Assimilation efficiencies of Cd and Zn in the common carp (Cyprinus carpio): effects of metal concentration, temperature and prey type

The impact of several factors on the assimilation efficiency (AE) of Cd and Zn from food in the common carp (Cyprinus carpio) was studied. Tested prey species were midge larvae (Chironomus riparius), zebra mussels (Dreissena polymorpha) and oligochaetes (Tubifex tubifex). The Cd load of the larvae did not affect the Cd AE in the carp. The Zn AE however, was negatively related to the Zn load of the prey. Food quantity and starvation of the carp did not significantly affect the Cd AE. For Zn, a significant decrease in AE was found when carp were fed ad libitum. Decreasing the temperature from 25 degrees C to 15 degrees C did not influence the Cd AE, while for Zn a significant decrease of the AE was measured. Carp assimilated Cd from both zebra mussels and oligochaetes with a significantly lower efficiency in comparison to the midge larvae, although Zn AEs was prey independent.     

Atrazine induction of a family 4 cytochrome P450 gene in Chironomus tentans (Diptera: Chironomidae)

Cytochrome P450-dependent microsomal monooxygenase (P450) activity was measured in control and atrazine-exposed third instar midge larvae, Chironomus tentans. Significantly elevated O-demethylase activity was observed in gut homogenates taken from midges exposed to atrazine concentrations from 1 to 10 ppm for 90 h. No significant induction was observed at atrazine concentrations below 1 ppm. A region of a cytochrome P450 family 4 gene was amplified and sequenced from C. tentans larvae. Alignments of inferred amino acid sequences with other insect CYP4 gene homologues indicate a high degree of similarity. Northern blot analysis employing the CYP4 gene fragment as a probe showed an over-expression in C. tentans exposed to atrazine. The results support the previously identified inducibility of cytochrome P450-dependent activity and provide insight into the potential consequences of atrazine exposure to aquatic organisms.     

Life cycle responses of the midge Chironomus riparius to polycyclic aromatic compound exposure

During acute exposure, polycyclic aromatic compounds (PACs) act mainly by narcosis, but during chronic exposure the same compounds may exert sublethal life cycle effects. The aim of this study was therefore to evaluate the chronic effects of sediment spiked PACs on the emergence of the midge Chironomus riparius. Three isomer pairs were selected, and 28-day LC50 values and 50% emergence times (EMt50) were determined. Concentration-response relationships were observed for phenanthrene, acridine, phenanthridine and acridone. Anthracene and phenanthridone had no effect on total emergence, but did cause a delay in emergence. Calculated porewater LC50 values correlated well with logKow values, suggesting narcosis as mode of action. In contrast, effect concentrations for delay in emergence (EMt50) deviated from narcosis, suggesting a specific mode of action during chronic exposure. It is concluded that emergence is a powerful endpoint to detect life cycle effects of PACs on a key sediment inhabiting invertebrate.     

Bioaccumulation, depuration and biotransformation of 4,4'-dibromodiphenyl ether in crucian carp (Carassius auratus)

Polybrominated diphenyl ethers (PBDEs) are extensively used as a class of flame retardants and have become ubiquitous environmental pollutants. Significant biotransformation of some PBDEs via reductive debromination has been observed. However, little is known about the fate of lower brominated BDEs in fish. In this study, the tissue distribution, excretion, depuration and biotransformation of 4,4′-dibromodiphenyl ether (BDE 15) were investigated in crucian carp (Carassius auratus) which were exposed to spiked water solution at different concentrations for 50 d, followed by a 14-d depuration period. Bioaccumulation parameters were calculated and the results showed that BDE 15 was mainly concentrated in the gill and liver. In particular, five biotransformation products of BDE 15 in carp were identified using GC-MS/MS. Besides two debrominated metabolites, three of the metabolites were mono-OH-BDE 15, diOH-BDE 15 and bromophenol. Our results unequivocally suggested that BDE 15 oxidation did occur via the formation of hydroxylated (OH-) metabolites in crucian carp exposed in vivo. These findings will be useful for determination of the metabolic pathways of PBDEs in freshwater fish, especially about their oxidation metabolism.     

