Effect of Previous Frozen Storage and Coating Medium on the Essential Macroelement and Trace Element Content of Canned Mackerel.

The effect of previous frozen storage (-18 °C for 6 months) and different coating media (aqueous: water and brine; oily: sunflower, refined olive, and extra-virgin olive oils) on the essential macroelement and trace element content of canned Atlantic mackerel (Scomber scombrus) was studied. Previous frozen storage led to an increased (p < 0.05) content of canned samples of K (oil-coated samples) and Ca (all coating conditions) and to a decreased (p < 0.05) content of P (aqueous-coating samples) and S (water- and oil-coated samples). For trace elements, a content increase (p < 0.05) in Cu and Se (brine-canned samples) and Mn (water- and refined-olive-oil-coated samples) was detected in canned fish muscle with frozen storage. Concerning the coating effect, aqueous-coating samples showed lower (p < 0.05) Mg, P, S, K, and Ca contents than their corresponding oil-coated samples. For trace elements, lower average contents were found for Co, Cu, Mn, Se, and Fe in aqueous-coating fish muscle when compared to their counterparts coated in oily media. Content changes in the different elements in canned fish muscle are discussed based on interactions with other tissue constituents and modifications that such constituents undergo during processing (i.e., protein denaturation, liquor losses from the muscle, lipid changes).

Major, minor and trace element concentrations in the muscle and liver of a pregnant female Pacific sharpnose shark (Rhizoprionodon longurio) and its embryos.

The Pacific sharpnose shark Rhizoprionodon longurio is an abundant shark consumed by human population in Mexico. This study aimed to determine and compare the concentrations of thirteen essential elements (K, S, P, Na, Ca, Mg, Fe, Zn, Se, Cu, Mn, Cr and Co) and eleven non-essential elements (As, Sr, Cd, V, Li, U, Tl, Ag, Sn, Sb and Pb) in the muscle and liver of a pregnant female, fished near the copper mine of Santa Rosalía, and their respective embryos. Major, minor and trace (essential and non-essential) elements were transferred during gestation to embryos. All major elements analyzed had significantly higher concentrations in embryos liver than the pregnant female, except for magnesium. Higher concentrations in embryo tissues than the pregnant female tissues were found for the non-essential trace elements of strontium, lithium, thallium and silver, which indicate an easy maternal transfer of these non-essential elements.

Faeces of marine birds and mammals as substrates for microbial plankton communities.

The chemical composition of the seawater soluble fraction (WSF) of yellow-legged gulls and harbour seal faeces and their impact on microbial plankton communities from an eutrophic coastal area have been tested. After characterisation of the C:N:P stoichiometry, trace metals content and organic molecular composition of the faeces, significant differences between species have been observed in all parameters. Seagull faeces present about three times larger N content than seal faeces and are also richer in trace elements except for Cu and Zn. Organic nitrogen in seagull faeces is dominated by uric acid, while the proteins are the main N source in seal faeces. It is remarkable that seagull faeces are five times more soluble in seawater than seal faeces and present a much higher N content (48.0 versus 3.5 mg N in the WSF per gram of dry faeces), >85% of which as dissolved organic nitrogen, with C:N molar ratios of 2.4 and 13 for seagull and seal faeces, respectively. Based on this contrasting N content, large differences were expected in their impact on microbial populations. To test this hypothesis, a 3-day microcosm incubation experiment was performed, in which coastal seawater was amended with realistic concentrations of the WSF of seagull or seal faeces. A significant and similar increase in bacterial biomass occurred in response to both treatments. In the case of phytoplankton, the impact of the treatment with seagull faeces was significantly larger that the effect of the treatment with seal faeces. Our data suggest that the distinct competitive abilities of phytoplankton and bacteria largely influence the potential impact of distinct animal faeces on primary productivity in coastal ecosystems. Impacts on the microbial plankton communities do not affect only this trophic level, but the whole trophic chain, contributing to nutrient recycling in coastal areas where large populations of these species are settled.

Essential and non-essential trace element concentrations in muscle and liver of a pregnant Munk's pygmy devil ray (Mobula munkiana) and its embryo.

