Metal contamination and fractionation in sediments from the lower basin of the Vale do Ribeira (SE, Brazil).

The sediment quality of Ribeira de Iguape River is affected by former Pb extraction mining. Some studies affirm the restoration status of the basin, however, mobility of metals and its associated risk is still questioned. This study integrates the metal concentrations in the lower part of the basin with different contamination source to determine the existence of risks associated with the mobile fractions of the geochemical matrix. Despite concentrations of metals were low and the environmental risk factor values were negative, our results indicated that As, Mn, Pb, and V were present in the most labile forms. The multivariate analysis conducted using metal concentrations, environmental risk factor values and speciation suggested that any risk would be associated with the labile fractions of the analyzed elements, especially for Pb. The station from Registro was stressed by Co, Pb and Zn; with Pb under the reactive fraction that could be associated with high mobility and potential bioavailability.

The use of a Weight-of-Evidence approach to address sediment quality in the Odiel River basin (SW, Spain).

The fluvial systems of the Iberian Pyrite Belt (SW Iberian Peninsula) are affected by acid mine drainage (a lixiviate residue product of mining activities derived from sulfide oxidation). The high acidity and high concentrations of sulfates and metal(loid)s are the main causes of the environmental degradation of the Odiel River basin. The use of weight of evidence approach in areas of the Odiel River basin implies the integration of different lines of evidence (chemistry, toxicity and bioaccumulation) using the freshwater clam Corbicula fluminea as target species. The integration of the results from the application of the different lines of evidence showed that the index of pollution was higher downvalley (Ptriad=12,312), moderate after mining effluent discharges (Ptriad=13.9) and very low where the Odiel River sources (Ptriad=6.31). The multivariate analysis indicated that variables and chemicals were associated with geochemical matrix and background levels (% of fines and toxic metal(loid) concentrations), toxic effects, and metal(loid) bioaccumulation reflecting the geographical distribution of the contamination towards the estuary. Metal(loid) thresholds were calculated for the study area as site-specific values of interim freshwater sediment quality values: As≥171; Cd≥0.48; Co≥8.82; Cr≥38.4; Cu≥451; Ni≥18.4; Pb≥377; Sb≥17.7; Zn≥221mg/kg of freshwater fluvial dry sediment. These results revealed the possibility of using the TEL values proposed by the USEPA and the NOAA for sediments from this site, and proposed lower PEL values for the Iberian Pyrite Belt as result of toxicity effects found in the Asian clam due to the combination of extreme acidity and high metal(loid) concentrations.

General stress, detoxification pathways, neurotoxicity and genotoxicity evaluated in Ruditapes philippinarum exposed to human pharmaceuticals.

A battery of biomarkers was evaluated on Ruditapes philippinarum exposed during 14 days to caffeine, ibuprofen, carbamazepine and novobiocin (0.1, 1, 5, 10, 15, and 50µgL(-1)). The battery included general stress (lysosomal membrane stability - LMS) analysed in the hemolymph, and biochemical biomarkers analysed in digestive gland tissues including: biomarkers of phase I (etoxyresorufin O-deethylase - EROD, dibenzylfluorescein dealkylase - DBF), phase II (gluthathione-S-transferase - GST), oxidative stress (gluthathione reductase - GR, gluthathione peroxidase - GPX, lipid peroxidation - LPO), neurotoxicity (acetylcholinesterase activity - AChE), and genotoxicity (DNA damage). Pharmaceuticals tested induced the sublethal responses (even at the environmental range 0.1µgL(-1)). At this low concentration; caffeine, ibuprofen and carbamazepine decreased the LMS significantly compared with controls (p<0.05). The four compounds induced significantly the detoxification metabolism and oxidative stress (p<0.05). Neurotoxicity was noticed in clams exposed to caffeine and carbamazepine (p<0.05). Ibuprofen, carbamazepine and novobiocin produced genotoxic effects (p<0.05). Results from this research validate the use of biomarkers when assessing the effects of pharmaceuticals within a marine environmental risk assessment framework, using as a laboratory bioassay model the species R. philippinarum.

