pȱ <ȱ 0.05)ȱ resultsȱ
comparedȱ toȱ thoseȱ obtainedȱ inȱ theȱ referenceȱ areȱ consideredȱ asȱ toxic)ȱ [22]ȱ andȱ
antioxidantȱ responsesȱ underȱ laboratoryȱ assaysȱ withȱ crabsȱ andȱ clamsȱ (GR);ȱ theȱ
effectsȱ onȱ theȱ benthicȱ communityȱ areȱ shownȱ asȱ anȱ alterationȱ ofȱ theȱ diversityȱ
andȱ percentageȱ ofȱ molluscs.ȱ Theseȱ correlationsȱ suggestȱ theȱ presenceȱ ofȱ someȱ
stressȱinȱtheȱenvironmentȱdueȱtoȱaȱsourceȱorȱsourcesȱofȱmetalsȱ(Zn,ȱCuȱandȱNi)ȱ
andȱorganicȱcompoundsȱ(PAHs)ȱdifferentȱtoȱtheȱsourceȱexplainedȱbyȱFactorȱ#1.ȱ
Inȱthisȱcase,ȱFactorȱ#3ȱpresentsȱaȱpositiveȱeffectȱinȱsitesȱAȱ(1.2)ȱinȱtheȱAINPȱandȱFȱ
(1.3)ȱinȱtheȱBayȱofȱCormeȬLaxeȱ(Figureȱ2).ȱPreviousȱstudiesȱinȱtheȱareaȱofȱCormeȬ
LaxeȱhaveȱdetectedȱsevereȱcontaminationȱbyȱCuȱinȱtheȱsedimentsȱ[28],ȱhoweverȱ
aȱ fuelȱ oilȱ originȱ wasȱ unlikelyȱ andȱ aȱ majorȱ sourceȱ ofȱ Cuȱ relatedȱ toȱ antifoulingȱ
- 320 -
paintsȱ fromȱ theȱ hullsȱ ofȱ fishingȱ vesselsȱ wasȱ suggestedȱ [32].ȱ Althoughȱ
contaminationȱ byȱ Cuȱ andȱ Znȱ wasȱ observedȱ inȱ theȱ uppermostȱ layerȱ inȱ theȱ
Prestigeȱ shipwreckȱ areaȱ ofȱ theȱ Northeastȱ Atlanticȱ Oceanȱ [33,ȱ 34],ȱ thisȱ
contaminationȱ shouldȱ notȱbeȱrelatedȱtoȱ theȱshipwreck,ȱbecauseȱlevelsȱofȱCuȱinȱ
theȱ fuelȱ oilȱ carriedȱ byȱ theȱ Prestigeȱ wereȱ relativelyȱ lowȱ (3.39ȱ mgȱ kgȬ1)ȱ andȱ
previousȱstudiesȱhaveȱshownȱthatȱinputsȱfromȱterrestrialȱsourcesȱofȱmetalsȱareȱ
probablyȱ higherȱ thanȱ inputsȱ fromȱ theȱ spilledȱ fuelȱ oilȱ [35,ȱ 36].ȱ Someȱ ofȱ theȱ
studiedȱ variablesȱ demonstrateȱ theȱ stressȱ ofȱ theseȱ contaminantsȱ andȱ theȱ effectsȱ
onȱ theȱ benthicȱ community;ȱ however,ȱ theȱ resultsȱ mostlyȱ pointȱ toȱ chronicȱ
contaminationȱ withȱ lowȱ bioavailability,ȱandȱpotentialȱ butȱlargelyȱunconfirmedȱ
biologicalȱrisk.ȱInȱthisȱsenseȱpreviousȱstudiesȱ[37]ȱconsideredȱthatȱdespiteȱaȱhighȱ
percentageȱ ofȱ theȱ totalȱ contentȱ ofȱ traceȱ metalsȱ inȱ sedimentsȱ fromȱ theȱ Galicianȱ
coastȱpresentedȱaȱreactivityȱandȱbioavailabilityȱwereȱveryȱlow,ȱtheȱhighȱdegreeȱ
ofȱpyritizationȱfoundȱforȱsomeȱofȱtheȱmostȱtoxicȱtraceȱmetalsȱmayȱfavourȱtheirȱ
releaseȱ byȱ oxidationȱ ofȱ theȱ sulphidesȱ thatȱ theyȱ form,ȱ thusȱ makingȱ themȱ
bioavailableȱtoȱbenthicȱfauna.ȱ
Significantȱdifferencesȱamongȱstationsȱ
Fromȱ theȱ resultsȱ obtained,ȱ theȱ authorsȱ consideredȱ stationȱ C,ȱ whichȱ
presentedȱanȱabsenceȱofȱPAHsȱcontaminationȱandȱtheȱlowestȱbiologicalȱeffects,ȱ
asȱaȱsuitableȱsiteȱtoȱuseȱasȱreferenceȱstation.ȱTakingȱthisȱintoȱaccount,ȱtheȱfactorȱ
loadingsȱ obtainedȱ inȱ theȱ MAAȱ wereȱ submittedȱ toȱ ANOVAȱ andȱ Tukeyȱ testȱ inȱ
orderȱ toȱ determineȱ theȱ significantȱ differencesȱ betweenȱ theȱ stationsȱ andȱ theȱ
referenceȱsiteȱforȱeachȱofȱtheȱthreeȱdisplayedȱfactorsȱtoȱidentifyȱtheȱcauseȱwhichȱ
isȱproducingȱ(orȱnot)ȱpollutionȱinȱeveryȱsingleȱstudyȱsiteȱ(Figureȱ3).ȱ
- 321 -
A
Factor 3
B
Factor 1
Factor 3
Factor 1
Factor 2
Factor 2
D
Factor 3
Factor 1
Factor 2
E
Factor 3
F
Factor 1
Factor 3
Factor 2
Factor 1
Factor 2
ȱ
Figureȱ 3.ȱ Pieȱ chartsȱ whichȱ representȱ theȱ significantȱ differencesȱ ofȱ theȱ
factorsȱscoreȱinȱeveryȱstudyȱsiteȱrelatedȱtoȱtheȱreferenceȱsiteȱCȱ(dotted:ȱpȱ<ȱ0.01;ȱ
slightlyȱdotted:ȱpȱ<ȱ0.05;ȱnotȱdotted:ȱnoȱsignificantȱdifferences,ȱp>0.05).ȱȱȱ
ȱ
ȱ
ȱ
- 322 -
Noneȱ ofȱ theȱ sedimentsȱ fromȱ Ciesȱ Islandȱ inȱ theȱ AINPȱ presentedȱ
significantȱ differencesȱ inȱ Factorȱ #1ȱ whichȱ meansȱ thatȱ theȱ effectsȱ ofȱ theȱ Prestigeȱ
oilȱspillȱareȱnotȱstillȱoccurringȱinȱtheȱarea.ȱHowever,ȱsiteȱAȱpresentsȱsignificantȱ
differencesȱ(pȱ<ȱ0.01)ȱwithȱtheȱselectedȱreferenceȱstationȱ(C)ȱrelatedȱtoȱFactorȱ#ȱ2ȱ
andȱ#ȱ3ȱwhichȱsuggestsȱtheȱpotentialȱriskȱandȱtheȱenvironmentalȱstressȱcausedȱ
byȱnonȬmeasuredȱsubstancesȱcomingȱfromȱotherȱsourcesȱapartȱfromȱtheȱtankerȱ
Prestige.ȱ Siteȱ Bȱ presentsȱ significantȱ differencesȱ (pȱ <ȱ 0.05)ȱ withȱ theȱ referenceȱ
stationȱ accordingȱ toȱ Factorȱ #3,ȱ whatȱ meansȱ thatȱ theȱ presenceȱ ofȱ someȱ
contaminantsȱ inȱ theȱ areaȱ areȱ consideredȱ aȱ potentialȱ risk,ȱ althoughȱ inȱ general,ȱ
theȱ sedimentsȱ presentȱ aȱ relativelyȱ goodȱ environmentalȱ quality.ȱ Onȱ theȱ otherȱ
hand,ȱsedimentsȱfromȱCormeȬLaxeȱD,ȱEȱandȱFȱshowȱsignificantȱdifferencesȱ(pȱ<ȱ
0.01)ȱ withȱ theȱ referenceȱ stationȱ forȱ Factorȱ #1ȱ meaningȱ theȱ remainingȱ
contaminantsȱ fromȱ theȱ Prestigeȱ oilȱ spillȱ areȱ stillȱ producingȱ subȬlethalȱ effectsȱ toȱ
theȱbiotaȱofȱtheȱbay.ȱInȱaddition,ȱtheȱsignificantȱdifferencesȱ(pȱ<ȱ0.01)ȱshownȱforȱ
Factorȱ#2ȱinȱDȱfocusȱtoȱotherȱunknownȱsourcesȱofȱcontaminantsȱresponsibleȱofȱ
biologicalȱ stressȱ inȱ theȱ studyȱ site,ȱ whereasȱ inȱ theȱ caseȱ ofȱ siteȱ Fȱ aȱ mixtureȱ ofȱ
metalsȱandȱPAHsȱfromȱdifferentȱsourcesȱcouldȱbeȱconsideredȱaȱpotentialȱriskȱinȱ
theȱareaȱasȱitȱisȱshownȱinȱFactorȱ#3ȱ(pȱ<ȱ0.01).ȱ
Itȱ isȱ wellȱ knownȱ thatȱ biomarkersȱ haveȱ beenȱ shownȱ toȱ beȱ usefulȱ “earlyȱ
warning”ȱ toolsȱ inȱ characterizingȱ theȱ healthȱ statusȱ ofȱ animalsȱ fromȱ impactedȱ
areasȱ [38,ȱ 39],ȱ suchȱ asȱ oilȱ affectedȱ placesȱ [40],ȱ whereȱ complexȱ mixturesȱ ofȱ
pollutantsȱ areȱ usuallyȱ present.ȱ Inȱ theȱ presentȱ studyȱ itȱ hasȱ beenȱ showȱ howȱ theȱ
useȱ ofȱ theȱ setȱ ofȱ biomarkersȱ asȱ partȱ ofȱ aȱ WOEȱ approachȱ designedȱ toȱ assessȱ
contaminatedȱsedimentsȱcontributesȱaddedȱvalueȱtoȱtheȱclassicalȱLOEsȱoriginalȱ
ideaȱ andȱ allowsȱ forȱ theȱ characterizationȱ ofȱ theȱ environmentalȱ statusȱ ofȱ theȱ
studiedȱareaȱinȱaȱmoreȱpreciselyȱandȱaccuratelyȱway.ȱInȱadditionȱtheȱinclusionȱ
ofȱ chronicȱ bioassaysȱ withȱ twoȱ invertebrateȱ speciesȱ notȱ onlyȱ underȱ laboratoryȱ
- 323 -
conditionsȱbutȱalsoȱinȱfieldȱdeploymentsȱfavourȱtoȱelucidateȱdifferentȱsourcesȱofȱ
contaminantsȱapartȱfromȱtheȱsedimentsȱpermittingȱaȱmoreȱrealisticȱapproachȱtoȱ
theȱoriginalȱsituationȱofȱtheȱecosystem,ȱandȱtheȱpotentialȱecologicalȱrisks.ȱȱ
4.ȱConclusionsȱ
Thereȱisȱevidenceȱthatȱ4ȱyearsȱafterȱtheȱimpactȱofȱtheȱPrestigeȱoilȱspillȱtheȱ
fuelȱ isȱ notȱ producingȱ acuteȱ toxicȱ effectsȱ onȱ theȱ environmentȱ [9]ȱ butȱ subȬlethalȱ
responsesȱ haveȱ beenȱ detectedȱ inȱ theȱ areaȱ ofȱ CormeȬLaxe,ȱ relatedȱ toȱ PAHs,ȱ Pbȱ
andȱ Hg;ȱ noȱ effectsȱ ofȱ thisȱ spillȱ wereȱ observedȱ inȱ theȱ studyȱ sitesȱ locatedȱ inȱ theȱ
AINPȱ althoughȱ aȱ contaminationȱ byȱ metals,ȱ speciallyȱ Zn,ȱ Cuȱ andȱ Niȱ wasȱ
observedȱinȱsomeȱsitesȱonȱCíesȱIsland.ȱTheȱcoastalȱanthropogenicȱinfluenceȱhasȱ
madeȱ evidentȱ inȱ bothȱ areasȱ dueȱ toȱ anȱ inputȱ ofȱ aȱ mixtureȱ ofȱ pollutantsȱ thatȱ
shouldȱ beȱ consideredȱ aȱ potentialȱ risk.ȱ Inȱ theȱ caseȱ ofȱ CormeȬLaxe,ȱ aȱ possibleȱ
impactȱofȱtheȱmusselȱraftsȱwasȱdetected.ȱȱ
Theȱ useȱ ofȱ biomarkersȱ hasȱ demonstratedȱ havingȱ higherȱ sensitivityȱ thanȱ
acuteȱ toxicityȱ approachesȱ asȱ partȱ ofȱ aȱ moreȱ completeȱ andȱ integratedȱ studyȱ
basedȱ onȱ aȱ weightȬofȬevidenceȱ approachȱ [9].ȱ Theȱ presentȱ studyȱ hasȱ
demonstratedȱ theȱ feasibilityȱ ofȱ incorporatingȱ aȱ fourthȱ lineȱ ofȱ evidenceȱ toȱ theȱ
classicalȱ methodologyȱ employedȱ inȱ theȱ Sedimentȱ Qualityȱ Triadȱ andȱ theȱ aȱ
suitabilityȱ ofȱ biomarkersȱ asȱ aȱ toolȱ toȱ assessȱ metallicȱ andȱ petrogenicȱ
contaminatedȱ sedimentsȱ asȱ wellȱ asȱ unknownȱ mixturesȱ ofȱ compounds,ȱ byȱ
carryingȱ outȱ bioassaysȱ underȱ fieldȱ andȱ laboratoryȱ conditionsȱ whichȱ helpȱ toȱ
distinguishȱ possibleȱ sourcesȱ ofȱ pollutantsȱ inȱ theȱ environmentȱ andȱ providesȱ
informationȱaboutȱecologicalȱrisks.ȱȱȱ
ȱ
- 324 -
5.ȱAcknowledgementsȱ
Theȱ workȱ describedȱ wasȱ partlyȱ supportedȱ byȱ theȱ projectsȱ VEM2003Ȭ
20563/INTER,ȱ andȱ CTM2005Ȭ07282ȬC03Ȭ01/TECNOȱ financedȱ byȱ theȱ Spanishȱ
