Drastic Change in Zinc Speciation during Anaerobic Digestion and Composting: Instability of Nanosized Zinc Sulfide.

Zinc (Zn) is a potentially toxic trace element that is present in large amounts in organic wastes (OWs) spread on agricultural lands as fertilizer. Zn speciation in OW is a crucial parameter to understand its fate in soil after spreading and to assess the risk associated with agricultural recycling of OW. Here, we investigated changes in Zn speciation from raw OWs up to digestates and/or composts for a large series of organic wastes sampled in full-scale plants. Using extended X-ray absorption fine structure, we show that nanosized Zn sulfide (nano-ZnS) is a major Zn species in raw liquid OWs and a minor species in raw solid OWs. Whatever the characteristics of the raw OW, anaerobic digestion always favors the formation of nano-ZnS (>70% of zinc in digestates). However, after 1 to 3 months of composting of OWs, nano-ZnS becomes a minor species (<10% of zinc). In composts, Zn is mostly present as amorphous Zn phosphate and Zn sorbed to ferrihydrite. These results highlight (i) the influence of OW treatment on Zn speciation and (ii) the chemical instability of nano-ZnS formed in OW in anaerobic conditions.

Respiratory hazard of Li-ion battery components: elective toxicity of lithium cobalt oxide (LiCoO2) particles in a mouse bioassay.

Rechargeable Li-ion batteries (LIB) are increasingly produced and used worldwide. LIB electrodes are made of micrometric and low solubility particles, consisting of toxicologically relevant elements. The health hazard of these materials is not known. Here, we investigated the respiratory hazard of three leading LIB components (LiFePO4 or LFP, Li4Ti5O12 or LTO, and LiCoO2 or LCO) and their mechanisms of action. Particles were characterized physico-chemically and elemental bioaccessibility was documented. Lung inflammation and fibrotic responses, as well as particle persistence and ion bioavailability, were assessed in mice after aspiration of LIB particles (0.5 or 2 mg); crystalline silica (2 mg) was used as reference. Acute inflammatory lung responses were recorded with the 3 LIB particles and silica, LCO being the most potent. Inflammation persisted 2 m after LFP, LCO and silica, in association with fibrosis in LCO and silica lungs. LIB particles persisted in the lungs after 2 m. Endogenous iron co-localized with cobalt in LCO lungs, indicating the formation of ferruginous bodies. Fe and Co ions were detected in the broncho-alveolar lavage fluids of LFP and LCO lungs, respectively. Hypoxia-inducible factor (HIF) -1α, a marker of fibrosis and of the biological activity of Co ions, was upregulated in LCO and silica lungs. This study identified, for the first time, the respiratory hazard of LIB particles. LCO was at least as potent as crystalline silica to induce lung inflammation and fibrosis. Iron and cobalt, but not lithium, ions appear to contribute to LFP and LCO toxicity, respectively.

Effect of phytoliths for mitigating water stress in durum wheat.

The role of silicon (Si) in alleviating biotic and abiotic stresses in crops is well evidenced by empirical studies; however, the mechanisms by which it works are still poorly known. The aim of this study is to determine whether or not phytolith composition and distribution in wheat are affected by drought and, if so, why. Durum wheat was grown using hydroponics in the presence of polyethylene glycol (PEG)-6000 to perform a water-stress simulation. We developed an original method for in situ analysis of phytoliths in leaves via X-ray imaging. PEG was efficient in inhibiting water uptake by roots and creating stress, and prevented a small fraction of Si from being accumulated in the shoots. The application of Si with PEG maintained shoot and root fresh weights (FW) and relative water content at higher values than for plants without Si, especially at PEG 12%. Our data show that, under water stress in the presence of Si, accumulation of phytoliths over the veins provides better support to the leaf, thus allowing for a better development of the whole plant than in the absence of Si. The development of silicified trichomes in durum wheat depends primarily on the availability of Si in soil and is not an adaptation to water stress.

Silver Nanoparticles and Wheat Roots: A Complex Interplay.