Multiple Biomarker Responses in Corbicula fluminea Exposed to Copper in Laboratory Toxicity Tests.

This study evaluated the alteration of the enzymatic system of the freshwater Asian clam exposed to different copper concentrations. Individuals of Corbicula fluminea were exposed to different concentrations of dissolved Cu (0.5, 1, and 2 mg L(-1)) for 7 days, then, biomarkers of oxidative stress (GST, GPx, GR), exposure (MTs), effect (AChE), and damage (LPO, DNA strand breaks) were quantified. Results showed positive correlations between dissolved metal concentrations and GPx, MTs, and DNA damage, and negative correlation with GST and AChE. In contrast, no clear trend was found for GR and LPO. In general, the established mechanisms of protection might have a beneficial effect on the decreasing ROS attack on membrane and the activation of the metallothioneins. Integrated biomarker analysis revealed that the measured alterations are well correlated with the levels of increasing dissolved copper concentrations in water, demonstrating the effectiveness of this organism for biomonitoring approach purposes.

Studying the effect of CO2-induced acidification on sediment toxicity using acute amphipod toxicity test.

Carbon capture and storage is increasingly being considered one of the most efficient approaches to mitigate the increase of CO2 in the atmosphere associated with anthropogenic emissions. However, the environmental effects of potential CO2 leaks remain largely unknown. The amphipod Ampelisca brevicornis was exposed to environmental sediments collected in different areas of the Gulf of Cádiz and subjected to several pH treatments to study the effects of CO2-induced acidification on sediment toxicity. After 10 days of exposure, the results obtained indicated that high lethal effects were associated with the lowest pH treatments, except for the Ría of Huelva sediment test. The mobility of metals from sediment to the overlying seawater was correlated to a pH decrease. The data obtained revealed that CO2-related acidification would lead to lethal effects on amphipods as well as the mobility of metals, which could increase sediment toxicity.

Application of neutral red retention assay to caged clams (Ruditapes decussatus) and crabs (Carcinus maenas) in the assessment of dredged material.

Dredged material management is a key issue for the protection of aquatic environments. The in situ approach using caged bioindicator species has been chosen lately as a new methodology for the assessment of dredged material. In a tier testing approach, neutral red retention (NRR) assay has been applied as a screening tool to detect adverse changes in health status associated with contamination. Nevertheless, to authors' knowledge, little is known about the application and validation of this technique in sediment bioindicator species and under field conditions. Caged Ruditapes decussatus and Carcinus maenas were exposed during 28 days to potentially contaminated sediments at three sites in Algeciras Bay (SW Spain) and one site in Cádiz Bay (SW Spain). Lysosomal membrane stability was measured over time in haemolymph samples of exposed clams and crabs using the NRR assay. Sediment characterization of the study sites was performed in parallel. NRR time did not vary significantly (p > 0.05) over time in organisms from Cádiz Bay. Conversely, significant differences (p < 0.05) in NRR time were found in clams and crabs exposed to sediments from Algeciras Bay, which exhibited a 30-70% decrease in haemocyte lysosome membrane stability compared to day 0. Statistical analysis showed a strong correlation between the drop of haemocyte lysosome membrane stability, in both crabs and clams, and the presence of metals (p < 0.05) and PAHs (p < 0.01) in the studied sediments. The results obtained confirmed the use of NRR assay as a suitable and sensitive method to be used in the assessment of sediment quality using as bioindicator species the clam R. philippinarum and the crab C. maenas.

Hepatic proteome changes in Solea senegalensis exposed to contaminated estuarine sediments: a laboratory and in situ survey.

