The effects of ammonium sulphate and urea upon egg hatching and miracidial survival of Schistosoma mansoni

Abstract

The effects of various concentrations of ammonium sulphate and urea on egg hatching and miracidial survival of S. mansoni were tested in order to determine if the use of these fertilizers in the ricelands of the Republic of Cameroon could affect the transmission of schistosomiasis. Results indicate that hatching of eggs and survival of miracidia varied with concentrations of tested chemicals and times of exposure. Exposure of S. mansoni eggs to 0.20%‐1.00% ammonium sulphate or to 0.50%‐4.00% urea reduced their ability to hatch and produce miracidia. A chemical concentration of 1.00% ammonium sulphate or 4.00% urea was found to be sufficient to produce complete inhibition of hatching. High concentrations of both chemicals not only inhibited miracidial emmergence but also may be ovocidal. Results obtained from miracidial survival tests indicated that LC₅, LC₅₀ and LC₉₅ values for ammonium sulphate were 0.07%, 0.80% and 10.61% after 2 hours of exposure; 0.03%, 0.16% and 0.90% after 4 hours of exposure; and 0.30%, 0.20% and 0.40% after 6 hours of exposure respectively. Similar statistical analyses revealed that the LC₅, LC₅₀ and LC₉₅ values for urea were 0.22%, 1.90% and 16.25% after 2 hours of exposure; 0.28%, 0.57% and 1.14% after 4 hours of exposure; and 0.13%, 0.27% and 0.57%” after 6 hours of exposure respectively. Although the two fertilizers exerted some adverse effects on S. mansoni eggs and miracidia at relatively high concentrations, neither of them was found to be of practical value. Ammonium sulphate and urea concentrations effective in killing S. mansoni eggs and miracidia were about one to two orders of magnitude greater than the actual field application rates.     

The effects of bayluscide and malathion on the survival of Schistosoma mansoni miracidia

Laboratory experiments were conducted to assess the toxic effects of bayluscide and malathion against Schistosoma mansoni miracidia. The results indicate that survival of miracidia varied with times of exposure and concentrations of tested chemicals. Statistical analyses reveal that LC5, LC50 and LC95 for bayluscide were 0.04 ppm, 0.06 ppm and 0.12 ppm after 2 hours of exposure; 0.02 ppm, 0.03 ppm and 0.06 ppm after 4 hours of exposure; and 0.01 ppm, 0.02 ppm and 0.04 ppm after 6 hours of exposure respectively. These data indicate that bayluscide is much more toxic to the first stage larvae of schistosomes than to snail intermediate hosts cited in the literature. Application of lower concentrations of molluscicide in the transmission sites is thus expected to curtail the survival of miracidia; therefore controlling schistosomiasis at relatively low costs. Such applications also reduce the risk of toxicity to non target organisms present in the aquatic environment. Statistical analysis of the results of tests using malathion gave LC5, LC50 and LC95 values of 83.38 ppm, 153.11 ppm and 245.85 ppm after 2 hours of exposure; and 76.86 ppm, 116.48 ppm and 172.04 ppm after 4 hours of exposure respectively. These data indicate that the use of malathion as an insecticide in tropical ecosystems may also affect the survival and viability of schistosome miracidia. Such uses could help reducing the risk of schistosomiasis transmission in these particular locations.     

Methane and nitrous oxide emissions from an irrigated rice of North India

Upland rice was grown in the kharif season (June-September) under irrigated condition in New Delhi, India (28 degree 40'N and 77 degree 12'E) to monitor CH4 and N2O emission, as influenced by fertilizer urea, ammonium sulphate and potassium nitrate alone (at 120 kg ha-1) and mixed with dicyandiamide (DCD), added at 10% of applied N. The experimental soil was a typic ustochrept (Inceptisol), clay loam, in which rice (Oryza sativa L., var. Pusa-169, duration: 120-125 days) was grown and CH4 and N2O was monitored for 105 days by closed chamber method, starting from the 5 days and 1 day after transplanting, respectively. Methane fluxes had a considerable temporal variation (CV=52-77%) and ranged from 0.05 (ammonium sulphate) to 3.77 mg m-2 h-1 (urea). There was a significant increase in the CH4 emission on the application of fertilizers while addition of DCD with fertilizers reduced emissions. Total CH4 emission (105 days) ranged from 24.5 to 37.2 kg ha-1. Nitrous oxide fluxes were much lower than CH4 fluxes and had ranged from 0.18 to 100.5 g m-2 h-1 with very high temporal variation (CV=69-143%). Total seasonal N2O emission from different treatments ranged from 0.037 to 0.186 kg ha-1 which was a N loss of 0.10-0.12% of applied N. All the fertilizers significantly increased seasonal N2O emission while application of DCD reduced N2O emissions significantly in the range of 10-53%.     