During gestation, essential and non-essential trace elements are transferred from the pregnant females to embryos. This study aimed to determine and compare the concentrations of seven essential trace elements (Fe, Zn, Se, Cu, Mn, Cr, Co) and six non-essential trace elements (As, Cd, V, U, Tl, Ag) in the muscle and the liver of a Munk's pygmy devil ray pregnant female, Mobula munkiana, and its embryo. Transfer evidence of essential and non-essential trace elements was detected in M. munkiana tissues. Arsenic was found in elevated concentrations in the pregnant female and the embryo tissues. Elevated levels of Cd, V, U, and Ag were found in the pregnant female liver, but were minimal in the embryo tissue. This is the first study to investigate maternal transfer of essential and non-essential trace elements in these species and their reproductive strategy.

Implications of kinetically-hindered metals in ecotoxicological studies: Effect of platinum spike aging on its toxicity to Dunaliella salina.

Platinum (Pt) is considered an emerging environmental micro-contaminant due to its increasing use in anthropogenic activities during the past decades. However, there are still important gaps in the understanding of its biogeochemical behavior in the aquatic environment - e.g. its speciation, reactivity and fate - mainly as a result of the analytical challenge of the determination of its typical ultra-trace environmental concentrations. Also, Pt is a kinetically-hindered metal displaying slow reaction kinetics, which has important implications regarding eco-toxicological studies. That is, investigation of its toxicity under laboratory-controlled conditions may therefore require ensuring that equilibrium speciation conditions are reached before starting the experiments. In order to shed further light on this issue, in this study we have monitored the speciation changes during aging of the Pt(IV) spikes in controlled media (seawater) using an UV-Vis spectrophotometry. Platinum toxicity to the green microalgae Dunaliella salina was then compared, using standardized tests, with fresh and aged Pt(IV) spikes at the mg L-1 concentration range. Following 96-hour exposure, ecotoxicological assays consisting in spectrometric measurements of chlorophyll-a concentrations and Effective Concentrations (EC) of Pt resulting in the inhibition of 10% and 50% of algae growth rate were calculated (EC10 and EC50, respectively). Daily monitoring of Pt speciation reflected the transition from PtCl62- (spike) to hydrolyzed species, probably in the form [PtCl3-n(OH)3+n]2-, n = 0-3. Exposure experiments showed that after a short period of aging (10 days), Pt(IV) toxicity increased one order of magnitude compared to freshly spiked media. These results confirm the relevance of considering spike aging to ensure that speciation equilibrium conditions are attained in order to produce environmental realistic eco-toxicological data.

Nutritional and Healthy Value of Chemical Constituents Obtained from Patagonian Squid (Doryteuthis gahi) By-Products Captured at Different Seasons.

This study focuses on the extraction of nutritional and healthy constituents of whole by-products resulting from the commercialisation of Patagonian squid (Doriteuthis gahi). By-products corresponding to squid individuals captured at different seasons were comparatively analysed for proximate composition, lipid classes content, fatty acid (FA) profile, and macroelement and trace element composition. As a result, moisture, lipid, protein, and ash values were included in the ranges 829.0-842.8, 17.5-21.8, 106.0-123.7, and 9.3-13.3 g·kg-1 by-products, respectively. Phospholipids showed to be the most abundant lipid class (359.2-463.5 g·kg-1 lipids), while triacylglycerols were only present in a 9.5-13.1 g·kg-1 lipids range. Valuable levels were detected for α-tocopherol (539.6-973.3 mg·kg-1 lipids), polyunsaturated fatty acids (PUFA; 50.5-52.6 g·100 g-1 FA), ω3 PUFA (47.0-48.6 g·100 g-1 FA), PUFA/saturated FA ratio (1.4-1.6), and ω3/ω6 ratio (12.1-13.4). Among macroelements, S, P, and Na showed to be more abundant than K, Mg, and Ca. Profitable levels of Co, Cu, Fe, Mn, Se, and Zn were detected in all kinds of individuals. In spite of content variations found as a result of the capture season of Patagonian squid individuals, whole by-products of this cephalopod species can be considered as a profitable source to provide the food and pharmaceutical industries with useful value-added constituents.

Ultra-trace interference-free analysis of palladium in natural waters by ICP-MS after on-line matrix separation and pre-concentration.