Agricultural soils are major sinks of silver nanoparticles in the environment, and crops are directly exposed to these emerging contaminants. A clear picture of their chemical transformations, uptake and transport mechanisms, and phytotoxic impacts is still lacking. In this work, wheat plants were exposed to pristine metallic (Ag-NPs) and sulfidized (Ag2S-NPs) silver nanoparticles and ionic Ag. Data on Ag distribution and speciation, phytotoxicity markers, and gene expression were studied. A multi-technique and multi-scale approach was applied, combining innovating tools at both the laboratory and synchrotron. Various chemical transformations were observed on the epidermis and inside roots, even for Ag2S-NPs, leading to an exposure to multiple Ag forms, which likely evolve over time. Genes involved in various functions including oxidative stress, defense against pathogens, and metal homeostasis were impacted in different ways depending upon the Ag source. This study illustrates the complexity of the toxicity pattern for plants exposed to Ag-NPs, the necessity of monitoring several markers to accurately evaluate the toxicity, and the interest of interpreting the toxicity pattern in light of the distribution and speciation of Ag.

Nanoparticle Uptake in Plants: Gold Nanomaterial Localized in Roots of Arabidopsis thaliana by X-ray Computed Nanotomography and Hyperspectral Imaging.

Terrestrial plants can internalize and translocate nanoparticles (NPs). However, direct evidence for the processes driving the NP uptake and distribution in plants is scarce at the cellular level. Here, NP-root interactions were investigated after 10 days of exposure of Arabidopsis thaliana to 10 mg·L-1 of negatively or positively charged gold NPs (∼12 nm) in gels. Two complementary imaging tools were used: X-ray computed nanotomography (nano-CT) and enhanced dark-field microscopy combined with hyperspectral imaging (DF-HSI). The use of these emerging techniques improved our ability to detect and visualize NP in plant tissue: by spectral confirmation via DF-HSI, and in three dimensions via nano-CT. The resulting imaging provides direct evidence that detaching border-like cells (i.e., sheets of border cells detaching from the root) and associated mucilage can accumulate and trap NPs irrespective of particle charge. On the contrary, border cells on the root cap behaved in a charge-specific fashion: positively charged NPs induced a higher mucilage production and adsorbed to it, which prevented translocation into the root tissue. Negatively charged NPs did not adsorb to the mucilage and were able to translocate into the apoplast. These observations provide direct mechanistic insight into NP-plant interactions, and reveal the important function of border cells and mucilage in interactions of plants with charged NPs.

Nanoscale Coloristic Pigments: Upper Limits on Releases from Pigmented Plastic during Environmental Aging, In Food Contact, and by Leaching.

The life cycle of nanoscale pigments in plastics may cause environmental or human exposure by various release scenarios. We investigated spontaneous and induced release with mechanical stress during/after simulated sunlight and rain degradation of polyethylene (PE) with organic and inorganic pigments. Additionally, primary leaching in food contact and secondary leaching from nanocomposite fragments with an increased surface into environmental media was examined. Standardized protocols/methods for release sampling, detection, and characterization of release rate and form were applied: Transformation of the bulk material was analyzed by Scanning Electron Microscopy (SEM), X-ray-tomography and Fourier-Transform Infrared spectroscopy (FTIR); releases were quantified by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), single-particle-ICP-MS (sp-ICP-MS), Transmission Electron Microscopy (TEM), Analytical Ultracentrifugation (AUC), and UV/Vis spectroscopy. In all scenarios, the detectable particulate releases were attributed primarily to contaminations from handling and machining of the plastics, and were not identified with the pigments, although the contamination of 4 mg/kg (Fe) was dwarfed by the intentional content of 5800 mg/kg (Fe as Fe2O3 pigment). We observed modulations (which were at least partially preventable by UV stabilizers) when comparing as-produced and aged nanocomposites, but no significant increase of releases. Release of pigments was negligible within the experimental error for all investigated scenarios, with upper limits of 10 mg/m2 or 1600 particles/mL. This is the first holistic confirmation that pigment nanomaterials remain strongly contained in a plastic that has low diffusion and high persistence such as the polyolefin High Density Polyethylene (HDPE).

Effect of pH and Pressure on Uranium Removal from Drinking Water Using NF/RO Membranes.

