How do replanted mangroves affect phosphorus dynamics in their soils?

Phosphorus is a limiting element for the productivity of mangroves, which in turn are important ecosystems in regulating nutrients cycle and climate change by sequestering carbon (C). Despite this, there is an intense process of degradation in these environments. In addition to providing socio-environmental services, mangrove replanting can also alter the dynamics of nutrients in soils. Therefore, this study aims to understand the changes in soil phosphorus (P) fractions after a mangrove restoration. Soil samples from an unvegetated area (NV), a mature mangrove (R) and 7 and 9 year old replanted mangroves at SE-Brazil (APA Guapi-mirim, Rio de Janeiro state) were collected and analyzed to characterize the redox conditions (Eh), pH, and iron (Fe) fractionation, Total Organic Carbon (TOC) contents and P fractionation (exchangeable P; P associated with reducible Fe and Mn oxyhydroxides; associated with Al silicates and hydroxides; associated with humic acids; associated with Ca and Mg; associated with humin). The results indicate an increase in TOC as the age of the mangrove restoration increases (from 8.6 to 17.9%). The pH values were significantly lower, reaching very acidic values, associated with an increase in Eh. Both parameters also showed strong seasonal variation, with a drop in Eh during the wet period (from 165% to -46%) and an increase in pH in the same period (from 6.0 to 6.7). Regarding P fractionation, the main P pool was organic P forms, which showed the highest concentrations in all studied sites. Unvegetated areas showed higher organic P forms (NV: 108.8 µg g-1) than vegetated areas (M7: 55.7 µg g-1, M9: 83.6 µg g-1, R: 87.3 µg g-1). Vegetated sites also showed lower levels of the PEx, PFeMn and Papatite fractions (total forest mean: 2.4 µg g-1, 5.8 µg g-1, 3.0 µg g-1, respectively). Besides no clear trend on P fractionation through seasons and forest age, pseudo-total P increased following the forest recovery (e.g. M7

Distribution of acid-volatile sulfides and simultaneously extracted metals in Guanabara Bay: combination of anthropogenic, sedimentological, and geochemical processes.

Coastal sedimentary systems are affected by continental and marine metal pollutant inputs associated with different hydrodynamic characteristics and geochemical processes. These include the formation of acid-volatile sulfides (AVS) within sediments, which affects metal bioavailability and associated aquatic biota toxicity risks. Physicochemical changes in these environments in the face of extreme natural or man-made environmental influences can dramatically alter metal bioavailability and toxicity through metal binding and immobilization as insoluble sulfides. Surface sediments from Guanabara Bay, river mouths, and two mangrove areas were collected, and AVS and simultaneously extracted metals Cd, Cu, Fe, Mn, Ni, Pb, and Zn and ΣSEM were determined to assess sediment quality. A severe eutrophication history favored AVS concentrations exceeding or close to the sum-SEM concentrations, demonstrating that AVS play an important role in making trace metals unavailable for assimilation by living organisms, mitigating the risks of contamination for the local biota. This eutrophication-driven sulfide accumulation may attenuate the sediment toxicity in sites heavily polluted by metals, while some fewer eutrophic sites became more exposed to metals in excess to AVS.

Phosphate buffering in mangrove sediment pore water under eutrophication and deforestation influences.

Phosphorus (P) behavior was evaluated in mangrove wetlands impacted by urban sewage, including a deforested site. Sediment cores were analyzed for grain size, organic carbon, total nitrogen, stable isotopes (δ13C and δ15N), P contents, and pore water PO43- concentrations and net consumption/production rates. Under stronger eutrophication influence, significantly higher P (1390 vs. <1000 µg/g), δ15N (8.9 vs. <6.7 ‰) and algal material contents (with lower C/N ratio and heavier δ13C) occurred. Depth-integrated PO43- consumption rates in eutrophicated sites were up to two orders of magnitude higher (at the deforested site) than in a moderately preserved mangrove. The whole core of the moderately preserved site presented no saturation of PO43- buffering capacity, while more eutrophicated sites developed buffering zones saturated at ∼18-26 cm depth. Contrasting to nearby subtidal environments, eutrophication did not cause larger pore water PO43- concentration, evidencing the role of PO43- buffering on P filtering by mangrove wetlands.