Assessing toxicity of contaminated estuarine sediments poses a challenge to ecotoxicologists due to the complex geochemical nature of sediments and to the combination of multiple classes of toxicants. Juvenile Senegalese soles were exposed for 14 days in the laboratory and in situ (field) to sediments from three sites (a reference plus two contaminated) of a Portuguese estuary. Sediment characterization confirmed the combination of metals, polycyclic aromatic hydrocarbons and organochlorines in the two contaminated sediments. Changes in liver cytosolic protein regulation patterns were determined by a combination of two-dimensional electrophoresis with de novo sequencing by tandem mass spectrometry. From the forty-one cytosolic proteins found to be deregulated, nineteen were able to be identified, taking part in multiple cellular processes such as anti-oxidative defence, energy production, proteolysis and contaminant catabolism (especially oxidoreductase enzymes). Besides a clear distinction between animals exposed to the reference and contaminated sediments, differences were also observed between laboratory- and in situ-tested fish. Soles exposed in the laboratory to the contaminated sediments failed to induce, or even markedly down-regulated, many proteins, with the exception of a peroxiredoxin (an anti-oxidant enzyme) and a few others, when compared to reference fish. In situ exposure to the contaminated sediments revealed significant up-regulation of basal metabolism-related enzymes, comparatively to the reference condition. Down-regulation of basal metabolism enzymes, related to energy production and gene transcription, in fish exposed in the laboratory to the contaminated sediments, may be linked to sediment-bound contaminants and likely compromised the organisms' ability to deploy adequate responses against insult.

Assessment of the genotoxic potential of contaminated estuarine sediments in fish peripheral blood: laboratory versus in situ studies.

Juvenile Senegalese soles (Solea senegalensis) were exposed to estuarine sediments through 28-day laboratory and in situ (field) bioassays. The sediments, collected from three distinct sites (a reference plus two contaminated) of the Sado Estuary (W Portugal) were characterized for total organic matter, redox potential, fine fraction and for the levels of metals, polycyclic aromatic hydrocarbons (PAHs) and organochlorines, namely polychlorinated biphenyls (PCBs) and dichloro diphenyl tricholoethane plus its main metabolites (DDTs). Genotoxicity was determined in whole peripheral blood by the single-cell gel electrophoresis (SCGE or "comet") assay and by scoring erythrocytic nuclear abnormalities (ENA). Analysis was complemented with the determination of lipid peroxidation in blood plasma by the thiobarbituric acid reactive substances (TBARS) protocol and cell type sorting. The results showed that exposure to contaminated sediments induced DNA fragmentation and clastogenesis. Still, laboratory exposure to the most contaminated sediment revealed a possible antagonistic effect between metallic and organic contaminants that might have been enhanced by increased bioavailability. The laboratory assay caused a more pronounced increase in ENA whereas a very significant increase in DNA fragmentation was observed in field-tested fish exposed to the reference sediment, which is likely linked to increased lipid peroxidation that probably occurred due to impaired access to food. Influence of natural pathogens was ruled out by unaltered leukocyte counts. The statistical integration of data correlated lipid peroxidation with biological variables such as fish length and weight, whereas the genotoxicity biomarkers were more correlated to sediment contamination. It was demonstrated that laboratory and field bioassays for the risk assessment of sediment contamination may yield different genotoxicity profiles although both provided results that are in overall accordance with sediment contamination levels. While field assays may provide more ecologically relevant data, the multiple environmental variables may produce sufficient background noise to mask the true effects of contamination.

Sediment-quality assessment using the polychaete Arenicola marina: contamination, bioavailability, and toxicity.

