Marta Martins

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.

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.

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.

The joint effect of polycyclic aromatic hydrocarbons on fish behavior.

Polycyclic aromatic hydrocarbons (PAHs) are one of the most widespread organic environmental pollutants that pose a potential risk to marine biota. Although they occur as mixtures in the marine environment, only little information exists about their joint action on fish behavior. In 4-day tests with juvenile gilthead seabream (Sparus aurata) concentration-response analyses were performed for three PAH compounds--fluorene (FE), phenanthrene (PHE), and pyrene (PY). Responses of fish to these compounds were assessed by recording visually the changes in their locomotory activities and social behaviors. Based on these concentration-response data, mixture effects were predicted by applying the model of concentration addition. The mixture was tested using a fixed-ratio design, and the resulting effects were compared to the predictions. The single compounds and the mixture were accumulated in fish muscle and produced a clear change in the overall behavioral performance of fish, as all individual parameters were affected in a dose-response way. For lethargy and swimming, we determined regression fits for all single compounds, with PY the most potent (EC(10)=0.031 microM for swimming and 0.039 microM for lethargy) and FE the least (EC(10)=0.29 microM for swimming and 0.26 microM for lethargy). Also, changes in the number of lethargic fish were always the most sensitive parameter and social interaction the least. The mixture study revealed for lethargy and swimming a good agreement between the predicted and observed effect changes, and statistically significant deviations could not be identified.

Influence of diffuse sources on levels and distribution of polychlorinated biphenyls in the Guadiana River estuary, Portugal

Particulate material was collected along the Guadiana River estuary at different river flow and tidal conditions: coarser material eroded by flood in January 2001 at one station in the river; fresh surface sediments after storm water runoff at 7 sites; 24 surface sediments at 10 cross sections in March 2001; one sediment core from the adjoining coastal area in the same period; 124 suspended particulate matter (SPM) samples at low- and high-tide of neap and spring tides in May and October 2001. Polychlorinated biphenyl (PCB) congeners were analysed in all materials. Interpretative parameters, like salinity and suspended particulate matter concentration, organic carbon, Al and Si were also determined. Low levels of PCB in sediments are in conformity with the absence of industrial activities in the drainage basin. Both coarser material and fresh surface sediments transported by the flood contained slightly higher PCB concentrations (0.8–1.8 ng g−1) than surface sediments (0.1–1.1 ng g−1). Furthermore, PCB concentrations were higher in suspended particulate matter transported by moderate river flows (October), particularly of upstream limit of salinity (0.4 to 30.1 ng g−1) than in May (0.8 and 8.2 ng g−1). Alterations were also recorded in PCB composition. Less chlorinated compounds (higher ratio between tri+tetra-CB and hexa-CB) were enriched in coarser and fresh surface sediments, as well as in a sediment layer of the core taken outside the estuary. This ratio increased also in suspended particulate matter of the upstream limit of salinity collected in October. Changes of PCB concentrations and proportions of congeners evidence the importance of storm water runoff to the PCB input into this low-contaminated estuarine system.

The Comet Assay and its applications in the field of ecotoxicology: a mature tool that continues to expand its perspectives

Since Singh and colleagues, in 1988, launched to the scientific community the alkaline Single Cell Gel Electrophoresis (SCGE) protocol, or Comet Assay, its uses and applications has been increasing. The thematic areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. It has been applied to a wide range of experimental models: bacteria, fungi, cells culture, arthropods, fishes, amphibians, reptiles, mammals, and humans. This document is intended to be a comprehensive review of what has been published to date on the field of ecotoxicology, aiming at the following main aspects: (i) to show the most relevant experimental models used as bioindicators both in the laboratory and in the field. Fishes are clearly the most adopted group, reflecting their popularity as bioindicator models, as well as a primary concern over the aquatic environment health. Amphibians are among the most sensitive organisms to environmental changes, mainly due to an early aquatic-dependent development stage and a highly permeable skin. Moreover, in the terrestrial approach, earthworms, plants or mammalians are excellent organisms to be used as experimental models for genotoxic evaluation of pollutants, complex mix of pollutants and chemicals, in both laboratory and natural environment. (ii) To review the development and modifications of the protocols used and the cell types (or tissues) used. The most recent developments concern the adoption of the enzyme linked assay (digestion with lesion-specific repair endonucleases) and prediction of the ability to repair of oxidative DNA damage, which is becoming a widespread approach, albeit challenging. For practical/technical reasons, blood is the most common choice but tissues/cells like gills, sperm cells, early larval stages, coelomocytes, liver or kidney have been also used. (iii) To highlight correlations with other biomarkers. (iv) To build a constructive criticism and summarize the needs for protocol improvements for future test applications within the field of ecotoxicology. The Comet Assay is still developing and its potential is yet underexploited in experimental models, mesocosmos or natural ecosystems.

