The challenge of studying TiO2 nanoparticle bioaccumulation at environmental concentrations: crucial use of a stable isotope tracer.

The ecotoxicity of nanoparticles (NPs) is a growing area of research with many challenges ahead. To be relevant, laboratory experiments must be performed with well-controlled and environmentally realistic (i.e., low) exposure doses. Moreover, when focusing on the intensively manufactured titanium dioxide (TiO2) NPs, sample preparations and chemical analysis are critical steps to meaningfully assay NP's bioaccumulation. To deal with these imperatives, we synthesized for the first time TiO2 NPs labeled with the stable isotope (47)Ti. Thanks to the (47)Ti labeling, we could detect the bioaccumulation of NPs in zebra mussels (Dreissena polymorpha) exposed for 1 h at environmental concentrations via water (7-120 µg/L of (47)TiO2 NPs) and via their food (4-830 µg/L of (47)TiO2 NPs mixed with 1 × 10(6) cells/mL of cyanobacteria) despite the high natural Ti background, which varied in individual mussels. The assimilation efficiency (AE) of TiO2 NPs by mussels from their diet was very low (AE = 3.0 ± 2.7%) suggesting that NPs are mainly captured in mussel gut, with little penetration in their internal organs. Thus, our methodology is particularly relevant in predicting NP's bioaccumulation and investigating the factors influencing their toxicokinetics in conditions mimicking real environments.

Seasonal and PAH impact on DNA strand-break levels in gills of transplanted zebra mussels.

Genotoxicity endpoints are useful tools to biomonitor the physicochemical and biological quality of aquatic ecosystems. A caging study on the freshwater bivalve Dreissena polymorpha was planned to run over four seasons in the Seine River basin in order to assess whether DNA damage measured in transplanted mussels to polluted area vary according to seasonal changes. Three sites were chosen along the Seine River, one upstream from Paris and two downstream, corresponding to a chemical gradient of water contamination. The DNA strand break (comet assay) and chromosomal damage (micronucleus test) were measured in caged mussels at each site and in winter, spring and summer, along with PAH water contamination, PAH bioaccumulation, the mussel condition index (CI), the gonado-somatic index (GSI) and the filtration rate (FR). The level of DNA strand break measured in winter was low and increased in spring, concomitantly with FR and GSI. Over the same period, micronucleus (MN) frequency and PAH bioaccumulation decreased significantly in caged mussels, with both parameters positively correlated to each other. DNA strand-break levels and MN frequencies showed inter-site variations corresponding to the chemical contamination gradient. These two genotoxicity endpoints usefully complement each other in field studies. These results show that the MN test and comet assay, when applied to gill cells of caged zebra mussels, are sensitive tools for freshwater genotoxicity monitoring.

Spatio-temporal variability of solid, total dissolved and labile metal: passive vs. discrete sampling evaluation in river metal monitoring.

In order to obtain representative dissolved and solid samples from the aquatic environment, a spectrum of sampling methods are available, each one with different advantages and drawbacks. This article evaluates the use of discrete sampling and time-integrated sampling in illustrating medium-term spatial and temporal variation. Discrete concentration index (CI) calculated as the ratio between dissolved and solid metal concentrations in grab samples are compared with time-integrated concentration index (CI) calculated from suspended particulate matter (SPM) collected in sediment traps and labile metals measured by the diffusive gel in thin films (DGT) method, collected once a month during one year at the Seine River, upstream and downstream of the Greater Paris Region. Discrete CI at Bougival was found to be significantly higher than at Triel for Co, Cu, Mn, Ni and Zn, while discrete metal partitioning at Marnay was found to be similar to Bougival and Triel. However, when using time-integrated CI, not only was Bougival CI significantly higher than Triel CI, CI at Marnay was also found to be significantly higher than CI at Triel which was not observed for discrete CI values. Since values are time-averaged, dramatic fluctuations were smoothed out and significant medium-term trends were enhanced. As a result, time-integrated concentration index (CI) was able to better illustrate urbanization impact between sites when compared to discrete CI. The impact of significant seasonal phenomenon such as winter flood, low flow and redox cycles was also, to a certain extent, visible in time-integrated CI values at the upstream site. The use of time-integrated concentration index may be useful for medium- to long-term metal studies in the aquatic environment.