Educationȱ andȱ Scienceȱ Ministryȱ andȱ byȱ CISȱ fundedȱ byȱ theȱ Ministryȱ ofȱ
Environment.ȱ Carmenȱ MoralesȬCasellesȱ thanksȱ theȱ Ministryȱ ofȱ Educationȱ andȱ
Scienceȱforȱfundingȱherȱresearchȱfellowshipȱ(FPU).ȱAuthorsȱwouldȱlikeȱtoȱthankȱ
theȱmembersȱofȱtheȱCISȱforȱtheirȱsupportȱandȱhelpȱinȱtheȱchemicalȱanalysisȱandȱ
theȱ benthicȱ communityȱ information;ȱ specialȱ thanksȱ areȱ givenȱ toȱ Lauraȱ Martín,ȱ
Nuriaȱ Fernández,ȱ Augustoȱ César,ȱ Pabloȱ Vidalȱ andȱ Antonioȱ Moreno.ȱ Authorsȱ
appreciateȱ theȱ helpȱ ofȱ theȱ reviewersȱ andȱ theȱ supportȱ ofȱ CSLMȱ inȱ theȱ Englishȱ
revision.ȱ
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Theȱapplicationȱofȱaȱweightȱofȱevidenceȱapproachȱtoȱcompareȱtheȱ
qualityȱofȱcoastalȱsedimentsȱaffectedȱbyȱacuteȱ(Prestigeȱ2002)ȱandȱ
chronicȱ(BayȱofȱAlgeciras)ȱoilȱspillsȱ
CarmenȱMoralesȬCaselles1,2,*,ȱInmaculadaȱRiba1,2,ȱCarmenȱSarasquete1,ȱT.ȱÁngelȱ
DelValls1,2ȱ
1
ȱUnidadȱAsociadaȱdeȱCalidadȱAmbientalȱyȱPatologíaȱ(CSICȱ&ȱUCA)ȱinstitutoȱdeȱ
CienciasȱMarinasȱdeȱAndalucíaȱ(ICMANȬCSIC),ȱAvda.ȱRepúblicaȱSaharauiȱs/n,ȱPuertoȱ
Realȱ11510,ȱCádiz,ȱSpainȱ
UNESCOȱUNITWIN/WiCop,ȱFacultadȱdeȱCienciasȱdelȱMarȱyȱAmbientales,ȱ
2ȱ
UniversidadȱdeȱCádiz,ȱPolígonoȱRíoȱSanȱPedroȱs/n,ȱPuertoȱRealȱ11510,ȱCádiz,ȱSpain.ȱ
Abstractȱ
Toȱ evaluateȱ sedimentȱ qualityȱ inȱ differentȱ areasȱ affectedȱ byȱ oilȱ spillsȱ aȱ
weightȱ ofȱ evidenceȱ approachȱ wasȱ employedȱ byȱ includingȱ aȱ completeȱ setȱ ofȱ
parametersȱ asȱ partȱ ofȱ 4ȱ differentȱ linesȱ ofȱ evidence:ȱ sedimentȱ contamination,ȱ
biologicalȱ effectsȱ andȱ bioaccumulationȱ underȱ laboratoryȱ conditions,ȱ toxicityȱ inȱ
fieldȱ conditionsȱ andȱ benthicȱ alteration.ȱ Theȱ methodologyȱ wasȱ appliedȱ toȱ
sedimentsȱfromȱtheȱBayȱofȱAlgecirasȱ(SȱSpain)ȱchronicallyȱimpactedȱbyȱdifferentȱ
oilȱ spills,ȱ andȱ theȱ Galicianȱ Coastȱ (NWȱ Spain)ȱ acutelyȱ impactedȱ byȱ anȱ oilȱ spillȱ
(Prestigeȱ 2002).ȱ Resultsȱ obtainedȱ haveȱ elucidatedȱ theȱ sourcesȱ andȱ fatesȱ ofȱ
pollutantsȱ andȱ theȱ typeȱ ofȱ riskȱ involvedȱ forȱ theȱ ecosystem.ȱ Itȱ hasȱ beenȱ
demonstratedȱ thatȱ theȱ impactȱ associatedȱ withȱ chronicȱ eventȱ ofȱ contaminationȱ
byȱoilȱspillsȱareȱsignificantlyȱmoreȱdangerousȱandȱpollutedȱthanȱthoseȱrelatedȱtoȱ
acuteȱeffects.ȱInȱtheȱacuteȱeventsȱitȱhasȱbeenȱshownȱthatȱtheȱoriginalȱpollutionȱisȱ
recoveredȱ yearsȱ latersȱ whereasȱ theȱ pollutionȱ stillȱ inȱ thoseȱ chronicȱ affectedȱ
environments.ȱȱ
Keywords:ȱsedimentȱcontamination,ȱsedimentȱtoxicity,ȱSedimentȱQualityȱTriad,ȱ
bioaccumulation,ȱsublethal,ȱbenthicȱalteration.ȱ
ȱEnvironmentalȱPollutionȱ(aceptadoȱconȱrevisiones)
- 331 -
Capsule:ȱChronicȱinputsȱdueȱtoȱtheȱcontinuousȱentranceȱofȱcontaminantsȱresultȱ
muchȱ moreȱ harmfulȱ inȱ coastalȱ ecosystemsȱ thanȱ majorȱ butȱ preciseȱ environmentalȱ
impactsȱ
1.ȱIntroductionȱ
Nowadays,ȱhumanȱactivitiesȱinȱcoastalȱareasȱinvolveȱaȱhighȱpressureȱandȱ
aȱ sourceȱ ofȱ differentȱ contaminantsȱ toȱ theȱ naturalȱ environmentȱ thatȱ becomesȱ
evidentȱinȱtheȱdecreasedȱqualityȱofȱcoastalȱsediments.ȱSedimentsȱactȱasȱaȱtrapȱofȱ
contaminantsȱ andȱ mayȱ becomeȱ sufficientlyȱ pollutedȱ toȱ disruptȱ naturalȱ
biologicalȱ communitiesȱ (Adamsȱ etȱ al.ȱ 1992;ȱ Tolunȱ etȱ al.ȱ 2001).ȱ Substancesȱ
introducedȱintoȱtheȱenvironmentȱmayȱbeȱmoreȱorȱlessȱbioavailableȱtoȱorganismsȱ
dependingȱ onȱ theirȱ chemicalȱ form,ȱ modifyingȱ factorsȱ inȱ theȱ environment,ȱ theȱ
environmentalȱ compartmentȱ theyȱ occupy,ȱ andȱ theȱ reactionsȱ (behaviouralȱ andȱ
physiological)ȱ ofȱ exposedȱ biotaȱ (Chapmanȱ etȱ al.,ȱ 2003;ȱ Chapman,ȱ 2007).ȱ Theȱ
biologicalȱ effectsȱ canȱ beȱ establishedȱ basedȱ onȱ laboratoryȱ testsȱ thatȱ determineȱ
toxicȱresponses,ȱbesides,ȱfieldȱdataȱonȱtheȱcommunitiesȱlivingȱinȱtheȱsedimentsȱ
allowȱ toȱ establishȱ whetherȱ thereȱ isȱ observableȱ pollutionȬinducedȱ degradationȱ
effectȱinȱtheȱbiotaȱ(Chapmanȱetȱal.,ȱ1991).ȱ
Integratedȱ studiesȱ useȱ differentȱ linesȱ ofȱ evidenceȱ (LOEs)ȱ whichȱ addressȱ
differentȱquestionsȱaboutȱtheȱpresenceȱofȱcontaminants,ȱtheirȱbioavailabilityȱandȱ
theirȱadverseȱbiologicalȱeffectsȱ(Ribaȱetȱal.,ȱ2004)ȱinȱaȱweightȱofȱevidenceȱ(WOE)ȱ
framework.ȱInȱtheȱpresentȱstudyȱaȱWOEȱfollowingȱ4ȬLOEsȱhasȱbeenȱappliedȱtoȱ
compareȱtheȱsedimentȱqualityȱofȱtwoȱareasȱofȱtheȱSpanishȱCoastȱaffectedȱbyȱoilȱ
spills.ȱ Theȱ Bayȱ ofȱ Algecirasȱ (Sȱ Spain)ȱ hasȱ sufferedȱ aȱ chronicȱ impactȱ lastingȱ
severalȱ decades,ȱ causedȱ byȱ theȱ inputȱ ofȱ oilȱ andȱ otherȱ contaminantsȱ fromȱ theȱ
variousȱindustriesȱlocatedȱinȱtheȱareaȱandȱfromȱaccidentalȱspillsȱandȱdeliberateȱ
dischargesȱ fromȱ commercialȱ shippingȱ activitiesȱ (MoralesȬCasellesȱ etȱ al.,ȱ 2007),ȱ
- 332 -
whereasȱtheȱGalicianȱCoastȱwasȱimpactedȱbyȱtheȱsinkingȱofȱtheȱtankerȱPrestigeȱ
(2002),ȱ whichȱ spiltȱ aboutȱ 63,000ȱ tonnesȱ ofȱ heavyȱ fuelȱ oilȱ (MariñoȬBalsaȱ etȱ al.,ȱ
2003;ȱBlancoȱetȱal.,ȱ2006;ȱFernándezȱetȱal.,ȱ2006).ȱInȱadditionȱaȱthirdȱareaȱlocatedȱ
inȱ theȱ Bayȱ ofȱ Cádizȱ (SWȱ Spain)ȱ andȱ widelyȱ characterizedȱ byȱ differentȱ
ecotoxicologicalȱstudiesȱwasȱselectedȱasȱtheȱreferenceȱsiteȱ(DelȱVallsȱetȱal.,ȱ1998,ȱ
Ribaȱetȱal.,ȱ2004,ȱMartínȬDíazȱetȱal.,ȱ2005;ȱMoralesȬCasellesȱetȱal.,ȱ2007).ȱȱ
Theȱ aimȱ ofȱ thisȱ studyȱ are:ȱ (a)ȱ toȱ determineȱ theȱ feasibilityȱ ofȱ usingȱ theȱ
selectedȱ parametersȱ asȱ partȱ ofȱ 4ȱ LOEsȱ toȱ assessȱ sedimentsȱ contaminatedȱ byȱ
differentȱtypesȱofȱoilȱspills;ȱ(b)ȱtoȱestablishȱtheȱenvironmentalȱdegradationȱinȱtheȱ
studiedȱ areas;ȱ andȱ (c)ȱ toȱ elucidateȱ whatȱ isȱ moreȱ harmfulȱ toȱ theȱ environment:ȱ
acuteȱorȱchronicȱimpactsȱassociatedȱwithȱoilȱspills.ȱȱ
2.ȱMethodologyȱ
2.1.ȱApproachȱ
Fig.ȱ 1ȱ showsȱ theȱ 6ȱ sedimentȱ samplingȱ stationsȱ locatedȱ inȱ theȱ areaȱ ofȱ
Galiciaȱ (NWȱ Spain),ȱ threeȱ stationsȱ inȱ theȱ Atlanticȱ Islandsȱ Nationalȱ Parkȱ (A,ȱ Bȱ
andȱC)ȱandȱthreeȱstationsȱinȱtheȱ BayȱofȱCormeȬLaxeȱ(D,ȱE,ȱF).ȱBothȱareasȱwereȱ
importantlyȱ affectedȱ byȱ theȱ Prestigeȱ oilȱ spillȱ andȱ areȱ consideredȱ ofȱ highȱ
ecologicalȱ importance.ȱ Inȱ theȱ Gulfȱ ofȱ Cádizȱ (Sȱ Spain)ȱ threeȱ stationsȱ wereȱ
selectedȱ inȱ theȱ areaȱ ofȱ theȱ Bayȱ ofȱ Algecirasȱ (GR3’,ȱ GR4,ȱ P1)ȱ whichȱ isȱ highlyȱ
industrializedȱ placeȱ whereȱ itȱ takeȱ placeȱ aȱ largeȱ numberȱ ofȱ petrochemicalȱ
activitiesȱthatȱcompriseȱseveralȱaccidentalȱoilȱspills;ȱbesides,ȱaȱreferenceȱsiteȱwasȱ
chosenȱinȱaȱcleanȱareaȱinȱtheȱBayȱofȱCádizȱ(CA)ȱ(Ribaȱetȱal.,ȱ2003).ȱȱ
ȱ
ȱ
ȱ
- 333 -
ȱ
ȱ
Atlantic Islands
National Park
ȱ
•C
ƒF ƒE
ƒD
ȱ
•A
•B
Corme-Laxe
ȱ
ȱ
Spain
ȱ
ȱ
Bay of
Cádiz
•GR3
•GR4
•P1
ȱ
ȱ
•CA
Bay of
Algeciras
N
E
W
S
ȱ
Figureȱ1.ȱMapȱofȱtheȱcoastalȱareaȱofȱGalicia,ȱtheȱBayȱofȱAlgecirasȱandȱtheȱ
BayȱofȱCádizȱshowingȱtheȱgeneralȱareasȱsampledȱandȱlocationsȱofȱtheȱsamplingȱ
stations.ȱ A,ȱ Bȱ andȱ Cȱ areȱ theȱ stationsȱ locatedȱ inȱ theȱ Ciesȱ Islandȱ inȱ theȱ Atlanticȱ
IslandsȱNationalȱParkȱ(GalicianȱCoast);ȱD,ȱEȱandȱFȱareȱtheȱsitesȱfromȱtheȱBayȱofȱ
CormeȬLaxeȱ (Galicianȱ Coast);ȱ GR3,ȱ GR4ȱ andȱ P1ȱ areȱ locatedȱ inȱ theȱ Bayȱ ofȱ
AlgecirasȱwhereasȱtheȱreferenceȱstationȱCAȱisȱplacedȱinȱtheȱBayȱofȱCádiz.ȱ
ȱ
ȱ
ȱ
- 334 -
2.1.ȱTheȱWOEȱcomponentsȱ
Aȱ weightȬofȬevidenceȱ approachȱ (WOE)ȱ wasȱ conductedȱ inȱ theȱ sitesȱ
selectedȱthatȱincludesȱ4ȱlinesȱofȱevidenceȱ(LOEs)ȱincorporatingȱtheȱnextȱanalysisȱ
(Figureȱ2):ȱȱ
ȱ
Contamination
- PAHs
- metals
ȱ
ȱ
Effects in laboratory
ȱ Benthic alteration
- abundance-biomass analysis,
species richness, diversity,
dominance, % polychaete,
ȱ
% crustacean, % molluscs
SEDIMENT
QUALITY
ȱ
- Acute: Microtox® (bioluminiscence),
Arenicola marina (biaccumulation and
mortality), Corophium volutator (mortality).