Copper and zinc isotope systematics in different bivalve mollusk species from the French coastline: Implications for biomonitoring.

Zinc (Zn) and copper (Cu) stable isotopic compositions have been analyzed in various species of bivalve mollusks worldwide, but no comprehensive systematic interspecies comparison exists. Thus, we assessed isotope differences between species harvested in emblematic French coastal ecosystems to unveil biologically driven Cu and Zn isotope fractionation patterns. Inter-species isotopic variability of Cu is larger than Zn, with organisms that regulate internal concentrations displaying preferential bioaccumulation of heavy isotopes. The degree of internal isotope fractionation decreases from mussels > clams > oysters, affecting Cu more than Zn. The less pronounced Zn inter-specie variability helps preserve source information more reliably. Spatial analysis of a single oyster species denotes thus an important isotope variability of environmental Zn sources, including natural, anthropogenic and dietary components. Overall, results highlight the importance of considering systematic offset in Cu and Zn isotope values when comparing data from different bivalve species.

Geochemical fractionation of trace metals and ecological risk assessment of surface sediments in Sepetiba Bay, Brazil.

The Sepetiba Bay (Southeast Brazil) is a known Cd- and Zn-contaminated site that received spills of a large slag pile leachate from a Zn smelter. With important harbors, Sepetiba Bay demands periodic dredging operations which affect the mobility of the metals. The main goal of this work was to assess metal mobility in sediments and its associated toxicity in a fictive dredging area, to evaluate the risks of the operation. To achieve this goal, 18 superficial sediment samples were collected and characterized for pH and Eh. Sediments were analyzed for grain size, organic carbon, and total nitrogen, and metal mobility was evaluated with a sequential extraction procedure, proposed by the European Community Bureau of Reference (BCR). The results demonstrate that Cd and Zn are mainly associated with the exchangeable fraction (mean concentrations 1.4 mg kg-1 and 149.4 mg kg-1, respectively) and reducible fractions (mean concentrations 0.3 mg kg-1 and 65.5 mg kg-1, respectively), while Fe, Cr, Cu, Ni, and Al were associated with the residual fraction. Metals in the residual fraction are probably associated with the mineral lattice of the sediment and should not represent an environmental risk for the biota. The application of the enrichment factor and three risk assessment indexes (Risk Assessment Code, Risky Pollution Index, and Bioavailability Risk Assessment Index) show that the sediments are considerably enriched in metals that constitute a relevant risk for the sediment biota. In the case of dredging operations, Cd and Zn should be released to the overlying waters and be available to organisms, threatening the whole ecosystem. The proposed approach was shown to be much more precise than what is frequently presented in the Environmental Impact Assessments that only consider the threshold limits of the legislation.

The French Mussel Watch Program reveals the attenuation of coastal lead contamination over four decades.

The mid-20th century industrial peak caused severe global lead (Pb) marine contamination. Although Europe initiated Pb emission reduction regulations in the 1980s, the short- and long-term impacts remain unclear. This study investigates the evolution of Pb contamination on the French coast through elemental and isotope analysis in oysters and mussels from the French "Mussel Watch" Program. Observations at 114 monitoring stations over four decades have shown decreasing Pb levels in these bivalve mollusks. In 1988, 95 % exceeded the background reference values; this level had dropped to 39 % by 2021. The Pb isotope ratios in bivalves from eight target sites revealed a reduction in bioaccumulated anthropogenic Pb, albeit without complete elimination. The long residence time of legacy Pb combined with inputs from diffuse urban sources likely explains the persistent presence of anthropogenic Pb on the French coast. This study endorses the importance of continuous biomonitoring to evaluate environmental regulations and policies.

Antimicrobial resistance and biotechnological potential of plastic-associated bacteria isolated from an urban estuary.

Plastics have quickly become one of the major pollutants in aquatic environments worldwide and solving the plastic pollution crisis is considered a central goal of modern society. In this study, 10 different plastic samples, including high- and low-density polyethylene and polypropylene, were collected from a deeply polluted urban estuary in Brazil. By employing different isolation and analysis approaches to investigate plastic-associated bacteria, a predominance of potentially pathogenic bacteria such as Acinetobacter, Aeromonas, and Vibrio was observed throughout all plastic samples. Bacteria typically found in the aquatic environment harboured clinically relevant genes encoding resistance to carbapenems (blaKPC ) and colistin (such as mcr-3 and mcr-4), along with genetic determinants associated with potentially active gene mobilization. Whole genome sequencing and annotation of three plastic-associated Vibrio strains further demonstrated the carriage of mobile genetic elements and antimicrobial resistance and virulence genes. On the other hand, bacteria isolated from the same samples were also able to produce esterases, lipases, and bioemulsifiers, thus highlighting that the plastisphere could also be of special interest from a biotechnological perspective.

