João M. Oliveira

Genotoxic endpoints in the earthworms sub-lethal assay to evaluate natural soils contaminated by metals and radionuclides

Eisenia andrei was exposed, for 56 days, to a contaminated soil from an abandoned uranium mine and to the natural reference soil LUFA 2.2. The organisms were sampled after 0, 1, 2, 7, 14 and 56 days of exposure, to assess metals bioaccumulation, coelomocytes DNA integrity and cytotoxicity. Radionuclides bioaccumulation and growth were also determined at 0 h, 14 and 56 days of exposure. Results have shown the bioaccumulation of metals and radionuclides, as well as, growth reduction, DNA damages and cytotoxicity in earthworms exposed to contaminated soil. The usefulness of the comet assay and flow cytometry, to evaluate the toxicity of contaminants such as metals and radionuclides in earthworms are herein reported. We also demonstrated that DNA strand breakage and immune cells frequency are important endpoints to be employed in the earthworm reproduction assay, for the evaluation of soil geno and cytotoxicity, as part of the risk assessment of contaminated areas. This is the first study that integrates DNA damage and cytotoxicity evaluation, growth and bioaccumulation of metals and radionuclides in a sub lethal assay, for earthworms exposed to soil contaminated with metals and radionuclides.

Evaluation of the sensitivity of genotoxicity and cytotoxicity endpoints in earthworms exposed in situ to uranium mining wastes

Earthworms were exposed for 56 days to a contaminated soil from an abandoned uranium mine and to the natural reference soil LUFA 2.2. The exposure occurred in situ: the containers with contaminated soil were placed near the mine pit; the containers with reference soil were placed in a reference site. For the assessment of metals bioaccumulation, DNA damages, cell-to-cell variation in DNA content, Median Fluorescence Intensity (MFI), coelomocytes frequency and proliferation, organisms were sampled after 0, 1, 2, 7, 14 and 56 days of exposure. For the assessment of radionuclides bioaccumulation, animals were sampled after 0, 14 and 56 days of exposure. As for growth, organisms were sampled after 0, 14, 28 and 56 days of exposure. The reproduction assay was performed according to the OECD (2004) guideline. DNA damages were assessed by comet assay and flow cytometry was used to determine cell-to-cell variation in DNA content, Median Fluorescence Intensity (MFI), coelomocytes frequency and proliferation. Results have shown a myriad of effects in the organisms exposed to the contaminated soil, namely: the inhibition of reproduction, growth reduction, DNA damages, cytotoxicity, changes in eleocytes fluorescence intensity, coelomocytes proliferation and bioaccumulation of metals and radionuclides. Our results showed that the evaluation of genotoxicity and cytotoxicity endpoints, along with other parameters at an individual level in standard reproduction assays conducted in situ, are important to improve the risk assessment process of areas contaminated with uranium and other radioactive mining wastes.

Factors affecting 210Po and 210Pb activity concentrations in mussels and implications for environmental bio-monitoring programmes.

The activity of (210)Po and (210)Pb was determined in mussels of the same size (3.5-4.0 cm shell length) sampled monthly over a 17-month period at the Atlantic coast of Portugal. Average radionuclide concentration values in mussels were 759±277 Bq kg(-1) for (210)Po (range 460-1470 Bq kg(-1) dry weight), and 45±19 Bq kg(-1) for (210)Pb (range 23-96 Bq kg(-1) dry weight). Environmental parameters and mussel biometric parameters were monitored during the same period. Although there was no seasonal variation of radionuclide concentrations in sea water during the study period, the concentration of radionuclide activity in mussels varied seasonally displaying peaks of high concentrations in winter and low concentrations in summer. Analysis of radionuclide data in relation to the physiological Condition Index of mussels revealed that (210)Po and (210)Pb activities in the mussel (average activity per individual) remained nearly constant during the investigation period, while mussel body weight fluctuated due to fat storage/expenditure in the soft tissues. Similar variation of radionuclide concentrations was observed in mussels transplanted from the sea coast into the Tejo Estuary. However, under estuarine environmental conditions and with higher food availability throughout the year, transplanted mussel Condition Index was higher than in coastal mussels and average radionuclide concentrations were 210±75 Bq kg(-1) (dry weight) for (210)Po and 10±4 Bq kg(-1) (dry weight) for (210)Pb, therefore lower than in coastal mussels with similar shell length. It is concluded that the apparent seasonal fluctuation and inter-site difference of radionuclide concentrations were mostly caused by mussel body weight fluctuation and not by radionuclide body burden fluctuation. This interpretation can be extended to the apparent seasonal fluctuation in concentrations of lipophilic and lipophobic contaminants in mussels, and provides an explanation for occasional high concentrations of (210)Po and man-made contaminants measured in mussels far from pollution sources.

