Physicochemical surface properties of Chlorella vulgaris: a multiscale assessment, from electrokinetic and proton uptake descriptors to intermolecular adhesion forces.

Given the growing scientific and industrial interests in green microalgae, a comprehensive understanding of the forces controlling the colloidal stability of these bioparticles and their interactions with surrounding aqueous microenvironment is required. Accordingly, we addressed here the electrostatic and hydrophobic surface properties of Chlorella vulgaris from the population down to the individual cell levels. We first investigated the organisation of the electrical double layer at microalgae surfaces on the basis of electrophoresis measurements. Interpretation of the results beyond zeta-potential framework underlined the need to account for both the hydrodynamic softness of the algae cells and the heterogeneity of their interface formed with the outer electrolyte solution. We further explored the nature of the structural charge carriers at microalgae interfaces through potentiometric proton titrations. Extraction of the electrostatic descriptors of interest from such data was obscured by cell physiology processes and dependence thereof on prevailing measurement conditions, which includes light, temperature and medium salinity. As an alternative, cell electrostatics was successfully evaluated at the cellular level upon mapping the molecular interactions at stake between (positively and negatively) charged atomic force microscopy tips and algal surface via chemical force microscopy. A thorough comparison between charge-dependent tip-to-algae surface adhesion and hydrophobicity level of microalgae surface evidenced that the contribution of electrostatics to the overall interaction pattern is largest, and that the electrostatic/hydrophobic balance can be largely modulated by pH. Overall, the combination of multiscale physicochemical approaches allowed a drawing of some of the key biosurface properties that govern microalgae cell-cell and cell-surface interactions.

Photodegradation of disposable polypropylene face masks: Physicochemical properties of debris and implications for the toxicity of mask-carried river biofilms.

COVID-19 outbreak led to a massive dissemination of protective polypropylene (PP) face masks in the environment, posing a new environmental risk amplified by mask photodegradation and fragmentation. Masks are made up of a several kilometres long-network of fibres with diameter from a few microns to around 20 µm. After photodegradation, these fibres disintegrate, producing water dispersible debris. Electrokinetics and particle stability observations support that photodegradation increases/decreases the charge/hydrophobicity of released colloidal fragments. This change in hydrophobicity is related to the production of UV-induced carbonyl and hydroxyl reactive groups detectable after a few days of exposure. Helical content, surface roughness and specific surface area of mask fibres are not significantly impacted by photodegradation. Fragmentation of fibres makes apparent, at the newly formed surfaces, otherwise-buried additives like TiO2 nanoparticles and various organic components. Mortality of gammarids is found to increase significantly over time when fed with 3 days-UV aged masks that carry biofilms grown in river, which is due to a decreased abundance of microphytes therein. In contrast, bacteria abundance and microbial community composition remain unchanged regardless of mask degradation. Overall, this work reports physicochemical properties of pristine and photodegraded masks, and ecosystemic functions and ecotoxicity of freshwater biofilms they can carry.

Kinetics of metal detection by luminescence-based whole-cell biosensors: connecting biosensor response to metal bioavailability, speciation and cell metabolism.

Luminescent whole-cell metal biosensors are genetically engineered cells used for the detection of metals in e.g. aqueous solutions. Herein, we detail the quantitative connections between time-response of luminescent bacterial metal sensors and the bioavailability of free and complexed metal species. To that end, we formulate the biophysicochemical dynamics of metal partitioning at a biosensor/solution interface and integrate the required metabolism contribution to cell response. The formalism explains the ways in which cell signal depends on: coupled Eigen kinetics of metal complexation and diffusion of metal species to/from the interface; kinetics of metal excretion, Michaelis-Menten bioaccumulation and ensuing metal depletion from bulk solution; and kinetics of bioluminescence production following intracellular metal sequestration by regulatory metalloproteins. In turn, an expression is derived for the time-dependent cell signal as a function of interrelated (bioavai)lability of metal species and (thermo)dynamic descriptors of extra/intracellular metal complexation. Quantitative criteria are elaborated to identify scenarios where equilibrium modeling of metal speciation is incorrect, bulk metal depletion is operative, metal biouptake kinetics is governed by metal diffusion, or labile metal complexes fully contribute to cell response. Remarkably, in agreement with experiments, the theory predicts time-shifts of bioluminescence peaks with increasing concentration of biosensor and/or metal ligand in solution. We show that these shifts originate from the crosstalk between activation kinetics of cell photoactivity and speciation-dependent kinetics of bulk metal depletion. Overall, the work paves the way for the elaboration of new strategies to exploit the bioluminescence response of metal lux-biosensors at a dynamic level and evaluate metal bioavailability properties in environmental or biological aqueous samples.