- Chronic: Carcinus maenas and Ruditapes
philippinarum (biomarkers: GR, GST, GPX,
EROD, FRAP)
In situ effects
- Carcinus maenas and Ruditapes
philipinarum (biomarkers: GR, GST, GPX,
EROD, FRAP)
ȱ
Figureȱ 2.ȱ Summarizedȱ descriptionȱ ofȱ theȱ 4ȱ linesȱ ofȱ evidenceȱ selectedȱ inȱ
theȱ weighȱ ofȱ evidenceȱ approachȱ usingȱ theȱ schematicȱ representationȱ
modificationȱofȱtheȱclassicalȱtriad.ȱ
(a)ȱ sedimentȱ contamination:ȱ includesȱ theȱ concentrationȱ ofȱ totalȱ PAHsȱ
(acenaphtalene,ȱ
acenaphtylene,ȱ
benzo(a)pyrene,ȱ
benzo(k)fluoranthene,ȱ
anthracene,ȱ
benzo(b)fluoranthene,ȱ
chrysene,ȱ
dibenzo(a,h)ȱ
- 335 -
benzo(a)anthracene,ȱ
benzo(g,h,i)ȱ
perylene,ȱ
anthracene,ȱ
fenanthrene,ȱ
fluoranthene,ȱfluorene,ȱindeneȱ(1,2,3,cdȱ)pyrene,ȱnaphthalene,ȱandȱpyrene)ȱandȱ
traceȱ metalsȱ (Zn,ȱ Pb,ȱ Cu,ȱ Ni,ȱ Coȱ andȱ V).ȱ Sedimentȱ characterizationȱ byȱ organicȱ
carbonȱ andȱ percentageȱ ofȱ finesȱ isȱ alsoȱ includedȱ inȱ thisȱ sectionȱ (methodologiesȱ
describedȱinȱMoralesȬCasellesȱetȱal.,ȱ2006);ȱȱ
(b)ȱsedimentȱtoxicityȱunderȱlaboratoryȱconditions:ȱincludingȱtheȱbacteriaȱ
assayȱ Microtox®ȱ (MoralesȬCasellesȱ etȱ al.,ȱ 2007),ȱ theȱ amphipodȱ mortalityȱ testȱ
withȱ Corophiumȱ volutatorȱ (MoralesȬCasellesȱ etȱ al.,ȱ 2007)ȱ andȱ theȱ ȱ polychaetaȱ
mortalityȱ andȱ bioaccumulationȱ assayȱ (CasadoȬMartínezȱ etȱ al.,ȱ inȱ press)ȱ withȱ
Arenicolaȱmarina;ȱsublethalȱassaysȱwereȱalsoȱconductedȱbasedȱonȱbiomarkersȱbyȱ
usingȱtwoȱinvertebrateȱspecies,ȱtheȱcrabȱCarcinusȱmaenasȱandȱtheȱclamȱRuditappesȱ
philippinarum,ȱ andȱ aȱ suiteȱ ofȱ biomarkersȱ measuredȱ afterȱ 28ȱ daysȱ ofȱ exposure:ȱ
Ethoxyresorufinȱ OȬdeethylaseȱ (EROD),ȱ phaseȱ Iȱ detoxificationȱ enzymeȱ
implicatedȱ inȱ monooxygenationȱ reactionsȱ ofȱ dioxinsȱ andȱ PAHs;ȱglutathioneȬSȬ
transferaseȱ (GST)ȱ phaseȱ IIȱ detoxificationȱ enzymeȱ butȱ alsoȱ implicatedȱ inȱ
oxidativeȱstressȱevents;ȱglutathioneȱperoxidaseȱ(GPX)ȱandȱglutathioneȱreductaseȱ
(GR),ȱantioxidantȱenzymesȱ(MartínȬDíazȱetȱal.,ȱ2007);ȱFerricȱreducingȱabilityȱofȱ
plasmaȱ (FRAP)ȱ assayȱ asȱ aȱ measureȱ ofȱ antioxidantȱ capacityȱ (Benzieȱ andȱ Strain,ȱ
1996);ȱandȱtheȱvitellogeninȱvariationȱinȱcrabsȱ(MartínȬDíaz,ȱ2004).ȱ
(c)ȱ Fieldȱ bioassaysȱ wereȱ carriedȱ outȱ toȱ determineȱ theȱ “inȱ situ”ȱ effects.ȱ
Theseȱ toxicityȱ testsȱ wereȱ performedȱ usingȱ fieldȱ deploymentsȱ inȱ cagesȱ ofȱ theȱ
crabȱ Carcinusȱ maenasȱ andȱ theȱ clamȱ Ruditappesȱ philippinarum.ȱ Theȱ sameȱ suiteȱ ofȱ
biomarkersȱ describedȱ aboveȱ andȱ usedȱ underȱ laboratoryȱ conditionsȱ wasȱ
employedȱtoȱdetermineȱsublethalȱeffectsȱinȱtheȱorganismsȱexposedȱduringȱaȱ28Ȭ
dȱperiodȱ(MartínȬDíazȱetȱal.,ȱ2007).ȱ
(e)ȱ‘inȱsituȱalteration’:ȱBenthicȱalterationȱwasȱselectedȱandȱdeterminedȱbyȱ
measuringȱ parametersȱ inȱ situȱ basedȱ inȱ taxonomicȱ identificationsȱ andȱ
- 336 -
communityȱdescriptiveȱstatisticsȱ(abundanceȬbiomassȱanalysis,ȱspeciesȱrichness,ȱ
diversity,ȱdominanceȱandȱproportionsȱofȱtheȱmajorȱtaxonomicȱgroups).ȱ
2.2.ȱDataȱintegrationȱȱ
Theȱ integrationȱ ofȱ theȱ dataȱ obtainedȱ fromȱ theȱ 4ȬLOEsȱ wasȱ performedȱ
throughȱ aȱ multivariateȱ analysisȱ approachȱ basedȱ onȱ linkingȱ allȱ theȱ variablesȱ
obtainedȱ whichȱ determinesȱ theȱ environmentalȱ degradationȱ ofȱ theȱ studiedȱ
ecosystemsȱ (Ribaȱ etȱ al.,ȱ 2004)ȱ andȱ (b)ȱ aȱ representationȱ usingȱ pieȱ chartsȱ byȱ anȱ
ANOVAȱapproachȱandȱbyȱmeansȱofȱtheȱdeterminationȱofȱdifferentȱfactorsȱ(Ribaȱ
etȱ al.,ȱ 2004;ȱ MoralesȬCaselles,ȱ accepted).ȱ Theȱ multivariateȱ analysisȱ wasȱ
performedȱ usingȱ principalȱ componentsȱ analysisȱ (PCA)ȱ inȱ orderȱ toȱ deriveȱ aȱ
reducedȱnumberȱofȱnewȱvariablesȱ(factors)ȱasȱlinearȱcombinationsȱofȱtheȱoriginalȱ
variables.ȱ Thisȱ providesȱ aȱ descriptionȱ ofȱ theȱ structureȱ ofȱ theȱ dataȱ withȱ theȱ
minimumȱ lossȱ ofȱ informationȱ (Ribaȱ etȱ al.,ȱ 2003).ȱ Pieȱ chartsȱ wereȱ obtainedȱ byȱ
conductingȱanȱANOVAȱandȱTukeyȱtestsȱwhichȱidentifiedȱsignificantȱdifferencesȱ
(pȱ <ȱ 0.05;ȱ pȱ <ȱ 0.01)ȱ inȱ sensitivityȱ amongȱ stationsȱ andȱ theȱ referenceȱ stationȱ forȱ
eachȱ ofȱ theȱ factorȱ scoresȱ obtainedȱ fromȱ theȱ PCAȱ (MoralesȬCasellesȱ etȱ al.,ȱ
accepted).ȱ
3.ȱResultsȱ
Tableȱ 1ȱ showsȱ theȱ summarizedȱ resultsȱ ofȱ theȱ differentȱ parametersȱ
analyzedȱ inȱ theȱ study.ȱ ȱ Inȱ general,ȱ noȱ organicȱ contaminationȱ wasȱ observedȱ inȱ
theȱreferenceȱsiteȱwhereasȱtheȱhighestȱlevelsȱofȱPAHsȱwereȱdetectedȱinȱstationsȱ
fromȱ theȱ Bayȱ ofȱ Algeciras.ȱ Theȱ concentrationȱ ofȱ metalsȱ inȱ sedimentsȱ variesȱ
amongȱ theȱ sitesȱ andȱ theȱ organicȱ carbonȱ andȱ finesȱ contentsȱ areȱ higherȱ inȱ
sedimentsȱ collectedȱ onȱ Algeciras.ȱ Itȱ wasȱ notȱ observedȱ aȱ generalȱ patternȱ inȱ
biologicalȱ parametersȱ betweenȱ theȱ samplingȱ sitesȱ exceptȱ forȱ siteȱ GR3ȱ whichȱ
- 337 -
presentedȱ theȱ highestȱ mortalityȱ inȱ theȱ acuteȱ bioassaysȱ andȱ alsoȱ aȱ remarkableȱ
benthicȱ alteration.ȱ Toȱ elucidateȱ theȱ matrixȱ ofȱ dataȱ theȱ PCAȱ wasȱ performedȱ toȱ
linkȱ theȱ variablesȱ includedȱ inȱ theȱ 4ȱ LOEsȱ (contamination,ȱ effectsȱ underȱ
laboratoryȱ conditions,ȱ inȱ situȱ effectsȱ andȱ benthicȱ alteration)ȱ appliedȱ toȱ
determineȱtheȱsedimentȱ qualityȱ ofȱtheȱtwoȱareasȱaffectedȱbyȱoilȱspillsȱweȱhaveȱ
obtainedȱfiveȱnewȱfactorsȱthatȱaccountȱforȱallȱtheȱvariablesȱandȱhaveȱaȱdifferentȱ
influenceȱforȱeachȱsamplingȱsiteȱ(tableȱ2).ȱTheseȱfactorsȱexplainȱanȱ82.7ȱ%ȱofȱtheȱ
varianceȱ inȱ theȱ originalȱ dataȱ set,ȱ andȱ negativeȱ loadingȱ areȱ consideredȱ asȱ
importantȱasȱpositiveȱvalues.ȱTheȱpredominantȱfactor,ȱFactorȱ#1,ȱaccountsȱforȱaȱ
34.5ȱ%ȱofȱtheȱvarianceȱandȱshowsȱtheȱrelationshipȱbetweenȱtheȱconcentrationȱofȱ
Pb,ȱNiȱandȱPAHsȱinȱsediment,ȱtheȱpercentageȱofȱorganicȱcarbonȱandȱfines,ȱtheȱ
lethalȱ toxicityȱ determinedȱ byȱ theȱ amphipodȱ bioassay,ȱ theȱ bioaccumulationȱ ofȱ
PAHsȱ inȱ A.ȱ marina,ȱ theȱ inductionȱ ofȱ GPXȱ (crabs),ȱ ERODȱ (crabsȱ andȱ clams),ȱ
vitellogeninȱ variationȱ (crab)ȱ andȱ FRAPȱ (clam)ȱ activityȱ underȱ laboratoryȱ
conditionsȱ andȱ theȱ inductionȱ ofȱ ERODȱ activityȱ (clams)ȱ afterȱ fieldȱ exposures;ȱ
besidesȱ allȱ theȱ variablesȱ relatedȱ withȱ benthicȱ alterationȱ (abundance,ȱ speciesȱ
richness,ȱ diversity,ȱ dominanceȱ andȱ proportionsȱ ofȱ molluscs,ȱ polychaeteȱ andȱ
crustacean)ȱ areȱ gatheredȱ inȱ Factorȱ #1.ȱ Takingȱ thisȱ intoȱ accountȱ thisȱ factorȱ
representsȱenvironmentalȱdegradationȱbyȱPAHs,ȱPbȱandȱNi.ȱ
Factorȱ#2ȱ(18.0ȱ%)ȱcombines,ȱwithȱnegativeȱloading,ȱtheȱpresenceȱofȱHgȱinȱ
theȱsedimentsȱwithȱaȱsetȱofȱbiomarkersȱ(GPXȱandȱGSTȱactivityȱinȱcrabȱandȱGRȱ
inductionȱinȱclamsȱunderȱlaboratoryȱandȱfieldȱconditions,ȱGPX,ȱGRȱandȱGSTȱinȱ
crabsȱ afterȱ fieldȱ exposures,ȱ GRȱ andȱ FRAPȱ activityȱ inȱ clamsȱ underȱ laboratoryȱ
conditionsȱ andȱ inverseȱ relationȱ withȱ GSTȱ inȱ clamȱ inȱ laboratoryȱ studiesȱ andȱ
FRAPȱinȱcrabsȱunderȱfieldȱdeployments)ȱandȱtheȱvariablesȱofȱbenthicȱalterationȱ