Assessment of eutrophication from phosphorus remobilization after resuspension of coastal sediments from an urban tropical estuary.

Dredging activities cause sediment resuspension, which can change the bioavailability of nutrients such as phosphorus (P) in aquatic ecosystems due to remobilization. This study evaluated the remobilization of P in the solid and dissolved phase before and after sediment resuspension in the Meriti and Iguaçu River estuaries and the Rio de Janeiro and Niterói harbor in Guanabara Bay (Rio de Janeiro, Brazil). Three water and sediment samples were collected at each point. Dissolved phosphorus (DP), total phosphorus (TP), organic phosphorus (OP), and inorganic phosphorus (IP) were analyzed before and after resuspension. Resuspension directly impacted the fine-grained samples, causing the release of P into the water column after resuspension, increasing eutrophication of the estuary and risk to biota. The phosphorus enrichment index (PEI) was calculated in the four areas, and in all areas, the index was above 1, which means high ecological risk. The area with sandy granulometry and a lower percentage of organic matter showed an increase in the index after resuspension. The resuspension may impact the increase of eutrophication in some areas, due to the remobilization of the sediment and the adsorbed contaminants.

Copper and lead isotope records from an electroplating activity in sediments and biota from Sepetiba Bay (southeastern Brazil).

An old electroplating plant in Sepetiba Bay discharged metal-enriched wastes into the surrounding mangroves for 30 years (from the 1960s to 1990s), resulting in a hotspot zone of legacy sediments highly concentrated in toxic trace metals. This study applies Cu and Pb isotope systems to investigate the contributions of past punctual sources relative to emerging modern diffuse sources. The electroplating activity imprinted particular isotopic signatures (average δ65CuSRM-976: 0.4 ‰ and 206Pb/207Pb: 1.14) distinct from the natural baseline and urban fluvial sediments. The isotopic compositions of tidal flat sediments show intermediate isotope compositions reflecting the mixing of Cu and Pb from the hotspot zone and terrigenous materials carried by rivers. Oyster isotope fingerprints match legacy sediments, attesting that anthropogenic Cu and Pb are bioavailable to the biota. These findings confirm the interest in combining two or more metal isotope systems to discriminate between modern and past metal source emissions in coastal environments.

Spatial distribution of total mercury and methylmercury in the sediment of a tropical coastal environment subjected to heavy urban inputs.

Jurujuba Cove is located in Guanabara Bay (adjacent to highly populated city of Rio de Janeiro, Brazil), which receives diffuse sources of contaminants along with two main freshwater inputs (the Cachoeira and Icaraí rivers), and hosts mussel farms. The main goal of this work was to evaluate the total mercury (THg) and methylmercury (MeHg) concentrations distributions in the sediments of the cove and their associations with physical and chemical parameters, thereby assessing their geochemical behavior. Twenty samples of surface sediments were collected and characterized for grain size, pH, redox potential, organic carbon, total phosphorus, THg and MeHg. Spatial distribution maps were produced for each parameter and a principal components analysis was carried out, to assess THg and MeHg behavior and their relationships with other parameters. The principal components analysis showed that grain size functions as the main diluting agent. The highest THg concentrations were observed in the mussel-farm area (656.1 ng g-1), and were related to fine grain size and elevated organic carbon values. High MeHg concentrations also occurred in the center of the cove, probably favored by high organic carbon content (low-energy environment). Total phosphorus concentrations indicate that Cachoeira River is a possible source of sewage, but little mercury seems to come from it. The results showed that although total mercury concentrations are elevated, with exception of a few locations, small methylmercury convertion rates were recorded in the sediments.

Mercury methylation upon coastal sediment resuspension: a worst-case approach under dark conditions.