Comparative Analysis of Cell Metabolic Activity Sensing by Escherichia coli rrnB P1-lux and Cd Responsive-Lux Biosensors: Time-Resolved Experiments and Mechanistic Modelling

Whole-cell bacterial sensors are used in medical/environmental applications to detect chemicals, and to assess medium toxicity or stress. Non-specific constitutive biosensors generally serve the latter purpose, whereas chemical detection is performed with biosensors involving a specific chemical-inducible promoter. Herein, we show that functioning principles of specific and non-specific whole-cell biosensors are not exclusive as both can probe modulations of cell metabolic activity under stressing conditions. The demonstration is based on (i) time-resolved measurements of bioluminescence produced by constitutive rrnB P1-luxCDABE Escherichia coli biosensor in media differing with respect to carbon source, (ii) theoretical reconstruction of the measured signals using a here-reported theory for bioluminescence generated by constitutive cells, (iii) comparison between time-dependent cell photoactivity (reflecting metabolic activity) retrieved by theory with that we reported recently for cadmium-inducible PzntA-luxCDABE E. coli in media of similar compositions. Whereas signals of constitutive and non-constitutive biosensors differ in terms of shape, amplitude and peak number depending on nutritional medium conditions, analysis highlights the features shared by their respective cell photoactivity patterns mediated by the interplay between stringent response and catabolite repressions. The work advocates for the benefits of a theoretical interpretation for the time-dependent response of biosensors to unravel metabolic and physicochemical contributions to the bioluminescence signal.

Exploiting Catabolite Repression and Stringent Response to Control Delay and Multimodality of Bioluminescence Signal by Metal Whole-Cell Biosensors: Interplay between Metal Bioavailability and Nutritional Medium Conditions.

The time-dependent response of metal-detecting whole-cell luminescent bacterial sensors is impacted by metal speciation/bioavailability in solution. The comprehensive understanding of such connections requires the consideration of the bacterial energy metabolism at stake and the effects of supplied food on cells' capability to convert bioaccumulated metals into light. Accordingly, we investigated the time response (48 h assay) of PzntA-luxCDABE Escherichia coli Cd biosensors in media differing with respect to sources of amino acids (tryptone or Lysogeny Broth) and carbon (glucose, xylose and mixtures thereof). We show that the resulting coupling between the stringent cell response and glucose/xylose-mediated catabolite repressions lead to well-defined multimodalities and shapes of the bioluminescence signal over time. Based on a recent theory for the time-response of metal-sensing luminescent bacteria, successful theoretical reconstructions of the bioluminescence signals are reported under all Cd concentrations (0-20 nM) and nutritive conditions examined. This analysis leads to the evaluation of time-dependent cell photoactivity and qualitative information on metal speciation/bioavailability in solution. Biosensor performance and the position, shape, number, and magnitude of detected peaks are discussed in relation to the metabolic pathways operative during the successive light emission modes identified here over time. Altogether, the results clarify the contributions of metal/nutrient bio-availabilities and food quality to cell response typology.

Osmotic stress and vesiculation as key mechanisms controlling bacterial sensitivity and resistance to TiO2 nanoparticles.

Toxicity mechanisms of metal oxide nanoparticles towards bacteria and underlying roles of membrane composition are still debated. Herein, the response of lipopolysaccharide-truncated Escherichia coli K12 mutants to TiO2 nanoparticles (TiO2NPs, exposure in dark) is addressed at the molecular, single cell, and population levels by transcriptomics, fluorescence assays, cell nanomechanics and electrohydrodynamics. We show that outer core-free lipopolysaccharides featuring intact inner core increase cell sensitivity to TiO2NPs. TiO2NPs operate as membrane strippers, which induce osmotic stress, inactivate cell osmoregulation and initiate lipid peroxidation, which ultimately leads to genesis of membrane vesicles. In itself, truncation of lipopolysaccharide inner core triggers membrane permeabilization/depolarization, lipid peroxidation and hypervesiculation. In turn, it favors the regulation of TiO2NP-mediated changes in cell Turgor stress and leads to efficient vesicle-facilitated release of damaged membrane components. Remarkably, vesicles further act as electrostatic baits for TiO2NPs, thereby mitigating TiO2NPs toxicity. Altogether, we highlight antagonistic lipopolysaccharide-dependent bacterial responses to nanoparticles and we show that the destabilized membrane can generate unexpected resistance phenotype.