describedȱbyȱ speciesȱ richnessȱ andȱ percentageȱ ofȱ crustacean.ȱ Thisȱ factorȱ canȱ beȱ
explainedȱasȱpollutionȱproducedȱbyȱtheȱpresenceȱofȱHgȱinȱtheȱsediments.ȱ
- 338 -
Tableȱ 1.ȱ Summarizedȱ resultsȱ ofȱ physicochemicalȱ analysisȱ (mgKgȬ1ȱ forȱ
metals,ȱ ΐgKgȬ1ȱ forȱ PAHs,ȱ percentageȱ ofȱ organicȱ carbonȱ –o.c.Ȭȱ andȱ finesȱ inȱ
sediment)ȱ theȱ acuteȱ toxicityȱ testsȱ (Corophiumȱ andȱ Arenicola:ȱ %ȱ mortality;ȱ
Microtox:ȱIC50;ȱbioaccumulationȱofȱPAHs:ȱΐgKgȬ1),ȱbiomarkerȱresponsesȱunderȱ
fieldȱ andȱ laboratoryȱ conditionsȱ (glutathioneȱ peroxidaseȱ activityȱ GPX:ȱ
nmol/min/mgȱ prot,ȱ glutathioneȱ transferaseȱ GSTȱ activityȱ nmol/min/mgȱ prot,ȱ
glutathioneȱreductaseȱGRȱactivityȱnmol/min/mgȱprot,ȱferricȱreducingȱabilityȱofȱ
plasmaȱ FRAPȱ activityȱ ΐM/mg/min,ȱ ERODȱ activityȱ pmol/mg/minȱ andȱ
vitellogeninȱ variationȱ ngȱ100mLȬ1)ȱ andȱtheȱalterationȱ parametersȱforȱsedimentsȱ
fromȱtheȱGalicianȱCoastȱȬAINPȱ(A,ȱB,ȱC)ȱandȱCormeȬLaxeȱ(D,ȱE,ȱF)ȬȱandȱtheȱGulfȱ
ofȱCádizȱ–BayȱofȱAlgecirasȱ(GR3,ȱGR4,ȱP1)ȱandȱtheȱBayȱofȱCádizȱ–CAȬ.ȱn.d.:ȱnotȱ
detected;ȱn.a.:ȱnotȱavailable.ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
- 339 -
ȱȱ
Biomarkersȱ(laboratory)ȱ
Toxicityȱtestsȱ Physicochemicalȱanalysesȱ
ȱȱ
Aȱ
Biomarkersȱ(field)ȱ
Cȱ
Dȱ
Eȱ
Fȱ
GR3ȱ
GR4ȱ
P1ȱ
CAȱ
Znȱȱ
377ȱ
91ȱ
164ȱ
25ȱ
19.9ȱ
271ȱ
138ȱ
35.3ȱ
56.7ȱ
21.3ȱ
Pbȱ
1.5ȱ
0.9ȱ
0.85ȱ
3.7ȱ
7.3ȱ
5.9ȱ
21.6ȱ
6.21ȱ
12.3ȱ
2.28ȱ
Cuȱ
5.2ȱ
1.4ȱ
1.4ȱ
0.7ȱ
0.43ȱ
4.2ȱ
5.01ȱ
3.67ȱ
75.2ȱ
6.98ȱ
Niȱ
13.3ȱ
2.4ȱ
4.5ȱ
1.7ȱ
1.5ȱ
5.7ȱ
74.7ȱ
13.1ȱ
13.3ȱ
0.06ȱ
Hgȱ
0.7ȱ
0.8ȱ
0.6ȱ
2ȱ
2.1ȱ
3.4ȱ
1.04ȱ
0.25ȱ
0.65ȱ
n.d.ȱ
PAHȱ
108ȱ
67ȱ
n.d.ȱ
38ȱ
52ȱ
323ȱ
2961ȱ
802ȱ
641ȱ
n.d.ȱ
O.C.ȱ
0.28ȱ
0.26ȱ
0.30ȱ
0.31ȱ
0.37ȱ
0.65ȱ
2.15ȱ
3.19ȱ
3.86ȱ
1.07ȱ
Finesȱ
4.32ȱ
2.81ȱ
2.76ȱ
3.79ȱ
5.50ȱ
5.95ȱ
69.35ȱ
59.33ȱ
35.44ȱ
2.5ȱ
Corophiumȱ
23ȱ
20ȱ
17ȱ
10ȱ
17ȱ
20ȱ
100ȱ
75ȱ
20ȱ
0ȱ
Arenicolaȱ
28ȱ
28ȱ
22ȱ
39ȱ
17ȱ
17ȱ
30ȱ
17ȱ
46ȱ
0ȱ
Microtoxȱ
5631ȱ
9422ȱ
1801ȱ
3977ȱ
21041ȱ
4398ȱ
235ȱ
249ȱ
1642ȱ
6013ȱ
BioaccumulationȱPAHȱ
2927ȱ
2573ȱ
2666ȱ
2616ȱ
3912ȱ
3285ȱ
GPXȬcrabȬlabȱ
11.6ȱ
9.7ȱ
8.2ȱ
19.3ȱ
19.5ȱ
15.9ȱ
18.8ȱ
9.1ȱ
12.4ȱ
6.3ȱ
GPXȬclamȬlabȱ
2.1ȱ
2.9ȱ
4.5ȱ
6.1ȱ
3.1ȱ
4.2ȱ
3.8ȱ
2.7ȱ
2.5ȱ
5.1ȱ
5158.9ȱ 4809.1ȱ 4097.0ȱ 2421.0ȱ
GRȬcrabȬlabȱ
1.1ȱ
0.7ȱ
0.9ȱ
0.9ȱ
0.6ȱ
1.5ȱ
1.1ȱ
0.5ȱ
1.0ȱ
0.9ȱ
GRȬclamȬlabȱ
2.1ȱ
1.6ȱ
2.3ȱ
3.4ȱ
11.7ȱ
4.0ȱ
2.8ȱ
1.4ȱ
1.5ȱ
2.7ȱ
GSTȬcrabȬlabȱ
140ȱ
218ȱ
407ȱ
430ȱ
684ȱ
1071ȱ
294.6ȱ
203.2ȱ
377.7ȱ
611.2ȱ
GSTȬclamȬlabȱ
1634.5ȱ 1117.7ȱ 1542.1ȱ
1293ȱ
839ȱ
1624ȱ
1199ȱ
910ȱ
848ȱ
901.1ȱ
ERODȬcrabȬlabȱ
0.1ȱ
0.1ȱ
0.1ȱ
0.0ȱ
0.1ȱ
0.1ȱ
0.3ȱ
0.3ȱ
0.0ȱ
0.0ȱ
ERODȬclamȬlabȱ
0.3ȱ
0.3ȱ
0.4ȱ
0.4ȱ
0.4ȱ
0.2ȱ
1.2ȱ
0.7ȱ
0.8ȱ
0.1ȱ
FRAPȬcrabȬlabȱ
3.9ȱ
2.1ȱ
2.6ȱ
2.9ȱ
2.9ȱ
1.6ȱ
2.1ȱ
n.a.ȱ
6.1ȱ
1.7ȱ
FRAPȬclamȬlabȱ
10.6ȱ
7.8ȱ
4.0ȱ
13.7ȱ
12.1ȱ
6.4ȱ
15.4ȱ
3.6ȱ
8.7ȱ
9.5ȱ
VITȬcrabȬlabȱ
Benthicȱalterationsȱ
Bȱ
0.0936ȱ 0.0380ȱ 0.0879ȱ 0.1130ȱ 0.0889ȱ 0.1440ȱ 0.3074ȱ 0.2332ȱ 0.4301ȱ 0.0755ȱ
GPXȬcrabȬfieldȱ
17.8ȱ
23.1ȱ
15.9ȱ
41.4ȱ
193.1ȱ
125.7ȱ
4.8ȱ
6.5ȱ
4.0ȱ
GPXȬclamȬfieldȱ
10.5ȱ
3.6ȱ
4.0ȱ
25.5ȱ
3.2ȱ
7.0ȱ
2.9ȱ
2.8ȱ
3.6ȱ
5.1ȱ
GRȬcrabȬfieldȱ
0.7ȱ
1.4ȱ
1.4ȱ
9.9ȱ
9.5ȱ
23.4ȱ
0.8ȱ
1.5ȱ
0.5ȱ
0.9ȱ
6.3ȱ
GRȬclamȬfieldȱ
2.9ȱ
1.3ȱ
3.8ȱ
9.7ȱ
14.7ȱ
8.0ȱ
1.9ȱ
1.1ȱ
0.8ȱ
2.7ȱ
GSTȬcrabȬfieldȱ
1098ȱ
1564ȱ
690ȱ
1489ȱ
7523ȱ
6073ȱ
443.4ȱ
1352.7ȱ
592.7ȱ
611.2ȱ
GSTȬclamȬfieldȱ
2061ȱ
372ȱ
1199ȱ
3366ȱ
131ȱ
1558ȱ
997.8ȱ
1031.6ȱ
876.5ȱ
1542.1ȱ
ERODȬcrabȬfieldȱ
0.1ȱ
3.0ȱ
0.0ȱ
8.5ȱ
0.4ȱ
0.5ȱ
0.0ȱ
0.0ȱ
0.0ȱ
0.0ȱ
ERODȬclamȬfieldȱ
0.2ȱ
0.1ȱ
0.1ȱ
0.6ȱ
0.1ȱ
0.1ȱ
0.3ȱ
0.3ȱ
0.3ȱ
0.1ȱ
FRAPȬcrabȬfieldȱ
2.7ȱ
n.a.ȱ
n.a.ȱ
2.4ȱ
n.a.ȱ
n.a.ȱ
1.9ȱ
3.5ȱ
4.2ȱ
1.7ȱ
FRAPȬclamȬfieldȱ
10.4ȱ
3.1ȱ
2.6ȱ
23.6ȱ
2.0ȱ
6.6ȱ
2.4ȱ
7.7ȱ
4.1ȱ
9.5ȱ
VITȬcrabȬfieldȱ
0.0049ȱ 0.0099
0.0879
0.0047
0.0426
0.0098
0.0189ȱ 0.1652ȱ 0.0303
speciesȱNºȱ
28.5ȱ
42.4ȱ
28.6ȱ
32.1ȱ
48.2ȱ
0.67ȱ
4.67ȱ
4.67ȱ
14ȱ
33.9ȱ
0.0721
specificȱrichnessȱ
5.1ȱ
5ȱ
4.3ȱ
3ȱ
3ȱ
2.9ȱ
0ȱ
1.21ȱ
1.25ȱ
2.57ȱ
Diversityȱ
15.3ȱ
28.4ȱ
39.1ȱ
30ȱ
40.1ȱ
15.4ȱ
0ȱ
1.29ȱ
1.24ȱ
1.64ȱ
Dominanceȱ
0.50ȱ
0.10ȱ
0.06ȱ
0.15ȱ
0.19ȱ
0.20ȱ
0ȱ
0.72ȱ
0.68ȱ
0.66ȱ
%ȱMolluscaȱ
15.3ȱ
28.4ȱ
39.1ȱ
30.0ȱ
40.1ȱ
15.4ȱ
0.0ȱ
34.4ȱ
25.4ȱ
78.5ȱ
%ȱPolychaeteȱ
20.0ȱ
21.5ȱ
21.7ȱ
20.0ȱ
22.2ȱ
23.1ȱ
100.0ȱ
45.3ȱ
64.4ȱ
12.7ȱ
%ȱCrustaceaȱ
37.0ȱ
41.0ȱ
39.1ȱ
50.0ȱ
51.4ȱ
61.5ȱ
0.0ȱ
20.3ȱ
10.2ȱ
8.8ȱ
ȱ
- 340 -
Tableȱ 2.ȱ Sortedȱ rotatedȱ factorȱ loadingsȱ forȱ theȱ fiveȱ principalȱ factorsȱ
obtainedȱafterȱ applyingȱ theȱ principalȱcomponentsȱanalysisȱtoȱtheȱ originalȱdataȱ
setȱofȱ41ȱparametersȱincludedȱinȱtheȱweightȱofȱevidenceȱapproach.ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
- 341 -
ȱȱ
Znȱȱ
Pbȱȱ
Cuȱȱ
Niȱȱ
Hgȱ
PAHȱȱ
O.C.ȱ
Finesȱ
Corophiumȱ
Arenicolaȱȱ
Microtoxȱ
BioaccumulationȱPAHsȱ
GPXȬcrabȬlabȱ
GPXȬclamȬlabȱ
GRȬcrabȬlabȱ
GRȬclamȬlabȱ
GSTȬcrabȬlabȱ
GSTȬclamȬlabȱ
ERODȬcrabȬlabȱ
ERODȬclamȬlabȱ
FRAPȬcrabȬlabȱ
FRAPȬclamȬlabȱ
VTGȬcrabȬlabȱ
GPXȬcrabȬfieldȱ
GPXȬclamȬfieldȱ
GRȬcrabȬfieldȱ
GRȬclamȬfieldȱ
GSTȬcrabȬfieldȱ
GSTȬclamȬfieldȱ
ERODȬcrabȬfieldȱ
ERODȬclamȬfieldȱ
FRAPȬcrabȬfieldȱ
FRAPȬclamȬfieldȱ
VTGȬcrabȬfieldȱ
speciesȱNºȱ
specificȱrichnessȱ
Diversityȱ
Dominanceȱ
%ȱMolluscȱ
%ȱPolychaetaȱ
%ȱCrustaceaȱ
Factor 1
34.5ȱ
Factor 2
18.0ȱ
Factor 3
14.0ȱ
Factor 4
9.0ȱ
Factor 5
7.1ȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.78ȱ
0.90ȱ
ņȱ
0.95ȱ
ņȱ
0.98ȱ
0.45ȱ
0.86ȱ
0.92ȱ
ņȱ
ņȱ
0.83ȱ
0.47ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.85ȱ
0.88ȱ
ņȱ
0.45ȱ
0.55ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.30ȱ
ņȱ
ņȱ
ņȱ
0.96ȱ
0.52ȱ
0.93ȱ
0.93ȱ
0.55ȱ
0.89ȱ
ņȱ
ņȱ
ņȱ
Ȭ0.81ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
Ȭ0.46ȱ
ņȱ
ņȱ
Ȭ0.87ȱ
Ȭ0.72ȱ
0.50ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
Ȭ0.96ȱ
ņȱ
Ȭ0.76ȱ
Ȭ0.88ȱ
Ȭ0.94ȱ
ņȱ
ņȱ
ņȱ
0.47ȱ
ņȱ
ņȱ
ņȱ
0.42ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
Ȭ0.35ȱ
ņȱ
ņȱ
0.68ȱ
Ȭ0.57ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
Ȭ0.45ȱ
ņȱ
ņȱ
Ȭ0.94ȱ
ņȱ
ņȱ
ņȱ
Ȭ0.73ȱ
Ȭ0.89ȱ
Ȭ0.86ȱ
ņȱ
Ȭ0.93ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.35ȱ
0.93ȱ
ņȱ
ņȱ
ņȱ
0.67ȱ
ņȱ
ņȱ
0.70ȱ
Ȭ0.30ȱ
ņȱ
ņȱ
Ȭ0.31ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.38ȱ
0.77ȱ
ņȱ
0.77ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
0.34ȱ
0.65ȱ
ņȱ
ņȱ
ņȱ
0.36ȱ
ņȱ
ņȱ
ņȱ
0.38ȱ
ņȱ
ņȱ
ņȱ
0.43ȱ
ņȱ
Ȭ0.38ȱ
ņȱ
ņȱ
0.43ȱ
Ȭ0.47ȱ
ņȱ
ņȱ
ņȱ
0.64ȱ
ņȱ
ņȱ
Ȭ0.49ȱ
ņȱ
ņȱ
0.39ȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
ņȱ
Ȭ0.80ȱ
ņȱ
Ȭ0.54ȱ
ņȱ
ņȱ
0.70ȱ
ņȱ
0.53ȱ
0.60ȱ
ņȱ
ņȱ
Ȭ0.35ȱ
- 342 -
ȱ
Theȱ thirdȱ factor,ȱ Factorȱ #3,ȱ accountsȱ forȱ aȱ 14.0ȱ %ȱ ofȱ theȱ varianceȱ andȱ
links,ȱ withȱ negativeȱ loading,ȱ theȱ toxicityȱ detectedȱ byȱ theȱ Arenicolaȱ assay,ȱ withȱ
theȱ inductionȱ ofȱ someȱ biomarkersȱ mainlyȱ underȱ fieldȱ conditionsȱ (GPXȱ andȱ
FRAPȱ inȱ clamȱ inȱ fieldȱ andȱ laboratoryȱ exposures;ȱ GSTȱ inductionȱ inȱ clamȱ andȱ
ERODȱactivityȱinȱclamȱandȱcrabsȱunderȱfieldȱconditions;ȱoppositeȱlinkȱwithȱGPXȱ
activityȱ inȱ crabsȱ fromȱ theȱ laboratoryȱ experiments).ȱ Thisȱ factorȱ isȱ relatedȱ toȱ anȱ
unknownȱstressorȱwhichȱisȱproducingȱaȱgeneralȱstressȱtoȱtheȱexposedȱorganismsȱ