Toxicity of acid-sulphate soil leachate and aluminium to the embryos and larvae of Australian bass (Macquaria novemaculeata) in estuarine water

The toxicity of leachate water from acid-sulphate soil to the early life stages of Australian bass, Macquaria novemaculeata, incubated in seawater was evaluated. Acid-sulphate soil leachate water (pH> or =6.8) delayed the hatching of fertilised eggs, but after 48 h the per cent hatching was normal. In comparison, acidic saline water (25 per thousand salinity) at pH 4.0 or less prevented embryos from hatching. The survival of yolk-sac larvae exposed to acid-sulphate soil leachate water at a concentration of 32% in seawater and an initial pH of 7.2, was significantly different to controls after 96 hours. In corresponding tests with only acidified saline water (20 per thousand salinity), pH levels equal to or below 5.0 killed yolk-sac larvae after 96 h exposure. Aluminum showed a pH dependent toxicity to yolk-sac larvae, with added aluminium as low as 200 microg litre(-1) having a significant effect on larval survival at pH 5.5, and concentrations of 600-800 microg litre(-1) having a significant effect on larval survival at an initial pH range of 6.0 < pH < 6.8. It was concluded that significant mortality of the early life stages of Australian bass would occur if they are exposed to acid-sulphate soil leachate that results in a pH in the receiving estuarine water below 5.5, or when the pH is below 6.8 and aluminium is present at a total concentration of 800 microg litre(-1) or greater.     

Influence of the DMPP (3,4-dimethyl pyrazole phosphate) on nitrogen transformation and leaching in multi-layer soil columns

The application of nitrogen fertilizers leads to various ecological problems such as nitrate leaching. The use of nitrification inhibitors as nitrate leaching retardants is a proposal that has been suggested for inclusion in regulations in many countries. In this study, using a multi-layer soil column device, the influence of new nitrification inhibitor DMPP (3,4-dimethyl pyrazole phosphate) was studied for understanding the nitrogen vertical transformation and lowering the nitrate leaching at different soil profile depths. The results indicated that, within 60 d of experiment, the regular urea added 1.0% DMPP can effectively inhibit the ammonium oxidation in the soil, and improve the ammonium concentration in soil solution over the 20cm depths of soil profile, while decline the concentrations of nitrate and nitrite. No obvious difference was found on ammonium concentrations in soil solution collected from deep profile under 20cm depths between regular urea and the urea added 1.0% DMPP. There was also no significant difference for the nitrate, ammonium and nitrite concentrations in the soil solution under 40cm depths of soil profile with the increasing nitrogen application level, among the treatments of urea added 1.0% DMPP within 60 d. It is proposed that DMPP could be used as an effective nitrification inhibitor in some region to control ammonium oxidation and decline the ion-nitrogen leaching, minimizing the shallow groundwater pollution risk and being beneficial for the ecological environment.     

Effects of point-source PCB contamination on breeding performance and post-fledging survival in the dipper Cinclus cinclus

