Bacterial removal and protozoan grazing in biological sand filters

The objective of the study was to investigate the importance of protozoan predation as a biological removal mechanism in sand filters used for purification of bacteria from wastewater. Eleven sand filter columns were seeded with a high dose of wastewater (70 mm d(-1)) and a high concentration (10(8) colony forming units [CFU] mL(-1)) of Aeromonas hydrophila (American Type Culture Collection [ATCC] 14715) for a period of 30 d. Water samples from three filter outlets were analyzed for the concentration of A. hydrophila. In addition, one filter column was sacrificed each sampling day for the quantification of A. hydrophila, culturable bacteria (heterotrophic plate counts, HPC), total bacterial counts, and protozoa in the sand. The mean removal efficiency of A. hydrophila in the sand filter columns was 4.7 log units. The concentration of A. hydrophila in the sand filter effluent, however, had a clearly time-dependent pattern from high (log 6) and unstable concentrations to low and more stable levels (log 2). The removal efficiency of A. hydrophila correlated significantly (P = 0.0005, r2 = 0.6) with numbers of protozoa in the sand filters. Significantly higher (P < 0.05) concentrations of A. hydrophila were observed in sand filter effluents from columns treated with the protozoan inhibitor cycloheximide, compared with nontreated columns. Results from the present study show that protozoan grazing plays an important role as a bacterial removal mechanism in sand infiltration systems.     

Cadmium and zinc in vegetation and litter of a voluntary woodland that has developed on contaminated sediment-derived soil

Vegetation that develops spontaneously on metal-contaminated soils presents an opportunity to evaluate both metal bioavailability and the risks posed to biota. The behavior of Cd and Zn in the species of a spontaneously developed woodland, colonizing a canal embankment, has been investigated. Nitric-acid-extractable metal concentrations in the sediment-derived substrate ranged between 5.0 to 376 mg kg(-1)dry wt. Cd and 83.0 to 784 mg kg(-1)dry wt. Zn. The woodland is dominated by Willow (Salix) species. Salix caprea selectively accumulated Cd in all stem tissues, in contrast to S. viminalis, which regulated tissue Cd content. Both species showed an effective regulation of tissue Zn. Cadmium uptake by S. caprea was correlated with differences in soil pH, while Zn uptake was not. There was no relationship between tissue metal concentrations and soil metal nitric acid-extractable concentrations. Other aspects of ecosystem function appeared unaffected by the elevated Cd flux in S. caprea; leaf litter organisms present represented all major groups and there was no accumulation of organic matter. The woodland represents a potentially sustainable option for remediating a low value site with difficult access that does not involve removal of the contaminated material to a landfill or making a permanent inert cover.     

Multivariate statistics to investigate metal contamination in surface soil

The modification of soil composition in the urbanized area of Ankara due to wet-dry deposition and pollution-derived particles from the atmosphere is investigated by analyzing 120 surface soil samples, collected from the urbanized area and its un-urbanized surrounding, for major, minor and trace elements. Concentrations of elements from human activity (e.g. Cd, Pb, Cr, Zn, Cu and Ca) in the urbanized area were higher than their corresponding concentrations in global average soil and soil in un-urbanized areas outside the urbanized area. Metal contents in soil were very high in densely populated districts and around some industrial facilities. The only exception was Pb distribution, which was more dispersed, due to the nature of motor vehicle emissions. Alteration of the Cd, Zn, Cu and Cr content of soil was confined to the inhabited and industrial areas, whereas enrichment factors of these elements were close to unity in the remaining study area. Factor analysis identified two polluted soil factor associations. One factor includes elements, such as Zn and Cd, which had high factor scores in inhabited areas and the other factor (high loading of Pb) represents soil polluted by motor vehicle emissions.     