butȱ notȱ aȱ benthicȱ alterationȱ neitherȱ aȱ pollutionȱ norȱ degradationȱ inȱ theȱ benthicȱ
environment.ȱ
Factorȱ #4ȱ (9.0ȱ %)ȱ isȱ aȱ combinationȱ ofȱ theȱ concentrationȱ ofȱ Pbȱ andȱ Cuȱ inȱ
theȱ sediments,ȱ withȱ theȱ percentageȱ ofȱ organicȱ carbon,ȱ toxicityȱ inȱ theȱ Arenicolaȱ
toxicityȱtest,ȱinȱsituȱalterationȱofȱtheȱpolychaeteȱpopulationȱandȱtheȱvariationȱofȱ
someȱbiomarkersȱ(ERODȱinȱclamsȱandȱFRAPȱinȱcrabsȱunderȱlaboratoryȱanȱfieldȱ
conditions,ȱ Vitellogeninȱ variationȱ inȱ crabsȱ andȱ GPXȱ activityȱ inȱ clamsȱ underȱ
laboratoryȱ conditions).ȱ Inȱ general,ȱ Factorȱ #4ȱ canȱ beȱ relatedȱ toȱ aȱ contaminationȱ
byȱCuȱandȱPbȱthatȱcanȱbeȱconsideredȱaȱpotentialȱriskȱtoȱtheȱenvironmentȱbutȱnotȱ
associatedȱwithȱpollution.ȱ
Factorȱ #5ȱ representsȱ aȱ 7.1ȱ %ȱ ofȱ theȱ varianceȱ andȱ groupsȱ theȱ metalsȱ Znȱ
andȱHgȱboundȱtoȱsedimentȱwithȱtoxicȱresponsesȱinȱtheȱArenicolaȱtoxicityȱtest,ȱtheȱ
antioxidantȱ activityȱ determinedȱ byȱ theȱ inductionȱ ofȱ GRȱ andȱ FRAPȱ inȱ crabsȱ inȱ
laboratoryȱ exposuresȱ andȱ theȱ alterationȱ ofȱ theȱ molluscȱ population.ȱ Otherȱ
variablesȱ presentȱ oppositesȱ behaviourȱ suchȱ asȱ theȱ GSTȱ inȱ clamsȱ andȱ
vitellogeninȱ variationȱ inȱ crabs,ȱ specificȱ richnessȱ andȱ theȱ percentageȱ ofȱ
crustacean.ȱ Inȱ thisȱ sense,ȱ thisȱ factorȱ couldȱ beȱ explainedȱ asȱ aȱ contaminationȱ byȱ
Znȱ andȱ Hgȱ whichȱ isȱ producingȱ someȱ stressȱ inȱ theȱ environmentȱ butȱ notȱ
pollution.ȱ
Inȱorderȱtoȱestablishȱtheȱmeaningȱofȱeachȱfactorȱinȱtheȱareaȱofȱstudy,ȱtheȱ
factorȱscoresȱhaveȱbeenȱrepresentedȱforȱeveryȱsingleȱstationȱ(Figureȱ3).ȱFactorȱ#1ȱȱ
- 343 -
ȱ
3
2
GR3
Factor 1
Factor 2
1
2
A
CA
B
C
GR3
GR4
P1
0
1
D
GR4
-1
F
0
D
-1
CA
A
B
E
P1
F
-2
E
C
-3
2
P1
3
Factor 4
Factor 3
1
B
CA
E
C
F
A
GR3
GR4
P1
2
0
1
-1
D
E
GR4
0
-2
A
-1
-3
D
B
CA
C
F
GR3
-2
-4
2
Factor 5
A
1
F
B
GR3
C
P1
D
0
E
-1
CA
-2
GR4
Figureȱ 3.ȱ Estimatedȱ factorȱ scoresȱ forȱ theȱ threeȱ factorsȱ inȱ eachȱ ofȱ theȱ 10ȱ
cases.ȱTheȱfactorȱscoresȱquantifyȱtheȱprevalenceȱofȱeachȱfactorȱforȱeveryȱstation.ȱ
relatedȱ toȱ theȱ environmentalȱ degradationȱ byȱ PAHs,ȱ Pbȱ andȱ Niȱ presentsȱ aȱ
positiveȱ loadingȱmainlyȱinȱ theȱstationȱGR3ȱ(2.7)ȱfollowedȱbyȱtheȱsiteȱGR4ȱ(0.6)ȱ
fromȱ theȱ Bayȱ ofȱ Algeciras.ȱ Theȱ secondȱ factorȱ whichȱ explainsȱ withȱ negativeȱ
loadingȱ theȱ stressȱ producedȱ byȱ theȱ presenceȱ ofȱ Hgȱ inȱ theȱ sedimentsȱ showsȱ
prevalenceȱinȱtheȱstudyȱsitesȱlocatedȱinȱtheȱBayȱofȱCormeȬLaxeȱEȱ(Ȭ2.2)ȱ>ȱFȱ(Ȭ1.4)ȱ
>ȱDȱ(Ȭ0.3).ȱTheȱunknownȱstressorȱdescribedȱbyȱFactorȱ#3ȱwithȱnegativeȱloadingȱ
hasȱ onlyȱ prevalenceȱ inȱ theȱ stationȱ Dȱ (Ȭ2.8)ȱ fromȱ CormeȬLaxe.ȱ Inȱ theȱ caseȱ ofȱ
Factorȱ #4ȱ relatedȱ toȱ aȱ contaminationȱ byȱ Cuȱ thatȱ whichȱ couldȱ beȱ consideredȱ aȱ
- 344 -
potentialȱrisk,ȱtheȱaffectedȱareȱslightlyȱstationsȱDȱ(0.2)ȱandȱEȱ(0.1)ȱlocatedȱinȱtheȱ
areaȱofȱCormeȬLaxeȱandȱmainlyȱstationȱP1ȱ(2.8)ȱlocatedȱinȱtheȱBayȱofȱAlgeciras.ȱ
Finally,ȱ theȱ prevalenceȱ ofȱ Factorȱ #5,ȱ whichȱ explainsȱ theȱ stressȱ causedȱ byȱ theȱ
contaminationȱbyȱZnȱandȱHg,ȱisȱdetectedȱinȱtheȱstationsȱlocatedȱinȱtheȱAINPȱAȱ
(1.3),ȱBȱ(0.6)ȱandȱCȱ(0.2),ȱtheȱsiteȱFȱ(1.1)ȱinȱCormeȬLaxeȱandȱGR3ȱ(0.6)ȱinȱtheȱBayȱ
ofȱAlgeciras.ȱȱ
Figureȱ 4ȱ showsȱ theȱ significantȱ differencesȱ betweenȱ theȱ stationsȱ andȱ theȱ
referenceȱ siteȱ forȱ eachȱ ofȱ theȱ fiveȱ studiedȱ factors.ȱ Significantȱ differencesȱ (pȱ <ȱ
0.01)ȱfromȱtheȱreferenceȱ(CA)ȱwereȱobservedȱforȱFactorȱ#1ȱandȱ#2ȱinȱtheȱstationsȱ
fromȱCormeȱLaxeȱ(D,ȱE,ȱF)ȱandȱtheȱBayȱofȱAlgecirasȱ(GR3,ȱGR4ȱandȱP1).ȱFactorȱ
#3ȱwasȱsignificantlyȱdifferentȱforȱB,ȱCȱ(pȱ<ȱ0.05)ȱinȱCies,ȱDȱ(pȱ<ȱ0.01),ȱEȱ(pȱ<ȱ0.01)ȱ
inȱCormeȬLaxeȱandȱP1ȱ(pȱ<ȱ0.05)ȱinȱAlgeciras.ȱOnȱtheȱotherȱhandȱFactorsȱ#4ȱandȱ
#5ȱ resultedȱ toȱ beȱ significantlyȱ differentȱ (pȱ <ȱ 0.01ȱ exceptȱ forȱ F#4ȱ inȱ Cȱ whichȱ
presentedȱpȱ<ȱ0.05)ȱtoȱtheȱreferenceȱforȱallȱtheȱstudiedȱstationsȱexceptȱforȱFactorȱ
#5ȱ inȱ P1ȱ whichȱ didȱ notȱ presentȱ theseȱ differences.ȱ Accordingȱ toȱ theȱ Clusterȱ
analysisȱ (Figureȱ 5)ȱ theȱ stationsȱ wereȱ groupedȱ inȱ aȱ wayȱ similarlyȱ toȱ theirȱ realȱ
locationȱinȱfield.ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
ȱ
- 345 -
ȱ
ȱ
A
ȱF 5
B
F1
F5
C
F1
F5
F1
ȱ
F4
F2
F4
F2
F4
F2
ȱ
F3
F3
F3
D
E
F
ȱ
ȱ
ȱ
F5
F1
F5
F1
F5
F1
ȱ
F4
F2
F4
F2
F4
F2
ȱ
F3
F3
GR3
GR4
F3
ȱ
F5
F1
F5
ȱ
P1
F1
F5
F1
ȱ
F4
F2
F4
F2
F4
F2
ȱ
F3
F3
F3
ȱ
Figureȱ 4.ȱ Pieȱ chartsȱ whichȱ representȱ theȱ significantȱ differencesȱ ofȱ theȱ
factorsȱscoreȱinȱeveryȱstudyȱsiteȱrelatedȱtoȱtheȱreferenceȱsiteȱCAȱ(dotted:ȱpȱ<ȱ0.01;ȱ
slightlyȱdotted:ȱpȱ<ȱ0.05;ȱnotȱdotted:ȱnoȱsignificantȱdifferences,ȱp>0.05).ȱȱȱ
ȱ
ȱ
- 346 -
Tree Diagram for 20 Cases
Single Linkage
Percent disagreement
1.0
Linkage D istance
0.8
0.6
0.4
0.2
0.0
GR3-2 GR4-2
P1-2
F-2
E-2
D-2
B-2
C-2
A-2
CA-2
GR3-1 GR4-1
P1-1
F-1
E-1
D-1
B-1
C-1
A-1
CA-1
ȱFigureȱ 5.ȱ Treeȱ diagramȱ classificationȱ ofȱ theȱ 10ȱ stationsȱ (inȱ duplicate)ȱ
basedȱinȱClusterȱanalysisȱ(CA:ȱreferenceȱstation;ȱA,ȱBȱandȱC:ȱAINP;ȱD,ȱEȱandȱF:ȱ
CormeȬLaxe;ȱGR3,ȱGR4ȱandȱP1:ȱBayȱofȱAlgeciras).ȱȱ
4.ȱDiscussionȱ
Inȱtheȱpresentȱstudyȱtheȱintegrationȱofȱ4ȱLOEsȱasȱpartȱofȱaȱWOEȱapproachȱ
toȱassessȱoilȱcontaminatedȱsedimentsȱisȱproposed.ȱTheȱdifferentȱlinesȱemployedȱ
includeȱ aȱ setȱ ofȱ 41ȱ variablesȱ relatedȱ toȱ contamination,ȱ toxicityȱ andȱ
bioaccumulationȱunderȱlaboratoryȱconditions,ȱsedimentȱtoxicityȱmeasureȱunderȱ
fieldȱ conditionsȱ andȱ benthicȱ alterationȱ analyzingȱ theȱ macrobenthicȱ structureȱ
parameters.ȱ Theȱ useȱ ofȱ sublethalȱ bioassaysȱ validatedȱ bothȱ inȱ laboratoryȱ andȱ
fieldȱ exposuresȱ byȱ usingȱ biomarkersȱ contributesȱ toȱ aȱ betterȱ understandingȱ ofȱ
theȱ toxicȱ processesȱ ofȱ theȱ contaminantsȱ andȱ suppliesȱ theȱ lackȱ ofȱ informationȱ
oftenȱ shownȱ byȱ acuteȱ toxicityȱ testsȱ performedȱ alone.ȱ Theȱ applicationȱ ofȱ thisȱ
methodologyȱ toȱ sedimentsȱ affectedȱ byȱ oilȱ spillsȱ inȱ differentȱ mannersȱ hasȱ
allowedȱdeterminingȱtheȱenvironmentalȱqualityȱofȱtheȱimpactedȱareasȱasȱwellȱasȱ
differentiatingȱtheȱmostȱprobableȱcausesȱofȱtheȱenvironmentalȱdegradation.ȱȱȱȱ
- 347 -
TheȱMultivariateȱanalysesȱhaveȱdemonstratedȱtheȱsuitableȱuseȱofȱtheȱsiteȱ
CAȱasȱreferenceȱstation.ȱResultsȱhaveȱshownȱthatȱtheȱGalicianȱCoastȱwhichȱwasȱ
affectedȱ byȱ theȱ oilȱ spillȱ fromȱ theȱ tankerȱ Prestigeȱ inȱ 2002ȱ doesȱ notȱ presentȱ anȱ
environmentalȱdegradationȱdueȱtoȱhydrocarbonsȱwhenȱcomparingȱwithȱtheȱBayȱ
ofȱ Algecirasȱ fourȱ yearsȱ afterȱ theȱ spill;ȱ howeverȱ significantȱ differencesȱ (P<0.01)ȱ
wereȱdetectedȱ withȱ theȱ referenceȱstationȱregardingȱ toȱ sedimentsȱpollutionȱ dueȱ
toȱfuelȱoilȱinȱtheȱBayȱofȱCormeȬLaxeȱ(MoralesȬCasellesȱetȱalȱsubmitted).ȱOnȱtheȱ
otherȱ handȱ theȱ studyȱ sitesȱ locatedȱ inȱ theȱ Ciesȱ Islandȱ inȱ theȱ AINPȱ presentȱ
absenceȱ ofȱ pollutionȱ dueȱ toȱ fuelȱ oilȱ stemȱ fromȱ theȱ tankerȱ Prestigeȱ althoughȱ anȱ
environmentalȱ riskȱ causedȱ byȱ aȱ metallicȱ contaminationȱ ofȱ Cu,ȱ Znȱ andȱ Hgȱ isȱ