Despite widespread information on the incidence and biochemical effects of polychlorinated biphenyls (PCBs) in birds, field studies of effects on population processes are still scarce. This is particularly so in passerines. We therefore assessed breeding performance and post-fledging survival in dippers, Cinclus cinclus, breeding along the Afon Mule, a Welsh river where previous work indicated locally elevated PCB concentrations. We hypothesised that marked toxicological effects, including endocrine disruption, would be detectable as altered clutch-size, hatching success, nest success, brood-size, post-fledging survival or recruitment to the breeding population. Congener-specific analysis of PCBs in failed and deserted dipper eggs during 1990-93 revealed that geometric mean concentrations were four to 20 times higher along the Mule than elsewhere in Wales, UK. PCB signatures also varied highly significantly from other locations, with congeners 138, 153 and 180 dominant at the contaminated site, but 118 or 170 more frequent in eggs elsewhere. Subsequent analysis of surface runoff confirmed a small breaker's yard as a point source. Neither laying dates, clutch-sizes, brood-sizes, nest failure nor hatching failure along the contaminated Mule (n=46 breeding attempts and 218 eggs) differed significantly from dippers along adjacent reference rivers (n=82 breeding attempts and 315 eggs) or Welsh rivers as a whole (n=332 breeding attempts and 1534 eggs). Subsequent recaptures of breeding dippers ringed initially as nestlings were nearly identical between the Mule (7.7% of 182 nestlings), the reference set (7.5% of 323 nestlings) and all other Welsh rivers (7.3% of 2821 nestlings). These data illustrate the value of congener-specific analysis of bird's eggs in indicating local PCB sources. The data show also that PCBs can occur in dipper eggs at total concentrations of 0.49 (geometric mean)-1.29 (upper quartile) microg g(-1) wet mass without effects on breeding performance and survival. These values are below those at which biochemical or reproductive effects on other passerines have been detected, but above current concentrations in any other population of European dippers for which PCB data are available. Population effects by PCBs on European dippers are therefore unlikely. Survivors from locally contaminated sites like that in our study might allow field assessments of second-generation effects on breeding performance in wild birds that have developed under PCB exposure.     

Effect of water pH and salinity on the survival of eggs and larvae of the euryhaline teleost, Gasterosteus aculeatus L

The effect of water acidification and salinity on the survival of the eggs and larvae of the euryhaline teleost, Gasterosteus aculeatus, was studied using tap water that was low in calcium and high in total aluminium as the basic medium. The survival of eggs from fertilisation to hatching increased with an increase in pH from 4.5 to 6.5. Survival was higher at a given pH at higher salinities (equivalent to 5% and 10% seawater) than in tap water. The salinity experienced by the female prior to spawning also had an effect on egg survival. Survival for 7 days after hatching was high except at the lowest pH levels. The salinity experienced by the female had no significant effect on larval survival.     

Heavy metals incidence in the application of inorganic fertilizers and pesticides to rice farming soils

The concentrations of Cd, Co, Cu, Ni, Pb, Zn, Fe and Mn in different inorganic fertilizers (urea, calcium superphosphate, iron sulphate and copper sulphate) and in pesticides (two herbicides and one fungicide) are evaluated together with the contribution of these metals in soils from their use. The study was made in rice farming areas to the north of Albufera Natural Park (Valencia, Spain). The results obtained show that superphosphate is the fertilizer that contains the highest concentrations of Cd, Co, Cu and Zn as impurities. Copper sulphate and iron sulphate have the most significant concentrations of Pb, and are the only fertilizers in which Ni was detected. The three pesticides analysed show similar Cd contents and the highest levels of Fe, Mn, Zn, Pb and Ni are found in the herbicides. The most significant additions of heavy metals as impurities that soil receives from agricultural practices, are Mn, Zn, Co and Pb. Three contamination indexes have been applied to provide a basis for comparison of potential heavy metal toxicity. These results denote the potential toxicity of heavy metals in the studied soils.     

Effects of point-source PCB contamination on breeding performance and post-fledging survival in the dipper Cinclus cinclus