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.

Validation of Arenicola marina in field toxicity bioassays using benthic cages: Biomarkers as tools for assessing sediment quality

Sediment toxicity assessments using caged organisms present advantages over using laboratory and native community studies. The use of caged Arenicola marina in sediment toxicity assessments was evaluated. Lugworms were exposed in situ to sediments from coastal and port areas in Spain for seven days, and the activities of the biotransformation enzymes ethoxyresorufin O-deethylase, dibenzylfluorescein dealkylase and glutathione S-transferase, the activities of the antioxidant enzymes glutathione reductase and glutathione peroxidase and lipid peroxidation were then analyzed as biomarkers. Biomarker results and sediment physicochemical data were integrated. Cádiz Bay (SW Spain) sediments presented metal contamination that was not linked to a biochemical response. In LPGC Port (SW Spain), Pb contamination exhibited a moderate toxic potential, while PAHs, and presumably pharmaceuticals, provoked biochemical responses that efficiently prevented lipid peroxidation. In Santander Bay (N Spain), exposure to PAHs and, presumably, pharmaceuticals induced biomarker responses, but lipid peroxidation occurred nevertheless. These results indicated that caged A. marina were effective for the assessment of sediment quality and that the selected biomarkers were sufficiently sensitive to identify chemical exposure and toxicity.

The comet assay in Environmental Risk Assessment of marine pollutants: applications, assets and handicaps of surveying genotoxicity in non-model organisms

Determining the genotoxic effects of pollutants has long been a priority in Environmental Risk Assessment (ERA) for coastal ecosystems, especially of complex areas such as estuaries and other confined waterbodies. The acknowledged link between DNA damage, mutagenicity and carcinogenicity to the exposure to certain toxicants has been responsible to the growing interest in determining the genotoxic effects of xenobiotics to wildlife as a measure of environmental risk. The comet assay, although widely employed in in vivo and in vitro toxicology, still holds many constraints in ERA, in large part owing to difficulties in obtaining conclusive cause-effect relationships from complex environments. Nevertheless, these challenges do not hinder the attempts to apply the alkaline comet assay on sentinel organisms, wild or subjected to bioassays in or ex situ (from fish to molluscs) as well to standardise protocols and establish general guidelines to the interpretation of findings. Fish have been regarded as an appealing subject due to the ease of performing the comet assay in whole blood. However, the application of the comet assay is becoming increasingly common in invertebrates (e.g. in molluscan haemocytes and solid tissues such as gills). Virtually all sorts of results have been obtained from the application of the comet assay in ERA (null, positive and inconclusive). However, it has become clear that interpreting DNA damage data from wild organisms is particularly challenging due to their ability to adapt to continuous environmental stressors, including toxicants. Also, the comet assay in non-model organisms for the purpose of ERA implies different constraints, assumptions and interpretation of findings, compared with the in vitro procedures from which most guidelines have been derived. This paper critically reviews the application of the comet assay in ERA, focusing on target organisms and tissues; protocol developments, case studies plus data handling and interpretation.

Evaluation of the potential of the common cockle (Cerastoderma edule L.) for the ecological risk assessment of estuarine sediments: bioaccumulation and biomarkers

Common cockles (Cerastoderma edule, L. 1758, Bivalvia: Cardiidae) were subjected to a laboratory assay with sediments collected from distinct sites of the Sado Estuary (Portugal). Cockles were obtained from a mariculture site of the Sado Estuary and exposed through 28-day, semi-static, assays to sediments collected from three sites of the estuary. Sediments from these sites revealed different physico-chemical properties and levels of metals and organic contaminants, ranging from unimpacted (the reference site) to moderately impacted, when compared to available sediment quality guidelines. Cockles were surveyed for bioaccumulation of trace elements (Ni, Cu, Zn, As, Cd and Pb) and organic contaminants (PAHs, PCBs and DDTs). Two sets of potential biomarkers were employed to assess toxicity: whole-body metallothionein (MT) induction and digestive gland histopathology. The bioaccumulation factor and the biota-to-soil accumulation factor were estimated as ecological indices of exposure to metals and organic compounds. From the results it is inferred that C. edule responds to sediment-bound contamination and might, therefore, be suitable for biomonitoring. The species was found capable to regulate and eliminate both types of contaminants. Still, the sediment contamination levels do not account for all the variation in bioaccumulation and MT levels, which may result from the moderate metal concentrations found in sediments, the species’ intrinsic resistance to pollution and from yet unexplained xenobiotic interaction effects.