Paula Sánchez-Marín

Effect of dissolved organic matter (DOM) of contrasting origins on Cu and Pb speciation and toxicity to Paracentrotus lividus larvae

Water samples of contrasting origin, including natural seawater, two sediment elutriates and sewage-influenced seawater, were collected and obtained to examine the effect of the dissolved organic matter (DOM) present on metal bioavailability. The carbon content (DOC) and the optical properties (absorbance and fluorescence) of the coloured DOM fraction (CDOM) of these materials were determined. Cu and Pb complexation properties were measured by anodic stripping voltammetry (ASV) and the effect of DOM on Cu and Pb bioavailability was studied by means of the Paracentrotus lividus embryo-larval bioassay. Sediment elutriates and sewage-influenced water (1) were enriched 1.4-1.7 times in DOC; (2) absorbed and reemitted more light; and (3) presented higher Cu complexation capacities (L(Cu)) than the natural seawater used for their preparation. L(Cu) varied from 0.08 microM in natural seawater to 0.3 and 0.5 microM in sediment elutriates and sewage-influenced water, respectively. Differences in DOC, CDOM and Cu complexation capacities were reflected in Cu toxicity. DOM enriched samples presented a Cu EC(50) of 0.64 microM, significantly higher than the Cu EC(50) of natural and artificial seawater, which was 0.38 microM. The protecting effect of DOM on Cu toxicity greatly disappeared when the samples were irradiated with high intensity UV-light. Cu toxicity could be successfully predicted considering ASV-labile Cu concentrations in the samples. Pb complexation by DOM was only detected in the DOM-enriched samples and caused little effect on Pb EC(50). This effect was contrary for both elutriates: one elutriate reduced Pb toxicity in comparison with the control artificial seawater, while the other increased it. UV irradiation of the samples caused a marked increase in Pb toxicity, which correlated with the remaining DOC concentration. DOM parameters were related to Cu speciation and toxicity: good correlations were found between DOC and Cu EC(50), while L(Cu) correlated better with the fluorescence of marine humic substances. The present results stress the importance of characterizing not only the amount but also the quality of seawater DOM to better predict ecological effects from total metal concentration data.

Dependence of Cu, Pb and Zn remobilization on physicochemical properties of marine sediments.

The resuspension of 65 marine sediments was simulated in the laboratory with elutriates from 30 different sites from the north coast of Spain. The partitioning of Cu, Pb and Zn between sediment and elutriate was studied as a function of different physicochemical characteristics of the sediment: organic matter (OM), fine fraction (FF), redox potential and acid volatile sulfides (AVS). Mean remobilization factors (RF) -calculated as metal concentration in the elutriate (μg/L) divided by metal concentration in the sediment (μg/g dry weight)- were 0.072 for Cu, 0.012 for Pb and 0.071 for Zn. Remobilization of Pb was significantly lower than that of Cu and Zn. Although AVS, OM and FF presented a strong intercorrelation, OM explained great part of the variability on Cu and Pb remobilization while AVS did it for Zn. A multiple regression model considering both OM and AVS explained slightly better the remobilization of Pb and Cu, but not that of Zn.

Cu and Pb accumulation by the marine diatom Thalassiosira weissflogii in the presence of humic acids

Metal complexation by dissolved organic matter, as humic acids, is considered to decrease metal bioavailability by lowering the free metal ion concentration. However, dissolved organic matter adsorption on cell surfaces can modify cell membrane properties, which can also influence metal uptake. Copper and lead accumulation and internalisation by the marine diatom Thalassiosira weissflogii were studied in the absence and presence of humic acids, and adsorption of humic acids to cell surfaces was evaluated. Both Pb and Cu intracellular concentrations decreased in the presence of humic acids according to labile metal concentrations measured by anodic stripping voltammetry, whereas total (intracellular plus adsorbed) metal content was enhanced in the presence of humic acids, probably owing to enhanced metal plus humics adsorption to cell surfaces. The results of the present work stress the importance of differentiating between intracellular and total cellular metal in bioavailability studies, and suggest that the silica frustule of diatoms represents a barrier against humic acids effects on cell membranes.

Pb uptake by the marine mussel Mytilus sp. Interactions with dissolved organic matter.

