Oscar Nieto

Ecotoxicological evaluation of polycyclic aromatic hydrocarbons using marine invertebrate embryo-larval bioassays

The toxicity of polycyclic aromatic hydrocarbons (PAHs) was determined using mussel, sea-urchin and ascidian embryo-larval bioassays. Fluorescent light exposure enhanced phenanthrene, fluoranthene, pyrene and hydroxypyrene toxicity in comparison with dark conditions, but not naphthalene and fluorene toxicity. The toxicity of PAHs was inversely related to their K(OW) values following QSAR models derived for baseline toxicity of general narcotics, whereas the obtained regression using toxicity data from photoactivated PAHs significantly departed from the general narcosis model. Also, the mixture toxicity of five PAHs to the larval growth of the sea-urchin was compared with predictions derived from the concentration addition concept, indicating less than additive effects. Finally, we compared our toxicity data with worst-case environmental concentrations in order to provide a preliminary estimate of the risk to the marine environment. Naphthalene, fluorene and pyrene are not considered to pose a risk to sea-urchin, mussel or ascidian larvae, whilst phenanthrene and fluoranthene may pose a risk for mussel and sea-urchin. Moreover, a higher risk for those species is expected when we consider the photoactivation of the PAHs.

Toxicity and phototoxicity of water-accommodated fraction obtained from Prestige fuel oil and Marine fuel oil evaluated by marine bioassays

Acute toxicity and phototoxicity of heavy fuel oil extracted directly from the sunken tanker Prestige in comparison to a standard Marine fuel oil were evaluated by obtaining the water-accommodated fraction (WAF) and using mussel Mytilus galloprovincialis and sea urchin Paracentrotus lividus embryogenesis bioassays, and copepod Acartia tonsa and fish Cyprinodon variegatus survival bioassays. Aromatic hydrocarbon (AH) levels in WAF were measured by gas chromatography. Prestige WAF was not phototoxic, its median effective concentrations (EC50) were 13% and 10% WAF for mussel and sea urchin respectively, and maximum lethal threshold concentrations (MLTC) were 12% and 50% for copepod and fish respectively. Marine WAF resulted phototoxic for mussel bioassay. EC50s of Marine WAF were 50% for sea urchin in both treatments and 20% for mussel under illumination. Undiluted Marine WAF only caused a 20% decrease in mussel normal larvae. Similar sensitivities were found among sea urchins, mussels and copepods, whilst fish were less sensitive. Unlike Marine WAF, Prestige WAF showed EC50 values at dilutions below 20%, and its toxicity was independent of lighting conditions. The differences in toxicity between both kinds of fuel could not be explained on the basis of total AH content.

Anodic stripping voltammetry measures copper bioavailability for sea urchin larvae in the presence of fulvic acids.

Copper speciation in the presence of fulvic acids (FAs) was studied in chemically defined seawater by square wave anodic stripping voltammetry (SQWASV). A simple complexation model assuming a single type of ligand and a 1:1 reaction stoichiometry successfully explained the measured data. A conditional stability constant of 5.80 +/- 0.07 and a complexing capacity of 610 +/- 80 micromol Cu/g FAs were obtained. This conditional stability constant would correspond to 7.23 on a free copper ion basis. The complexation kinetics was easily followed by anodic stripping voltammetry (ASV) measurements, and complexation equilibria were not reached until approximately 12 h. Coupled to speciation studies, the toxicity of copper in the presence of FAs was studied with the sea urchin Paracentrotus lividus embryogenesis bioassay. A clear protective effect of FAs was observed on Cu toxicity, and this effect could be accurately explained by SQWASV Cu speciation measurements. Additional experiments in the presence of high Cu and FA concentrations were performed, and no extra toxicity of Cu-FA complexes was observed. These results are in agreement with metal bioavailability models and also underline the usefulness of ASV for determining copper bioavailability in the presence of natural organic matter.

Complexation properties of the metallothionein fragment Lys-Cys-Thr-Cys-Cys-Ala [56-61] MT I with zinc using square wave voltammetry

Abstract An electrochemical study of Zn complexation with the peptide Lys-Cys-Thr-Cys-Cys-Ala thionein fragment [56-61] MT I (FT) of mouse liver was performed using square wave voltammetry in order to enable comparisons between the behaviour of Cd,Zn metallothioneins with that of this less complex molecule, intrinsic to the metallothionein structure. Optimum instrumental parameters were used in order to get the best resolution of the voltammetric peaks of both free and complexed zinc; these being low square wave frequencies and low square wave amplitudes. Complexation equilibria of the Zn-FT system as a function of metal-ligand proportion and, for a given metal-ligand ratio, as a function of pH have been studied. From the results obtained, the existence of a main complex with a stoichiometry of 1:1, in which the three thiolate groups are involved in the complexation reaction, is suggested. Another complex of a different structure and having different characteristics to the main one, probably also exists.

Electrochemical behaviour of a metallothionein related peptide in the presence of cadmium using cyclic voltammetry

Abstract The electrochemical study on the peptidic fragment Lys-Cys-Thr-Cys-Cys-Ala [56–61] MT I (FT) in the presence of cadmium was performed by cyclic voltammetry using a hanging mercury drop electrode in order to elucidate the diffusion-adsorption processes. The influence of scan range, number of cycles, scan rate and concentration was investigated. The variation of the initial and final potentials modify the voltammetric features strongly due to changes of concentrations of species near the electrode surface. For the 1st cycle, linear relationships were found between the peak current of the different peaks and the square root of the scan rate indicating that the electrochemical mechanism is mainly diffusion-controlled. The peak current as a function of the total concentration is also linear, at least for a given range of concentration. For concentrations of the mixture FT:Cd=1:1 higher than c ∼30 μM, an adsorption pre-peak was detected during the negative scan. The electrochemical systems are reversible. The evolution of voltammograms was investigated for the sequential addition of cadmium to the peptidic fragment.