Landfill cover soil, soil solution, and vegetation responses to municipal landfill leachate applications

Municipal solid waste landfill leachate must be removed and treated to maintain landfill cover integrity and to prevent contamination of surface and ground waters. From 2003 to 2007, we studied an onsite disposal system in Ottawa County, Michigan, where leachate was spray irrigated on the vegetated landfill cover. We established six 20-m-diameter circular experimental plots on the landfill; three were spray irrigated as part of the operational system, and three remained as untreated control plots. We quantified the effects of leachate application on soil properties, soil solution chemistry, vegetative growth, and estimated solute leaching. The leachate had high mean levels of electrical conductivity (0.6-0.7 S m(-1)), Cl (760-900 mg L(-1)), and NH(4)-N (290-390 mg L(-1)) but was low in metals and volatile organic compounds. High rates of leachate application in 2003 (32 cm) increased soil electrical conductivity and NO(3)-N leaching, so a sequential rotation of spray areas was implemented to limit total leachate application to <9.6 cm yr(-1) per spray area. Concentrations of NO(3)-N and leaching losses remained higher on irrigated plots in subsequent years but were substantially reduced by spray area rotation. Leachate irrigation increased plant biomass but did not significantly affect soil metal concentrations, and plant metal concentrations remained within normal ranges. Rotating spray areas and timing irrigation to conform to seasonal capacities for evapotranspiration reduced the localized impacts of leachate application observed in 2003. Careful monitoring of undiluted leachate applications is required to avoid adverse impacts to vegetation or soils and elevated solute leaching losses.     

Impact of grazing on soil in the southern guinea savanna zone of Nigeria

Summary This study examines the effects of traditional sedentary grazing on soils in the southern guinea savanna ecosystem in Nigeria. The characteristics of soil in grazed plots are compared with those of similar soil in ungrazed plots in a savanna forest reserve in a nearby locality, in order to infer the effects of grazing. In the 0–10 cm layer of the soil, organic carbon, total nitrogen, exchangeable calcium, magnesium, potassium, sodium, cation exchange capacity and available phosphorus levels are significantly lower in the grazed plots. Decline in the organic carbon and nutrient levels of the grazed plots is mainly due to soil exposure resulting from grazing and savanna burning and the attendant processes of accelerated organic matter decomposition and nutrient loss through leaching and erosion. Low soil nutrient levels in the grazed plots will reduce primary production and hence the rate of herbage production for livestock. It could also lead to some adverse ecological changes in the ecosystem with the disappearance of certain plant species. The ecosystem consequently becomes less diverse and resilient as soil nutrient status becomes increasingly impoverished. It is suggested that herdsmen should practise rotational grazing and that livestock should be fed with a supplementary fodder of legumes, such as Gliricidia sepiumand Leucaena leucocephalaplanted in natural grazing areas, in order to minimise the problems of overgrazing which are frequently experienced during the dry season. Dr A.O. Aweto is a Senior Lecturer in Biogeography in the Department of Geography at the University of Ibadan, and Mr D.O. Adejumobi is a geographer serving on the Nigerian National Youth Service Corps.     

RDX loss in a surface soil under saturated and well drained conditions

On military training ranges, low-order, incomplete detonations deposit RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) into surface soils. In this study, we evaluated RDX biodegradation in surface soils obtained from a military training range in Alaska. Two factors were compared: (i) soil water potential during the incubations; and (ii) the use of acetonitrile (ACN) as an RDX carrier to spike samples. Organic solvents have been used in laboratory studies to dissolve slightly water-soluble contaminants before addition to soil. We added ACN to obtain final soil ACN concentrations of 0 mg kg(-1) (0%), 1000 mg kg(-1) (0.1%) and 10 000 mg kg(-1) (1%). We then compared RDX attenuation in the soil under saturated and unsaturated conditions. RDX fell below the limit of detection within 3 wk of study initiation under the saturated condition. A maximum degradation rate of 0.15 mg RDX L(-1) d(-1) was measured. Under the unsaturated condition, 42% of the original RDX was still present at study termination (5 wk). The addition of acetonitrile at 0.1 or 1.0% had no affect on RDX loss in the saturated soil. In the unsaturated soil, however, ACN at 1.0% inhibited RDX loss by as much as 25%. These findings indicate that soil water potential and carrier solvent concentrations can impact the rate and extent to which RDX is attenuated in a surface soil.     

