Integration of chemical and biological measurements in mussels, passive samplers and sediments to investigate the effects of chemical pollution in ports of the north coast of Spain Nestor Etxebarria 1, Mª Jesus Belzunce 2, Joxe Mikel Garmendia 2, Óscar Nieto 3 1 Kimika Analitikoa Saila, Zientzia eta Teknologia Fakultatea. Euskal Herriko Unibersitatea / Universidad del País Vasco. 644 P.K. E‐48080 Bilbao 2 AZTI Tecnalia. Unidad de Investigación Marina. Herrera Kaia Portualdea z/g E‐20110 Pasaia 3 Departamento de Química Analítica y Alimentaria. Facultad de Ciencias del Mar. Universidade de Vigo. E‐36310 Vigo. XV Seminario Ibérico de Química Marina Vigo Febrero 2010

OBJECTIVES OF THE PROJECT -Integrative assesment of pollution in harbour sediments by means of chemical analysis, biomarkers and ecotoxicological bioassays. - Implementation of active monitorization systems. - Harmonization of analytical methodologies. - Pre-normative research, quality assesment criteria.

Sampling stations Water and sediment samples were collected in 2007, 2008 and Water: pH, salinity, temp., dissolved oxigen (Winkler and electrochemical), phosphates (spectrophotometry). Sediments: granulometry, redox potential, oxidized organic carbon (titration). - Accumulation of chemical pollutants in transplanted mussels (M. galloprovincialis) and semi-permeable membrane devices. - In situ bioassays.

Sediment analysis Cd, Zn, Pb, Hg & Cu

PAHs & PCBs

Sea Urchin (P. lividus) toxicity bioassays in elutriates Vigo Bilbao Pasaia

Amphipod mortality tests in sediments

Chemical analysis in the water column Metals

Sea Urchin (P. lividus) toxicity bioassays in water column Filtered water In situ bioassays

Mussel transplantation and instalation of passive monitors (SMPD)

Accumulation of pollutants in transplanted mussels

Accumulation of pollutants in the SMPDs

Biomarkers

 Normalization/smoothing of results: ERL, log C  Principal component analysis (PCA)  Partial least squares regression (PLS) Unscrambler® v9.2 Finally

Normalization (ERL) The biological responses are correlated versus a ratio given by a concentration which is considered toxic; and not versus the total concentration of the pollutant. Advantage: high concentrations of non toxic substances are not statistically significant. Disadvantage: lack of reference values for all the substances studied. Smoothing (log C) The variation in the biological responses only correlate with those substances whose concentrations vary in a similar way. Advantage: can be applied to all substances. Disadvantage: good correlations could be found between not toxic substances and the biological responses. Different biological responses have been found in the ports studied. Therefore, this is a plausible option.

Principal component analysis, sediments log C ERL

Partial least square regressions, sediments ERL Log C

Principal component analysis, mussels

Partial least square regression, mussels

CONCLUSIONS Sediments: Comparable results are obtained in PCA and PLS when the concentration of pollutants are normalized with ERL or smoothed with log C. Microtox® assay responds to chemical pollutants different than those to amphipods mortality and sea urchin embryo toxicity tests. Better correlations could be obtained if the biological responses were represented versus the bioavaliable fraction of chemical pollutants instead of their total concentrations. Mussels PCA confirms the different predominant pollutants found in the ports of Vigo, Bilbao and Pasaia. PLS confirms that biomarkers GST and GPx respond to the different type of contaminants, previously characterized by PCA.

Muchas gracias por su atención Muito obrigado pela sua atenção Moitas grazas pola súa atención Spanish Ministry of Science and Innovation: Project CTM C03-01/02/03 (SEDIPORT) Port Authorities of Vigo, Bilbao and Pasaia Acknowledgements