Changes in plasma thyroid hormones and cortisol levels in crucian carp (Carassius auratus) exposed to the extracted microcystins

The endocrine response of crucian carp injected intraperitoneally with extracted microcystins (MC) was investigated in this study. Fish were injected intraperitoneally either with 0.75% NaCl (control) and Microcystis extract corresponding to 150 and 600 microg microcystins per kg body weight. The plasma levels of triiodothyronine (T(3)), thyroxine (T(4)), free triiodothyronine (FT(3)), free thyroxine (FT(4)), and cortisol were determined at 0, 1, 3, 12, 24, and 48h post-administration of MC-containing extract. Treated fish displayed abnormal behaviors, such as a startle response and disoriented swimming, as well as changes in ventilation rates. Plasma cortisol concentrations of fish in both dose groups significantly increased after administration of extracted MC and remained high throughout the experiment, which suggested that MC elicited a stress response in treated fish. The profiles of cortisol changes in treated fish appeared to be dose dependent, indicating that fish in the high dose group experienced greater MC-induced disturbance. Mortality occurred after 12h in the high dose group. Plasma levels of T(4), T(3), FT(4), and FT(3) did not vary significantly between the control fish. In contrast to this, fish exposed to MC-containing extract showed significant declines in T(3), FT(4), and FT(3) levels in a dose-dependent manner throughout the experiment. Plasma T(4) levels, however, did not vary significantly in the low dose group, whereas they decreased significantly at 48h post injection in the high dose group. This study demonstrates that administration of microcystins-containing extract causes a stress response and reduces the plasma levels of thyroid hormones in crucian carp. These results illustrate that microcystins exerted potent effects on the endocrine system of crucian carp, through activating their hypothalamus-pituitary- interrenal axis and disturbing thyroid function.     

60Co accumulation from sediment and planktonic algae by midge larvae (Chironomus luridus)

This paper reports the results of several experiments carried out to evaluate uptake and retention by a limicolous midge larva of 60Co retained in sediment, either adsorbed on mineral particles or bound to planktonic algae. In order to determine their relative contributions in radionuclide accumulation, the different vectors (water, algae and sediment) were first labelled individually and then simultaneously. 60Co accumulation from water and from algae results in a maximum concentration factor of 30 and in a mean trophic transfer factor of 4.5 x 10(-3). The level of contamination of midge larvae from sediment is markedly influenced by the presence of endogenous organic matter. Thus the radionuclide transfer factor is about twice as high for larvae placed in labelled raw sediment than for larvae placed in labelled incinerated sediment, in the presence as in the absence of contaminated planktonic algae. Irrespective of the contamination conditions, 60Co depuration from midge larvae is a very rapid phenomenon that corresponds, in all cases, to a radionuclide half-life of only a few days.     

Perfluorooctane sulfonic acid and organohalogen pollutants in liver of three freshwater fish species in Flanders (Belgium): relationships with biochemical and organismal effects

A perfluorooctane sulfonic acid (PFOS) assessment was conducted on gibel carp (Carassius auratus gibelio), carp (Cyprinus carpio), and eel (Anguilla anguilla) in Flanders (Belgium). The liver PFOS concentrations in fish from the Ieperlee canal (Boezinge, 250-9031 ng/g wet weight, respectively) and the Blokkersdijk pond (Antwerp, 633-1822 ng/g wet weight) were higher than at the Zuun basin (Sint-Pieters-Leeuw, 11.2-162 ng/g wet weight) and among the highest in feral fish worldwide. Eel from the Oude Maas pond (Dilsen-Stokkem) and Watersportbaan basin (Ghent) had PFOS concentrations ranging between 212 and 857 ng/g wet weight. The hepatic PFOS concentration was significantly and positively related with the serum alanine aminotransferase activity, and negatively with the serum protein content in eel and carp. The hepatic PFOS concentration in carp correlated significantly and negatively with the serum electrolyte concentrations whereas a significant positive relation was found with the hematocrit in eel. Although 13 organochlorine pesticides, 22 polychlorinated biphenyl (PCB) congeners and 7 polybrominated diphenyl ethers (PBDEs) were also measured in the liver tissue, only PCB 28, PCB 74, gamma-hexachlorocyclohexane (gamma-HCH) and hexachlorobenzene (HCB) were suggested to contribute to the observed serological alterations in eel.     