Fast automated processing of AFM PeakForce curves to evaluate spatially resolved Young modulus and stiffness of turgescent cells.

Atomic Force Microscopy (AFM) is a powerful technique for the measurement of mechanical properties of individual cells in two (x × y) or three (x × y × time) dimensions. The instrumental progress makes it currently possible to generate a large amount of data in a relatively short time, which is particularly true for AFM operating in so-called PeakForce tapping mode (Bruker corporation). The latter corresponds to an AFM probe that periodically hits the sample surface while the pico-newton level interaction force is recorded from cantilever deflection. The method provides unprecedented high-resolution (a few tens of nm) imaging of the mechanical features of soft biological samples (e.g. bacteria, yeasts) and of hard abiotic surfaces (e.g. minerals). The rapid conversion of up to several tens of thousands spatially resolved force curves typically collected in AFM PeakForce tapping mode over a given cell surface area into comprehensive nanomechanical information requires the development of robust data analysis methodologies and dedicated numerical tools. In this work, we report an automated algorithm for (i) a rapid and unambiguous detection of the indentation regimes corresponding to non-linear and linear deformations of bacterial surfaces upon compression by the AFM probe, (ii) the subsequent evaluation of the Young modulus and cell surface stiffness, and (iii) the generation of spatial mappings of relevant nanomechanical properties at the single cell level. The procedure involves consistent evaluation of the contact point between the AFM probe and sample biosurface and that of the threshold indentation value marking the transition between non-linear and linear deformation regimes. For comparison purposes, the former regime is here analyzed on the basis of Hertz and Sneddon models corrected or not for effects of finite sample thickness. Analysis of AFM measurements performed on a selected Escherichia coli strain is detailed to demonstrate the feasibility, rapidity and robustness of the here-proposed PeakForce data treatment process. The flexibility of the algorithm allows consideration of force curve parameterizations other than that detailed here, which may be desired for investigation of e.g. eukaryotes nanomechanics. The performance of the adopted Hertz-based and Sneddon-based contact mechanics formalisms in recovering experimental data and in identifying nanomechanical heterogeneities at the bacterium scale is further thoroughly discussed.

Pleiotropic effects of rfa-gene mutations on Escherichia coli envelope properties.

Mutations in the rfa operon leading to severely truncated lipopolysaccharide (LPS) structures are associated with pleiotropic effects on bacterial cells, which in turn generates a complex phenotype termed deep-rough. Literature reports distinct behavior of these mutants in terms of susceptibility to bacteriophages and to several antibacterial substances. There is so far a critical lack of understanding of such peculiar structure-reactivity relationships mainly due to a paucity of thorough biophysical and biochemical characterizations of the surfaces of these mutants. In the current study, the biophysicochemical features of the envelopes of Escherichia coli deep-rough mutants are identified from the molecular to the single cell and population levels using a suite of complementary techniques, namely microelectrophoresis, Atomic Force Microscopy (AFM) and Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) for quantitative proteomics. Electrokinetic, nanomechanical and proteomic analyses evidence enhanced mutant membrane destabilization/permeability, and differentiated abundances of outer membrane proteins involved in the susceptibility phenotypes of LPS-truncated mutants towards bacteriophages, antimicrobial peptides and hydrophobic antibiotics. In particular, inner-core LPS altered mutants exhibit the most pronounced heterogeneity in the spatial distribution of their Young modulus and stiffness, which is symptomatic of deep damages on cell envelope likely to mediate phage infection process and antibiotic action.

Toxicity mechanisms of ZnO UV-filters used in sunscreens toward the model cyanobacteria Synechococcus elongatus PCC 7942.