Groundwater is becoming an increasingly important drinking water source. However, the use of groundwater for potable purposes can lead to chronic human exposure to geogenic contaminants, for example, uranium. Nanofiltration (NF) and reverse osmosis (RO) processes are used for drinking water purification, and it is important to understand how contaminants interact with membranes since accumulation of contaminants to the membrane surface can lead to fouling, performance decline and possible breakthrough of contaminants. During the current study laboratory experiments were conducted using NF (TFC-SR2) and RO (BW30) membranes to establish the behavior of uranium across pH (3-10) and pressure (5-15 bar) ranges. The results showed that important determinants of uranium-membrane sorption interactions were (i) the uranium speciation (uranium species valence and size in relation to membrane surface charge and pore size) and (ii) concentration polarization, depending on the pH values. The results show that it is important to monitor sorption of uranium to membranes, which is controlled by pH and concentration polarization, and, if necessary, adjust those parameters controlling uranium sorption.

Nanometer-long Ge-imogolite nanotubes cause sustained lung inflammation and fibrosis in rats.

BACKGROUND: Ge-imogolites are short aluminogermanate tubular nanomaterials with attractive prospected industrial applications. In view of their nano-scale dimensions and high aspect ratio, they should be examined for their potential to cause respiratory toxicity. Here, we evaluated the respiratory biopersistence and lung toxicity of 2 samples of nanometer-long Ge-imogolites. METHODS: Rats were intra-tracheally instilled with single wall (SW, 70 nm length) or double wall (DW, 62 nm length) Ge-imogolites (0.02-2 mg/rat), as well as with crocidolite and the hard metal particles WC-Co, as positive controls. The biopersistence of Ge-imogolites and their localization in the lung were assessed by ICP-MS, X-ray fluorescence, absorption spectroscopy and computed micro-tomography. Acute inflammation and genotoxicity (micronuclei in isolated type II pneumocytes) was assessed 3 d post-exposure; chronic inflammation and fibrosis after 2 m. RESULTS: Cytotoxic and inflammatory responses were shown in bronchoalveolar lavage 3 d after instillation with Ge-imogolites. Sixty days after exposure, a persistent dose-dependent inflammation was still observed. Total lung collagen, reflected by hydroxyproline lung content, was increased after SW and DW Ge-imogolites. Histology revealed lung fibre reorganization and accumulation in granulomas with epithelioid cells and foamy macrophages and thickening of the alveolar walls. Overall, the inflammatory and fibrotic responses induced by SW and DW Ge-imogolites were more severe (on a mass dose basis) than those induced by crocidolite. A persistent fraction of Ge-imogolites (15% of initial dose) was mostly detected as intact structures in rat lungs 2 m after instillation and was localized in fibrotic alveolar areas. In vivo induction of micronuclei was significantly increased 3 d after SW and DW Ge-imogolite instillation at non-inflammatory doses, indicating the contribution of primary genotoxicity. CONCLUSIONS: We showed that nm-long Ge-imogolites persist in the lung and promote genotoxicity, sustained inflammation and fibrosis, indicating that short high aspect ratio nanomaterials should not be considered as innocuous materials. Our data also suggest that Ge-imogolite structure and external surface determine their toxic activity.

Hyper-accumulation of vanadium in animals: Two sponges compete with urochordates.

Vanadium (V) concentrations in organisms are usually very low. To date, among animals, only some urochordate and annelid species contain very high levels of V in their tissues. A new case of hyper-accumulation of V in a distinct animal phylum (Porifera), namely, the two homoscleromorph sponge species Oscarella lobularis and O. tuberculata is reported. The measured concentrations (up to 30 g/kg dry weight) exceed those reported previously and are not found in all sponge classes. In both Oscarella species, V is mainly accumulated in the surface tissues, and in mesohylar cells, as V(IV), before being partly reduced to V(III) in the deeper tissues. Candidate genes from Bacteria and sponges have been identified as possibly being involved in the metabolism of V. This finding provides clues for the development of bioremediation strategies in marine ecosystems and/or bioinspired processes to recycle this critical metal.

Physico-chemical control over the single- or double-wall structure of aluminogermanate imogolite-like nanotubes.

It is known that silicon can be successfully replaced by germanium atoms in the synthesis of imogolite nanotubes, leading to shorter and larger AlGe nanotubes. Beside the change in morphology, two characteristics of the AlGe nanotube synthesis were recently discovered. AlGe imogolite nanotubes can be synthesized at much higher concentrations than AlSi imogolite. AlGe imogolite exists in the form of both single-walled (SW) and double-walled (DW) nanotubes, whereas DW AlSi imogolites have never been observed. In this article, we give details on the physicochemical control over the SW or DW AlGe imogolite structure. For some conditions, an almost 100% yield of SW or DW nanotubes is demonstrated. We propose a model for the formation of SW or DW AlGe imogolite, which also explains why DW AlSi imogolites or higher wall numbers for AlGe imogolite are not likely to be formed.