Analysis of soils to demonstrate sustained organic carbon removal during soil aquifer treatment

Soil samples from column studies using five soil types and from a field site were analyzed to assess the ability of soil aquifer treatment to sustain removal of organic carbon. The soil types used in the column studies were chosen to represent a wide range of soil properties that might be used for soil aquifer treatment. Soil samples were analyzed for total organic matter, and a subset of samples was sequentially extracted to determine the effects of soil aquifer treatment. For both column studies and the field site, no accumulation of organic matter was observed below a depth of 8 cm. Near the surface, biological activity at the soil-water interface resulted in an accumulation of biomass and associated organic matter. For the column studies, the accumulation of organic matter in the top 8 cm of soil was <20% of the total organic matter applied to the columns. Soils at depths greater than 8 cm had total organic matter levels less than the original soils before soil aquifer treatment. Significant changes in extractable iron and manganese oxides were observed at the field site, which had been in operation for >10 yr with extended periods of low redox conditions. However, these changes had no apparent effect on the removal of organic carbon in the system. This study provides evidence that soil aquifer treatment can remove organic carbon without accumulation from adsorption that might eventually lead to breakthrough.     

Storage and dynamics of carbon and nitrogen in soil after grazing exclusion in Leymus chinensis grasslands of northern China

Land-use change can lead to changes in soil carbon (C) and nitrogen (N) storage. This study aimed to determine the impact of long-term grazing exclusion (GE) on soil organic C and total N (TN) storage in the Leymus chinensis grasslands of northern China and to estimate the dynamics of recovery after GE. We investigated the aboveground biomass and soil organic C and TN storage in six contiguous plots along a GE chronosequence comprising free grazing, 3-yr GE, 8-yr GE, 20-yr GE, 24-yr GE, and 28-yr GE. Grazing exclusion for two decades increased the soil C and N storage by 35.7 and 14.6%, respectively, in the 0- to 40-cm soil layer. The aboveground net primary productivity and soil C and N storage were the highest with 24-yr GE and the lowest with free grazing. The storage increased logarithmically with the duration of GE; after an initial rapid increase after the introduction of GE, the storage attained equilibrium after 20 yr. A logarithmic regression analysis revealed 86.8 and 87.1% variation in the soil C storage and 74.2 and 80.7% variation in the soil N storage in the 0- to 10-cm and 0- to 40-cm soil layers, respectively. Based on these results, we suggest that two decades of GE would restore the L. chinensis grasslands from being lightly degraded to a stable productive condition with good soil C and N storage capacity. Our results demonstrated that by implementing GE, the temperate grasslands of northern China could facilitate significant C and N storage on decade scales in the context of mitigating global climate change.     

Fatty acid methyl ester analysis to identify sources of soil in surface water

Efforts to improve land-use practices to prevent contamination of surface waters with soil are limited by an inability to identify the primary sources of soil present in these waters. We evaluated the utility of fatty acid methyl ester (FAME) profiles of dry reference soils for multivariate statistical classification of soils collected from surface waters adjacent to agricultural production fields and a wooded riparian zone. Trials that compared approaches to concentrate soil from surface water showed that aluminum sulfate precipitation provided comparable yields to that obtained by vacuum filtration and was more suitable for handling large numbers of samples. Fatty acid methyl ester profiles were developed from reference soils collected from contrasting land uses in different seasons to determine whether specific fatty acids would consistently serve as variables in multivariate statistical analyses to permit reliable classification of soils. We used a Bayesian method and an independent iterative process to select appropriate fatty acids and found that variable selection was strongly impacted by the season during which soil was collected. The apparent seasonal variation in the occurrence of marker fatty acids in FAME profiles from reference soils prevented preparation of a standardized set of variables. Nevertheless, accurate classification of soil in surface water was achieved utilizing fatty acid variables identified in seasonally matched reference soils. Correlation analysis of entire chromatograms and subsequent discriminant analyses utilizing a restricted number of fatty acid variables showed that FAME profiles of soils exposed to the aquatic environment still had utility for classification at least 1 wk after submersion.     