Interference with xenobiotic metabolic activity by the commonly used vehicle solvents dimethylsulfoxide and methanol in zebrafish (Danio rerio) larvae but not Daphnia magna

Organic solvents, such as dimethylsulfoxide (DMSO) and methanol are widely used as vehicles to solubilise lipophilic test compounds in toxicity testing. However, the effects of such solvents upon innate detoxification processes in aquatic organisms are poorly understood. This study assessed the effect of solvent exposure upon cytochrome P450 (CYP)-mediated xenobiotic metabolism in Daphnia magna and zebrafish larvae (4 d post fertilisation). Adult D. magna were demonstrated to have a low, but detectable, metabolism of ethoxyresorufin in vivo and this activity was not modulated by pre-exposure to DMSO or methanol (24 h, up to 0.1% and 0.05% v/v, respectively). In contrast, the metabolism of ethoxyresorufin in zebrafish larvae was significantly reduced by both solvents (0.1% and 0.05% v/v, respectively) after 24 h of exposure. In zebrafish, these observed decreases in activity towards ethoxyresorufin were accompanied by decreased expression of a variety of genes coding for drug metabolising enzymes (corresponding to CYP1, CYP2, CYP3 and UDP-glucuronyl transferase [UGT] family enzymes), measured by quantitative PCR. Reduction of gene expression and CYP1 enzyme activities by methanol (0.05% v/v) in zebrafish larvae was partially reversed by co-exposure with Aroclor 1254 (100 μg L⁻¹). Overall this study suggests that relatively low concentrations of organic solvents can impact upon the biotransformation of certain xenobiotics in zebrafish larvae, and that this warrants consideration when assessing compounds for metabolism and toxicity in this species.     

Environmental factors in debromination activity of polybrominated diphenyl ethers by hepatic microsomes of freshwater fish

Although the debromination of polybrominated diphenyl ethers (PBDEs) in fish species has been studied, environmental factors, such as chemical contamination and habitat temperature, have not been well understood. This study compared debromination of BDE209 by hepatic microsomes of wild and cultured fish. PBDE concentrations in muscle tissue were lower in cultured fish than in wild fish. Debromination activity was high in wild common carp, followed by cultured common carp, moderate in cultured ayu sweetfish, and low in two cultured fish (rainbow trout and cherry salmon) and wild Japanese sea bass. Although common carps have been known as the species which have higher debromination ability, there were differences between wild and cultured common carps. First, wild common carp debrominated much more BDE209 than cultured common carp. Second was debromination of BDE209 lasted 96 h in wild carp but only 24 h in cultured carp. Wild carp were collected from warm wastewater effluent with consistently high concentrations of micropollutants. Cultured carp were collected from colder clean waters. Therefore, environmental factors in debromination include contamination or ambient temperature. To investigate the effects of habitat environment on debromination of PBDEs, we collected wild carp in summer and winter at two different locations with similar PBDE contamination levels. Carp collected from the natural river in winter had the highest BDE99 debromination activity. Although the results indicated seasonal difference of debromination of BDE209, we could not confirm whether habitat temperature or physiological cycle of carp affected to debromination ability. Thus, further investigation such as in vivo experiment is required.