Influence of the length of imogolite-like nanotubes on their cytotoxicity and genotoxicity toward human dermal cells.

Physical-chemical parameters such as purity, structure, chemistry, length, and aspect ratio of nanoparticles (NPs) are linked to their toxicity. Here, synthetic imogolite-like nanotubes with a set chemical composition but various sizes and shapes were used as models to investigate the influence of these physical parameters on the cyto- and genotoxicity and cellular uptake of NPs. The NPs were characterized using X-ray diffraction (XRD), small angle X-ray scattering (SAXS), and atomic force microscopy (AFM). Imogolite precursors (PR, ca. 5 nm curved platelets), as well as short tubes (ST, ca. 6 nm) and long tubes (LT, ca. 50 nm), remained stable in the cell culture medium. Internalization into human fibroblasts was observed only for the small particles PR and ST. None of the tested particles induced a significant cytotoxicity up to a concentration of 10(-1) mg·mL(-1). However, small sized NPs (PR and ST) were found to be genotoxic at very low concentration 10(-6) mg·mL(-1), while LT particles exhibited a weak genotoxicity. Our results indicate that small size NPs (PR, ST) were able to induce primary lesions of DNA at very low concentrations and that this DNA damage was exclusively induced by oxidative stress. The higher aspect ratio LT particles exhibited a weaker genotoxicity, where oxidative stress is a minor factor, and the likely involvement of other mechanisms. Moreover, a relationship among cell uptake, particle aspect ratio, and DNA damage of NPs was observed.

Adsorption of arsenic on polyaluminum granulate.

The kinetics and efficiencies of arsenite and arsenate removal from water were evaluated using polyaluminum granulates (PAG) with high content of aluminum nanoclusters. PAG was characterized to be meso- and macroporous, with a specific surface area of 35 ± 1 m(2) g(-1). Adsorption experiments were conducted at pH 7.5 in deionized water and synthetic water with composition of As-contaminated groundwater in the Pannonian Basin. As(III) and As(V) sorption was best described by the Freundlich and Langmuir isotherm, respectively, with a maximum As(V) uptake capacity of ~200 µmol g(-1) in synthetic water. While As(III) removal reached equilibrium within 40 h, As(V) was removed almost entirely within 20 h. Micro X-ray fluorescence and electron microscopy revealed that As(III) was distributed uniformly within the grain, whereas As(V) diffused up to 81 µm into PAG. The results imply that As(V) is adsorbed 3 times faster while being transported 10(5) times slower than As(III) in Al hydroxide materials.

Uptake patterns of critical metals in alpine plant species growing in an unimpaired natural site.

The range of metals used for industrial purposes - electrical engineering, solar panels, batteries - has increased substantially over the last twenty years. Some of these emerging metals are the subject of geopolitical conflict and are considered critical as their unique properties make them irreplaceable. Many of these elements are poorly studied and their biogeochemical cycles still raise many questions. Aim of this study is to analyse the soil-to-plant transfer of some of these chemical elements and to shed light on their uptake pathways. For this purpose, the geological site of Jas Roux (France) was chosen as this alpine site is naturally rich in critical and potentially toxic elements such as As, Sb, Ba and Tl, but nevertheless is host to a high diversity of plants. Elemental concentrations were analysed in the topsoil and in 12 selected alpine plant species sampled in situ. Statistical tools were used to detect species dependent characteristics in elemental uptake. Our analyses revealed accumulation of rare earth elements by Saxifraga paniculata, selective oxyanion absorption by Hippocrepis comosa, accumulation of Tl by Biscutella laevigata and Galium corrudifolium and an exclusion strategy in Juniperus communis. These findings advance our understanding of the environmental behaviour of critical metals and metalloids such as V, As, Y, Sb, Ce, Ba and Tl and might bare valuable information for phytoremediation applications.

Multiscale imaging on Saxifraga paniculata provides new insights into yttrium uptake by plants.