The sediment quality of Cádiz Bay, Las Palmas de Gran Canaria (LPGC) Port, Santander Bay, Algeciras Bay, and Huelva Estuary (Spain) was evaluated by analysing a battery of biochemical biomarkers-activities of biotranformation enzymes ethoxyresorufin O-deethylase [EROD], dibenzylflourescein dealkylase [DBF], and glutathione S-transferase [GST]; activity of antioxidant enzyme glutathione reductase [GR]; and lipid peroxidation [LPO]-in the polychaete Arenicola marina after laboratory sediment exposure. Huelva Estuary polychaetes showed significantly (p < 0.05) enhanced LPO, GST, and EROD activities compared with control lugworms related to metals and presumably polychlorinated biphenyls. EROD activity significant (p < 0.05) induction was associated polycyclic aromatic hydrocarbons after Santander Bay sediment exposure. Nickel appeared to significantly (p < 0.05) induce GR activity and LPO in LPGC Port sediment-exposed organisms. DBF activity significantly (p < 0.05) increased in polychaetes exposed to sediments from sewage-contaminated areas. A. marina was sensitive at the biochemical level. Integration of sediment characterization and biomarker results allowed the identification of polluted sites as well as the cause of possible sediment toxicity.

An integrated approach to determine sediment quality in areas above CO2 injection and storage in agreement with the requirements of the international conventions on the protection of the marine environment.

The urgent need to reduce the greenhouse emissions to the atmosphere has leaded to study new systems to capture and store carbon dioxide (CO(2)). The sequestration of CO(2) in marine geological formations is one of these systems proposed at the international level to effectively reduce the concentration of atmospheric CO(2). Although permanent containment is expected, it is necessary to determine the risk of leakage to the marine environment. The integrated model for the evaluation of the environmental quality of the marine environment will contribute to determine the potential environmental pathways and effects that are relevant to the consideration of the potential consequences of the leakage of CO(2) and incidental associated substances from the geological formations to the marine environment. In addition, this model will satisfy the requirements for a safe CO(2) storage in sub-seabed geological formations set in the international conventions on the protection of the marine environment (1992 OSPAR Convention and 1996 London Protocol). The objective of this paper is to show how to adapt classical methodologies based on a weight-of-evidence approach to establish the impact of CO(2) leaks in the sediment quality. It is described how the classical methods should modify their application when acidification occurs related to CO(2) leaks being the main potential impact in these areas.

Biochemical endpoints on juvenile Solea senegalensis exposed to estuarine sediments: the effect of contaminant mixtures on metallothionein and CYP1A induction.

Juvenile Solea senegalensis were exposed to fresh sediments from three stations of the Sado estuary (Portugal) in 28-day laboratory assays. Sediments revealed distinct levels of total organic matter, fine fraction, redox potential, trace elements (arsenic, cadmium, chromium, copper, nickel, lead and zinc) and organic contaminants (polycyclic aromatic hydrocarbons, polychlorinated biphenyls and a pesticide: dichloro diphenyl trichloroethane). Organisms were surveyed for contaminant bioaccumulation and induction of two hepatic biochemical biomarkers: metallothionein (MT) and cytochrome P450 (CYP1A), as potential indicators of exposure to metallic and organic contaminants, respectively. Using an integrative approach it was established that, although bioaccumulation is in general accordance with sediment contamination, lethality and biomarker responses are not linearly dependent of the cumulative concentrations of sediment contaminants but rather of their bioavailability and synergistic effects in organisms. It is concluded that metals and organic contaminants modulate both MT and CYP1A induction and it is suggested that reactive oxygen species may be the link between responses and effects of toxicity.

A weight of evidence approach for quality assessment of sediments impacted by an oil spill: the role of a set of biomarkers as a line of evidence.

In an attempt to incorporate both line of evidence (LOE) and classical weight of evidence (WOE) approaches for the assessment of sediment quality, a set of biomarkers were analyzed in target tissues of two invertebrate species after 28 days of exposure to sediments impacted by oil (derived from the tanker Prestige (2002)). The integration of biomarkers with sediment contamination, acute toxicity and benthic alteration parameters provides an "early warning" tool which not only indicates the environmental quality of an area, but also constitutes an advisory tool for potential ecological risks. The selected biomarkers provide information about the first biological responses due to the presence of contaminants in the environment providing predictable reports about further effects to the ecosystem. The present study demonstrates that the use of a set of biomarkers as part of a WOE approach designed to assess contaminated sediments contributes added value to the classical LOE and allows characterization of the environmental status of the studied area in a more precise and accurate way.