The determination of palladium (Pd) in environmental samples by ICP-MS is challenging as all its isotopes are extensively interfered due to isobaric (e.g. 110Cd on 110Pd, 106Cd on 106Pd), polyatomic (e.g. 92Mo16O on 108Pd, 89Y16O on 105Pd) and doubly-charged (e.g. 208Pb2+ on 104Pd) species formed in the plasma from elements usually present at concentrations several orders of magnitude higher. As a result, the determination of Pd in natural waters is extremely scarce despite is has been proven that this metal is subject to a significant anthropogenic impact mainly linked to its use in catalytic converters in motor vehicles. In order to overcome this situation, we have developed an ultra-trace interference-free methodology for the determination of Pd in natural waters by ICP-MS after on-line matrix separation and preconcentration. The method is based on the strong affinity of Pd towards a commercially-available carboxymethylated polyethylenimine resin, which also has the ability to retain most of the transition metals. However, Pd is not eluted from the resin at typical elution conditions (e.g. 2 M HNO3, which removes all the interference-forming metals), but this can be attained by passing a diluted thiourea solution (10-3 M). Therefore, the interference-free on-line determination of Pd in natural waters was successfully achieved using a two-step elution procedure. Procedural blank values were 0.012 ±â€¯0.003 ng kg-1 (n = 6), which results in a detection limit of 0.010 ng kg-1, allowing the determination of dissolved Pd in natural samples at low, ambient concentrations. The optimized methodology was applied to determine the concentrations of Pd in the Gironde estuary, which represents the first dissolved Pd profile along an estuarine salinity gradient and one of the first dataset of Pd concentrations in natural waters at ambient levels in almost 4 decades. The simplicity of the preconcentration setup and the possibility for its automation offers new analytical opportunities, which will be useful for future studies aiming to improve our understanding of the behavior of Pd in natural waters.

Drivers of Rh and Pt variability in the water column of a hydrodynamic estuary: Effects of contrasting environments.

Rhodium and platinum are amongst the less studied elements in estuarine waters and the understanding of their speciation analysis and environmental fate remains limited. In this study, we address the occurrence and discrimination of soluble/insoluble Rh and Pt species in aquatic systems, as well as their potential transport. Particulate and dissolved (< 0.45 µm) rhodium (RhP and RhD) and platinum (PtP and PtD), respectively, were determined in the water column of contrasting environments during neap (NT) and spring (ST) tide semi-diurnal cycles: in the upper Tagus estuary (VFX) and near the mouth, close to a wastewater treatment plant outfall (ALC). Both elements were analyzed by AdCSV and ICP-MS, as well ancillary parameters were determined. Concentrations of Rh and Pt followed the tidal regime, presenting higher concentrations during low tide. Concentrations of RhP (0.1-5.1 ng g-1) and RhD (0.03-0.12 ng L-1) were lower than PtP (1.0-25.6 ng g-1) and PtD (0.1-11.7 ng L-1), respectively. Concentrations found in ALC were higher than VFX, except for RhD, mirroring anthropogenic inputs attributed to automotive catalytic converters and an additional Pt source originated in Pt-based compounds. Distribution coefficients (KD) of 104 were computed and were independent of the salinity gradient. The speciation analysis done at VFX during NT showed that truly dissolved forms measured by AdCSV represented 39 ± 9% of total Pt in the water column, while total filter-passing species measured by ICP-MS were higher, 65 ± 14%. Pt speciation was controlled by its dissolved forms, whereas particulate Rh forms represented the bulk value (> 65%). The potential transport evaluated at downstream station indicated recirculation within the estuary and export towards the Atlantic Ocean, with higher concentrations associated with the ebb opposing to the flood. These results show estuaries as important pathways to introduce PGE in coastal regions, transferring them towards the ocean.

Macroelements and Trace Elements Content in Brine-Canned Mackerel (Scomber colias) Subjected to High-Pressure Processing and Frozen Storage.