Yttrium (Y) has gained importance in high tech applications and, together with the other rare earth elements (REEs), is also considered to be an emerging environmental pollutant. The alpine plant Saxifraga paniculata was previously shown to display high metal tolerance and an intriguing REE accumulation potential. In this study, we analysed soil grown commercial and wild specimens of Saxifraga paniculata to assess Y accumulation and shed light on the uptake pathway. Laser ablation inductively coupled plasma mass spectrometry and synchrotron-based micro X-ray fluorescence spectroscopy was used to localise Y within the plant tissues and identify colocalized elements. Y was distributed similarly in commercial and wild specimens. Within the roots, Y was mostly located in the epidermis region. Translocation was low, but wild individuals accumulated significantly more Y than commercial ones. In plants of both origins, we observed consistent colocalization of Al, Fe, Y and Ce in all plant parts except for the hydathodes. This indicates a shared pathway during translocation and could explained by the formation of a stable organic complex with citrate, for example. Our study provides important insights into the uptake pathway of Y in S. paniculata, which can be generalised to other plants.

Filter-feeding bivalves store and biodeposit colloidally stable gold nanoparticles.

Nanoparticles resistant to salt-induced aggregation are continually being developed for biomedical and industrial applications. Because of their colloidal stability these functionalized nanoparticles are anticipated to be persistent aquatic contaminants. Here, we show that Corbicula fluminea, a globally distributed clam that is a known sentinel of aquatic ecosystem contamination, can uptake and biodeposit bovine serum albumin (BSA) stabilized gold nanoparticles. Nanoparticle clearance rates from suspension were dictated by diameter and concentration, with the largest particles cleared most quickly on a mass basis. Particle capture facilitates size-selective 'biopurification' of particle suspensions with nanoscale resolution. Nanoparticles were retained either within the clam digestive tract or excreted in feces. Our results suggest that biotransformation and biodeposition will play a significant role in the fate and transport of persistent nanoparticles in aquatic systems.

Accumulation, speciation and localization of silver nanoparticles in the earthworm Eisenia fetida.

The use of silver nanoparticles (AgNPs) in agriculture and many consumer products has led to a significant release of Ag in the environment. Although Ag toxicity in terrestrial organisms has been studied extensively, very little is known about the accumulation capacity and coping mechanisms of organisms in Ag-contaminated soil. In this context, we exposed Eisenia fetida earthworms to artificial OECD soil spiked with a range of concentrations of Ag (AgNPs or AgNO3). The main aims were to (1) identify the location and form of accumulation of Ag in the exposed earthworms and (2) better understand the physiological mechanisms involved in Ag detoxification. The results showed that similar doses of AgNPs or AgNO3 did not have the same effect on E. fetida survival. The two forms of Ag added to soil exhibited substantial differences in speciation at the end of exposure, but the Ag speciation and content of Ag in earthworms were similar, suggesting that biotransformation of Ag occurred. Finally, 3D images of intact earthworms obtained by X-ray micro-computed tomography revealed that Ag accumulated preferentially in the chloragogen tissue, coelomocytes, and nephridial epithelium. Thus, E. fetida bioaccumulates Ag, but a regulation mechanism limits its impact in a very efficient manner. The location of Ag in the organism, the competition between Ag and Cu, and the speciation of internal Ag suggest a link between Ag and the thiol-rich proteins that are widely present in these tissues, most probably metallothioneins, which are key proteins in the sequestration and detoxification of metals.

X-ray absorption spectroscopy evidence of sulfur-bound cadmium in the Cd-hyperaccumulator Solanum nigrum and the non-accumulator Solanum melongena.

It has been proposed that non-protein thiols and organic acids play a major role in cadmium phytoavailability and distribution in plants. In the Cd-hyperaccumulator Solanum nigrum and non-accumulator Solanum melongena, the role of these organic ligands in the accumulation and detoxification mechanisms of Cd are debated. In this study, we used X-ray absorption spectroscopy to investigate Cd speciation in these plants (roots, stem, leaves) and in the soils used for their culture to unravel the plants responses to Cd exposure. The results show that Cd in the 100 mg kg-1 Cd-doped clayey loam soil is sorbed onto iron oxyhydroxides. In both S. nigrum and S. melongena, Cd in roots and fresh leaves is mainly bound to thiol ligands, with a small contribution of inorganic S ligands in S. nigrum leaves. We interpret the Cd binding to sulfur ligands as detoxification mechanisms, possibly involving the sequestration of Cd complexed with glutathione or phytochelatins in the plant vacuoles. In the stems, results show an increase binding of Cd to -O ligands (>50% for S. nigrum). We suggest that Cd is partly complexed by organic acids for transportation in the sap.