Zinc oxide (ZnO) nanoparticles are commonly used in sunscreens for their UV-filtering properties. Their growing use can lead to their release into ecosystems, raising question about their toxicity. Effects of these engineered nanomaterials (ENMs) on cyanobacteria, which are important primary producers involved in many biogeochemical cycles, are unknown. In this study, we investigated by several complementary approaches the toxicological effects of two marketed ZnO-ENMs (coated and uncoated) on the model cyanobacteria Synechococcus elongatus PCC 7942. It was shown that despite the rapid adsorption of ENMs on cell surface, toxicity is mainly due to labile Zn released by ENMs. Zn dissipates cell membrane potential necessary for both photosynthesis and respiration, and induces oxidative stress leading to lipid peroxidation and DNA damages. It leads to global downregulation of photosystems, oxidative phosphorylation, and transcription/translation machineries. This also translates into significant decrease of intracellular ATP content and cell growth inhibition. However, there is no major loss of pigments and even rather an increase in exposed cells compared to controls. A proposed way to reduce the environmental impact of Zn would be the improvement of the coating stability to prevent solubility of ZnO-ENMs.

Decoding the Time-Dependent Response of Bioluminescent Metal-Detecting Whole-Cell Bacterial Sensors.

The signal produced by aqueous dispersions of bioluminescent, metal-responsive whole-cell bacterial sensors is indicative of the concentration of bioavailable metal ions in solution. The conventional calibration-based strategy followed for measuring this concentration is however inadequate to provide any quantitative prediction of the cell response over time as a function of, e.g., their growth features, their defining metal accumulation properties, or the physicochemical medium composition. Such an evaluation is still critically needed for assessing on a mechanistic level the performance of biosensors in terms of metal bioavailability and toxicity monitoring. Herein we report a comprehensive formalism unraveling how the dependence of bioluminescence on time is governed by the dynamics of metal biouptake, by the activation kinetics of lux-based reporter gene, and by the ensuing rate of luciferase production, the kinetics of light emission, and quenching. It is shown that the bioluminescence signal corresponds to the convolution product between two time-dependent functions, one detailing the dynamic interplay of the above micro- and nanoscale processes, and the other pertaining to the change in concentration of photoactive cell sensors over time. Numerical computations illustrate how the shape and magnitude of the bioluminescence peak(s) are intimately connected to the dependence of the photoactive cell concentration on time and to the magnitudes of Deborah numbers that compare the relevant time scales of the biointerfacial and intracellular events controlling light emission. Explicit analytical expressions are further derived for practical situations where bioluminescence is proportional to the concentration of metal ions in solution. The theory is further quantitatively supported by experiments performed on luminescent cadmium-responsive lux-based Escherichia coli biosensors.

Impact of intracellular metallothionein on metal biouptake and partitioning dynamics at bacterial interfaces.

Genetically engineered microorganisms are alternatives to physicochemical methods for remediation of metal-contaminated aquifers due to their remarkable bioaccumulation capacities. The design of such biosystems would benefit from the elaboration of a sound quantitative connection between performance in terms of metal removal from aqueous solution and dynamics of the multiscale processes leading to metal biouptake. In this work, this elaboration is reported for Escherichia coli cells modified to overexpress intracellular metallothionein (MTc), a strong proteinaceous metal chelator. Depletion kinetics of Cd(ii) from bulk solution following biouptake and intracellular accumulation is addressed as a function of cell volume fraction using electroanalytical probes and ligand exchange-based analyses. It is shown that metal biouptake in the absence and presence of MTc is successfully interpreted on the basis of a formalism recently developed for metal partitioning dynamics at biointerfaces with integration of intracellular metal speciation. The analysis demonstrates how fast sequestration of metals by intracellular MTc bypasses metal excretion (efflux) and enhances the rate of metal depletion to an extent such that complete removal is achieved at sufficiently large cell volume fractions. The magnitude of the stability constant of nanoparticulate metal-MTc complexes, as derived from refined analysis of macroscopic bulk metal depletion data, is further confirmed by independent electrochemical measurement of metal binding by purified MTc extracts.

The influence of salinity on the fate and behavior of silver standardized nanomaterial and toxicity effects in the estuarine bivalve Scrobicularia plana.