Complex size-dependent habitat associations in potamodromous fish species

Knowledge of the distribution of species life stages at multiple spatial scales is fundamental to both a proper assessment of species management and conservation programmes and the ability to predict the consequences of human disturbances for river systems. The habitat requirements of three native cyprinid species—the Iberian barbel Barbus bocagei Steindachner, the Iberian straight-mouth nase Pseudochondrostoma polylepis (Steindachner), and the Northern straight-mouth nase Pseudochondrostoma duriense (Coelho)—were examined at 174 undisturbed or minimally disturbed sites in 8 river catchments across western Iberia, by modelling occurrence and counts of species life stages at two spatial scales—large (regional) and instream (local)—using hurdle models. All the life stages of the barbel showed a negative association with upstream high-gradient river reaches, whereas juvenile P. duriense favoured such areas. Stream width and openness were negatively related with the occurrence of juvenile and small adult barbel, but not with large adults. Juvenile nase, on the other hand, were found to be mainly confined to fast-flowing habitats with high instream cover and coarser substrata. Advanced life stages of the barbel were mainly associated with the “pure” regional and shared components, whereas the purely local attributes accounted for much of the model variation among nases, in particular juveniles, and juvenile barbel. The results of this study are useful for setting or refining management goals, and highlight the need to separately consider life stages when performing conservation-related studies of species distribution.

Radioactivity from Fukushima nuclear accident detected in Lisbon, Portugal.

The radioactivity released from the Fukushima Dai-ichi nuclear accident was transported around the globe by atmospheric processes. Several artificial radionuclides were detected and measured in aerosols and atmospheric surface depositions in the Lisbon area during late March and early April 2011. The highest concentrations measured in aerosols were those of particulate (131)I, 1.39 ± 0.08 mBq m(-3). Cesium-134, (137)Cs and (132)Te were also determined but at lower concentrations. The total atmospheric depositions on the ground were higher on the first week of April with values for (131)I, (134)Cs, and (137)Cs of 0.92 ± 0.11, 0.59 ± 0.06, and 0.62 ± 0.12 Bq m(-2), respectively. The four artificial radionuclides measurable, (131)I, (132)Te (134)Cs, and (137)Cs, caused little radiation exposure to the members of the public, that was five orders of magnitude lower than the ionizing radiation effective dose limits for members of the public for one year (1 mSv y(-1)).

Histopathological changes in the earthworm Eisenia andrei associated with the exposure to metals and radionuclides

Earthworms were exposed for 56 d to a contaminated soil, from an abandoned uranium mine, and to the natural reference soil LUFA 2.2. Histological changes in earthworms body wall (epidermis, circular and longitudinal muscles) and gastrointestinal tract (chloragogenous tissue and intestinal epithelium) were assessed, after 0, 14 and 56 d of exposure. Results have shown alterations in all the studied tissues after 14 d of exposure (except for the intestinal epithelium), yet more severe effects were registered after 56 d of exposure. Herein we report histopathological alterations as a good biomarker for the evaluation of soil quality. We also demonstrate that morphological changes in the body wall and gastrointestinal tract, are important endpoints that could be added to earthworms standardized tests, for the evaluation of soil toxicity, as part of the risk assessment of contaminated areas.

Uranium isotopes in the Balkan's environment and foods following the use of depleted uranium in the war