It is well known that dissolved organic matter binds metal ions and buffers them in natural waters. Although it is believed that a decrease in metal ion concentration should lead to a decrease in metal bioavailability, previous work has shown that Pb uptake by Mytilus edulis gills is greatly enhanced in the presence of humic acids. In the present work, the effect of more soluble organic matter (fulvic acids and DOM extracted from river) on Pb uptake by mussels and their gills is studied. Pb complexation by these organic substances was measured by anodic stripping voltammetry (ASV) and it is proven that Pb uptake by mussel gills in the presence of fulvic acids can be successfully predicted according to ASV-labile Pb concentrations. However, Pb uptake by whole mussels in the presence of river DOM is slightly higher than predicted on the basis of ASV measurements. The possible reasons leading to different effects of DOM on Pb uptake by mussels are discussed according to physicochemical properties of DOM.

Copper and lead internalisation by freshwater microalgae at different carbonate concentrations

Environmental context Metal–carbonato complexes have been reported to contribute to metal uptake and toxicity in aquatic organisms. We show that in the presence of lead–carbonato complexes, Pb internalisation by the microalga Chlamydomonas reinhardtii is higher than that predicted on the basis of the free Pb2+ concentration. This effect, which was not observed for another microalga that takes up Pb more slowly, is attributed to the very high rates of Pb uptake by C. reinhardtii, which result in diffusion limitation. Abstract The possible contribution of metal–carbonato complexes to metal uptake or toxicity has been mentioned several times in the literature, often in studies where dissolved inorganic carbon (DIC) concentrations and pH were varied together, but a thorough study of the effect on DIC on metal bioavailability to aquatic organisms has not been done. By using closed systems – allowing changes in DIC concentrations at fixed pH – and ion selective electrodes to determine free metal ion concentrations, we show that lead internalisation by the unicellular alga Chlamydomonas reinhardtii in the presence of high DIC concentrations is higher than predicted by the free Pb2+ ion concentration at bulk [Pb2+] lower than 50nM, but not at higher [Pb2+]. This effect is not observed for another microalga, Chlorella vulgaris, which shows a lower rate of Pb internalisation. Copper internalisation by C. reinhardtii seems also to be slightly higher than predicted on the basis of free Cu2+ at low (20nM) bulk Cu2+ concentrations but not at higher ones. The possibility that Pb (and Cu) internalisation by C. reinhardtii is partially limited by diffusion from the bulk solution to the algal surface is identified and discussed as a possible explanation for these results.

Quantification of the increase in Pb bioavailability to marine organisms caused by different types of DOM from terrestrial and river origin

Dissolved organic matter (DOM) has metal binding properties and decreases the concentration of free metal ions in the aquatic environments. However, humic acids (HA) have been shown to increase Pb bioavailability for marine invertebrates, including Pb toxicity to sea urchin embryos. The low solubility and other properties of commercially available HA, which may not be the most representative fraction of DOM in natural waters, limit the environmental relevance of these findings. The present study tested the effect of more soluble DOM, fulvic acids (FA) and DOM extracted from the Suwannee River (SRDOM), on the acute toxicity of Pb to Paracentrotus lividus embryos. It was confirmed that FA and SRDOM, despite their Pb binding properties, also increased Pb toxicity for sea urchin embryos, even though this increase was not as high as that previously observed for HA. The enhancing effect of DOM on Pb bioavailavility was quantified using multiple regression equations for the present data, as well as for previously published data on Pb uptake by the gills of the marine mussel Mytilus sp. and Pb internalization by the marine microalgae Isochrysis galbana and Thalassiosira weissflogii. These results confirm that dissolved Pb bioavailability in seawater in the presence of DOM is higher than predicted using current bioavailability models based on chemical equilibrium chemistry. The experimental evidence suggests that the mechanisms by which DOM enhances Pb uptake and toxicity implies direct contact of the organic compounds with the plasma membrane.