Medium-term effects of Ag supplied directly or via sewage sludge to an agricultural soil on Eisenia fetida earthworm and soil microbial communities.

The widespread use of silver nanoparticles (AgNPs) in consumer products that release Ag throughout their life cycle has raised potential environmental concerns. AgNPs primarily accumulate in soil through the spreading of sewage sludge (SS). In this study, the effects of direct exposure to AgNPs or indirect exposure via SS contaminated with AgNPs on the earthworm Eisenia fetida and soil microbial communities were compared, through 3 scenarios offering increasing exposure concentrations. The effects of Ag speciation were analyzed by spiking SS with AgNPs or AgNO3 before application to soil. SS treatment strongly impacted Ag speciation due to the formation of Ag2S species that remained sulfided after mixing in the soil. The life traits and expression of lysenin, superoxide dismutase, cd-metallothionein genes in earthworms were not impacted by Ag after 5 weeks of exposure, but direct exposure to Ag without SS led to bioaccumulation of Ag, suggesting transfer in the food chain. Ag exposure led to a decrease in potential carbon respiration only when directly added to the soil. The addition of SS had a greater effect on soil microbial diversity than the form of Ag, and the formation of Ag sulfides in SS reduced the impact of AgNPs on E. fetida and soil microorganisms compared with direct addition.

Investigation of copper speciation in pig slurry by a multitechnique approach.

It is now well-known that copper (Cu) can accumulate on the surface of soils upon which pig slurry has been applied. This is due to the high quantity of Cu in pig slurry resulting from its use as a growth promoter in animal feeds. The mobility and bioavailability of Cu from pig slurry spreading can be better predicted by determining the speciation of this element in addition to its total concentration. The aim of this study was to present a multitechnique approach to investigate Cu speciation in pig slurry. First, size fractionation and chemical characterization of each size fraction were performed to complement results obtained in raw samples. Micro X-ray fluorescence spectroscopy (µXRF) highlighted the colocalization of Cu and sulfur (S). Finally, X-ray absorption near-edge structure spectroscopy (XANES) showed that Cu speciation in raw pig slurry and size fractions could be described by Cu(2)S and that its oxidation state is Cu(I). In addition, geochemical calculation demonstrated that chalcocite (Cu(2)S) was the major Cu species present under pig slurry lagoon physical-chemical conditions. This Cu speciation in pig slurry may be the main reason for the observed Cu accumulation at the soil surface.

Screening of Native Plants Growing on a Pb/Zn Mining Area in Eastern Morocco: Perspectives for Phytoremediation.

Screening of native plant species from mining sites can lead to identify suitable plants for phytoremediation approaches. In this study, we assayed heavy metals tolerance and accumulation in native and dominant plants growing on abandoned Pb/Zn mining site in eastern Morocco. Soil samples and native plants were collected and analyzed for As, Cd, Cu, Ni, Sb, Pb, and Zn concentrations. Bioconcentration factor (BCF), translocation factor (TF), and biological accumulation coefficient (BAC) were determined for each element. Our results showed that soils present low organic matter content combined with high levels of heavy metals especially Pb and Zn due to past extraction activities. Native and dominant plants sampled in these areas were classified into 14 species and eight families. Principal components analysis separated Artemisia herba-alba with high concentrations of As, Cd, Cu, Ni, and Pb in shoots from other species. Four plant species, namely, Reseda alba, Cistus libanotis, Stipa tenacissima, and Artemisia herba-alba showed strong capacity to tolerate and hyperaccumulate heavy metals, especially Pb, in their tissues. According to BCF, TF, and BAC, these plant species could be used as effective plants for Pb phytoextraction. Stipa tenacissima and Artemisia herba-alba are better suited for phytostabilization of Cd/Cu and Cu/Zn, respectively. Our study shows that several spontaneous and native plants growing on Pb/Zn contaminated sites have a good potential for developing heavy metals phytoremediation strategies.