presentȱ inȱ theȱ area.ȱ Previousȱ studiesȱ haveȱ demonstrateȱ sourcesȱ ofȱ metalsȱ
comingȱ fromȱ anthropogenicȱ sourcesȱ locatedȱ inȱ theȱ areaȱ closedȱ toȱ theȱ AINPȱ
(Carballeiraȱ etȱal.,ȱ 1997;ȱPérezȬLópezȱetȱal.,ȱ2003).ȱTheȱBayȱofȱCormeȬLaxeȱalsoȱ
presentsȱ environmentalȱ stressȱ dueȱ toȱ Cu,ȱ Znȱ andȱ Hgȱ comingȱ fromȱ
anthropogenicȱsourcesȱwhichȱcouldȱincludeȱdifferentȱkindȱofȱspillsȱcomingȱfromȱ
landȱ andȱ theȱ maritimeȱ traffic.ȱ Evenȱ thoughȱ noȱ signalsȱ ofȱ alterationȱ ofȱ theȱ
benthicȱcommunityȱhaveȱthenȱdetectedȱinȱtheȱarea,ȱaȱnonȱquantifiedȱstressorȱhasȱ
beenȱ determinedȱ asȱ potentiallyȱ toxic.ȱ Theȱ stressȱ wasȱ mainlyȱ detectedȱ underȱ
fieldȱ exposuresȱ whatȱ suggestsȱ thatȱ theȱ stressorȱ couldȱ cameȱ fromȱ theȱ water;ȱ aȱ
possibleȱ causeȱ couldȱbeȱ relatedȱtoȱtheȱpresenceȱofȱ industrialȱculturedȱofȱ cagedȱ
musselsȱspeciallyȱclosedȱtoȱstationȱDȱwhichȱimplyȱaȱsourceȱofȱorganicȱmatterȱtoȱ
theȱ waterȱ columnȱ providingȱ stressȱ toȱ theȱ organismsȱ exposed.ȱ ȱ Onȱ theȱ otherȱ
hand,ȱ theȱ resultsȱ observedȱ inȱ theȱ studyȱ sitesȱ fromȱ theȱ Bayȱ ofȱ Algecirasȱ haveȱ
shownȱ anȱ importantȱ environmentalȱ degradationȱ inȱ theȱ Guadarranqueȱ Riverȱ
dueȱ toȱ aȱ chronicȱ contaminationȱ byȱ aȱ mixtureȱ ofȱ contaminantsȱ thatȱ includeȱ
mainlyȱ Pb,ȱ Niȱ andȱ PAHs,ȱ allȱ ofȱ themȱ representativeȱ ofȱ hydrocarbonȱ
contaminationȱ inȱ theȱ ecosystemȱ andȱ previouslyȱ reportedȱ byȱ otherȱ studiesȱ
(CSIC,ȱ2003;ȱCSIC,ȱ2005).ȱAcuteȱandȱsublethalȱtoxicologicalȱresponsesȱbesidesȱanȱ
importantȱ alterationȱ onȱ theȱ biotaȱ wereȱ associatedȱ withȱ thisȱ kindȱ ofȱ pollution.ȱ
Theȱ presenceȱ ofȱ petrochemicalȱ industries,ȱ theȱ highȱ maritimeȱ trafficȱ andȱ theȱ
- 348 -
bunkeringȱ activitiesȱ areȱ theȱ mainȱ factorsȱ whichȱ involveȱ aȱ threatȱ toȱ theȱ marineȱ
ecosystemȱofȱtheȱBayȱofȱAlgecirasȱinȱadditionȱtoȱtheȱhumanȱriskȱrepresentedȱbyȱ
theȱcollectionȱofȱgoodsȱforȱconsumeȱinȱtheȱzone.ȱOtherȱcontaminantsȱsuchȱasȱCu,ȱ
Znȱ andȱ Hgȱ areȱ producingȱ stressȱ toȱ theȱ biotaȱ inȱ stationsȱ P1ȱ andȱ GR3;ȱ onȱ theȱ
contrary,ȱ theȱ siteȱ GR4,ȱ whichȱ isȱ locatedȱ inȱ theȱ mouthȱ ofȱ theȱ river,ȱ doesȱ notȱ
presentȱthisȱenvironmentalȱpressureȱbyȱmetalsȱwhatȱsuggestsȱthatȱtheȱpollutionȱ
byȱ theseȱ contaminantsȱ mightȱ comeȱ fromȱ directȱ spillsȱ toȱ theȱ riversȱ fromȱ theȱ
industriesȱlocatedȱinȱtheȱarea.ȱDespiteȱtheȱtideȱregimeȱofȱtheȱareaȱwhichȱimpliesȱ
anȱimportantȱwaterȱrenewal,ȱtheȱdegradationȱofȱtheȱecosystemȱinȱtheȱmouthȱofȱ
theȱ Riverȱ Palmonesȱ andȱ Guadarranqueȱ isȱ aȱ fact.ȱ Takingȱ thisȱ intoȱ account,ȱ itȱ isȱ
possibleȱ toȱ haveȱ otherȱ typesȱ ofȱ contaminantsȱ inȱ theȱ areaȱ whichȱ mayȱ beȱ
contributingȱ toȱ theȱ environmentalȱ impactȱ andȱ notȱ measuredȱ inȱ thisȱ studyȱ
(Antón,ȱ2007).ȱTheȱclusterȱanalysisȱhaveȱconfirmedȱtheȱdisparityȱofȱtheȱstationsȱ
fromȱtheȱBayȱofȱAlgecirasȱ>ȱCormeȬLaxeȱ>ȱAINPȱ>ȱreferenceȱstation.ȱȱ
Regardingȱ toȱ theȱ obtainedȱ results,ȱ theȱ recoveryȱ ofȱ theȱ Galicianȱ coastȱ
affectedȱbyȱtheȱPrestigeȱoilȱspillȱisȱsignificantlyȱnotableȱ(MoralesȬCasellesȱetȱal.,ȱ
accepted;ȱ MoralesȬCasellesȱ etȱ al.,ȱ submitted)ȱ althoughȱ otherȱ sourcesȱ ofȱ
contaminantsȱshouldȱ beȱ takenȱ intoȱconsiderationȱdueȱtoȱtheȱpotentialȱriskȱthatȱ
involve.ȱOnȱtheȱotherȱhand,ȱtheȱchronicȱpollutionȱinȱtheȱBayȱofȱAlgecirasȱwhichȱ
isȱ notȱ onlyȱ composedȱ byȱ hydrocarbonsȱ spillsȱ butȱ withȱ theȱ existanceȱ ofȱ aȱ
complexȱ mixtureȱ ofȱ contaminantsȱ inputs,ȱ isȱ producingȱ aȱ considerableȱ
additionallyȱdamageȱ toȱ theȱecosystem.ȱInȱthisȱsense,ȱchronicȱinputsȱdueȱtoȱtheȱ
continuousȱ entranceȱ ofȱ contaminantsȱ resultȱ muchȱ moreȱ harmfulȱ inȱ coastalȱ
ecosystemsȱ thanȱ majorȱ butȱ preciseȱ environmentalȱ impacts,ȱ asȱ confirmedȱ inȱ
previousȱstudiesȱ(Ribaȱetȱal.,ȱ2004).ȱ
5.ȱConclusionsȱ
Inȱ theȱ presentȱ studyȱ authorsȱ haveȱ successfullyȱ integratedȱ 4ȱ LOEsȱ inȱ aȱ
WOEȱapproachȱtoȱassessȱsedimentsȱaffectedȱbyȱoilȱspillsȱandȱdifferentȱsourcesȱofȱ
- 349 -
contaminants.ȱTheȱuseȱofȱphysicochemicalȱcharacterization,ȱbiologicalȱresponsesȱ
underȱlaboratoryȱandȱfieldȱconditionsȱandȱinȱsituȱalterationȱofȱtheȱbiotaȱasȱpartȱ
ofȱaȱWOEȱapproachȱisȱconsideredȱaȱsuitableȱtoolȱtoȱcarryȱoutȱsedimentȱqualityȱ
studies.ȱ Thisȱ methodologyȱ basedȱ onȱ theȱ evaluationȱ ofȱ aȱ completeȱ setȱ ofȱ
parametersȱ underȱ anȱ integratedȱ frameworkȱ goesȱ furtherȱ thanȱ theȱ classicalȱ
studiesȱ byȱ studyingȱ theȱ realȱ statusȱ ofȱ theȱ environmentȱ andȱ includingȱ earlyȱ
warningȱ signalsȱ ofȱ risk.ȱ Theȱ combinationȱ ofȱ fieldȱ andȱ laboratoryȱ analysisȱ
supposesȱanȱaddedȱvalueȱtoȱtheȱassessmentȱwhereasȱtheȱcompleteȱmethodologyȱ
employedȱhasȱelucidatedȱaboutȱtheȱcontaminantsȱsourcesȱandȱfates,ȱinȱadditionȱ
toȱtheirȱimplicationȱasȱanȱenvironmentalȱrisk.ȱ
Theȱ existenceȱ ofȱ aȱ wideȱ groupȱ ofȱ sourcesȱ inȱ theȱ Bayȱ ofȱ Algecirasȱ
includingȱ urbanȱ andȱ industrialȱ activitiesȱ inȱ additionȱ toȱ theȱ maritimeȱ trafficȱ
whichȱ involveȱ accidentalȱ spillsȱ makesȱ difficultȱ toȱ elucidateȱ theȱ mainȱ causeȱ ofȱ
theȱ environmentalȱ healthȱ decrease.ȱ Resultsȱ obtainedȱ indicateȱ thatȱ theȱ highȱ
environmentalȱ degradationȱ presentȱ inȱ theȱ Bayȱ ofȱ Algecirasȱ isȱ mainlyȱ dueȱ toȱ
continuousȱoilȱspills.ȱOnȱtheȱotherȱhand,ȱfourȱyearsȱafterȱtheȱPrestigeȱoilȱspill,ȱaȱ
generalȱ recoveryȱ ofȱ theȱ sedimentsȱ affectedȱ inȱ Atlanticȱ Islandsȱ Nationalȱ Parkȱ
(AINP)ȱandȱanȱimprovementȱinȱtheȱenvironmentalȱqualityȱinȱtheȱBayȱofȱCormeȬ
Laxeȱ wasȱ observed.ȱ Otherȱ inputsȱ ofȱ contaminantsȱ notȱ relatedȱ withȱ oilȱ spillsȱ
wereȱalsoȱdetectedȱinȱtheseȱareas;ȱatȱtheȱmomentȱtheseȱsourcesȱofȱstressȱareȱnotȱ
producingȱdamageȱtoȱtheȱbiotaȱalthoughȱtheyȱconstituteȱanȱenvironmentalȱriskȱ
thatȱshouldȱnotȱbeȱignored.ȱȱȱ
Toȱ sumȱ up,ȱ theȱ environmentȱ capacityȱ ofȱ recovermentȱ afterȱ aȱ majorȱ oilȱ
spillȱ episodeȱ suchȱ asȱ theȱ Prestigeȱ hasȱ beenȱ demonstratedȱ whereasȱ littoralȱ
sedimentsȱaffectedȱbyȱlowȱorȱmoderatedȱbutȱcontinuousȱoilȱspillsȱhaveȱresultedȱ
toȱ beȱ moreȱ degradated.ȱ Thisȱ conclusionȱ shouldȱ leadȱ toȱ theȱ reflectionȱ onȱ ourȱ
perceptionȱandȱmajorȱconcernsȱofȱtheȱenvironmentalȱpollution.ȱȱȱ
ȱ
- 350 -
6.ȱAcknowledgementsȱ
Theȱ workȱ describedȱ wasȱ partlyȱ supportedȱ byȱ theȱ projectsȱ VEM2003Ȭ
20563/INTER,ȱ andȱ CTM2005Ȭ07282ȬC03Ȭ01/TECNOȱ financedȱ byȱ theȱ Spanishȱ
Educationȱ andȱ Scienceȱ Ministryȱ andȱ byȱ CISȱ fundedȱ byȱ theȱ Ministryȱ ofȱ
Environment.ȱ Carmenȱ MoralesȬCasellesȱ thanksȱ theȱ Ministryȱ ofȱ Educationȱ andȱ
Scienceȱ forȱ fundingȱ herȱ researchȱ fellowshipȱ (FPU).ȱ Dr.ȱ Ribaȱ thanksȱ theȱ I3pȱ
programȱ forȱ supportingȱ herȱ participationȱ inȱ thisȱ research.ȱ Authorsȱ wouldȱ likeȱ
toȱ thankȱ theȱ membersȱ ofȱ theȱ CISȱ forȱ theirȱ supportȱ andȱ helpȱ inȱ theȱ chemicalȱ
analysisȱ andȱ theȱ benthicȱ communityȱ information;ȱ specialȱ thanksȱ areȱ givenȱ toȱ