Despite widespread information on the incidence and biochemical effects of polychlorinated biphenyls (PCBs) in birds, field studies of effects on population processes are still scarce. This is particularly so in passerines. We therefore assessed breeding performance and post-fledging survival in dippers, Cinclus cinclus, breeding along the Afon Mule, a Welsh river where previous work indicated locally elevated PCB concentrations. We hypothesised that marked toxicological effects, including endocrine disruption, would be detectable as altered clutch-size, hatching success, nest success, brood-size, post-fledging survival or recruitment to the breeding population. Congener-specific analysis of PCBs in failed and deserted dipper eggs during 1990–93 revealed that geometric mean concentrations were four to 20 times higher along the Mule than elsewhere in Wales, UK. PCB signatures also varied highly significantly from other locations, with congeners 138, 153 and 180 dominant at the contaminated site, but 118 or 170 more frequent in eggs elsewhere. Subsequent analysis of surface runoff confirmed a small breaker's yard as a point source. Neither laying dates, clutch-sizes, brood-sizes, nest failure nor hatching failure along the contaminated Mule (n=46 breeding attempts and 218 eggs) differed significantly from dippers along adjacent reference rivers (n=82 breeding attempts and 315 eggs) or Welsh rivers as a whole (n=332 breeding attempts and 1534 eggs). Subsequent recaptures of breeding dippers ringed initially as nestlings were nearly identical between the Mule (7.7% of 182 nestlings), the reference set (7.5% of 323 nestlings) and all other Welsh rivers (7.3% of 2821 nestlings). These data illustrate the value of congener-specific analysis of bird's eggs in indicating local PCB sources. The data show also that PCBs can occur in dipper eggs at total concentrations of 0.49 (geometric mean)–1.29 (upper quartile) μg g⁻¹ wet mass without effects on breeding performance and survival. These values are below those at which biochemical or reproductive effects on other passerines have been detected, but above current concentrations in any other population of European dippers for which PCB data are available. Population effects by PCBs on European dippers are therefore unlikely. Survivors from locally contaminated sites like that in our study might allow field assessments of second-generation effects on breeding performance in wild birds that have developed under PCB exposure.     

Endosulfan and diazinon toxicity to the freshwater rotifer Brachionus calyciflorus.

The acute toxicity of endosulfan and diazinon to the freshwater rotifer Brachionus calyciflorus was determined after 24 hours exposure to these toxicants. The mean 24 hr-LC50 values were 5.15 and 29.22 mg/L for endosulfan and diazinon respectively. Based on these results, four sublethal concentrations were chosen to determined the median lethal time (LT50) at each concentration of toxicant tested. We also used a control with the solvent (acetone). The concentration tested were 1/5, 1/4, 1/2 and 2/3th LC50 (24hr) for both pesticides. We found a decrease in the median lethal time (LT50) with increasing pesticide concentrations. The LT50 values ranged from 6.49 to 3.48 days after endosulfan treatment, and from 6.96 to 2.49 days after diazinon exposure. No effects on survival were observed in control animals exposed to the solvent.     

Effects of nickel chloride and oxygen depletion on behaviour and vitality of zebrafish (Danio rerio, Hamilton, 1822) (Pisces, Cypriniformes) embryos and larvae

We examined acute (2 h exposure of 5-day-old larvae) and subchronic (exposure from fertilization up to an age of 11 days) effects of NiCl(2).6H2O on embryos and larvae of zebrafish (Danio rerio), both alone and in combination with oxygen depletion. The following endpoints were recorded: acute exposure: locomotory activity and survival; subchronic exposure: hatching rate, deformations, locomotory activity (at 5, 8 and 11 days) and mortality. In acute exposures nickel chloride (7.5-15 mg Ni/L) caused decreasing locomotory activity. Oxygen depletion (or=10 mg Ni/L resulted in delayed hatching at an age of 96 h, in decreased locomotory activity at an age of 5 days, and increased mortality at an age of 11 days (LC20=9.5 mg Ni/L). The observed LOEC for locomotory activity (7.5 mg Ni/L) is in the range of environmentally relevant concentrations. Since locomotory activity was already affected by acute exposure, this parameter is recommended to supplement commonly recorded endpoints of toxicity.     

Effect of ammonia on the survival of Zacco platypus (Temminck and Schlegel) at each developmental stage

The survival rates were studied of fertilized eggs, larvae, fry, and adults of pale chub Zacco platypus (Temminck and Schlegel) exposed to various concentrations of ammonia. There was a decrease in hatching rate and an increase in deformed alevins at a concentration of 0.9 mg liter(-1) of ammonia. All alevins were deformed at a concentration of 1.6 mg liter(-1) of ammonia and no eggs hatched at a concentration of 3.1 mg liter(-1) of ammonia. No larvae survived for 84, 24 and 12 h at concentrations of 1.2, 2.0 and 4.7 mg liter(-1) of ammonia, respectively. Eighty percent of the fry died after 168 h and no fry survived for 24 h at concentrations of 1.6 and 2.1 mg liter(-1) of ammonia, respectively. Eighty percent of the adults died after 24 h and no adult survived for 12 h at concentrations of 1.0 and 1.9 mg liter(-1), respectively. Larger adults were the most susceptible to ammonia. The order of resistance to ammonia was eggs, fry, larvae, and adults.     

