Miguel Caetano

Histological biomarkers in liver and gills of juvenile Solea senegalensis exposed to contaminated estuarine sediments: A weighted indices approach

Young juvenile Solea senegalensis were exposed to three sediments with distinct contamination profiles collected from a Portuguese estuary subjected to anthropogenic sources of contamination (the Sado estuary, western Portugal). Sediments were surveyed for metals (cadmium, chromium, copper, nickel, lead and zinc), a metalloid (arsenic) and organic contaminants (polycyclic aromatic hydrocarbons, polychlorinated biphenyls and a pesticide, dichloro-diphenyl-trichloroethane plus its metabolites), as well as total organic matter, redox potential and particle fine fraction. The fish were exposed to freshly collected sediments in a 28-day laboratorial assay and collected for histological analyses at days 0 (T(0)), 14 (T(14)) and 28 (T(28)). Individual weighted histopathological indices were obtained, based on presence/absence data of eight and nine liver and gill pathologies, respectively, and on their biological significance. Although livers sustained more severe lesions, the sediments essentially contaminated by organic substances caused more damage to both organs than the sediments contaminated by both metallic and organic contaminants, suggesting a possible synergistic effect. Correlation analyses showed that some alterations are linked, forming distinctive histopathological patterns that are in accordance with the severity of lesions and sediment characteristics. The presence of large eosinophilic bodies in liver and degeneration of mucous cells in gills (a first-time described alteration) were some of the most noticeable alterations observed and were related to sediment organic contaminants. Body size has been found to be negatively correlated with histopathological damage in livers following longer term exposures. It is concluded that histopathological indices provide reliable and discriminatory data even when biomonitoring as complex media as natural sediments. It is also concluded that the effects of contamination may result not only from toxicant concentrations but also from their interactions, relative potency and sediment characteristics that ultimately determine bioavailability.

Metal Remobilisation during Resuspension of Anoxic Contaminated Sediment: Short-Term Laboratory Study

The sediments of the Tagus estuary North Channel arecharacterised by high concentrations of trace metals andmonosulphides. During dredging operations Cd, Cu and Pb wereanalysed in water and suspended sediments collected 50 to 100 m around the dredging point. Concentrations in bothfractions fluctuated randomly: 2–3 fold for Cd and Cu and 10 for Pb. Since sampling in the dredging point reflectsintegration of rapid chemical reactions, a short-termlaboratory experiment was conducted to follow the geochemicalalterations occurring in the highest turbidity sites. Theexperiment was monitored as a function of time over a period of4 hr in short time intervals. Dissolved oxygen, pH, EH,AVS, SO42-, Cl- and metals were monitored in theslurry samples. Iron, Mn, Cd, Pb and Cu were determined in thedissolved fraction (<0.45 μm), in the reactive solid phaseand in the total fraction. Resuspension resulted in asignificant release of Fe, Mn, Cd, Cu and Pb from the solids.Following the release Pb and Cu were almost totally scavengedin the 4 hr by the newly precipitated Fe oxyhydroxides, while more than 50% of the mobilised Cd remained in the dissolved fraction. The less efficient removal of Cd from solution implies a prolonged availability of this metal in the environment.

Stock and losses of trace metals from salt marsh plants.

Pools of Zn, Cu, Cd and Co in the leaf, stem and root tissues of Sarcocornia fruticosa, Sarcocornia perennis, Halimione portulacoides and Spartina maritima were analysed for a Tagus estuary (Portugal) salt marsh. Pools of Cu and Cd in the salt marsh were higher in spring/summer, indicating a net uptake of these metals during the growing season. Standing stocks of Zn, Cu, Cd and Co in the leaf and stem biomass of S. fruticosa, S. perennis and H. portulacoides showed a strong seasonal variation, with higher values recorded in autumn. The metal-containing leaves and stems that shed in the autumn become metal-containing detritus. The amount of this material washed out from the total marsh area (200 ha) was estimated as 68 kg of Zn, 8.2 kg of Cu, 13 kg of Co and 0.35 kg of Cd. The high tidal amplitude, a branched system of channels and semi-diurnal tidal cycle greatly favour the export of the organic detritus to adjoining marsh areas.