Because of their antibacterial properties, silver (Ag) engineered nanomaterials are included in many products. The present study used a standardized Ag nanomaterial (NM-300K, 20 nm) supplied with a stabilizing agent. The aim was to investigate the behavior of Ag nanomaterial in an estuarine-like medium at 2 salinities (15 psu and 30 psu). Uptake as well as sublethal effects of Ag nanomaterial (10 µg Ag/L), its stabilizing agent, and AgNO3 (10 µg Ag/L) were assessed in the clam Scrobicularia plana, after 7 d of exposure. The release of soluble Ag from Ag nanomaterial in the experimental media was quantified by using diffusive gradient in thin films and ultrafiltration. A multibiomarker approach was employed to reveal responses of clams at subindividual and individual levels. The bioaccumulation of Ag was significantly greater at 15 psu versus 30 psu, which could be explained by differences in Ag speciation. In conclusion, the present study showed different impacts of Ag nanomaterial that were not always explained by the release of Ag ions in clams at both salinities; such impacts were particularly characterized by induction of oxidative stress, cell damage, and impairment of energetic levels. Burrowing of clams was affected by the stabilizing agent depending on the salinity tested, with stronger effects at 15 psu. Finally, the present study highlighted salinity-dependent changes in the physiology of estuarine bivalves. Environ Toxicol Chem 2016;35:2550-2561. © 2016 SETAC.

Integrated assessment of ceria nanoparticle impacts on the freshwater bivalve Dreissena polymorpha.

Exposures in realistic environmental conditions are essential to properly assess the effects of emerging pollutants on ecosystems. While ceria nanoparticles (nCeO2) production and use are expanding quickly, ecotoxicity studies remain very scarce. In this study, we set up experimental systems reproducing a simplified ecosystem to assess the effects of a chronic exposure to citrate-coated nCeO2 (ci-CeO2) and bare nCeO2 (ba-CeO2) on the freshwater mussel Dreissena polymorpha using an integrated multibiomarker approach. The fate of nanoparticles was tightly monitored to properly characterize the exposure. Organisms were exposed for 3 weeks and sampled weekly for biomarker analysis. Mussel filter-feeding activity resulted in significant removal of nCeO2 from the water column. At the same time, bioaccumulation was low, reaching its maximum in the first week. Mussels bioaccumulated ci-CeO2 three times more than ba-CeO2, probably due to coating-related differences in their behavior in the water column and in organisms. Meanwhile, biomarker results were integrated and synthesized using linear discriminant analysis, highlighting that pi-glutathione-S-transferase (piGST) mRNA, catalase (CAT) activity and lysosomal system were the most impacted of the seven biomarkers singled out by the discriminant analysis. These biomarker responses indicated that mussels exposed to both forms of nCeO2 were stressed and differentiate from the controls. Moreover, they responded differently to ba-CeO2 and ci-CeO2 exposure. However, biomarkers used in the experimental conditions of this study did not indicate severe nCeO2 toxicity on mussels, as cellular damage biomarkers and mussel filtering activity were left unimpaired. However, further studies are needed to investigate if the slight perturbations observed could lead to populational impacts in the long term.

Toxicity of CeO2 nanoparticles on a freshwater experimental trophic chain: A study in environmentally relevant conditions through the use of mesocosms.

The toxicity of CeO2 NPs on an experimental freshwater ecosystem was studied in mesocosm, with a focus being placed on the higher trophic level, i.e. the carnivorous amphibian species Pleurodeles waltl. The system comprised species at three trophic levels: (i) bacteria, fungi and diatoms, (ii) Chironomus riparius larvae as primary consumers and (iii) Pleurodeles larvae as secondary consumers. NP contamination consisted of repeated additions of CeO2 NPs over 4 weeks, to obtain a final concentration of 1 mg/L. NPs were found to settle and accumulate in the sediment. No effects were observed on litter decomposition or associated fungal biomass. Changes in bacterial communities were observed from the third week of NP contamination. Morphological changes in CeO2 NPs were observed at the end of the experiment. No toxicity was recorded in chironomids, despite substantial NP accumulation (265.8 ± 14.1 mg Ce/kg). Mortality (35.3 ± 6.8%) and a mean Ce concentration of 13.5 ± 3.9 mg/kg were reported for Pleurodeles. Parallel experiments were performed on Pleurodeles to determine toxicity pathways: no toxicity was observed by direct or dietary exposures, although Ce concentrations almost reached 100 mg/kg. In view of these results, various toxicity mechanisms are proposed and discussed. The toxicity observed on Pleurodeles in mesocosm may be indirect, due to microorganism's interaction with CeO2 NPs, or NP dissolution could have occurred in mesocosm due to the structural complexity of the biological environment, resulting in toxicity to Pleurodeles. This study strongly supports the importance of ecotoxicological assessment of NPs under environmentally relevant conditions, using complex biological systems.

