Carlos Barata

Influence of genetic and environmental factors on the tolerance of Daphnia magna Straus to essential and non-essential metals.

The ultimate aim of ecotoxicological studies is to predict how natural populations respond to contaminant exposure. Thus, it is crucial to understand how genetic and environmental factors in the field modify responses measured in the laboratory. In the present study the authors determine the genetic and environmental components of variability in acute responses among four Daphnia magna clones exposed to both essential (Zn and Cu) and non-essential (Cd and U) metals in waters with varying water hardness. The authors postulate that genotype acute responses to physiologically non-essential metals may be more variable than responses to essential metals and that this may be explained by hypothesizing that acute responses to non-essential substances are subject to intermittent selection (since the substances may not always be present in biologically significant amounts), whereas responses to essential substances are subject to continuous directional selection (since essential substances by definition co-occur with biota in biologically significant amounts). D. magna clones were exposed to single-metal solutions of varying concentration at two or three levels of water hardness (soft, moderate–hard and hard) for periods ranging from 12–96 h (12 h increments). LC50 values for each metal×genotype×water hardness×exposure period combination were determined for (i) total metal concentration and (ii) the free hydrated metal ion concentration (predicted using geochemical speciation modeling) and compared using analysis of covariance with environment (water hardness) and genotype (clone) as fixed factors and exposure time as a covariate. The results showed that Zn–Cd were consistent, but Cu–U inconsistent, with the essentiality hypothesis. In addition, the small, or even non-existent, genotype-environment interaction effect on the inheritance of metal tolerance supports the argument that genetic variability in resistance to trace metals is inherited through single major genes. Implications of these results for the prediction of population tolerance ranges are discussed in relation to the problem of predicting metal speciation and the biological effects of metals in fresh waters with varying hardness.

Determining the ecotoxicological mode of action of chemicals from measurements made on individuals: results from instar-based tests with Daphnia magna Straus

A short-term Daphnia toxicity test design was used to assess the ecotoxicological mode of action of pollutants. It was demonstrated that by exposing Daphnia females over a single instar to three toxic substances (3,4-dichloroaniline, cadmium and fluoranthene) it was possible to successfully measure both lethal effects on egg and adult stages and nonlethal effects on food acquisition and production rates. Dichloroaniline exposure reduced egg survival during development at concentrations below those which affected adult survival or production rates. For cadmium, however, concentration effects on production were almost an order of magnitude lower than those which affected adult survival, and no reductions in egg survival were observed. Responses to fluoranthene showed a different pattern again, with egg survival during development and production equally affected at concentrations which affected adult survival. Thus the three pollutants chosen showed contrasting dominant ecotoxicological modes of action, in terms of the relative importance of production and mortality effects, and this could be easily assessed using a novel toxicity test design. These results have important implications for risk assessment since with a relatively short, and thus cost-effective test, the functional relationship between effects on lethality and production rates and concentration can be determined.

Priority and emerging flame retardants in rivers: Occurrence in water and sediment, Daphnia magna toxicity and risk assessment

The occurrence, partitioning and risk of eight polybrominated diphenyl ethers (PBDEs), nine new brominated (NBFRs) and ten organophosphorus flame retardants (OPFRs) were evaluated in three Spanish rivers suffering different anthropogenic pressures (Nalón, Arga and Besòs). OPFRs were ubiquitous contaminants in water (ΣOPFRs ranging from 0.0076 to 7.2μgL(-1)) and sediments (ΣOPFRs ranging 3.8 to 824μgkg(-1)). Brominated flame retardants were not detected in waters, whereas ΣPBDEs ranged from 88 to 812μgkg(-1) and decabromodiphenyl ethane (DBDPE) reached 435μgkg(-1) in sediments from the River Besòs, the most impacted river. The occurrence of flame retardants in river water and sediment was clearly associated with human activities, since the highest levels occurred near urban and industrial zones and after wastewater treatment plants discharge. Daphnia magna toxicity was carried out for OPFRs, the most ubiquitous flame retardants, considering individual compounds and mixtures. Toxicity of nine tested OPFRs differed largely among compounds, with EC50 values ranging over three magnitude orders (0.31-381mgL(-1)). Results evidenced that these compounds act by non-polar narcosis, since their toxicity was proportional to their lipophilicity (Kow). Furthermore, their joint toxicity was additive, which means that single and joint toxicity can be predicted knowing their concentration levels in water using quantitative structure activity relationships (QSARs) and predictive mixture models. Based on these results, a risk assessment considering joint effect was performed calculating and summing risk quotients (RQs) for the water and sediment samples. No significant risk to D. magna (ΣRQs <1) was observed for any of the monitored rivers.

Multi-biomarker responses in the freshwater mussel Dreissena polymorpha exposed to polychlorobiphenyls and metals.