This study analysed the effect of prior high-pressure processing (HPP; 200-600 MPa, 2 min), freezing (-30 °C, 48 h), and frozen storage (-18 °C, 6 months) on the macroelement and trace element content in brine-canned mackerel (Scomber colias). Most elements (Na, Ca, Ba, Mn, Fe, Cu, Cd, Sn, As, S, and Se) showed an increased (p < 0.05) presence in mackerel muscle canned after freezing. A content increase (p < 0.05) was also observed for Na and Sn if prior frozen storage was also applied; on the contrary, Ca, Ba, Mn, Fe, Cd, S, and Se showed a content decrease (p < 0.05) as a result of such storage. Freezing, frozen storage, and canning led to lower values (p < 0.05) in canned fish for K, Mg, Pb, and P. Prior HPP led to relevant content decreases (p < 0.05) for K, Mg, Ca, Ba, Mn, Fe, Pb, and P contents in fish canned after the freezing step; HPP provoked additional decreases (p < 0.05) in Ca, Ba, and Mn levels in samples corresponding to 6-month frozen storage. On the contrary, prior HPP led to marked increases (p < 0.05) for Cd, S, and Se contents in all canned samples. Content changes are explained on the basis of modifications of other constituents and liquor losses from muscle.

Speciation analysis of Pt and Rh in urban road dust leachates.

Road dust is a major reservoir of anthropogenic Pt and Rh. However, information about how these elements are released to the aquatic systems under environmentally relevant conditions is scarce. In this work, an innovative combination of analytical strategies is used to provide insight into the speciation analysis of those elements. A composite sample of road dust thoroughly characterized was incubated over 7 days in synthetic rainwater and seawater. In the filtered (<0.45 µm) solutions, truly dissolved Pt and Rh concentrations were measured by adsorptive cathodic stripping voltammetry, while total concentrations were determined by inductively coupled plasma mass spectrometry. Truly dissolved species corresponded to a small fraction of total Pt and Rh in the road dust; accordingly, values of 0.01% and 0.1% were obtained in both media for Pt and Rh, respectively, which remained constant over time. The concentration of total filter-passing species predominates for both elements by a factor of 10 and 2-3 for Pt and Rh, respectively, evidencing that particulate species coexist with truly dissolved forms. Temporal variations were observed for Pt, as opposed to Rh. These findings contribute to the gap in knowledge regarding Pt and Rh mobility in aquatic systems.

Platinum and rhodium in Tagus estuary, SW Europe: sources and spatial distribution.

The spatial distribution of Pt and Rh was assessed in Tagus estuary and their sources discussed. Both elements were analysed in superficial sediment samples (n = 72) by adsorptive cathodic stripping voltammetry. Concentrations varied within the following ranges: 0.18-5.1 ng Pt g-1 and 0.02-1.5 ng Rh g-1. Four distinct areas were established: "reference"; waste- and pluvial water discharge; motorway bridges and industrialised areas. The calculated reference median concentrations were 0.55 ng Pt g-1 and 0.27 ng Rh g-1. Linear relationships were found between Pt and Al, Fe and LOI, whereas Rh depicted scattered patterns. The highest concentrations were found nearby industrialised areas and a motorway bridge, corresponding to the enrichment of 10 and 6 times the background of Pt and Rh, respectively. The main sources of contamination to the Tagus estuary derived from historical and present industrial activities and from automotive catalytic converters. Large variations of Pt/Rh ratio (0.48-39) point to different sources, reactivity and dilution effects.

Historical legacies of river pollution reconstructed from fish scales.

Many rivers have been impacted by heavy metal pollution in the past but the long-term legacies on biodiversity are difficult to estimate. The River Ulla (NW Spain) was impacted by tailings from a copper mine during the 1970-1980s but absence of baseline values and lack of subsequent monitoring have prevented a full impact assessment. We used archived fish scales of Atlantic salmon to reconstruct levels of historical copper pollution and its effects on salmon fitness. Copper bioaccumulation significantly increased over baseline values during the operation of the mine, reaching sublethal levels for salmon survival. Juvenile growth and relative population abundance decreased during mining, but no such effects were observed in a neighbouring river unaffected by mining. Our results indicate that historical copper exposure has probably compromised the fitness of this Atlantic salmon population to the present day, and that fish scales are suitable biomarkers of past river pollution.

Tracing platinum accumulation kinetics in oyster Crassostrea gigas, a sentinel species in coastal marine environments.