Impact of manufactured TiO2 nanoparticles on planktonic and sessile bacterial communities.

In the present study, we conducted a 2 week microcosm experiment with a natural freshwater bacterial community to assess the effects of titanium dioxide nanoparticles (TiO2-NPs) at various concentrations (0, 1, 10 and 100 mg/L) on planktonic and sessile bacteria under dark conditions. Results showed an increase of planktonic bacterial abundance at the highest TiO2-NP concentration, concomitant with a decrease from that of sessile bacteria. Bacterial assemblages were most affected by the 100 mg/L TiO2-NP exposure and overall diversity was found to be lower for planktonic bacteria and higher for sessile bacteria at this concentration. In both compartments, a 100 mg/L TiO2-NPs exposure induced a decrease in the ratio between the Betaproteobacteria and Bacteroidetes. For planktonic communities, a decrease of Comamonadaceae was observed concomitant with an increase of Oxalobacteraceae and Cytophagaceae (especially Emticicia). For sessile communities, results showed a strong decrease of Betaproteobacteria and particularly of Comamonadaceae.

Insight into the primary mode of action of TiO2 nanoparticles on Escherichia coli in the dark.

Large-scale production and incorporation of titanium dioxide nanoparticles (NP-TiO2 ) in consumer products leads to their potential release into the environment and raises the question of their toxicity. The bactericidal mechanism of NP-TiO2 under UV light is known to involve oxidative stress due to the generation of reactive oxygen species. In the dark, several studies revealed that NP-TiO2 can exert toxicological effects. However, the mode of action of these nanoparticles is still controversial. In the present study, we used a combination of fluorescent probes to show that NP-TiO2 causes Escherichia coli membrane depolarization and loss of integrity, leading to higher cell permeability. Using both transcriptomic and proteomic global approaches we showed that this phenomenon translates into a cellular response to osmotic stress, metabolism of cell envelope components and uptake/metabolism of endogenous and exogenous compounds. This primary mechanism of bacterial NP-TiO2 toxicity is supported by the observed massive cell leakage of K(+) /Mg(2+) concomitant with the entrance of extracellular Na(+), and by the depletion of intracellular ATP level.

Leaf-associated fungal diversity in acidified streams: insights from combining traditional and molecular approaches.

We combined microscopic and molecular methods to investigate fungal assemblages on alder leaf litter exposed in the benthic and hyporheic zones of five streams across a gradient of increasing acidification for 4 weeks. The results showed that acidification and elevated Al concentrations strongly depressed sporulating aquatic hyphomycetes diversity in both zones of streams, while fungal diversity assessed by denaturing gradient gel electrophoresis (DGGE) appeared unaffected. Clone library analyses revealed that fungal communities on leaves were dominated by members of Ascomycetes and to a lesser extent by Basidiomycetes and Chytridiomycetes. An important contribution of terrestrial fungi was observed in both zones of the most acidified stream and in the hyporheic zone of the reference circumneutral stream. The highest leaf breakdown rate was observed in the circumneutral stream and occurred in the presence of both the highest diversity of sporulating aquatic hyphomycetes and the highest contribution to clone libraries of sequences affiliated with aquatic hyphomycetes. Both methods underline the major role played by aquatic hyphomycetes in leaf decomposition process. Our findings also bring out new highlights on the identity of leaf-associated fungal communities and their responses to anthropogenic alteration of running water ecosystems.

Impaired leaf litter processing in acidified streams : learning from microbial enzyme activities.