Accumulation and biological cycling of heavy metal in four salt marsh species, from Tagus estuary (Portugal)

Pools of Zn, Cu, Cd and Co in leaf, stem and root tissues of Sarcocornia fruticosa, Sarcocornia perennis, Halimione portulacoides and Spartina maritima were analyzed on a bimonthly basis, in a Tagus estuary salt marsh. All the major concentrations were found in the root tissues, being the concentrations in the aboveground organs neglectable for sediment budget proposes, as seen by the low root-aboveground translocation. Metal annual accumulation, root turnovers and cycling coefficients were also assessed. S. maritima showed the higher root turnovers and cycling coefficients for most of the analyzed metals, making this a phytostabilizer specie. By contrast the low root turnover, cycling coefficient and low root necromass generation makes S. perennis the most suitable specie for phytoremediation processes. Although the high amounts of metal return to the sediments, due to root senescence, salt marshes can still be considered sinks of heavy metals, cycling heavy metals mostly between sediment and root.

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.

Tidal flushing of ammonium, iron and manganese from inter-tidal sediment pore waters

Concentrations of ammonium, manganese and iron were examined in sediment pore water and near-bottom water of inter-tidal sediments of Ria Formosa, a coastal lagoon in the South of Portugal, during the short period of time that water flooded the area. Short-sediment cores and overlying water retained during the ebb tide were sampled at air-exposed area. Immediately after the inundation sediment cores and flooding water were repeatedly collected over short periods of time, 20 min for ammonium and 60 min for iron and manganese. The pore water composition of the upper 2-cm sediment changed dramatically in NH4+, Mn and Fe during the first minutes of the flood. This decrease was recorded together with an increase of ammonium and manganese in the flooding water. The inundation causes a mixing of freshly tidal water in the interstices of sediment surface, pore water being mixed with sea water and oxygen supplied to anoxic layers of the sediment. Consequently NH4+ in pore water and weakly bound to sediment is exported to the water column, Mn(II) also escapes from the sediment, but Fe(II) is oxidised rapidly and iron is recycled inside the sediment. These processes occur on a semi-diurnal time scale that implies a continuously re-adjustment of the chemical of inter-tidal sediment surface of Ria Formosa.

Estuarine ecological risk based on hepatic histopathological indices from laboratory and in situ tested fish.

Juvenile Senegalese soles were exposed through 28-day laboratory and field (in situ) bioassays to sediments from three sites of the Sado estuary (W Portugal): a reference and two contaminated by metallic and organic contaminants. Fish were surveyed for ten hepatic histopathological alterations divided by four distinct reaction patterns and integrated through the estimation of individual histopathological condition indices. Fish exposed to contaminated sediments sustained more damage, with especial respect to regressive changes like necrosis. However, differences were observed between laboratory- and field-exposed animals, with the latest, for instance, exhibiting more pronounced fatty degeneration and hepatocellular eosinophilic alteration. Also, some lesions in fish exposed to the reference sediment indicate that in both assays unaccounted variables produced experimental background noise, such as hyaline degeneration in laboratory-exposed fish. Still, the field assays yielded results that were found to better reflect the overall levels of contaminants and physico-chemical characteristics of the tested sediments.

Mercury in contaminated sediments and pore waters enriched in sulphate (Tagus Estuary, Portugal)