Lauraȱ Martín,ȱ Nuriaȱ Fernández,ȱ Augustoȱ César,ȱ Pabloȱ Vidalȱ andȱ Antonioȱ
Moreno.ȱ
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- 354 -
ȱ
Capítuloȱ6.ȱ
Conclusionesȱ
1)ȱ Seȱ hanȱ determinadoȱ lasȱ concentracionesȱ deȱ metalesȱ yȱ contaminantesȱ
orgánicosȱenȱdiferentesȱsedimentosȱdeȱlaȱcostaȱGallegaȱlocalizadosȱenȱelȱParqueȱ
Nacionalȱ deȱ lasȱ Islasȱ Atlánticasȱ (islasȱ deȱ Onsȱ yȱ Cies)ȱ yȱ enȱ laȱ Bahíaȱ deȱ CormeȬ
Laxeȱ trasȱ elȱ vertidoȱ delȱ petroleroȱ Prestigeȱ yȱ seȱ hanȱ comparadoȱ conȱ nivelesȱ deȱ
estosȱ contaminantesȱ enȱ zonasȱ delȱ Golfoȱ deȱ Cádiz.ȱ Laȱ presenciaȱ deȱ nivelesȱ
elevadosȱ deȱ PAHsȱ enȱ sedimentosȱ juntoȱ conȱ losȱ nivelesȱ moderadosȱ deȱ metalesȱ
enȱalgunasȱdeȱlasȱzonasȱestudiadasȱenȱGalicia,ȱponenȱdeȱevidenciaȱlaȱinfluenciaȱ
delȱvertidoȱdeȱpetróleoȱenȱlaȱzona.ȱLosȱnivelesȱdeȱcontaminantesȱenȱlaȱzonaȱdeȱ
laȱBahíaȱdeȱAlgecirasȱconfirmanȱsuȱcarácterȱdeȱzonaȱcontaminadaȱenȱvariosȱdeȱ
losȱpuntosȱestudiados,ȱtantoȱporȱmetalesȱcomoȱporȱcontaminantesȱorgánicos.ȱLaȱ
estaciónȱ seleccionadaȱ enȱ laȱ Bahíaȱ deȱ Cádizȱ haȱ resultadoȱ serȱ adecuadaȱ comoȱ
referenciaȱ dadosȱ susȱ bajosȱ nivelesȱ deȱ contaminaciónȱ metálicaȱ yȱ laȱ ausenciaȱ deȱ
PAHsȱyȱPCBs.ȱȱȱ
2)ȱ Seȱ haȱ identificadoȱ yȱ cuantificadoȱ elȱ efectoȱ adversoȱ deȱ losȱ contaminantesȱ
presentesȱ enȱ elȱ fuelȱ procedenteȱ delȱ petroleroȱ Prestige,ȱ demostrandoȱ queȱ laȱ
ȱ
Ȭȱ355ȱȬȱ
Capítuloȱ6
especieȱAmpeliscaȱbrevicornisȱesȱadecuadaȱparaȱrealizarȱensayosȱdeȱtoxicidadȱaȱlaȱ
horaȱ deȱ determinarȱ laȱ toxicidadȱ agudaȱ enȱ sedimentosȱ afectadosȱ porȱ
contaminaciónȱdeȱtipoȱorgánicoȱyȱmetálico.ȱ
3)ȱ Seȱ haȱ demostradoȱ laȱ ventajaȱ deȱ utilizarȱ elȱ poliquetoȱ Arenicolaȱ marinaȱ enȱ laȱ
caracterizaciónȱdeȱzonasȱafectadasȱporȱvertidosȱdeȱpetróleo,ȱyȱqueȱaȱpesarȱdeȱserȱ
unaȱ especieȱ menosȱ sensibleȱ queȱ otrasȱ A.ȱ marinaȱ puedeȱ estarȱ presenteȱ enȱ áreasȱ
polucionadasȱ loȱ queȱ permiteȱ realizarȱ estudiosȱ deȱ bioacumulación.ȱ Losȱ PAHȱ
procedentesȱdelȱfuelȱdelȱPrestigeȱqueȱmásȱseȱbioacumularonȱfueronȱfluoranteno,ȱ
pirenoȱ benzo(fluorantenoȱ yȱ benzo(k)fluorantenoȱ mientrasȱ queȱ fenantrenoȱ yȱ
antracenoȱ seȱ acumularonȱ inicialmenteȱ yȱ luegoȱ fueronȱ probablementeȱ
metabolizados.ȱ
4)ȱ Aȱ partirȱ deȱ losȱ ensayosȱ deȱ toxicidadȱ seȱ haȱ detectadoȱ unaȱ respuestaȱ agudaȱ
moderadaȬbajaȱdebidoȱprincipalmenteȱaȱlosȱPAHsȱoriginadosȱtrasȱelȱvertidoȱenȱ
losȱ sedimentosȱ delȱ Parqueȱ Nacionalȱ deȱ lasȱ islasȱ Atlánticas;ȱ porȱ otraȱ parte,ȱ laȱ
Bahíaȱ deȱ Algecirasȱ presentóȱ elevadaȱ toxicidadȱ agudaȱ mientrasȱ queȱ noȱ seȱ
detectaronȱ efectosȱ adversosȱ enȱ losȱ organismosȱ expuestosȱ aȱ sedimentosȱ
procedentesȱdeȱlaȱestaciónȱdeȱreferenciaȱlocalizadaȱenȱlaȱBahíaȱdeȱCádiz.ȱ
5)ȱ Losȱ resultadosȱ obtenidosȱ demuestranȱ queȱ existeȱ unaȱ disminuciónȱ deȱ laȱ
toxicidadȱagudaȱenȱlosȱsedimentosȱafectadosȱporȱelȱvertidoȱdelȱpetroleroȱPrestigeȱ
enȱ laȱ costaȱ deȱ Galicia;ȱ losȱ sedimentosȱdeȱCormeȬLaxeȱyȱelȱParqueȱNacionalȱdeȱ
lasȱ Islasȱ Atlánticasȱ noȱ presentaronȱ toxicidadȱ agudaȱ cuatroȱ añosȱ despuésȱ delȱ
vertidoȱ aunqueȱ laȱ presenciaȱ deȱ algunosȱ metalesȱ yȱ PAHsȱ enȱ losȱ sedimentosȱ esȱ
consideradaȱ comoȱ unȱ riesgoȱ potencialȱ paraȱ laȱ calidadȱ deȱ losȱ mismos.ȱ Laȱ
bioacumulaciónȱdeȱPAHsȱenȱpoliquetosȱexpuestosȱaȱsedimentosȱdeȱCormeȬLaxeȱ
indicaȱlaȱposibilidadȱdeȱqueȱseȱdenȱefectosȱsubletalesȱenȱlosȱorganismos.ȱ
- 356 -
Conclusionesȱ
6)ȱ Conȱ elȱ finȱ deȱ realizarȱ unȱ estudioȱ másȱ completoȱ deȱ laȱ toxicidadȱ deȱ losȱ
sedimentosȱ afectadosȱ porȱ vertidosȱ deȱ petróleoȱ seȱ hanȱ diseñadoȱ unaȱ serieȱ deȱ
bioensayosȱ conȱ invertebradosȱ yȱ vertebradosȱ marinosȱ queȱ medianteȱ
exposicionesȱ subletalesȱ yȱ medidasȱ deȱ diferentesȱ biomarcadoresȱ hanȱ permitidoȱ
diferenciarȱ “zonasȱ grises”ȱ enȱ laȱ clasificaciónȱ deȱ toxicidad.ȱ Losȱ resultadosȱ
obtenidosȱmuestranȱunaȱclaraȱdiferenciaciónȱentreȱlosȱsedimentosȱrecogidosȱenȱ
elȱ Parqueȱ Nacionalȱ deȱ lasȱ Islasȱ Atlánticas,ȱ laȱ Bahíaȱ deȱ CormeȬLaxe.ȱ Enȱ esteȱ
sentido,ȱestosȱensayosȱyȱmedidasȱutilizadasȱenȱlosȱmismosȱhanȱdemostradoȱserȱ
útilesȱparaȱlaȱclasificaciónȱdeȱlaȱcalidadȱdeȱlosȱsedimentosȱestudiados.ȱ
7)ȱ Seȱ hanȱ determinadoȱ losȱ efectosȱ adversosȱ deȱ losȱ contaminantesȱ enȱ losȱ
sedimentosȱexponiendoȱduranteȱ60ȱdíasȱjuvenilesȱdelȱpezȱSparusȱaurataȱaȱéstosȱ
medianteȱ laȱ aplicaciónȱ deȱ unȱ bioensayoȱ deȱ toxicidadȱ crónicaȱ yȱ analizandoȱ
diferentesȱ biomarcadoresȱ deȱ exposiciónȱ aȱ metalesȱ (Metalotioneinas)ȱ yȱ aȱ
contaminantesȱ orgánicosȱ (Actividadȱ EROD)ȱ yȱ biomarcadoresȱ deȱ efectoȱ
(histopatologíaȱ enȱ dosȱ tejidos,ȱ branquiasȱ eȱhígado).ȱ Losȱ resultadosȱ despuésȱ deȱ
losȱ 60ȱ díasȱ muestranȱ unaȱ correlaciónȱ conȱ losȱ datosȱ deȱ toxicidadȱ agudaȱ yȱ
confirmanȱ laȱ presenciaȱ deȱ elevadasȱ concentracionesȱ deȱ PAHsȱ enȱ sedimentosȱ
comoȱlaȱcausaȱdeȱlosȱefectosȱanalizadosȱtrasȱelȱvertidoȱenȱestacionesȱdelȱParqueȱ
NacionalȱdeȱlasȱIslasȱAtlánticasȱ
8)ȱ Seȱ haȱ caracterizadoȱ laȱ relaciónȱ deȱ losȱ biomarcadoresȱ deȱ exposiciónȱ aȱ
contaminantesȱorgánicosȱ(ActividadȱEROD)ȱyȱlaȱdeȱlosȱbiomarcadoresȱdeȱefectoȱ
(histopatología)ȱ aȱ loȱ largoȱ delȱ tiempoȱ medianteȱ laȱ aplicaciónȱ deȱ unȱ modeloȱ
cinético.ȱLaȱinducciónȱdeȱlaȱactividadȱERODȱaȱloȱlargoȱdelȱtiempoȱesȱmayorȱenȱ
lasȱestacionesȱconȱmayorȱconcentraciónȱdeȱPAHs.ȱElȱmodeloȱprediceȱlaȱrelaciónȱ
entreȱ laȱ inducciónȱ deȱ laȱ actividadȱ ERODȱ comoȱ elȱ primerȱ sistemaȱ deȱ defensaȱ
enzimáticoȱfrenteȱaȱlaȱpresenciaȱdeȱéstosȱcontaminantesȱyȱademásȱdeterminaȱsuȱ
eficienciaȱ frenteȱ aȱ laȱ apariciónȱ delȱ dañoȱ histopatológicoȱ provocandoȱ suȱ
Ȭ 357ȱȬ
Capítuloȱ6
inducciónȱ unȱ retrasoȱ enȱ laȱ apariciónȱ delȱ daño.ȱ Laȱ apariciónȱ deȱ estosȱ dañosȱ
histológicosȱ esȱ másȱ severaȱ unaȱ vezȱ queȱ laȱ inducciónȱ deȱ actividadȱ ERODȱ seȱ
estabilizaȱ oȱ disminuyeȱ enȱ lasȱ tresȱ estacionesȱ delȱ Parqueȱ deȱ lasȱ Islasȱ Atlánticasȱ
utilizadasȱenȱesteȱestudio.ȱ
9)ȱ Seȱ haȱ demostradoȱ comoȱ losȱ biomarcadoresȱ medidosȱ enȱ laȱ almejaȱ Ruditapesȱ
philippinarumȱ yȱ elȱ cangrejoȱ Carcinusȱ maenasȱ seȱ activanȱ enȱ funciónȱ delȱ tipoȱ yȱ elȱ
nivelȱdeȱcontaminaciónȱenȱlosȱsedimentos,ȱpermitiendoȱestablecerȱdiferenciasȱenȱ
funciónȱdelȱorigenȱyȱfuenteȱdeȱlosȱcontaminantes.ȱȱ
10)ȱ Trasȱ variosȱ añosȱ despuésȱ delȱ vertidoȱ delȱ petroleroȱ Prestigeȱ losȱ sedimentosȱ
delȱParqueȱNacionalȱdeȱlasȱIslasȱAtlánticasȱpresentaronȱlosȱnivelesȱmásȱbajosȱdeȱ
respuestaȱ subletal,ȱ mostrandoȱ unaȱ recuperaciónȱ deȱ laȱ zonaȱ trasȱ elȱ vertido;ȱ sinȱ
embargo,ȱlaȱpresenciaȱdeȱalgunosȱmetalesȱligadosȱalȱsedimentoȱpodríanȱacarrearȱ
ciertoȱ estrésȱ ambientalȱ enȱ elȱ parque.ȱ Losȱ organismosȱ expuestosȱ enȱ jaulasȱ
ancladasȱenȱlaȱzonaȱdeȱCormeȬLaxeȱhanȱmostradoȱnivelesȱelevadosȱdeȱesteȱtipoȱ