Exposure to pyriproxyfen (juvenile hormone agonist) does not alter maternal care and reproduction in the European earwig

Sublethal exposure to pesticides can alter the survival and reproduction of a wide range of non-target organisms. However, it remains unclear whether this exposure can alter behaviours that are often essential for long-term population dynamics and maintenance, such as parental care. In this study, we tested the effect of pyriproxyfen exposure (an insect growth regulator) on maternal care in the European earwig, an insect that is both used in pest control in pip-fruit orchards and considered a pest in stone fruit orchards. We exposed 424 females at doses either 10 times lower, equivalent or 10 times higher than normal application rates in French orchards. As maternal care can change over the weeks of family life, we exposed the earwig mothers at five different days before and after egg hatching. We then measured the expression of ten forms of maternal care towards eggs and juveniles, six non-caring behaviours, eggs and juvenile development, metabolic reserves in mothers at egg hatching and females’ production of a terminal clutch. First, our results revealed that the three tested doses of pyriproxyfen were non-lethal and confirmed that maternal care decreased throughout both pre- and post-hatching family life. However, we did not detect any effect of pyriproxyfen on maternal care and non-care behaviours, eggs and juvenile development, quantities of lipids, proteins and glycogen in mothers at egg hatching, and on the production of a future clutch. Overall, these findings suggest that the maximal doses of pyriproxyfen authorized in French orchards is likely to have limited effects on the short- and long-term maintenance of populations of the European earwig and raises fundamental questions about the nature of the link between juvenile hormone and parental care in insects.

     

Influence of water hardening of the chorion on cadmium accumulation in medaka (Oryzias latipes) eggs

This report describes a study in which in vitro fertilization methods were used to expose medaka (Oryzias latipes) eggs to cadmium (Cd(2+)). This approach was applied to address the differential sensitivity and cumulative potential of Cd(2+) when exposure was initiated early (before fertilization and water hardening of the chorion) versus later during embryo development (i.e., well after the chorion has undergone water hardening). Following range finding exposures (2.5, 10, 20, 40 or 80 mg/l) under artificially controlled experimental procedures, results from hatching success and embryo malformations showed the earlier exposure interval more sensitive than the assay involving only the embryonated egg. Subsequent accumulation studies have shown that the exposure initiated before fertilization apparently led to more Cd(2+) deposition in the chorion compared to the exposure during embryonated stages of the eggs. Similarly, values for total Cd(2+) indicated higher concentrations in those eggs exposed prior to--and during--water hardening. Results suggest an alteration of the properties of the zona radiata in the early-stage eggs, making it more permeable to the potential exit or entrance of waterborne agents even after water hardening. Ongoing studies must now address the development of more realistic exposure conditions of the gametes by using incubation media with osmolarities similar to surface waters, and by shortening duration for gamete exposure. Also, sensitive methods to localize Cd(2+) and to delineate the transfer from the chorion to the embryo are needed.     

Changes in hematological and serum biochemical values in jundiá Rhamdia quelen due to sub-lethal toxicity of cypermethrin

Jundiá (Rhamdia quelen, Quoy and Gaimard), a South American teleostean fish, was exposed to sub-lethal concentrations of cypermethrin (30% and 45% of the 48-h LC(50) value of 0.265 ppm) for 2, 4 or 8 days. Serum biochemical and hematological values and behavioral changes were studied. The 30% LC(50), 0.08 ppm, produced significant increases in Mg(2+), P, K(+), creatinine, urea, glucose, cholesterol, aspartate aminotransferase and alkaline phosphatase levels, and reduction in total proteins and triglycerides in serum. The 45% LC(50), 0.12 ppm, produced significant increase in Na(+), Mg(2+), P, K(+), creatinine, urea, glucose, cholesterol, and alkaline phosphatase, and reduction in triglycerides and alanine aminotransferase levels in serum. At this concentration, the fish showed behavior changes such as hyper-excitability, asphyxia, and widening of mouth and operculum. The hematological values remained normal, except for hemoglobin concentrations and the mean corpuscular hemoglobin concentration, which increased with exposure to 0.08 ppm and 0.12 ppm cypermethrin. Results of the present work show that biochemical analysis of serum can be useful to detect incipient cypermethrin intoxication of the shoal.     