Interaction of TiO2 nanoparticles with proteins from aquatic organisms: the case of gill mucus from blue mussel.

To better understand the mechanisms of TiO2 nanoparticle (NP) uptake and toxicity in aquatic organisms, we investigated the interaction of NPs with the proteins found in gill mucus from blue mussels. Mucus is secreted by many aquatic organisms and is often their first line of defense against pathogens, xenobiotics, and other sources of environmental stress. Here, five TiO2 NPs and one SiO2 NP were incubated with gill mucus and run out on a one-dimensional polyacrylamide gel for a comparative qualitative analysis of the free proteins in the mucosal solution and the proteins bound to NPs. We then used nanoscale liquid chromatography coupled with tandem mass spectrometry to identify proteins of interest. Our data demonstrated dissimilar protein profiles between the crude mucosal solution and proteins adsorbed on NPs. In particular, extrapallial protein (EP), one of the most abundant mucus proteins, was absent from the adsorbed proteins. After thermal denaturation experiments, this absence was attributed to the EP content in aromatic amino acids that prevents protein unfolding and thus adsorption on the NP. Moreover, although the majority of the protein corona was qualitatively similar across the NPs tested here (SiO2 and TiO2), a few proteins in the corona showed a specific recruitment pattern according to the NP oxide (TiO2 vs SiO2) or crystal structure (anatase TiO2 vs rutile TiO2). Therefore, protein adsorption may vary with the type of NP. Graphical abstract Proteins with adsorption selectivity as identified from isolated bands.

DGT as surrogate of biomonitors for predicting the bioavailability of copper in freshwaters: an ex situ validation study.

The present report is the companion study of our previous study in which we investigated the impact of the dissolved organic matter, water cationic composition and pH on the bioavailability and the bioaccumulation of copper (Cu) in aquatic mosses (Fontinalis antipyretica). The impact had been assessed under laboratory controlled conditions and modelled using a two-compartment model calibrated under a wide range of water compositions (Ferreira et al., 2008, 2009). Herein are reported the validation stage of the abovementioned approach for contrasted geochemical field conditions. Experiments were performed with aquatic mosses that were exposed for 7d to two nominal Cu concentrations (5 and 15µgL(-1)) in a flow-through field microcosm supplied with four contrasting natural waters. At the end of the exposure period, a 6-fold difference in the bioaccumulated Cu contamination levels was found among the four deployment sites, suggesting a significant control of the water quality on the metal bioaccumulation by aquatic mosses. In parallel, the so-called 'labile' Cu concentration for the same four field conditions was determined using a DGT device (Diffusive Gradient in Thin film). By coupling these DGT measurements and a cation competition model involving Ca(2+), Mg(2+), Na(+) and H(+), the time-dependent Cu concentrations in aquatic mosses were predicted; these simulation results were compared to the actual bioaccumulation of Cu in mosses. We found that any bioaccumulation model that ignores water characteristics is not suitable to predict the Cu accumulation by aquatic mosses under various water quality conditions. Instead, we found that our approach integrating DGT measurements and cationic composition was able to reproduce the Cu bioaccumulation kinetics by aquatic mosses for a wide range of water quality conditions. In conclusion, the DGT approach was demonstrated to be a dynamic in situ measuring technique that can be used as a surrogate of bioindicators if the cationic correction is taken into account.

Speciation and bioavailability of dissolved copper in different freshwaters: comparison of modelling, biological and chemical responses in aquatic mosses and gammarids.