Anthropogenic acidification in headwater streams is known to affect microbial assemblages involved in leaf litter breakdown. Far less is known about its potential effects on microbial enzyme activities. To assess the effects of acidification on microbial activities associated with decaying leaves, a 70-day litter bag experiment was conducted in headwater streams at six sites across an acidification gradient. The results revealed that microbial leaf decomposition was strongly and negatively correlated with total Al concentrations (r = -0.99, p < 0.001) and positively correlated with Ca(2+) concentrations (r = 0.94, p = 0.005) and pH (r = 0.93, p = 0.008). Denaturing gradient gel electrophoresis analyses showed that microbial assemblages differed between non-impacted and impacted sites, whereas fungal biomass associated with decaying leaves was unaffected. The nutrient content of leaf detritus and ecoenzymatic activities of carbon (C), nitrogen (N) and phosphorus (P) acquisition revealed that N acquisition was unaltered, while P acquisition was significantly reduced across the acidification gradient. The P content of leaf litter was negatively correlated with total Al concentrations (r = -0.94, p < 0.01) and positively correlated with decomposition rates (r = 0.95, p < 0.01). This potential P limitation of microbial decomposers in impacted sites was confirmed by the particularly high turnover activity for phosphatase and imbalanced ratios between the ecoenzymatic activities of C and P acquisition. The toxic form of Al has well-known direct effects on aquatic biota under acidic conditions, but in this study, Al was found to also potentially affect microbially mediated leaf processing by interfering with the P cycle. These effects may in turn have repercussions on higher trophic levels and whole ecosystem functioning.

Modifications of the bacterial reverse mutation test reveals mutagenicity of TiO(2) nanoparticles and byproducts from a sunscreen TiO(2)-based nanocomposite.

The bacterial reverse mutation test, recommended by the Organization for Economic Co-operation and Development (OECD) to determine genotoxicity of chemical compounds, has been recently used by several authors to investigate nanoparticles. Surprisingly, test results have been negative, whereas in vitro mammalian cell tests often give positive genotoxic responses. In the present study, we used the fluctuation test procedure with the Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 to determine the mutagenic potential of TiO(2) nanoparticles (NP-TiO(2)) and showed that, when it is used conventionally, this test is not suitable for nanoparticle genotoxicity assessment. Indeed, the medium used during exposure prevents electrostatic interactions between bacterial cells and nanoparticles, leading to false-negative responses. We showed that a simple pre-exposure of bacteria to NP-TiO(2) in a low ionic strength solution (NaCl 10mM) at a pH below the nanoparticle isoelectric points (pH 5.5) can strongly improve the accuracy of the test. Thus, based on these improvements, we have demonstrated the genotoxicity of the engineered NP-TiO(2) tested and a NP-TiO(2) byproduct from a sunscreen nanocomposite. It was also shown that strain TA102 is more sensitive than the other strains, suggesting an oxidative stress-mediated mechanism of genotoxicity.

Hemocyte responses of Dreissena polymorpha following a short-term in vivo exposure to titanium dioxide nanoparticles: preliminary investigations.

The widespread use of titanium-based nanoparticles and their environmental release may pose a significant risk to aquatic organisms within freshwater ecosystems. Suspension-feeder invertebrates like bivalve molluscs represent a unique target group for nanoparticle toxicology. The aim of this work was to investigate the short-term responses of Dreissena polymorpha hemocytes after in vivo exposure to titanium dioxide nanoparticles (TiO(2) NP). For this purpose, freshwater mussels were exposed to P25 TiO(2) NP at the concentrations of 0.1, 1, 5 and 25mg/L during 24h. Viability, phagocytosis activity and mitogen activated protein kinase (MAPK) phosphorylation level of ERK 1/2 and p38 in hemocytes extracted from exposed mussels were compared to those from control specimens. Results demonstrated an inhibition of the phagocytosis activity after exposure to TiO(2) NP at 0.1 and 1mg/L. Similar trends, albeit less pronounced, were reported for higher concentrations of NP. Transmission electron microscopy showed for the first time the internalization of TiO(2) NP into Dreissena polymorpha hemocytes. Besides, exposure to NP increased the ERK 1/2 phosphorylation levels in all treatments. Concerning the phosphorylation level of p38, only exposures to 5 and 25mg/L of NP induced significant p38 activation in comparison to that of the control. Finally, these short-term effects observed at environmentally relevant concentrations highlighted the need for further studies concerning ecotoxicological evaluation of nanoparticle release into an aquatic environment.