Mercury behavior upon resuspension of sediments from two impacted areas of Guanabara Bay was evaluated to assess worst-case methylmercury (MeHg) responses, under dark experimental conditions to prevent demethylation by photolysis. Study areas include the Rio de Janeiro Harbor (RJH) and the chlor-alkali plant-affected Meriti River (MR) estuary. Total mercury (THg) and MeHg concentrations were determined along 24-h experiments of sediment resuspension in the bay water in dark conditions. Fine-grained Meriti River (MR) estuary sediments had 8 times higher MeHg initial concentrations than sandy Rio de Janeiro Harbor (RJH) sediments (3.4 ± 0.29 vs. 0.41 ± 0.1 ng g-1, respectively). Though THg contents were uncorrelated with resuspension time, statistically significant correlations of MeHg (rs = 0.78) and %MeHg in relation to THg (rs = 0.86) with resuspension time were observed for RJH sediments, indicating net methylation only for this study site. These positive correlation trends correspond to a 2.8 times MeHg concentration increase (ΔMeHg = 0.75 ng g-1) and 4.4 times increase in %MeHg (Δ%MeHg = 1.0%), after 24 h of resuspension. This suggests that assessments of factors affecting the MeHg spatial-temporal variability and associated toxicity risks can be limited in some sites if concentration changes due to sediment resuspension-redeposition processes are not considered. Therefore, the inclusion of MeHg evaluation before and after sediment resuspension events is recommendable for the improvement of dredging licensing and monitoring activities.

Linking eutrophication to carbon dioxide and methane emissions from exposed mangrove soils along an urban gradient.

Mangroves are one of the most important but threatened blue carbon ecosystems globally. Rapid urban growth has resulted in nutrient inputs and subsequent coastal eutrophication, associated with an enrichment in organic matter (OM) from algal and sewage sources and substantial changes in greenhouse gas (GHG) emissions. However, the effects of nitrogen (N) and phosphorus (P) enrichment on mangrove soil OM composition and GHG emissions, such as methane (CH4) and carbon dioxide (CO2), are still poorly understood. Here, we aim to evaluate the relationships between CO2 and CH4 efflux with OM composition in exposed soils from three mangrove areas along watersheds with different urbanization levels (Rio de Janeiro State, Brazil). To assess spatial (lower vs. upper intertidal zones) and seasonal (summer vs. winter) variability, we measured soil-air CO2 and CH4 fluxes at low spring tide, analyzing elementary (C, N, and P), isotopic (δ13C and δ15N), and the molecular (n-alkanes and sterols) composition of surface soil OM. A general trend of OM composition was found with increasing urban influence, with higher δ15N (proxy of anthropogenic N enrichment), less negative δ13C, more short-chain n-alkanes, lower C:N ratio (proxies of algal biomass), and higher epicoprostanol content (proxies of sewage-derived OM). The CO2 efflux from exposed soils increased greatly in median (25/75 % interquartile range) from 4.6 (2.9/8.3) to 24.0 (21.5/32.7) mmol m-2 h-1 from more pristine to more urbanized watersheds, independent of intertidal zone and seasonality. The CO2 fluxes at the most eutrophicated site were among the highest reported worldwide for mangrove soils. Conversely, CH4 emissions were relatively low (three orders of magnitude lower than CO2 fluxes), with high peaks in the lower intertidal zone during the rainy summer. Thus, our findings demonstrate the influence of coastal eutrophication on global warming potentials related to enhanced heterotrophic remineralization of blue carbon within mangrove soils.

Mercury distribution in water masses of the South Atlantic Ocean (24°S to 20°S), Brazilian Exclusive Economic Zone.

Mercury (Hg) is a toxic globally spread pollutant that has been found at increasing concentrations in the South Atlantic Ocean. The present work provides the first insight into the total mercury (HgT, unfiltered waters) content in the water of the Brazilian Exclusive Economic Zone (BEEZ), within a 24°S to 20°S. Water samples were collected from surface to 3400 m depth along transects, and analyzed with atomic fluorescence. The mean HgT concentration for the Tropical Water mass (TW) was 6.3 ± 1.4 pM (n = 16), for the South Atlantic Central Water (SACW), 5.9 ± 0.7 pM (n = 8), for the Antarctic Intermediate Water (AAIW), 5.0 ± 0.6 pM (n = 2), for the Upper Circumpolar Deep Water (UCDW), 6.5 pM (n = 1), and for the North Atlantic Deep Water (NADW), 5.7 ± 0.9 pM (n = 12). HgT concentrations were highest throughout the BEEZ in comparison with other parts of the Atlantic Ocean, farther from the coast.