Triphenyltin induces imposex in Nucella lapillus through an aphallic route

Triphenyltin (TPT) was used until recently as a biocide in antifouling systems and nowadays is still applied as an agriculture pesticide in some countries. This compound is known to cause imposex (the imposition of male characters in females of gastropod molluscs) in a very limited number of species, when compared with tributyltin (TBT), the universally recognized imposex-causing agent. In this study, we tested if TPT could induce imposex in females of the dog-whelk Nucella lapillus. Experimental groups of 40 females were injected with a volume of 2μL/g of soft tissue wet weight (ww) of one of the following treatments, using DMSO as a solvent carrier: DMSO (solvent control); 1μg/g ww of TBT (positive control); 0.2, 1 and 5μg/g ww of TPT and a non-injected group (negative control). Concentrations were confirmed in the organism tissues by means of chemical analyses of a pool of 10 specimens at T0 and then after the imposex analysis at T56days. After 8-week trial, results pointed out statistically significant differences between treatments, with both TPT and TBT positively inducing imposex. However, imposex development in TPT-injected females differed from that of TBT, since females that developed imposex presented an aphallic condition (no penis development) while the TBT-treated females developed standard imposex (with penis formation). These results suggest that TPT and TBT act differently in the sequential process of female masculinization, casting new insights about the hypothetical pathways underlying imposex development.

Copper uptake by the marine mussel Mytilus edulis in the presence of fulvic acids

Copper uptake and accumulation by the marine mussel Mytilus edulis were studied at different Cu concentrations in chemically defined artificial seawater in the presence and absence of fulvic acids. Both short-term uptake of Cu by excised mussel gills and Cu accumulation in whole mussels after 24 h of exposure decreased in the presence of fulvic acids compared with their absence at similar dissolved Cu concentrations. Calculations of Cu speciation based on previous measurements of labile Cu by anodic stripping voltammetry demonstrated that Cu uptake and accumulation depended on the concentration of labile Cu, in agreement with the free ion activity model. No evidence of a significant uptake of Cu-fulvic acid complexes was observed.

Shotgun proteomics analysis discards alkali labile phosphate (ALP) as a reliable method to assess vitellogenin levels in Mytilus galloprovincialis

Vitellogenin, the egg yolk precursor, is a well-known biomarker of endocrine disruption in oviparous vertebrates. In invertebrates, such as bivalves, it has been used in the last 10 years for the same purpose, despite the limited knowledge of invertebrate endocrinology. In bivalves, vitellogenin levels are usually estimated using an indirect technique, alkali labile phosphate (ALP), that assumes that vitellogenin is the most abundant phosphorylated protein in the analyzed tissue. In this study, we applied shotgun proteomics for the identification and quantification of vitellogenin in marine mussel gonads and compared the results with those obtained with the ALP method. The proteomic analysis revealed that vitellogenin is only detected in female gonads with expression levels that are rather variable among female mussels at different stages of gonad development. ALP analysis, on the contrary, detected similar amounts of phosphorylated proteins regardless of sex or gonad development stage. These results show evidence that the ALP method is not providing reliable information about Vtg levels, at least in marine mussel gonads. ALP is not a good proxy to assess Vtg levels in marine mussels, and careful verification of the adequacy of the procedure should be done before ALP is further assumed as a proxy of Vtg in other bivalve mollusks.

Determination of trace metals accumulated and internalized by marine phytoplankton; interferences with colloidal organic matter

In the present work, we study the effects that aggregation of humic acids in seawater, and its adsorption to surfaces, can have on the determination of Cu and Pb accumulation by phytoplankton within the context of metal bioavailability studies, using the diatom Thalassiosira weissflogii. Two methods commonly used for collection of algae to analyse metal content, filtration and centrifugation, were compared, and a procedure to differentiate the metal internalized by the alga from that adsorbed to cell walls-washing with 0.01 M ethylenediaminetetraacetic acid (EDTA)- was optimized for its use in seawater and in the presence of humic acids. The present results highlight the possibility of obtaining biased results if filtration is used to isolate the algae, and show that centrifugation is a more suitable procedure minimizing contribution of particulate and colloidal forms of metals to the measured cellular and intracellular metal contents. Thus a combination of centrifugation to isolate the algae from the medium with a 10 minutes washing with a washing agent composed of EDTA 0.01 M at pH 8 and NaCl and KCl to 0.5 M ionic strength is recommended to extract metals adsorbed to the surfaces of marine phytoplankton for a seawater containing humic substances. The importance of discriminating between internalized metal and total cellular metal in bioavailability studies is noted, given the different effects of humic substances on metal accumulation in the different cellular pools.