deȱ estrés,ȱ loȱ cualȱ noȱ seȱ observóȱ enȱ losȱ ensayosȱ deȱ laboratorioȱ loȱ queȱ revelaȱ elȱ
impactoȱdeȱotrasȱfuentesȱdeȱcontaminaciónȱenȱlaȱzona.ȱEnȱelȱcasoȱdeȱlaȱBahíaȱdeȱ
Algecirasȱ laȱ inducciónȱ deȱ losȱ biomarcadoresȱ fueȱ significativaȱ bajoȱ condicionesȱ
deȱ laboratorioȱ mientrasȱ queȱ enȱ lasȱ exposicionesȱ enȱ campoȱ seȱ observóȱ unaȱ
disminuciónȱdeȱlosȱmismos,ȱposiblementeȱrelacionadaȱconȱlaȱinfluenciaȱmarealȱ
oȱ elȱ efectoȱ deȱ “lavado”ȱ deȱ laȱ contaminación,ȱ queȱ disminuiríaȱ laȱ
biodisponibilidadȱdeȱlosȱcontaminantes.ȱEnȱelȱcasoȱdeȱlaȱvitelogeninaȱmedidaȱenȱ
cangrejos,ȱ paraȱ todasȱ lasȱ estacionesȱ éstaȱ mostróȱ mayorȱ respuestaȱ bajoȱ
condicionesȱ deȱ laboratorio,ȱ mientrasȱ queȱ lasȱ exposicionesȱ enȱ jaulasȱ resultaronȱ
menosȱ sensiblesȱ aȱ laȱ toxicidadȱ deȱ losȱ sedimentos.ȱ Losȱ resultadosȱ obtenidosȱ
ponenȱ deȱ manifiestoȱ laȱ importanciaȱ deȱ realizarȱ exposicionesȱ enȱ campoȱ
complementariasȱaȱaquellosȱensayosȱdeȱlaboratorio.ȱȱ
- 358 -
Conclusionesȱ
11)ȱ Losȱ resultadosȱ deȱ biomarcadoresȱ obtenidosȱ enȱ losȱ diferentesȱ organismosȱ
expuestosȱ aȱ sedimentoȱ deȱ laȱ Bahíaȱ deȱ Cádiz,ȱ confirmanȱ suȱ elecciónȱ comoȱ
referenciaȱ enȱ estudiosȱ deȱ toxicidad,ȱ dadaȱ laȱ ausenciaȱ deȱ respuestasȱ biológicasȱ
adversas.ȱ Yȱ confirmanȱ estaȱ zonaȱ comoȱ deȱ referenciaȱ paraȱ estudiosȱ queȱ
impliquenȱ evaluacionesȱ deȱ toxicidadȱ medianteȱ ensayosȱ crónicosȱ yȱ utilizandoȱ
medidasȱsubletalesȱcomoȱlosȱbiomarcadores.ȱ
12)ȱ Losȱ biomarcadoresȱ analizadosȱ enȱ cangrejosȱ yȱ almejasȱ seȱ indujeronȱ deȱ
maneraȱ significativaȱ duranteȱ laȱ primeraȱ semanaȱ deȱ exposición.ȱ Seȱ hanȱ
observadoȱrelacionesȱ entreȱ biomarcadoresȱdeȱ laȱ faseȱ Iȱ yȱ IIȱ deȱ detoxificaciónȱ loȱ
queȱ sugiereȱ laȱ funcionalidadȱ delȱ esteȱ sistemaȱ enȱ ambasȱ especiesȱ deȱ
invertebradosȱ marinos.ȱ ȱ Elȱ estudioȱ deȱ biomarcadoresȱ aȱ loȱ largoȱ delȱ tiempoȱ haȱ
ayudadoȱaȱidentificarȱtantoȱlasȱfuentesȱcomoȱlaȱmaneraȱdeȱactuarȱyȱdetoxificarȱ
losȱ contaminantes,ȱ asíȱ comoȱ deȱ identificarȱ respuestasȱ biológicasȱ frenteȱ aȱ
contaminantesȱqueȱnoȱhanȱsidoȱanalizados.ȱȱȱ
13)ȱ Seȱ haȱ demostradoȱ comoȱ varíaȱ laȱ biologíaȱ delȱ poliquetoȱ Arenicolaȱ marinaȱ
expuestoȱ aȱ diferentesȱ nivelesȱ deȱ contaminación.ȱ Seȱ hanȱ establecidoȱ
correlacionesȱ entreȱ contaminantesȱ orgánicos,ȱ metalesȱ yȱ respuestasȱ biológicas,ȱ
incluyendoȱ actividadȱ antioxidante,ȱ inmuneȱ yȱ químicoȬsensorialȱ siendoȱ elȱ
biomarcadorȱ másȱ destacableȱ elȱ dañoȱ deȱ ADNȱ medidoȱ porȱ elȱ ensayoȱ Cometa.ȱ
Porȱ vezȱ primeraȱ seȱ haȱ seleccionadoȱ yȱ aplicadoȱ unaȱ bateríaȱ deȱ biomarcadoresȱ
conȱ elȱ poliquetoȱ A.ȱ marinaȱ yȱ losȱ resultadosȱ obtenidosȱ muestranȱ nuevasȱ
herramientasȱparaȱsuȱaplicaciónȱenȱestudiosȱmedioambientales.ȱȱȱ
14)ȱ Seȱ haȱ demostradoȱ laȱ recuperaciónȱ deȱ laȱ faunaȱ bentónicaȱ añosȱ despuésȱ delȱ
vertidoȱ delȱ Prestigeȱ enȱ laȱ zonaȱ deȱ Galicia.ȱ Seȱ haȱ demostradoȱ unaȱ importanteȱ
alteraciónȱ biológicaȱ enȱ lasȱ estacionesȱ localizadasȱ enȱ laȱ bahíaȱ deȱ Algecirasȱ yȱ seȱ
hanȱ relacionadoȱ conȱ alȱ concentraciónȱ deȱ PAHsȱ enȱ elȱ sedimento.ȱ Aȱ suȱ vez,ȱ lasȱ
Ȭ 359ȱȬ
Capítuloȱ6
comunidadesȱ bentónicasȱ deȱ CormeȬLaxeȱ yȱ Algeciras,ȱ puedenȱ verseȱ afectadasȱ
porȱ laȱ presenciaȱ deȱ metalesȱ enȱ losȱ sedimentos.ȱ Laȱ estructuraȱ deȱ laȱ comunidadȱ
bentónicaȱ enȱ laȱ zonaȱ deȱ Cádizȱ seȱ establecióȱ comoȱ normalȱ noȱ asociándoseȱ conȱ
alteraciónȱsignificativa,ȱalȱmenosȱenȱlaȱestaciónȱelegidaȱcomoȱreferencia.ȱ
15)ȱ Seȱ haȱ llevadoȱ aȱ caboȱ unaȱ nuevaȱ mejoraȱ enȱ laȱ metodologíaȱ integradaȱ deȱ
evaluaciónȱ deȱ laȱ calidadȱ deȱ losȱ sedimentosȱ dentroȱ delȱ marcoȱ delȱ “Weightȱ ofȱ
Evidenceȱ approach”ȱ queȱhaȱpermitidoȱobtenerȱresultadosȱmásȱobjetivos.ȱSeȱhaȱ
demostradoȱcomoȱtrasȱelȱvertidoȱlosȱPAHsȱfueronȱelȱprincipalȱcontaminanteȱdeȱ
laȱ costaȱ gallega.ȱ Seȱ haȱ identificadoȱ laȱ existenciaȱ deȱ fuentesȱ deȱ metalesȱ enȱ elȱ
Parqueȱ Nacionalȱ deȱ lasȱ Islasȱ Atlánticasȱ yȱ deȱ laȱ bahíaȱ deȱ CormeȬLaxeȱ queȱ
aparentementeȱ noȱ estánȱ produciendoȱ efectosȱ biológicosȱ deȱ tipoȱ agudo.ȱ Laȱ
poluciónȱhaȱdisminuidoȱenȱlosȱúltimosȱañosȱenȱambasȱzonasȱdeȱGalicia,ȱaunqueȱ
aúnȱexisteȱciertoȱestrésȱambientalȱprincipalmenteȱenȱlasȱzonasȱestudiadasȱdeȱlaȱ
bahíaȱdeȱCormeȬLaxe.ȱȱ
16)ȱSeȱhanȱdemostradoȱlasȱventajasȱdeȱincorporarȱlosȱbiomarcadoresȱcomoȱlíneaȱ
deȱ evidenciaȱ dentroȱ deȱ unȱ estudioȱ integrado,ȱ éstosȱ hanȱ mostradoȱ unaȱ mayorȱ
sensibilidadȱenȱlosȱresultadosȱaȱlaȱhoraȱdeȱcuantificarȱlaȱpoluciónȱeȱidentificarȱlaȱ
misma;ȱ elȱ usoȱ deȱ losȱ biomarcadoresȱ obtenidosȱ enȱ exposicionesȱ deȱ campoȱ yȱ
laboratorioȱdentroȱdelȱ“WeightȱofȱEvidenceȱapproach”ȱhaȱayudadoȱaȱrelacionarȱ
lasȱfuentesȱdeȱcontaminaciónȱyȱlosȱefectosȱinclusoȱcuandoȱelȱcontaminanteȱnoȱhaȱ
sidoȱ analizado.ȱ Aȱ pesarȱ queȱ variosȱ añosȱ despuésȱ delȱ vertidoȱ noȱ seȱ hanȱ
detectadoȱ efectosȱ agudosȱ significativosȱ enȱ elȱ áreaȱ deȱ CormeȬLaxe,ȱ seȱ hanȱ
observadoȱ respuestasȱ subletalesȱ relacionadasȱ conȱ lasȱ concentracionesȱ deȱ
contaminantesȱcomoȱlosȱPAHs,ȱyȱlosȱmetalesȱPbȱyȱHg.ȱLaȱpresenciaȱdeȱciertosȱ
metalesȱcomoȱZn,ȱCuȱyȱNiȱenȱlasȱislasȱCíesȱpodríanȱsuponerȱunȱriesgoȱaunqueȱ
porȱelȱmomentoȱnoȱseȱhanȱdetectadoȱefectosȱbiológicosȱenȱlaȱzonaȱasociadosȱconȱ
estosȱcontaminantes.ȱTrasȱlosȱresultadosȱobtenidosȱenȱlaȱBahíaȱdeȱCormeȬLaxe,ȱ
- 360 -
Conclusionesȱ
seȱ sospechaȱ queȱ laȱ presenciaȱ deȱ lasȱ bateasȱ deȱ cultivoȱ deȱ mariscosȱ puedaȱ
suponerȱunaȱfuenteȱdeȱestrésȱimportanteȱenȱlaȱzona.ȱ
17)ȱLaȱexistenciaȱdeȱdistintasȱfuentesȱdeȱcontaminaciónȱdaȱlugarȱaȱlaȱpresenciaȱ
deȱunaȱmezclaȱcomplejaȱdeȱcontaminantesȱenȱlaȱBahíaȱdeȱAlgeciras,ȱincluyendoȱ
vertidosȱ industriales,ȱ urbanosȱ yȱ derivadosȱ delȱ tráficoȱ marítimoȱ yȱ deȱ lasȱ
actividadesȱdeȱbunkering.ȱTodoȱelloȱseȱreflejaȱenȱunaȱaltaȱdegradaciónȱambientalȱ
debidaȱ aȱ laȱ entradaȱ continuadaȱ deȱ estosȱ vertidos.ȱ Porȱ otroȱ lado,ȱ cuatroȱ añosȱ
despuésȱ delȱ vertidoȱ delȱ petroleroȱ Prestigeȱ seȱ observaȱ unaȱ recuperaciónȱ
generalizadaȱ deȱ losȱ sedimentosȱ delȱ Parqueȱ Nacionalȱ deȱ lasȱ islasȱ Atlánticasȱ yȱ
unaȱ mejoraȱ enȱ laȱ calidadȱ deȱ laȱ Bahíaȱ deȱ CormeȬLaxe.ȱ Elȱ métodoȱ integradoȱ haȱ
demostradoȱ laȱ recuperaciónȱ delȱ sistemaȱ afectadoȱ enȱ laȱ costaȱ deȱ Galicia,ȱ laȱ
poluciónȱ enȱ laȱ zonaȱ deȱ laȱ Bahíaȱ deȱ Algecirasȱ yȱ laȱ condiciónȱ deȱ zonaȱ deȱ
referenciaȱenȱlaȱestaciónȱelegidaȱenȱlaȱBahíaȱdeȱCádiz.ȱȱ
18)ȱ Seȱ haȱ demostradoȱ laȱ capacidadȱ ambientalȱ deȱ recuperaciónȱ trasȱ unȱ granȱ
vertidoȱ deȱ petróleoȱ comoȱ elȱ ocurridoȱ enȱ Galiciaȱ enȱ 2002ȱ mientrasȱ queȱ
sedimentosȱ litoralesȱ queȱ seȱ venȱ afectadosȱ porȱ moderadasȱ dosisȱ deȱ vertidosȱ
duranteȱunȱlargoȱperiodoȱdeȱtiempoȱyȱqueȱenȱprincipioȱnoȱdesatanȱtantaȱalarmaȱ
socialȱ puedenȱ resultarȱ notablementeȱ másȱ degradadosȱ comoȱ esȱ elȱ casoȱ deȱ laȱ
BahíaȱdeȱAlgeciras.ȱȱ
ȱ
ȱ
Ȭ 361ȱȬ