Contaminant related changes in behavioral, phase I and II metabolizing enzymes and pro-oxidant/antioxidant processes in the freshwater mussels Dreissena polymorpha exposed to metals and PCBs were assessed. Behavioral and biochemical responses including filtering rates, key phase I, II and antioxidant enzymes and levels of metallothioneins, glutathione, lipid peroxidation and DNA strand breaks were determined in digestive glands of mussels after being exposed to sublethal levels of mercury chloride, methyl mercury, cadmium and Aroclor 1260 during 5 days. In 7 out of 12 responses analyzed, mussels showed significant differences across treatments. Unusual properties of measured ethoxyresorufin-O-deethylase (EROD) activities indicated that mussels lack an inducible CYP1A enzymatic activity. Despite of using similar exposure levels, inorganic and organic mercury showed different biomarker patterns of response with methyl mercury being more bio-available and unable to induce metallothionein proteins. Mussels exposed to Cd presented higher levels of metallothioneins and an enhanced metabolism of glutathione, whereas those exposed to Aroclor showed their antioxidant glutathione peroxidase related enzyme activities inhibited. Although there was evidence for increased lipid peroxidation under exposure to inorganic and organic mercury, only mussels exposed to Aroclor had significant greater levels than those in controls.

Single and combined toxicity of pharmaceuticals and personal care products (PPCPs) on the rainbow trout liver cell line RTL-W1

The toxicological implications of the presence of pharmaceuticals and personal care products (PPCPs) in the aquatic environment remain largely unknown. Acute toxicity tests have generally failed to detect the subtle action elicited by those compounds at environmentally relevant concentrations and they have often overlooked the fact that toxicity can be influenced by additive and synergistic effects. The aim of this study was to further assess the cytotoxicity of different pharmaceuticals and synthetic musks as well as their mixtures on the rainbow trout liver cell line RTL-W1. Eleven pharmaceuticals from different therapeutic classes (anti-inflammatory drugs, serotonin re-uptake inhibitors and lipid regulators) and five synthetic musks from the two major groups (nitro- and polycyclic musks) were selected for the study. Two fluorescent dyes were used to monitor cell viability. Among the tested compounds, estimated EC50s (effective concentration causing 50% decline of cell viability) denoted that polycyclic musks (7-25 microM) followed by anti-depressives (7-50 microM) showed the highest potential to induce cytotoxicity, whereas lipid regulators (20-380 microM), anti-inflammatory drugs (160-260 microM) and nitromusks (100-240 microM) had the lowest toxicity. Within a given therapeutic class, combined toxicity of mixtures was additive, following in most cases the concentration addition concept. However, the combined toxicity was higher than additive for those mixtures that included one compound from each class (i.e. dissimilar mixtures). Overall, this study shows that in the aquatic environment, toxicity of PPCPs on non-target organisms may occur at concentrations lower than expected due to synergistic effects between the different toxicants.

Analysis of 17 polar to semi-polar pesticides in the Ebro river delta during the main growing season of rice by automated on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry.

This work describes the optimization of a fully automated method based on on-line solid-phase extraction-liquid chromatography-electrospray-tandem mass spectrometry (SPE-LC-ESI-MS/MS) for the simultaneous determination of 17 medium to polar pesticides in water. The list of target analytes included organophosphates, triazines, phenylureas, anilides, chloroacetanilides, acidic herbicides and thiocarbamates. Detection limits achieved were below 5 ng/L for all compounds except metolachlor (12 ng/L), alachlor (17 ng/L), malathion (36 ng/L) and fenitrothion (50 ng/L). The method developed was used to investigate the occurrence of the target pesticides in a total of 52 water samples collected monthly (from May to August 2005) at 14 selected locations in the rice cultivation area of the Ebro river delta. The study showed high levels, in the microg/L range, of bentazone, MCPA, propanil, molinate and atrazine, in basically all the samples investigated. The remaining pesticides were present at lower levels (<0.1 microg/L) or only detected sporadically (e.g. fenitrothion and malathion). The sampling campaign performed in July showed comparatively higher levels than the other three campaigns.

Contaminant accumulation and multi-biomarker responses in field collected zebra mussels (Dreissena polymorpha) and crayfish (Procambarus clarkii), to evaluate toxicological effects of industrial hazardous dumps in the Ebro river (NE Spain).