Sediment quality assessment in the Guadalquivir River (SW, Spain) using caged Asian clams: A biomarker field approach.

This study assesses the sediment quality of the Guadalquivir River watercourse between the Alcalá del Río dam and the city of Seville. The main objective of this work is to address sediment quality in the area using an integrative approach that links sediment contamination and toxicity using the Asiatic clam (Corbicula fluminea) under field conditions. This is the first study conducted in the area that use of a battery of biomarkers from exposure (GST, GPx) to adverse biological effect (DNA and histopathological damage) to identify the contamination adverse effects in a river area affected by a cocktail of different anthropogenic activities (urban, industrial, agricultural, etc.). The sediment quality characterized in the area shows a significant biological stress related to metal(loid)s at station located in Alcalá del Río in the river upper part of the studied area, being this stress toxic when approaching the city of Seville. The sediments located nearby this city showed toxicity by means of positive values in the biomarkers of effects measured in the caged clams and related to contaminants with an industrial and urban discharge origins. These results have shown the useful and strength of the biomarker approach used in this study that combines biomarker responses from exposure to effects and allows identifying the contamination adverse effects by means of using caging individuals of the Asian clam. It has been proved in the different experiments how once the exposure biomarkers reach a maximum value of their system the detoxification ability of the organisms is collapsed and then the biomarkers of effect are measured significantly in the different tissues. The use of field surveys using tolerant specie such as the Asian clam is recommendable to determine sediment quality under an integrative point of view as here reported.

Genotoxic damage in Solea senegalensis exposed to sediments from the Sado Estuary (Portugal): effects of metallic and organic contaminants.

Juvenile Solea senegalensis (Senegalese sole) were exposed to freshly collected sediments from three sites of the Sado Estuary (West-Portuguese coast) in 28-day laboratory assays in order to assess the ecological risk from sediment contaminants, by measuring two genotoxicity biomarkers in peripheral blood: the percentage of Erythrocyte Nuclear Abnormalities (ENA) by use of an adaptation of the micronucleus test, and the percentage of DNA strand-breakage (DNA-SB) with the Comet assay. Sediments were surveyed for metallic (Cr, Ni, Cu, Zn, As, Cd and Pb) and organic (PAHs (polycyclic aromatic hydrocarbons), PCBs (polychlorinated biphenyls) and DDTs (dichloro-diphenyl-trichloroethane)) contaminants. Sediments from site A (farthest from hotspots of contamination) were found to be the least contaminated and weaker inducers of genotoxic damage, whereas sediments from sites B (urban influence) and C (affected by industrial effluents and agricultural runoffs) were responsible for a very significant increase in both ENA and DNA-SB, site B being most contaminated with metals and site C mainly with organic pollutants, especially PAHs and PCBs . Analysis of genotoxic effects showed a strong correlation between the concentrations of PAHs and PCBs and both biomarkers at sampling times T(14) and T(28), while the amounts of Cu, As, Cd and Pb were less strongly correlated, and at T(28) only, with ENA and DNA-SB. These results show that organic contaminants in sediment are stronger and faster acting genotoxic stressors. The results also suggest that metals may have an inhibitory effect on genotoxicity when interacting with organic contaminants, at least during early exposure. ENA and DNA-SB do not show a linear relationship, but a strong correlation exists between the overall increase in genotoxicity caused by exposure to sediment, confirming that they are different, and possibly non-linked effects that respond similarly to exposure. Although the Comet assay showed enhanced sensitivity, the two analyses are complementary and suitable for the biomonitoring of sediment contaminants in a benthic species like S. senegalensis.