Three sediment cores, collected nearby the effluent of a chlor-alkali industry, were sliced in 0.5-cm layers and centrifuged for pore water extraction. Mercury, Fe and Mn were determined in the solids as total concentration, hydroxylamine extractable fraction and HCl extractable fraction. Sulphur was determined in the HCl extraction. Total and reactive mercury, chlorinity, S(2-), SO(4)(2-), total Fe, and total Mn were measured in pore waters. The solids contained 3.0-60 nmol g(-1) of total Hg and pore waters 70-5800 pM of total Hg and 1.8-76 pM of reactive mercury. Pore waters presented 2.3-94 times more sulphate than the overlying estuarine waters due to the input from the industry. In layers where hydroxylamine extractable Fe exhibited a broad maximum (precipitation of Fe-oxides) sulphate was reduced to S(2-). The competition between the high content of SO(4)(2-) and Fe(III) as electron acceptors, in chemical reactions occurring in the upper sediments, may explain the co-existence of S(2-) and Fe-oxides in the same layers. Mercury was detected in the hydroxylamine extracts (20-29 nmolg(-1)) in the layers where Fe-oxides were formed, and reactive dissolved Hg showed minimum concentrations. The excess of sulphate in pore waters favoured the abundant Fe-oxides in the upper solid sediments, which appear to work as a barrier limiting the escape of mercury to the water column.

Retention of arsenic and phosphorus in iron-rich concretions of Tagus salt marshes

Iron-rich concretions are frequently found around plant roots in Tagus estuary (Portugal) where radial delivery of O2 takes place. Salt marsh sediments exhibit cracks that are an additional feature to introduce O2 and other solutes in the upper sediments. Metal concentrations in salt marsh sediments are clearly above the background levels reflecting the anthropogenic sources from a large city with 2.5 million inhabitants, and several industrial centres. In order to evaluate how both oxidised structures influences the redistribution of redox sensitive elements in salt marsh sediments, concretions were collected from roots of Halimione portucaloides below the oxygenated zone. These tubular cylindrical structures were analysed for Fe, Al, Mn, As, and P along 1-cm radial transect in a millimetre scale from the inner part to the adjacent anoxic sediment. In addition, oxidised cracks were analysed for the same spatial resolution, from the sediment–water interface to anoxic layers (2-cm transept). The parallelism between Fe, As, and P concentrations at this microscale is the most noticeable aspect. Iron and As presented very high concentrations in the 4-mm concretions (3.4 mmol g−1 and 3.1 μmol g−1, respectively) and decreased sharply to the host sediment. Oxygen released from roots oxidise the solid sulphides, and the reduced Fe and As are transported towards the root by both diffusion and pore water flow associated with the root water uptake. Subsequently, Fe(III) precipitates and As is retained by sorption and/or coprecipitation. These elements are also enriched in the first 2-mm of oxidised cracks, but in lower concentrations (50% and 30%, respectively). Manganese concentrations in concretions were low (11.8 μmol g−1), indicating that Fe dominates the sediment chemistry. Phosphorus and iron concentrations in the ascorbate fraction were higher in the oxidising surfaces of concretions (10.7 μmol g−1 and 1.6 mmol g−1, respectively) and of cracks (5.1 μmol g−1 and 0.47 mmol g−1). The parallelism of Fe and As distributions includes not only their similar redox chemistries, but also that to phosphate, including control by coprecipitation of the host iron phases. The mechanisms involved in the mobilisation of As and P are however different, whereas As comes from the oxidation of iron sulphides; dissolved P derives from reduction of ferri-hydroxide phases.

Assessment of contaminants and biomarkers of exposure in wild and farmed seabass

This study aimed to investigate the relationships between levels of organochlorine compounds (PCBs and DDTs), polycyclic aromatic hydrocarbons (PAHs), metals and biomarkers of exposure (EROD and GST) and oxidative stress biomarkers, in wild and cultured European seabass (Dicentrarchus labrax). Additionally, the fatty acid profile was determined in muscle to assess differences between cultivated and wild specimens of marketable size. Although results pointed to relatively low concentration of contaminants, wild specimens presented enhanced accumulation of metals and cultivated specimens higher levels of organic compounds. Biomarkers were correlated with contaminants in tissues, showing that higher levels can cause effects at the biochemical level. Wild specimens have presented higher content on omega-3 fatty acids in muscle. Besides the useful application of biomarkers in wild species, this study has shown that they can also be applied to monitor biological effects in the fish farming process.