Comparisons of soil surface sealing methods to reduce fumigant emission loss

State and federal regulatory agencies depend on quality field data for determining the effects of agricultural management practices on fumigant emissions to develop sound, science-based policies and regulations on preplant soil fumigants. Field plot tests, using growers' standard field operation procedures, were used to simultaneously determine the effectiveness of several commonly proposed emission reduction methods, in a trial involving shank injection of Telone II [a.i. 1,3-dichloropropnene (1,3-D)] to a sandy loam soil to a target rate of 372 kg ha(-1). The experiment was conducted in late September 2008 in the San Joaquin Valley of California. Fumigant emissions were captured using dynamic flux chambers. The results showed that virtually impermeable film (VIF) reduced emissions >95% when compared to bare soil, and the glue joints in the film did not significantly affect the tarp performance. The VIF also created a more uniform distribution of gaseous fumigant in the soil profile, which would likely benefit pest control efficacy. Standard high-density polyethylene (HDPE) tarp reduced total 1,3-D emissions about 50% (higher than most reported values) in this trial, whereas postfumigation intermittent water treatments (seals) reduced cumulative emission losses by approximately 20%. Adding 49.4 Mg ha (equivalent to 20 tons per acre) of composted dairy manure to surface soils did not reduce 1,3-D emissions during this experiment. Use of VIF was the most promising technique in reducing emissions and has the potential to allow lower application rates while providing satisfactory pest control.     

Simple protocols to determine dust potentials from cattle feedlot soil and surface samples

Cattle feedlot dust is an annoyance and may be a route for nutrient transport, odor emission, and pathogen dispersion, but important environmental factors that contribute to dust emissions are poorly characterized. A general protocol was devised to test feedlot samples for their ability to produce dust under a variety of environmental conditions. A blender was modified to produce dust from a variety of dried feedlot surface and soil samples and collect airborne particles on glass fiber filters by vacuum collection. A general blending protocol optimized for sample volume (150-175 cm3), blending time (5 min of pre-blending), and dust collection time (15 s) provided consistent dust measurements for all samples tested. The procedure performed well on samples that varied in organic matter content, but was restricted to samples containing less than 200 to 700 g H2O kg(-1) dry matter (DM). When applied to field samples, the technique demonstrated considerable spatial variability between feedlot pen sites. Mechanistically, dust potential was related to moisture and organic matter content. An alternative protocol also demonstrated differences within pen sites in maximum dust potential and dust airborne residence time. The two protocols were not intended, nor are they suitable, for predicting actual particulate matter emissions from agricultural sources. Rather, the protocols rapidly and inexpensively compared the potential for dust emission from samples of differing composition under a variety of environmental conditions.     

Nitrate removal in riparian wetlands: interactions between surface flow and soils

Riparian wetlands containing springs are thought to be ineffective at removing nitrate because contact times between the upwelled ground water and the underlying microbially active soils are short. Tracer experiments using lithium bromide (LiBr) and nitrate (NO3-N) injected at the surface were used to quantify residence times and NO3-N removal in a riparian swale characteristic of New Zealand hill-country pasture. An experimental enclosure was used with collecting trays at the downstream end to measure flow and concentration, shallow wells to measure subsurface concentrations, and an array of logging conductivity probes to monitor tracer continuously. The majority of added tracer reached the outlet more slowly than could be explained by surface flow, but more quickly than could be explained by Darcy seepage flow. There was evidence from the wells of tracer diffusing vertically to a depth of at least 5 cm into the surface soil layer, which was permanently saturated and highly porous. During dry weather 24 +/- 9% of added NO3-N was removed over a distance of 1.5 m largely by denitrification. The net uptake length coefficient for this wetland (K = 0.08 +/- 0.03 m(-1)) is slightly higher than the range (K = 0.01-0.07 m(-1)) measured in a small stream channel infested with macrophytes. Nitrate removal is expected to decrease with increasing flow. Seepage flow is estimated to have removed only 7 +/- 4% of the added NO3-N and we hypothesize that vertical diffusion substantially increases NO3-N removal in this type of wetland. Riparian wetlands with springs and surface flows should not be dismissed as having low NO3-N removal potential without checking whether there is significant vertical mixing.     

Flocculant in wastewater affects dynamics of inorganic N and accelerates removal of phenanthrene and anthracene in soil

Recycling of municipal wastewater requires treatment with flocculants, such as polyacrylamide. It is unknown how polyacrylamide in sludge affects removal of polycyclic aromatic hydrocarbons (PAH) from soil. An alkaline-saline soil and an agricultural soil were contaminated with phenanthrene and anthracene. Sludge with or without polyacrylamide was added while emission of CO₂ and concentrations of NH₄⁺, NO₃⁻, NO₂⁻, phenanthrene and anthracene were monitored in an aerobic incubation experiment. Polyacrylamide in the sludge had no effect on the production of CO₂, but it reduced the concentration of NH₄⁺, increased the concentration of NO₃⁻ in the Acolman soil and NO₂⁻ in the Texcoco soil, and increased N mineralization compared to the soil amended with sludge without polyacrylamide. After 112 d, polyacrylamide accelerated the removal of anthracene from both soils and that of phenanthrene in the Acolman soil. It was found that polyacrylamide accelerated removal of phenanthrene and anthracene from soil.     