Platinum Group Elements (PGEs) are extremely scarce in the Earth's Crust and of strong interest for high-end technologies due to their specific properties. They belong to the Technology Critical Elements (TCEs) for which use is forecast to increase, implying growing emissions into the environment in the following years. In particular, with the intensive use of platinum (Pt) in car catalytic converters, the anthropogenic geochemical cycle of this element has surpassed the natural cycle. Yet, environmental Pt levels are still in the sub picomolar range, making its analytical detection a challenge. Few studies cover the behavior of Pt in marine waters in terms of speciation, reactivity and possible transfer to the biota. In this study, oysters (Crassostrea gigas) from an unpolluted estuary were exposed to the stable isotope 194Pt in seawater at a range of concentrations during 35days. Seawater was renewed daily and spiked to three nominal Pt concentrations (50, 100, and 10,000ng·L-1) for two replicate series. In addition, control conditions were monitored. Five oysters from each tank were dissected after 3, 7, 14, 21, 28, 35days of Pt exposure, and analyzed by ICP-MS. Accuracy of this analytical method applied to biological matrix was checked by an inter-method comparison with a voltammetrical technique. A concentration-dependent accumulation of Pt in oysters increasing with exposure time occurred. After 28days, oyster Pt accumulation from low and intermediate exposure conditions reached a plateau. This was not the case of the highest exposure condition for which oyster tissues showed increasing concentrations until the last day of the experiment. A linear correlation exists between seawater concentrations and Pt content in oysters for low and intermediate exposure concentrations i.e. closer to environmental concentrations. By showing high Pt accumulation potential, oysters may serve as sentinels, ensuring biomonitoring of Pt concentrations in marine coastal waters.

Historical record of trace elements (1983-2007) in scales from Atlantic salmon (Salmo salar): Study of past metal contamination from a copper mine (Ulla River, NW Iberian Peninsula).

The chemical composition of fish scales has been reported to reflect the composition of the waters in which fish have been resident, therefore having the potential for the assessment of temporal trends in watershed water quality. Here we studied the historical (1983-2007) metal contamination in the Ulla river (NW Iberian Peninsula) watershed - impacted by a Cu mine that was in operation from 1973 until 1988 - by means of the analysis of major and trace elements in salmon scales. Results indicate the presence of a significant contamination for several metals (especially Cu, Au, Ag, Sb, Zn) during the 1980's. Concentrations of Cu in salmon scales during the influence of the mine (1983-1990) were 20 ± 5 µg/g, exceeding the values for the recent years (1995-2007): 1.8 ± 0.4 µg/g. Concentrations for Au in these two periods were 31 ± 12 and 2.1 ± 1.2 ng/g; for Ag: 21 ± 4 and 4 ± 2 ng/g; for Sb: 48 ± 21 and 15 ± 4 ng/g; and for Zn: 133 ± 16 and 93 ± 10 µg/g. The estimated concentrations of dissolved copper during the operation of the mine indicate a scenario of toxic effects due to sensory impairments in the salmon, and a reduction in scales calcification. The results presented here demonstrate that the analysis of trace elements in archived fish scales is a suitable tool for the reconstruction of the past contamination in aquatic systems, and it can be also used as a non-lethal approach for biomonitoring purposes.

Improved voltammetric method for simultaneous determination of Pt and Rh using second derivative signal transformation - application to environmental samples.

The determination of Platinum-group elements (PGE) in relevant environmental matrices is a challenging task. Sensitive and accurate analytical procedures for simultaneous determination of Pt and Rh are still needed. In this study, we report for the first time on the use of second derivative signal transformation to the ultra-trace simultaneous determination of Pt and Rh by Adsorptive Cathodic Stripping Voltammetry (AdCSV). With that step, the ill-defined peaks typically observed in the original voltammograms are transformed into well-shaped peaks, resulting in accurate detection. The experimental conditions were investigated and optimised: a suitable electrolyte for both elements, with less reagents consumption, (0.25M H2SO4, 0.05M HCl, 0.01M FA and 0.5mM HZ), deposition time (td) and deposition potential (Ed). For td = 120s and Ed = -0.75V, linear relationships r > 0.999 were obtained in the concentration range up to 5.8ngL-1 (27 pM) for Pt and up to 3.4ngL-1 (34 pM) for Rh. Limits of detection were 0.2ngL-1 for Pt and 0.08ngL-1 for Rh. Lower values can be achieved by increasing the deposition time. Limits of quantification, LOQ, calculated as 3 times LOD, were 0.5ngL-1 for Pt and 0.2ngL-1 for Rh. The sensitivity of Pt was affected by elevated Zn concentrations, whereas a minor effect was observed for Rh. However, Pt and Rh determinations were not influenced using the standard addition method. Precision as intermediate precision and expressed as relative standard deviation, based on Pt and Rh spiked solutions and digested road dust CRM BCR-723 was 17% and 20% for Pt and Rh, respectively. Recoveries of CRM were around 90% for both elements. The method was successfully applied in the simultaneous determination of Pt and Rh in sediments from Tagus estuary and, for the first time, dissolved Rh was determined in water samples of a waste water treatment plant. Application of this technique in a multidisciplinary approach will be a relevant contribution to the current understanding of PGE cycle and fate in the environment.