Using a classical weight-of-evidence approach for 4-years' monitoring of the impact of an accidental oil spill on sediment quality.

In the present report, the successful application of a Weight of evidence approach (WOE) to sediment quality assessment during a four year impact period following an oil spill is discussed. The study assesses the sediment quality on the Galician Coast (NW Spain) which was impacted by an accidental spill associated with the sinking of the tanker Prestige (2002). The assessment is based on three lines of evidence: physicochemical characterization of the sediments; determination of acute toxicity by conducting sediment toxicity tests and benthic alteration including taxonomic identifications along with community descriptive statistics. The data obtained were integrated using a WOE approach by means of two different methodologies: multivariate analysis and ANOVA-based pie charts. Results confirm that PAHs related to the Prestige oil spill are the main contaminant associated with biological effects in the area which has since recovered from the initial acute impact. Also, the WOE allowed the identification of metal contamination not previously described in the area responsible for toxicity in sediments analyzed. In addition, the methodology proposed to link the 3 lines of evidence results shows the use for the first time of an objective indice based on factor analysis which allows pollution of the sediments studied to be qualitatively and quantitatively evaluated while demonstrating the WOE approach to be recommendable in monitoring environmental quality.

Sublethal responses in caged organisms exposed to sediments affected by oil spills.

This study was performed to determine sublethal responses of two invertebrate species by using field deployments in areas affected by oil spills, which are acute in the Galician Coast (NNW, Spain) and chronic in the Bay of Algeciras (SSW, Spain). The organisms employed were the crab Carcinus maenas and the clam Ruditapes philippinarum, and during 28 days the animals were exposed to contaminated sediments in cages under field conditions. Different biomarkers of exposure were determined after a 28-day period exposure: ethoxyresorufin O-deethylase (EROD), phase I detoxification enzyme, glutathione-S-transferase (GST) phase II detoxification enzyme but also implicated in oxidative stress events, glutathione peroxidase (GPX) and glutathione reductase (GR), both antioxidant enzymes. In addition, histopathological effects in target tissues of the deployed organisms were evaluated. Biomarker measurements were linked with the concentration of chemicals in the sediments in order to elucidate the type, source and bioavailability of contaminants that produce adverse effects in the bioindicator species. Results obtained in this study have shown how the application of the selected battery of biomarkers under field bioassays allows for the identification of alternative sources of stress that are not observable in laboratory experiments.

Is delta-aminolevulinic acid dehydratase activity in bivalves from south-west Iberian Peninsula a good biomarker of lead exposure?

The coast of Huelva is considered as a heavily contaminated area where the Tinto and Odiel rivers discharge after running through a metalliferous mining area in the Iberian Pyrite Belt and end in common estuary called Ría of Huelva. Lead is a highly toxic and widely distributed element in the aquatic environment; therefore there is a great interest in assessing the impact of this contaminant on aquatic organisms. To study the bioavailability and sources of lead, the bivalve species Chamelea gallina was collected from six sampling sites along the coast of Huelva. Besides the effect of this metal on the enzyme activity, delta-aminolevulinic acid dehydratase (ALA-D) was determined in the whole tissue. Results show the inverse relationship between the lead concentration and the activity of ALA-D measured in whole tissues (r2=0.7). Individuals collected from the Ría of Huelva estuary had the highest levels of total lead concentrations and the lowest activity of ALA-D. Lead isotope analyses demonstrated the different sources of lead contamination and also confirmed the influence of Ría of Huelva on the metal input to the marine environment.

In situ evaluation of sediment toxicity in Guadalete Estuary (SW Spain) after exposure of caged Arenicola marina.