Immediately after the Balkans war in 1999, there has been widespread international concern about the environmental contamination with depleted uranium (DU) from ammunitions used in the conflict. Exposure of military staff and local populations to uranium metal and to its ionizing radiation were feared as potential causes for leukemia and other diseases in that region. In January 2001 a scientific mission was carried out by Portugal to evaluate those issues. A large number of environmental and food samples collected in Kosovo and Bosnia-Herzegovina, such as soils, water, aerosols, vegetables, bread, and meat were analyzed by radiochemistry and alpha spectrometry. Results of the analyses for total uranium and individual uranium isotopes are presented. Uranium in agriculture soils in Kosovo and Bosnia-Herzegovina averaged 1.8+/-0.8mgkg(-)(1) and 3+/-1mgkg(-)(1), while concentrations in drinking water from public supplies averaged 0.5+/-0.2microgL(-)(1) and 0.4+/-0.3microgL(-)(1), respectively. Results on soils indicated also that environmental contamination by DU was much localized and confined to the areas of ammunition impact. Concentrations of uranium in most of the environmental and food samples were comparable to concentrations of uranium measured in other European regions, such as Portugal and United Kingdom, and uranium isotopic ratios were in general compatible with isotopic ratios typical of natural uranium. However, a few samples displayed modified uranium isotopic ratios and could have been contaminated by DU. Implications of DU in radiation exposure of the population and in environmental contamination are discussed.

Modelling stream-fish functional traits in reference conditions: regional and local environmental correlates

Identifying the environmental gradients that control the functional structure of biological assemblages in reference conditions is fundamental to help river management and predict the consequences of anthropogenic stressors. Fish metrics (density of ecological guilds, and species richness) from 117 least disturbed stream reaches in several western Iberia river basins were modelled with generalized linear models in order to investigate the importance of regional- and local-scale abiotic gradients to variation in functional structure of fish assemblages. Functional patterns were primarily associated with regional features, such as catchment elevation and slope, rainfall, and drainage area. Spatial variations of fish guilds were thus associated with broad geographic gradients, showing (1) pronounced latitudinal patterns, affected mainly by climatic factors and topography, or (2) at the basin level, strong upstream-downstream patterns related to stream position in the longitudinal gradient. Maximum native species richness was observed in midsize streams in accordance with the river continuum concept. The findings of our study emphasized the need to use a multi-scale approach in order to fully assess the factors that govern the functional organization of biotic assemblages in ‘natural’ streams, as well as to improve biomonitoring and restoration of fluvial ecosystems.

Comparability of fish-based ecological quality assessments for geographically distinct Iberian regions.

In this work we compare two Iberian and a pan-European fish-based methods to assess ecological quality in rivers: the Fish-based Index of Biotic Integrity for Portuguese Wadeable Streams (F-IBIP), the Mediterranean Index of Biotic Integrity (IBIMED) and the pan-European Fish Index (EFI+). The results presented herein were developed in the context of the 2nd phase of the Intercalibration Exercise (IC), as required by the Water Frame Directive (WFD). The IC is aimed at ensuring comparability of the quality boundaries among the different WFD assessment methods developed by the Member States for each biological quality element. Although the two national assessment methods were developed for very distinct regions of Iberia (Western and Eastern Iberian Peninsula) they share the same methodological background: both are type-specific and guild-based multimetric indices. EFI+ is a multimetric guild-based model, but it is site-specific and uses a predictive modelling approach. The three indices were computed for all sites included in the Iberian Intercalibration database to allow the direct comparison, by means of linear regressions, of the resulting three quality values per site. The quality boundary harmonization between the two Iberian methods was only possible through an indirect comparison between the two indices, using EFI+ as a common metric. The three indices were also shown to be responsive to a common set of human induced pressures. This study highlights the need to develop general assessment methods adapted to wide geographical ranges with high species turnover to help intercalibrating assessment methods tailored for geographically more restricted regions.

A Preliminary Fishery Quality Index for Portuguese Streams

Abstract There is a need to quantify the multivariate quality of a recreational fishery at the site scale to better communicate the relative quality among sites to the public and anglers. Borrowing on the general approach of multimetric indices of biotic integrity (IBIs), we developed fishery quality indices (FQIs) from species quality indices (SQIs) based on measures of fish abundance and size structure for northern and central Portuguese streams. Our FQIs showed regional patterns indicating a range in fishery quality. Higher coldwater FQI scores were mostly found in the northwestern (Minho and Lima), northeastern Douro, and northern Tagus basins. Higher warmwater FQI scores occurred in the eastern Tagus basin. The species that contributed the most to warmwater FQI scores were largemouth bass Micropterus salmoides, pumpkinseed Lepomis gibbosus, the cyprinid Luciobarbus bocagei, chubs Squalius carolitertii and S. pyrenaicus, and nases Pseudochondrostoma duriense and P. polylepis. The chubs, nases, and bro...