Glutathione S-transferase, glutathione peroxidase and acetylcholinesterase activities in mussels transplanted to harbour areas

As part of an integrative monitoring campaign involving water and sediment chemistry, in situ bioassays, and mussel bioaccumulation and biomarkers, Mytilus galloprovincialis mussels of standard size were transplanted from a clean location to five sites in two important harbours from the Atlantic coast of Spain (Vigo and Pasaia). After a 30-day field exposure, the concentrations of major contaminants (trace metals, polychlorinated biphenyls and polycyclic aromatic hydrocarbons) accumulated in mussel tissues were measured at each site, and a mussel bioaccumulation index (MBI) was calculated. The enzymatic activity levels of glutathione S-transferase (GST), glutathione peroxidase (GPx) and acetylcholinesterase (AChE) were quantified in the gills of transplanted mussels (n=12). Mussels from the most polluted sites consistently exhibited significantly higher GST and GPx activities compared to the control site, whereas AChE activity was significantly inhibited. The responses of the GST and GPx activities were related to MBI, trace metals and PAH concentrations in mussels, whereas AChE activity was related to the trace metals concentrations in mussels. The above results suggest that GST and AChE activities can be used as potential biomarkers for active monitoring in marine coastal ecosystems. However, at this moment, GPx activity is not robust enough to be applicable to harbour areas.

Evaluation of artificially-weathered standard fuel oil toxicity by marine invertebrate embryogenesis bioassays.

wWeathering of petroleum spilled in the marine environment may not only change its physical and chemical properties but also its effects on the marine ecosystem. The objective of this study was to evaluate the toxicity of the water-accommodated fraction (WAF) obtained from a standard fuel oil following an environmentally realistic simulated weathering process for a period of 80 d. Experimental flasks with 40 g L(-1) of fuel oil were incubated at 18°C with a 14 h light:10 h dark photoperiod and a photosynthetically active radiation (PAR) intensity of 70 μE m(-2) s(-1). Samples were taken at four weathering periods: 24 h, 7, 21 and 80 d. WAF toxicity was tested using the sea urchin (Paracentrotus lividus) and mussel (Mytilus galloprovincialis) embryo-larval bioassays and the aromatic hydrocarbons levels (AH) in the WAF were measured by gas chromatography/mass spectrometry. In contrast with the classic assumption of toxicity decrease with oil weathering, the present study shows a progressive increase in WAF toxicity with weathering, being the EC(50) after 80d eightfold lower than the EC(50) at day 1, whereas AH concentration slightly decreased. In the long term, inoculation of WAF with bacteria from a hydrocarbon chronically-polluted harbor slightly reduced toxicity. The differences in toxicity between fresh and weathered fuels could not be explained on the basis of the total AH content and the formation of oxidized derivatives is suggested to explain this toxicity increase.

Electrochemical study of metallothioneins using cyclic voltammetry

The electrochemical study of two different metallothioneins (MT) from rabbit liver, one of them containing almost only cadmium and the other containing almost only zinc in the molecule, was performed by cyclic voltammetry using a hanging mercury drop electrode. The influence of several parameters, i.e. scan range, number of cycles, quiet time, scan rates and concentration was investigated in order to elucidate the diffusion-adsorption process. The variation of the initial potential, allowing or avoiding the oxidation of the mercury electrode in the presence of thiol groups of the MT complexed by cations, modifies the concentration of compounds near the electrode surface and consequently the features of the voltammograms, particularly in the case of the zinc-metallothionein. In fact, the behaviour of both cadmium-MT and zinc-MT as a function of the parameters studied is similar, but not identical. Peaks corresponding to the electrochemical system of the cadmium complexes behave as expected for an electrochemical reaction where the product is adsorbed weakly. The variation of the reduction potential for the zinc complexes is more dependent on the experimental conditions, which are determining whether the adsorption phenomenon and the accumulation process are or are not important. In the first cycle and at low concentration of compounds the electrochemical systems are mainly diffusion controlled.

Electrochemical study of a metallothionein related peptide in the presence of zinc using cyclic voltammetry

Abstract The electrochemical response of the peptidic fragment Lys-Cys-Thr-Cys-Cys-Ala [56–61] MT I (FT) in the presence of zinc was investigated by cyclic voltammetry using a hanging mercury drop electrode. For a given concentration at the ratio of [Zn]:[FT]=1:1, the influence of scan range, scan rate and concentration was studied. The variation of the initial potential, allowing or avoiding the oxidation of the mercury electrode in the presence of thiol groups complexed by zinc, modifies the concentration of compounds near the electrode and consequently the voltammogram features. The evolution of the complexation equilibrium, attained by successive additions of Zn 2+ to a sample of FT, was followed by the negative scan of voltammograms recorded at the first cycle and at an initial potential of −0.55 V at which the oxidation of the mercury is avoided.

Characterisation of human foetal liver Zn-metallothioneins using differential pulse polarography.

A study on electrochemical characterisation of three isoforms of human foetal liver Zn-metallothioneins, labelled MT-0, MT-1 and MT-2, has been performed by using differential pulse polarography (DPP). Two different peaks, attributed to two different Zn complexes with metallothioneins, have been detected. The electrochemical behaviour is similar for the three studied isoforms. Studies on the addition of Cd(2+) and Zn(2+) as well as studies as a function of pH have been carried out. The association and dissociation equilibria of metal ions with MTs are reversible in the studied pH range. The behaviour of Zn complexes in human foetal liver Zn-metallothioneins is comparable to the Cd complexes obtained using other mammalian Cd, Zn-metallothioneins, particularly as a function of pH.