Field-scale variation in microbial activity and soil properties in relation to mineralization and sorption of pesticides in a sandy soil

Pesticides applied to agricultural soils are subject to environmental concerns because leaching to groundwater reservoirs and aquatic habitats may occur. Knowledge of field variation of pesticide-related parameters is required to evaluate the vulnerability of pesticide leaching. The mineralization and sorption of the pesticides glyphosate and metribuzin and the pesticide degradation product triazinamin in a field were measured and compared with the field-scale variation of geochemical and microbiological parameters. We focused on the soil parameters clay and organic carbon (C) content and on soil respiratory and enzymatic processes and microbial biomass. These parameters were measured in soil samples taken at two depths (Ap and Bs horizon) in 51 sampling points from a 4-ha agricultural fine sandy soil field. The results indicated that the spatial variation of the soil parameters, and in particular the content of organic C, had a major influence on the variability of the microbial parameters and on sorption and pesticide mineralization in the soil. For glyphosate, with a co-metabolic pathway for degradation, the mineralization was increased in soils with high microbial activity. The spatial variability, expressed as the CV, was about five times higher in the Bs horizon than in the Ap horizon, and the local-scale variation within 100 m(2) areas were two to three times lower than the field-scale variation within the entire field of about 4 ha.     

Evaluation of phosphorus transport in surface runoff from packed soil boxes

Evaluation of phosphorus (P) management strategies to protect water quality has largely relied on research using simulated rainfall to generate runoff from either field plots or shallow boxes packed with soil. Runoff from unmanured, grassed field plots (1 m wide x 2 m long, 3-8% slope) and bare soil boxes (0.2 m wide and 1 m long, 3% slope) was compared using rainfall simulation (75 mm h(-1)) standardized by 30-min runoff duration (rainfall averaged 55 mm for field plots and 41 mm for packed boxes). Packed boxes had lower infiltration (1.2 cm) and greater runoff (2.9 cm) and erosion (542 kg ha(-1)) than field plots (3.7 cm infiltration; 1.8 cm runoff; 149 kg ha(-1) erosion), yielding greater total phosphorus (TP) losses in runoff. Despite these differences, regressions of dissolved reactive phosphorus (DRP) in runoff and Mehlich-3 soil P were consistent between field plots and packed boxes reflecting similar buffering by soils and sediments. A second experiment compared manured boxes of 5- and 25-cm depths to determine if variable hydrology based on box depth influenced P transport. Runoff properties did not differ significantly between box depths before or after broadcasting dairy, poultry, or swine manure (100 kg TP ha(-1)). Water-extractable phosphorus (WEP) from manures dominated runoff P, and translocation of manure P into soil was consistent between box types. This study reveals the practical, but limited, comparability of field plot and soil box data, highlighting soil and sediment buffering in unamended soils and manure WEP in amended soils as dominant controls of DRP transport.     

Fate of urine nitrogen in three soils throughout a grazing season

The fate of 15N-labeled cattle (Bos taurus) urine (52 g N m(-2)), applied to a 0.4-m2 surface area on three dates between May and October to three different pasture soils, was studied using 2-m2 lysimeters. Over a period of two years, the sward recovered most of the 15N, but the amount recovered decreased with application date (62% in spring to 17% in fall). However, N uptake by ryegrass (Lolium perenne L.) in Year 2 showed that some nitrogen came from the previous year's urine application. The largest leaching losses of urine N resulted from the late application date. These losses mainly occurred during the first winter despite the small amount of water drainage. Soil type largely determined 15N losses. The granitic Brunisol was the most freely draining and had the greatest leaching (up to 35% recovery of urinary N). In contrast, leaching in the silty loam Neoluvisol remained under 4% of 15N applied. The Calcosol appeared to be susceptible to all kinds of N losses with intermediate unaccounted-for N pool and leaching fractions and lesser utilization of urinary N by grass. Immobilization in soil organic matter, roots and litter, and stubble pools were not markedly influenced by the date of application or soil type. They amounted to 25 to 33, 2, and 2% of N applied as urine, respectively. In these climatic conditions with moderate drainage, leaching of water poor in quality for nitrate only occurred for late-season grazing or on the granitic Brunisol, which was very vulnerable to leaching.     