A 7 day in situ assay to assess sediment toxicity was carried out per replicate in two sites from Guadalete Estuary (Guad1 and Guad2) (South-West, Spain) using the polychaete Arenicola marina (n = 20) in benthic cages. After the exposure period, a battery of biomarkers were determined to asses the sediment toxicity. Five of them were exposure biomarkers [ethoxyresorufin O-deethylase (EROD), dibenzylfluorescein (DBF) dependant CytP450, gluthation reductase (GR), and gluthation-S-transferase (GST) activities] and one of them was an effect biomarker [lipid peroxidation (LPO)]. Toxicity due to metals bound to the sediment was found in Guad2. Metals provoke the inhibition of EROD, DBF, and GR and the induction of GST, which is showed to carry out antioxidative stress defense. Toxicity caused by metals and also PAH was detected in Guad1. The presence of these chemicals leads to GST induction in and presumably in consequence, to LPO inhibition. GST activity resulted to be the main protection mechanism against oxidative stress in Arenicola marina. We suggest further research in Guadalete Estuary focusing on the detection and toxicity assessment of pharmaceutical and personal care products.

Chronic bioassay in benthic fish for the assessment of the quality of sediments in different areas of the coast of Spain impacted by acute and chronic oil spills.

More and more, the coastal regions of the world suffer from the contamination of petroleum hydrocarbon [principally polycyclic aromatic hydrocarbons (PAHs)]. This contamination can be acute, as it happened in the Coast of Galicia (NW, Spain) by the oil spill from a tanker, or chronic by the existence of high maritime traffic and a lot of industries as it is the case of the Bay of Algeciras (BA) (SW, Spain). It is of a great concern due to the toxicity, especially in sediments and ecosystem associated to it. The objective of this study is to assess, through chronic bioassay, sediment toxicity in samples collected in different littoral areas of Spain and to compare the damage caused in benthic fish, Solea senegalensis, according to that which suffers acute spill (Coast of Galicia) or chronic spill (the BA) by means of histopathology methods and enzymatic activities studies. Organisms were exposed to different sediments from Galician Coast and the BA during 42 days and every sample was analyzed by triplicate in glass aquaria. At the end of the bioassay, histopathological diseases were analyzed in the gills, target organ. Likewise, stress parameters as ethoxyresorufin-O-deethylase (EROD) and the glutathione S-transferases (GSTs) activities were determined in the liver. Results showed a significant positive correlation between the biomarkers of exposure (EROD and GST), biomarkers of effect (histopathology), and PAHs concentrations in the sediments.

Acid mine drainage pollution in the Tinto and Odiel rivers (Iberian Pyrite Belt, SW Spain) and bioavailability of the transported metals to the Huelva Estuary.

The Tinto and Odiel rivers are seriously affected by acid mine drainage (AMD) from the long-term mining activities in Iberian Pyrite Belt (IPB). As a consequence, the Huelva estuary is heavily contaminated by metals and metalloids. This study presents an estimation of the seasonal variation, and the dissolved contaminant load transported by both rivers from February 2002 to September 2004. Besides, toxicity and bioaccumulation tests with the sediments of the estuary have been conducted in order to measure the mobility of the toxic metals. Results show that the Tinto and Odiel rivers transport enormous quantities of dissolved metals to the estuary: 7900 t yr(-1) of Iron (Fe), 5800 t yr(-1) Aluminium (Al), 3500 t yr(-1) Zinc (Zn), 1700 t yr(-1) Copper (Cu), 1600 t yr(-1) Manganese (Mn) and minor quantities of other metals and metalloids. These values represent 37% of the global gross flux of dissolved Zn transported by rivers in to the ocean, and 15% of the global gross flux of dissolved Cu. These metals and metalloids usually sink in the estuarine sediments due to pH and salinity changes. The increase of salinity in the estuary favours the adsorption and trapping of metals. For this reason, the mobility and bioavailability of metals such as Zn, Cd and Cu is higher in sediments located in the area of fresh water influence that in sediments located in the marine influenced area of the estuary, showing a higher percentage of fractionation and bioaccumulation of these metals in the station influenced by the fresh water environment.