Ana Isabel Gusmão Lima

Looking for suitable biomarkers in benthic macroinvertebrates inhabiting coastal areas with low metal contamination: Comparison between the bivalve Cerastoderma edule and the Polychaete Diopatra neapolitana

Metals accumulated in marine sediments are often a threat to benthic communities. With the recognized importance and wide use of stress biochemical responses as indicators of metal contamination it becomes essential to compare these markers between different species and verify their ubiquity and accuracy. Using wild Diopatra neapolitana and Cerastoderma edule, collected at several areas differing in metal contamination, this study aimed to assess the use of these two macrobenthic species as sentinel organisms and to determine the applicability of currently used biomarkers in benthic species exposed to a range of low metal and As concentrations. Total metal accumulation and intracellular partitioning was analyzed and metal-induced alterations were assessed through the analysis of several biochemical parameters in both organisms, including stress-induced reactive oxygen species (ROS), lipid peroxidation and protein content, the activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and of glutathione S-transferase (GST). Metal chelation by Metallothioneins (MTs) was also determined. Results revealed that D. neapolitana accumulated higher levels of metals when compared to C. edule, independently of the sediment concentration. Results also showed strong species-specific responses to metals and differences in the ability to sequester metals. Overall, C. edule showed to be more efficient metal chelator and precipitatior than D. neapolitana, which was less tolerant and presented oxidative stress. MTs proved to be a good predictor of metal accumulation in both species, even under low metal exposures. On the other hand, lipid peroxidation was a good indicator of oxidative damage, only observed in D. neapolitana, which was a result of higher metal retention in the soluble fraction.

Accumulation, distribution and cellular partitioning of mercury in several halophytes of a contaminated salt marsh

This work evaluates the role of a plant community in mercury (Hg) stabilization and mobility in a contaminated Portuguese salt marsh. With this aim, the distribution of Hg in below and aboveground tissues, as well as the metal partitioning between cellular fractions (soluble and insoluble) in four different species (Triglochin maritima L., Juncus maritimus Lam, Sarcocornia perennis (Miller) A.J. Scott, and Halimione portulacoides (L.) Aellen) was assessed. Mercury accumulation, translocation and compartmentation between organs and cellular fractions were related to the plant species. Results showed that the degree of Hg absorption and retention was influenced both by environmental parameters and metal translocation/partitioning strategies. Different plant species presented different allocation patterns, with marked differences between monocots (T. maritima and J. maritimus) and dicots (S. perennis, H. portulacoides). Overall, the two monocots, in particular T. maritima showed higher Hg retention in the belowground organs whereas the dicots, particularly S. perennis presented a more pronounced translocation to the aboveground tissues. Considering cellular Hg partitioning, all species showed a higher Hg binding to cell walls and membranes rather than in the soluble fractions. This strategy can be related to the high degree of tolerance observed in the studied species. These results indicate that the composition of salt marsh plant communities can be very important in dictating the Hg mobility within the marsh ecosystem and in the rest of the aquatic system as well as providing important insights to future phytoremediation approaches in Hg contaminated salt marshes.

Sensitivity of biochemical markers to evaluate cadmium stress in the freshwater diatom Nitzschia palea (Kützing) W. Smith.

Human activities have been increasing the cadmium levels in soils and waters, disturbing many organisms in the primary trophic levels such as microalgae. Toxic metal pollution is a focus point of serious concern and the examination and monitoring water quality are becoming essential procedures. Diatoms are important bioindicators to monitor the metal concentrations in diverse habitats. The present study was planned to determine the biochemical mechanisms used by freshwater diatoms to cope with cadmium stress and to identify biomarkers of metal stress. For this, Nitzschia palea (Kützing) W. Smith was grown under different concentrations of Cd (0.01-0.1 mg l(-1)) and the IC(50) determined. Three concentrations (0.1, 0.2 and 0.3 mg Cd l(-1)) and a control (no cadmium) were used to undergo the experimental assays which allowed the determination of cadmium accumulation and several biochemical markers currently used to assess metal stress. N. palea was sensitive to cadmium, as the IC(50) calculated was 0.0276 mg Cd l(-1). Cadmium accumulation increased sharply and was mainly associated to the frustule. Total protein content increased with cadmium exposure, inducing increases and decreases in polypeptide expression, indicating an attempt of N. palea cells to adjust to the new prevailing conditions induced by metal stress. In order to cope with cadmium stress, cells induced the synthesis of chelating molecules such as phytochelatins (PCs). The enzymatic (SOD and CAT) and non-enzymatic (glutathione and proline) ROS scavenging mechanisms were also induced. Our results indicate the existence of diverse metal stress-mediated mechanisms in order to lessen metal damages to the cell. PCs showed to be a suitable biomarker of metal stress; besides being metal specific and concentration respondent it also allows to infer about the level of stress imposed to cells, constituting a useful tool to complement the evaluation of diatom communities when accessing aquatic metal toxicity.