Biological and chemical measurements were performed in mesocosms to investigate the bioavailability of copper, with a greater emphasis on the effects of competing ions and copper speciation. Measurements were achieved in three different natural waters for two aquatic species (Gammarus pulex and Fontinalis antipyretica) along a copper gradient concentration: natural concentration, spiked at 5 and 15 µg L(-1). Aquatic mosses exhibited high enrichment rates that were above the background levels compared to gammarids. The accumulation of copper in F. antipyretica is better correlated to the weakly complexed copper concentrations measured using differential pulse anodic stripping voltammetry (DPASV) and diffusive gradient in thin film (DGT) than to the free copper concentration measured using an ion selective electrode (ISE). In unspiked natural waters, the presence of dissolved organic ligands strongly controls the metal speciation and consequently largely minimised the impact of competing cations on the accumulation of Cu in mosses. Furthermore, the BioMet Biotic Ligand Model (BLM) successfully describes the site-specific copper bioaccumulation for the freshwater mosses studied. However, the comparison of the results with a previous study appears to indicate that the adsorption/desorption of Cu in mosses is impacted by seasons. This highlights a limit of the BioMet model in which the physiological state of aquatic organisms is not considered. No toxic effect of Cu exposure on lipid peroxidation was observed in the mosses and gammarids regardless of the site and the concentration considered. However, the oxidative stress measured in the mosses via their guaiacol peroxidase (GPX) activity increased in the case where internalised Cu reached maximal values, which suggests a threshold effect on the GPX activity.

Diffuse urban pollution increases metal tolerance of natural heterotrophic biofilms.

This study is a first attempt to investigate the impact of urban contamination on metal tolerance of heterotrophic river biofilms using a short-term test based on ß-glucosidase activity. Tolerance levels to Cu, Cd, Zn, Ni and Pb were evaluated for biofilms collected at three sites along an urban gradient in the Seine river (France). Metallic pollution increased along the river, but concentrations remained low compared to environmental quality standards. Biofilm metal tolerance increased downstream from the urban area. Multivariate analysis confirmed the correlation between tolerance and contamination and between multi-metallic and physico-chemical gradients. Therefore, tolerance levels have to be interpreted in relation to the whole chemical and physical characteristics and not solely metal exposure. We conclude that community tolerance is a sensitive biological response to urban pressure and that mixtures of contaminants at levels lower than quality standards might have a significant impact on periphytic communities.

Bioavailability of particulate metal to zebra mussels: biodynamic modelling shows that assimilation efficiencies are site-specific.

This study investigates the ability of the biodynamic model to predict the trophic bioaccumulation of cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni) and zinc (Zn) in a freshwater bivalve. Zebra mussels were transplanted to three sites along the Seine River (France) and collected monthly for 11 months. Measurements of the metal body burdens in mussels were compared with the predictions from the biodynamic model. The exchangeable fraction of metal particles did not account for the bioavailability of particulate metals, since it did not capture the differences between sites. The assimilation efficiency (AE) parameter is necessary to take into account biotic factors influencing particulate metal bioavailability. The biodynamic model, applied with AEs from the literature, overestimated the measured concentrations in zebra mussels, the extent of overestimation being site-specific. Therefore, an original methodology was proposed for in situ AE measurements for each site and metal.

Modeling the effect of water chemistry on the bioaccumulation of waterborne cadmium in zebra mussels.

The present study aims at investigating the effects of Zn, Ca, and dissolved organic carbon (DOC) on the waterborne Cd bioaccumulation of a freshwater bivalve (Dreissena polymorpha). Mussels were exposed for 48 h at 3 µg/L of Cd in different media. Their physiological activities were assessed by separately measuring the filtration rate in the same exposure water. Increased Zn (from 3 to 89 µg/L) and Ca (from 37 to 131 mg/L) concentrations in water led to a threefold and sevenfold reduction of Cd bioaccumulation, whereas the effect of DOC varied greatly depending on its concentration. At low DOC concentrations (from 0.2 to 1.1 mg/L), the uptake of Cd increased, whereas at higher concentrations (from 1.1 to 17.1 mg/L), the uptake decreased. The filtration activity was not strongly influenced by either Zn or Ca concentration, whereas it was modified in enriched DOC media in the same manner as Cd uptake. A competitive model was built to predict the waterborne uptake rate constant of Cd (k (u)) as a function of Zn and Ca concentrations in the water. Over the range of DOC concentrations we tested, organic matter was shown to influence Cd bioaccumulation in two ways: by modifying Cd speciation and thus its bioavailability and its interaction with the biological membrane, and by affecting the mussel's physiology and therefore its sensitivity to metal. The present study provides a useful means of adjusting the toxicokinetic constant to the water's physicochemical characteristics and proposes a unifying model that takes into account the different geochemical and biological influences on bioaccumulation.