Ecological risks associated to trace metals of contaminated sediments from a densely urbanized tropical eutrophic estuary.

The sediment contamination by trace metals in coastal aquatic ecosystems is a worldwide environmental problem, since metals can be toxic, persistent, and bioaccumulated. In case of natural events, such as storms, or anthropogenic activities, like dredging, the sediment resuspension to the water column occurs and can solubilize metals, probably increasing their bioavailability and consequently the risk to aquatic life. This study evaluated the bioavailability on reactive trace metals (Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) in estuarine sediments from Iguaçu and Meriti Rivers, both in the drainage basin of Guanabara Bay (Rio de Janeiro, Brazil). Additionally, a discussion about the anthropogenic interference throughout time of six short sediments cores, calculating three different indexes (contamination factors, CF; potential ecological risk index for a single heavy metal, Eif for short; potential ecological risk, PERI) was performed. It was considered as reactive phase, the metal concentrations obtained using a weak acid extraction (in HCl 1 mol L-1 solution). Zn presented high concentrations after resuspension, being above effect range medium (ERM) (52.81 to 1337.4 mg kg-1). The CF indicated very high contamination degree for Cu (14.62 to 17.96) and Zn (27.80 to 35.85) for both rivers. The Eif for short presented higher risk to Cu and Zn for Iguaçu and Meriti rivers. PERI index classified Meriti River samples as severely contaminated (238.10 to 351.62).

Mangrove sediments as long-term mercury sinks: Evidence from millennial to decadal time scales.

The mercury (Hg) cycle in estuaries has been globally discussed, although Holocene deposition in mangrove sediments remains unknown. Herein, a sediment core from a mangrove system in southeastern Brazil was 14C-dated to evaluate millennial Hg deposition. The highest Hg concentrations (1010-2540 ng g-1) in surface sediments were explained by emissions from a chlor-alkali industry (1964 CE). However, Hg levels were also high in pre-industrial periods, associated to fine grain-size and algal organic deposition. Less anomalous Hg concentrations in bottom sediments indicate Holocene ages (~1940-3324 cal yr BP), potentially associated to Serra do Mar mountains weathering. This study reveals the capacity of mangrove to retain Hg over millennial time scales, acting as significant and long-term Hg sinks. Therefore, the use of Hg as an Anthropocene marker must be considered cautiously in coastal systems that act as Hg sinks in times when environmental changes were not caused by human activities.

Arsenic contamination in widely consumed Caribbean sharpnose sharks in southeastern Brazil: Baseline data and concerns regarding fisheries resources.

Although shark meat is consumed worldwide, elevated arsenic (As) concentrations have been increasingly reported. The Caribbean sharpnose shark (Rhizoprionodon porosus) is a widely consumed fishing resource in Brazil, with scarce information on As burdens to date. Herein, commercial-sized juvenile Caribbean sharpnose sharks from Rio de Janeiro (Brazil) were assessed in this regard, presenting significantly higher hepatic As concentrations in males (8.24 ± 1.20 mg kg-1 wet weight; n = 12) compared to females (6.59 ± 1.87 mg kg-1 w.w.; n = 8), and a positive correlation (r = 0.74) was noted between female muscle and liver As concentrations, indicating systemic inter-tissue transport not evidenced in males. Arsenic concentrations were over the maximum permissible As limit established in Brazilian legislation for seafood and calculated Target Hazard Quotients suggest consumption risks, although cancer risks were not evidenced. Therefore, Public Health concerns with regard to Caribbean sharpnose shark As burdens deserve careful attention.

Sediment contamination and toxicity of Marine Protected Areas in southeastern Brazilian coast: A baseline for management planning.