Thiobencarb toxicity and plasma AChE inhibition in the European eel

The acute toxicity of the herbicide thiobencarb (S-4-chlorobenzyl diethylthiocarbamate) was determined for the European eel (Anguilla anguilla). The 24, 48, 72 and 96 hours median lethal concentrations (LC50) were 25.7, 21.7, 17.0 and 13.2 mg/L, respectively. Fish were also exposed to a sublethal thiobencarb concentration (1/60 LC50-96 hr = 0.22 mg/L) during 96 hours in a flow-through system and then an elimination period of 192 hours in clean water was allowed. Eels were removed and blood samples taken out at each exposure time and recovery period in order to evaluate AChE activity. Thiobencarb induced significant inhibitory effects on plasma AChE activity of A. anguilla from the first contact time with the toxicant. This inhibition (under 50% activity) was maintained during the entire exposure period (96 hours) and even those animals transferred to clean water showed plasma AChE activity different from the controls. Differences between total and specific AChE activity were detected during the exposure period. Total AChE activity in the plasma from animals transferred to a medium free of toxicant recovered its normal value while specific AChE activity remained depressed (< 50%) until five days later.     

Latent effects of early life stage exposure to triclosan on survival in fathead minnows,Pimephales promelas

The objective of this study was to evaluate the effects of early life stage triclosan (5-chloro-2-(2,4, dichlorophenoxy)phenol, TCS) exposure on hatching, development, and survival in the fathead minnow, Pimephales promelas. Embryonic minnows were exposed to TCS (50 and 100 µg L^?1) for 10 days followed by 6 weeks depuration. Mortality and morphological deformities were recorded and quantified during exposure and at the end of depuration. No significant effects on embryonic survival, time to reach the eyed stage, or hatching were found. However, at the conclusion of the depuration period, survival was significantly reduced in TCS exposed fish depending on the concentration. Visual inspection of the exposed fish suggests that mortality is related to spinal deformities, emaciation, and reduced foraging ability. Triclosan exhibits deleterious effects in fish at lower concentrations over longer durations than previously reported. Further, mortality in exposed fish 6 weeks after exposure demonstrates the need for various exposure assays to evaluate effects of TCS.     

The response of Chenopodium album L. and Abutilon theophrasti Medik. to reduced doses of mesotrione

Limited toxicity data are available for estuarine and marine species and the widely used pyrethroid insecticide, permethrin. This study determined acute effects of permethrin on survival, lipid peroxidation, acetylcholinesterase activity, and splenocyte proliferation for two fish species found in South Carolina estuaries; juvenile red drum (Sciaenops ocellatus) and adult mummichog (Fundulus heteroclitus). Juvenile S. ocellatus were significantly more sensitive than adult F. heteroclitus to permethrin exposure, with a 96-h LC50 value of 8 μg/L determined for red drum compared to 23 μg/L for mummichog. Lipid peroxidation activity of the liver increased in permethrin-treated fish compared to control animals after 24 h and decreased after 96 h. Permethrin had no effect on acetylcholinesterase activity of the brain at the concentrations tested. Permethrin exposure significantly inhibited splenocyte proliferation, indicating an immunosuppressive effect. Most of the effects of permethrin on fish cellular stress enzymes and survival occurred at concentrations much higher than those typically measured in the environment. However, inhibition of splenocyte proliferation in juvenile red drum occurred at approximately twice that of measured permethrin concentrations in surface water. These findings may prove useful to the future management and regulation of pyrethroid insecticide use near estuarine habitats.     

Ammonia emission factors for N fertilizers applied to two contrasting grassland soils

Ammonia volatilization from nitrogen (N) fertilizer applied throughout the year to two soil types was measured using a system of small wind tunnels. Losses from urea ranged from 12 to 46% of the applied N. Small losses, averaging <1%, were measured from ammonium nitrate (AN) and calcium nitrate applications. Factors influencing these losses are discussed. Using these results and those from other workers, emission factors for urea and AN applications to grassland in the UK were determined as 23.0 and 1.6% of the applied N, respectively. Emission factors for these fertilizers when applied to arable land were estimated as 11.8 and 0.8%, respectively. The emission factor for all other applied N (as straight and compound fertilizers) was assumed to be similar to that for AN. Calculations showed that fertilizer applications to agricultural land in the UK contributes 34 kt NH3-N per year, equivalent to 17% of the total annual NH3 emission.