Large amounts of industrial waste containing high concentrations of mercury, cadmium and organochlorine residues were dumped in a reservoir adjacent to a chlorine-alkali plant in the village of Flix(Catalonia, Spain), situated at the shore of the lower Ebro river. Effects of these contaminants to aquatic river invertebrates were assessed by integrating analyses of metals and organochlorine residues in field collected zebra mussels and crayfish with a wide range of biomarkers. Biological responses included levels of metallothioneins, activities of ethoxyresorufin-O-deethylase, oxidative stress biomarkers (glutathione content, enzymatic activities of superoxide dismutase, catalase, glutathione s-transferase, glutathione peroxidise and glutathione reductase), levels of lipid peroxidation and of DNA strand breaks. The results obtained evidenced similar response patterns in mussels and crayfish with increasing toxic stress levels from upper parts of the river towards the meander located immediately downstream from the most polluted site, close to the waste dumps. The aforementioned stress levels could be related with concentrations of mercury, cadmium, hexachlorobenzene, polychlorobiphenyls and dichlorodiphenyltrichloroethanes from 4- to 195-fold greater than local background levels. The response of biomarkers to these pollutant concentrations differences was reflected in high activities and levels of antioxidant enzymes, metallothioneins, lipid peroxidation and DNA strand breaks and decreased levels of glutathione.

Procambarus clarkii as a bioindicator of heavy metal pollution sources in the lower Ebro River and Delta

In the Ebro River basin, point and diffuse pollution of heavy metals stems mainly from industry and agriculture. Bioaccumulation patterns were examined under different pollution sources (point and diffuse) using levels of heavy metals (As, Cd, Cr, Cu, Hg, Pb and Zn) in abdominal muscle tissue of Procambarus clarkii. P. clarkii captured under point source effects presented the highest concentrations of Hg, Pb and As; and were related with distance to the source of industrial waste sediments. Mean Hg levels in crayfish exposed to point sources of metals significantly exceeded legal allowed values established by the European Union legislation. In the Ebro Delta, high levels of As, Cr, Cu and Zn were associated with traditional agriculture activity (diffuse pollution) as well. These results demonstrate the potential of P. clarkii to bioaccumulate heavy metals from both point and diffuse sources and hence potentially transfer these metals to higher trophic levels.

Characterization of the multixenobiotic resistance (MXR) mechanism in embryos and larvae of the zebra mussel (Dreissena polymorpha) and studies on its role in tolerance to single and mixture combinations of toxicants

The study of the cellular mechanisms of tolerance of organisms to pollution is a key issue in aquatic environmental risk assessment. Recent evidence indicates that multixenobiotic resistance (MXR) mechanisms represent a general biological defense of many marine and freshwater organisms against environmental toxicants. In this work, toxicologically relevant xenobiotic efflux transporters were studied in early life stages of zebra mussels (Dreissena polymorpha). Expression of a P-gp1 (ABCB1) transporter gene and its associated efflux activities during development were studied, using qRT-PCR and the fluorescent transporter substrates rhodamine B and calcein-AM combined with specific transporter inhibitors (chemosensitizers). Toxicity bioassays with the model P-gp1 chemotherapeutic drug vinblastine applied singly and in combination with different chemosensitizers were performed to elucidate the tolerance role of the P-gp1 efflux transporter. Results evidenced that the gene expression and associated efflux activities of ABC transporters were low or absent in eggs and increased significantly in 1-3d old trochophora and veliger larvae. Specific inhibitors of Pgp1 and/or MRP transport activities including cyclosporine A, MK571, verapamil and reversin 205 and the musk celestolide resulted in a concentration dependent inhibition of related transport activities in zebra mussel veliger larvae, with IC50 values in the lower micromolar range and similar to those reported for mammals, fish and mussels. Binary mixtures of the tested transporter inhibitors except celestolide with the anticancer drug and P-gp1 substrate vinblastine increased the toxicity of the former compound more than additively. These results indicate that MXR transporter activity is high in early life-stages of the zebra mussel and that may play an important role in the tolerance to environmental contaminants.

Physiological responses to mercury in feral carp populations inhabiting the low Ebro River (NE Spain), a historically contaminated site

The low Ebro River course (Northeast Spain) is historically affected by mercury pollution due to a chlor-alkali plant operating at the town of Flix for more than a century. River sediments analysed during the last 10 years showed high mercury levels in the river section starting just downstream the factory and spanning some 90km, down to the river delta. The possible environmental impact was studied by a combination of field and laboratory studies. Mercury concentrations in liver, kidney and muscle of feral carp (Cyprinus carpio) sampled downstream Flix were one to two orders of magnitude higher than those from carps sampled upstream Flix. Elevated levels of mercury in these samples associated with significant increases on the concentration of reduced glutathione (GSH) in liver and on mRNA expression of two metallothionein genes, MT1 and MT2, in kidney and, partially, in scales, but not in liver. Conversely, no biochemical evidence for oxidative stress or DNA damage was found in these tissues. Non-contaminated carps subjected to intraperitoneal mercury injection resulted in a 20-fold increase of MT1 and MT2 mRNA levels in carp kidney, with minimal changes in liver levels. Our data suggests the coordinate increase of metallothionein mRNA in kidney and of GSH in liver constitutes an excellent marker of exposure to sub-toxic mercury levels in carps. This study also demonstrates that apparently healthy fish populations may exceed the mercury contamination acceptable for human consumption.