Phosphorus leaching in relation to soil type and soil phosphorus content

Phosphorus losses from arable soils contribute to eutrophication of freshwater systems. In addition to losses through surface runoff, leaching has lately gained increased attention as an important P transport pathway. Increased P levels in arable soils have highlighted the necessity of establishing a relationship between actual P leaching and soil P levels. In this study, we measured leaching of total phosphorus (TP) and dissolved reactive phosphorus (DRP) during three years in undisturbed soil columns of five soils. The soils were collected at sites, established between 1957 and 1966, included in a long-term Swedish fertility experiment with four P fertilization levels at each site. Total P losses varied between 0.03 and 1.09 kg ha(-1) yr(-1), but no general correlation could be found between P concentrations and soil test P (Olsen P and phosphorus content in ammonium lactate extract [P-AL]) or P sorption indices (single-point phosphorus sorption index [PSI] and P sorption saturation) of the topsoil. Instead, water transport mechanism through the soil and subsoil properties seemed to be more important for P leaching than soil test P value in the topsoil. In one soil, where preferential flow was the dominant water transport pathway, water and P bypassed the high sorption capacity of the subsoil, resulting in high losses. On the other hand, P leaching from some soils was low in spite of high P applications due to high P sorption capacity in the subsoil. Therefore, site-specific factors may serve as indicators for P leaching losses, but a single, general indicator for all soil types was not found in this study.     

Simulating sulfadimidine transport in surface runoff and soil at the microplot and field scale

To prevent residues of veterinary medicinal products (VMPs) from contaminating surface waters and ground water, an environmental impact assessment is required before a new product is allowed on the market. Physically based simulation models are advocated for the calculation of predicted environmental concentrations at higher tiers of the assessment process. However, the validation status of potentially useful models is poor for VMP transport. The objective of this study was to evaluate the dual-permeability model MACRO for simulation of transport of sulfonamide antibiotics in surface runoff and soil. Special focus was on effects of solute application in liquid manure, which may alter the hydraulic properties at the soil surface. To this end we used data from a microplot runoff experiment and a field experiment, both conducted on the same clay loam soil prone to preferential flow. Results showed that the model could accurately simulate concentrations of sulfadimidine and the nonreactive tracer bromide in runoff and in soil from the microplot experiments. The use of posterior parameter distributions from calibrations using the microplot data resulted in poor simulations for the field data of total sulfadimidine losses. The poor results may be due to surface runoff being instantly transferred off the field in the model, whereas in reality re-infiltration may occur. The effects of the manure application were reflected in smaller total and micropore hydraulic conductivities compared with the application in aqueous solution. These effects could easily be accounted for in regulatory modeling.     

Effect of mixing soil aggregates on the phosphorus concentration in surface waters

At any time, the phosphorus (P) concentration in surface waters is determined by a complex interaction of inputs of soluble P and sorption-desorption reactions of P with sediments. This study investigated what factors control P in solution when various soil aggregates were mixed, seen as being analogous to selective soil erosion events, transport, and mixing within river systems. Fifteen soils with widely differing properties were each separated into three aggregate size fractions (2-52 microm, 53-150 microm, and 151-2,000 microm). Resin P, water-soluble phosphorus (WSP), and the phosphorus buffer capacity (PBC = resin P/WSP) were measured for each aggregate size fraction and WSP was also measured for 11 mixes of the aggregate fractions. The smallest aggregates tended to be enriched with resin P relative to the larger aggregates and the whole soils, while the opposite was true for WSP. As the PBC was a function of resin P and WSP, the PBC was greatest in the 2- to 52-microm aggregate size fraction in most cases. When two aggregate size fractions were mixed, the measured WSP was always lower than the predicted WSP (i.e., the average of the WSP in the two individual aggregates), indicating that WSP released by one aggregate fraction could be resorbed by another aggregate fraction. This resorption of P may result in lower than expected solution P concentration in some surface waters. The strength with which an eroded aggregate can release or resorb P to or from solution is in part determined by that aggregate's PBC.