Osmium and Platinum Decoupling in the Environment: Evidences in Intertidal Sediments (Tagus Estuary, SW Europe).

Catalytic converters in automobiles have significantly increased the input of platinum group elements (PGE) to the environment, and their coupled geochemical behavior has been proposed. To check this hypothesis, Pt and Os concentrations and (187)Os/(188)Os ratios were determined in sediment cores and interstitial waters from the Tagus Estuary (SW Europe) affected by different traffic pressure. Platinum concentration in surface sediments nearby the high traffic zone (up to 40 ng g(-1)) indicated severe contamination. Although lower than Pt, Os enrichment was also observed in surface sediments, with lower (187)Os/(188)Os ratios than in deeper layers. Dissolved Pt and Os in interstitial waters, 0.1-0.7 pg g(-1) and 0.03-0.10 pg g(-1), respectively, were higher than in typical uncontaminated waters. Results indicate two sources of Pt and Os into the Tagus Estuary salt marshes: a regional input associated with industrial activities, fossil fuel combustions, and regional traffic and a local source linked to nearby traffic density emissions. Estimations of Os and Pt released by catalytic converters support this two-source model. Differences in geochemical reactivity and range of dispersion from their sources lead to a decoupled behavior of Os and Pt, questioning the use of Os isotopes as proxies of PGE sources to the environment.

Evidence of increased anthropogenic emissions of platinum: time-series analysis of mussels (1991-2011) of an urban beach.

The anthropogenic emissions of Pt to the environment have increased significantly over the past decades, especially after the introduction of the catalytic converters in motor vehicles. In order to check whether this is affecting the levels of this trace metal on living organisms, time-series analysis of freeze-dried soft tissue material of wild mussels (Mytilus galloprovincialis) covering the period from 1991 to 2001 and collected at an urban beach in the city of Vigo (NW Iberian Peninsula) was conducted. Concentrations ranged from 0.30 to 0.68 ng g(-1) with an average concentration of 0.47 ± 0.10 ng g(-1) (n=21); these concentrations were higher than those obtained for samples collected at a control location away from anthropogenic pressure (0.31 ± 0.10 ng g(-1); n=5). Platinum concentrations followed a statistically significant temporal trend (at the 0.020 level), and the excess of Pt in mussels over the 1991-2011 period compared to the control location were correlated with the European Pt autocatalyst demand (p=0.0006) and, especially, the car sales in Spain (p=0.0001). A bioaccumulation factor of ~5·10(3) was derived, which is greater than those previously calculated for Pt from exposure experiments, but 1-2 orders of magnitude lower than other trace elements (e.g. Zn, Cu, Pb, Cd).

Improving the voltammetric quantification of ill-defined peaks using second derivative signal transformation: example of the determination of platinum in water and sediments.

The determination of trace elements using stripping voltammetry may be seriously affected by the presence of intensive matrix background or interfering peaks, leading to poorer detection limits and/or inaccurate quantitative results. In this work, we have tested the use of signal transformation (e.g., second derivative) in the analysis of platinum in seawater and sediment digests by means of catalytic adsorptive stripping voltammetry. In natural waters, the limit of detection of Pt is affected by a broad background wave due to the formazone complex used in the sample matrix for its determination, while in sediment digests, the Pt peak may be interfered with due to the presence of elevated concentrations of Zn, affecting the accuracy of the determination. Results applying second derivative signal transformation revealed a significant improvement (2-3-fold) of the detection limit in water due to the minimization of background effects, therefore allowing shorter accumulation times and faster determinations. In the presence of interfering peaks, the inaccuracy resulting from erroneous baseline selection in the original signal is eliminated when the second derivative is used. Signal processing should be considered as a useful tool for other voltammetric methodologies where more accurate or faster determinations are needed.