Recent studies have shown that the effectiveness of establishing Marine Protected Areas (MPAs) is threatened by toxic effects of contaminants. São Paulo is the most economically developed state of Brazil, and its coast is protected by a system of MPAs, including the North Shore Marine Protected Area (NSMPA). The present study provides a first assessment of sediment quality in NSMPA and two reference sites. Concentrations of arsenic (As), metals (Al, Fe, Cd, Cr, Cu, Ni, Pb, and Zn) and polycyclic aromatic hydrocarbons (PAHs) and its sources were determined. Comparisons with sediment quality guidelines indicated low contamination, as confirmed by the Geoaccumulation Index, which classified the results as background levels. Whole-sediment exposures of the amphipod Tiburonella viscana were performed to estimate possible acute effects, but samples exhibited no toxicity. The results presented in this study represent a baseline for further monitoring programs of MPAs, supporting conservation actions of management plans.

Anthropogenic and environmental influences on nutrient accumulation in mangrove sediments.

Here we provide a global review on nutrient accumulation rates in mangroves which were derived from sixty-nine dated sediment cores, addressing environmental and anthropogenic influences. Conserved mangroves presented nitrogen and phosphorous accumulation rates near to 5.8 ± 2.1 and 0.8 ± 0.5 g m-2 yr-1, respectively. These values were significantly lower than those observed for mangroves impacted by coastal eutrophication, which were found to bury 21.5 ± 8.6 and 17.9 ± 2.4 g m-2 yr-1, of nitrogen and phosphorous respectively. Moreover, higher nutrient accumulation rates were found in mixed mangroves as compared to monospecific forests, and higher values were noted within vegetated areas as compared to mudflats. For South America and Asia, mangroves impacted by anthropogenic activities may result in up to seventeen-fold higher nitrogen and phosphorous accumulation rates in comparison with values under conserved conditions. For Oceania, these differences may be up to fivefold higher in impacted as compared to the conserved ecosystems in this region.

Metal Bioaccumulation by the Neotropical Clam Anomalocardia flexuosa to Estimate the Quality of Estuarine Sediments.

This investigation evaluated the bioaccumulation potential of the tropical estuarine bivalve Anomalocardia flexuosa for trace metals. To this aim, chemical and sedimentological analyses and bioaccumulation tests were performed. The sediments were mainly composed by fine-sands and mud, with variable levels of organic matter and CaCO3. Muddy sediments from a depositional site (P2) presented the highest concentrations of metals, despite SEM/AVS not indicating bioavailability. Bioaccumulation factors showed high ratios for Cd, Ni, and Zn, while associations between the contents of mud, organic matter, CaCO3 and metals in sediments and tissues of A. flexuosa were indicated by a principal component analysis. The SEM/AVS was not effective to predict the bioavailability through dissolved metals. The results showed that contaminants were bioavailable, while the performed bioaccumulation test proved to be a reliable technique for assessing sediment contamination in estuarine regions. Moreover, A. flexuosa was considered an adequate test organism for bioaccumulation studies.

Trace metal dynamics in an industrialized Brazilian river: A combined application of Zn isotopes, geochemical partitioning, and multivariate statistics.

The Paraiba do Sul (PSR) and Guandu Rivers (GR) water diversion system (120 km long) is located in the main industrial pole of Brazil and supplies drinking water for 9.4 million people in the metropolitan region of Rio de Janeiro. This study aims to discern the trace metals dynamics in this complex aquatic system. We used a combined approach of geochemical tools such as geochemical partitioning, Zn isotopes signatures, and multivariate statistics. Zinc and Pb concentrations in Suspended Particulate Matter (SPM) and sediments were considerably higher in some sites. The sediment partition of As, Cr, and Cu revealed the residual fraction (F4) as the main fraction for these elements, indicating low mobility. Zinc and Pb were mostly associated with the exchangeable/carbonate (F1) and the reducible (F2) fractions, respectively, implying a higher susceptibility of these elements to being released from sediments. Zinc isotopic compositions of sediments and SPM fell in a binary mixing source process between lithogenic (δ66/64ZnJMC ≈ + 0.30‰) and anthropogenic (δ66/64ZnJMC ≈ + 0.15‰) end members. The lighter δ66/64ZnJMC values accompanied by high Zn concentrations in exchangeable/carbonate fraction (ZnF1) enable the tracking of Zn anthropogenic sources in the studied rivers. Overall, the results indicated that Hg, Pb, and Zn had a dominant anthropogenic origin linked to the industrial activities, while As, Cr, and Cu were mainly associated with lithogenic sources. This work demonstrates how integrating geochemical tools is valuable for assessing geochemical processes and mixing source effects in anthropized river watersheds.