Mercury intracellular partitioning and chelation in a salt marsh plant, Halimione portulacoides (L.) Aellen: Strategies underlying tolerance in environmental exposure

In the presence of metal stress, plants can resort to a series of tolerance mechanisms. Therefore field studies should be undertaken in order to evaluate the real role of these mechanisms in stress coping. The aim of this paper was to clarify the biochemical processes behind mercury tolerance in Halimione portulacoides (L.) Aellen (Caryophyllales: Chenopodiaceae) collected in a mercury contaminated salt marsh. Different fractions of mercury were separated: buffer-soluble (mainly cytosolic) and insoluble mercury (mainly associated with membranes and cell walls). The amounts in each fraction of metal were compared and related to metal distribution within plant organs. Protein-mercury complexes were isolated and analysed for their thiol content in order to assess wether the tolerance of this salt marsh plant was associated with the induction of metal chelation by phytochelatins. Overall, the mercury tolerance strategies of the plant are likely to involve root cell wall immobilization as a major mechanism of metal resistance, rather than metal chelation in the cytosolic fraction. Nevertheless, phytochelatins were demonstrated to chelate mercury under environmental exposure.

Health concerns of consuming cockles (Cerastoderma edule L.) from a low contaminated coastal system.

Commercial and recreational harvesting of shellfish within the coastal systems is usually very extensive. Since these ecosystems are frequently subjected to contamination, namely from agricultural, urban and industrial activities, and shellfish generally display a high capacity to bioaccumulate metals, populations may be at risk in terms of toxic metal exposure as a consequence of the harvesting and ingestion of near shore coastal marine organisms. Shellfish is regularly tested for concentrations of metals and other contaminants by legal authorities for commercial purposes, but although health officials use total metal as standards of food safety, only a part of the metal accumulated in shellfish is available to be assimilated and to cause toxic effect. In order to elucidate these issues an investigation on cockles inhabiting the Aveiro estuary was conducted. Element levels in sediments and wild Cerastoderma edule from sampling areas with different levels of contamination were measured; total element burden of cockles was related to accessible fraction for assimilation (TAM); element concentrations in wild C. edule were compared to EFSA (European Food Safe Authorities), USFDA (United States Food and Drug Administration) and FSANZ (Food Standards Australia and New Zealand) maximum levels (MLs); and the amount of cockle flesh needed to be consumed to exceed provisional tolerable weekly intake (PTWI) was determined. The present work showed that although sediment metal and metalloid contamination in Aveiro estuary is low the concentration of elements in C. edule does not reflect the contamination of the sediment. Aluminium (Al) and mercury (Hg) were the less and nickel (Ni), lead (Pb), zinc (Zn) and cadmium (Cd) were the most bioaccumulated metals by cockles. Comparison of MLs from international organisations with the concentration of elements in C. edule showed that arsenic (As) and Pb exceeded standard levels. The ingestion of less than 1 kg for As and 1.5 kg for Pb of cockles would result in exceeding the PTWI threshold (0.015 and 0.025 mg kg⁻¹ week⁻¹ respectively) in any of the areas considered in the study. Cd and Al also appear to be limiting elements for human consumption. Indeed, consumption of more than 3.1 kg and 2.1 kg of whole cockle soft part from one of the study areas during a single week would lead to exceedance of the recommended PTWI value for Cd (0.007 mg kg⁻¹ week⁻¹) and Al (7 mg kg⁻¹ week⁻¹) respectively. The health concerns to humans from cockle consumption from Aveiro estuary are discussed.