     

Species-specific responses to metals in organically enriched river water, with emphasis on effects of humic acids

Invertebrate communities in polluted rivers are often exposed to a wide variety of compounds. Due to complex interactions, 'pollution tolerant' species are not necessarily the most tolerant species to toxicants tested under standard laboratory conditions. It was hypothesized that the distribution of species in polluted rivers is not only dependent on the tolerance of species to toxicants, but also on species-specific capacities to modify or compensate for negative effects of toxicants. To test this hypothesis, species-specific responses to metals in organically enriched river water were studied under controlled conditions. The zebra mussel Dreissena polymorpha and the midge Chironomus riparius were exposed to metal-polluted water from the River Dommel. Additionally, the (interactive) effects of metals and humic acids (HA) on both species were evaluated. In spite of a lower tolerance of Chironomus riparius to metals in laboratory studies, the midge was the most tolerant of the two test species to metal-polluted site water. The results indicated that the sensitivities of the two test species determined in laboratory tests were inversely related to their sensitivities to polluted river water. In accordance with these results, midge larvae were protected from copper (Cu) toxicity by HA, while metal toxicity was not reduced (Cu) or even amplified (cadmium) by HA for the zebra mussel. Thus, the presence of (naturally occurring) HA in site water may partly account for discrepancies between responses of species to bioassays and toxicity tests. It is suggested that these differences in responses to metals in site water are strongly influenced by species-specific preferences for organic compounds (like HA). It is concluded that the response to organic compounds present in site water largely determines whether a species is classified as 'pollution tolerant' or 'pollution sensitive'.     

Immunohistochemical detection of polychlorinated biphenyls in field collected damselfish (Abudefduf sordidus; Pomacentridae) embryos and larvae

Antibodies against polychlorinated biphenyls (PCBs) were used to determine if immunohistochemical methods could detect PCBs in embryos and larvae of a territorial coral reef fish (Abudefduf sordidus; Pomacentridae) collected from Johnston Atoll, Central Pacific Ocean. Sites with differing levels of contamination were sampled, one with relatively high sediment PCB concentrations of up to 389.0 ng/g and another with low PCB concentrations of only 0.5 ng/g. Immunostaining suggested that PCB concentrations were higher in fish larvae from the PCB contaminated site and that PCB concentrations within abnormal embryos were higher than normal embryos from the same nest. This technique will be useful for detecting exposed populations in the field and assessing correlations with adverse effects, particularly in potential indicator organisms such as Abudefduf sordidus.     

Perfluorooctane sulfonate (PFOS) and other fluorochemicals in fish blood collected near the outfall of wastewater treatment plant (WWTP) in Beijing

Perfluorinated compounds (PFCs) were measured in zooplankton and five fish species collected from Gaobeidian Lake, which receives discharge from wastewater treatment plant (WWTP) in Beijing, China. The mean total PFCs in five fish were in the order: crucian carp > common carp > leather catfish > white semiknife carp > tilapia. Perfluorooctane sulfonate (PFOS) occurred at the greatest concentrations, with mean concentrations ranging from 5.74 to 64.2 ng/ml serum. Perfluorodecanoic acid (PFDA) was the second dominant PFC in fish samples except for common carp in which perfluorooctane sulfonamide (PFOSA) was dominant. A positive linear relationship (r² = 0.85, p < 0.05) was observed between ln PFOS concentrations (ln ng/ml) and trophic level (based on δ¹⁵N) if tilapia was excluded. The risk assessment showed that PFOS might not pose an immediate risk to fish in Gaobeidian Lake.     

Acute and Chronic Toxicity of Chlorine Dioxide (ClO2) and Chlorite (ClO2ˉ) to Rainbow Trout (Oncorhynchus mykiss) (4 pp)

Goal, Scope and Background Chlorite (ClO2ˉ) is a primary decomposition product when chlorine dioxide (ClO2) is added during water treatment; therefore the toxic effects of both compounds on aquatic organisms are possible. Limited data are available concerning their toxicity to fish. The aim of this study was to investigate sensitivity of rainbow trout to acute and chronic toxicity of chlorine dioxide and chlorite, and to estimate the Maximum-Acceptable-Toxicant-Concentration (MATC) of those compounds in fish. Methods The acute and chronic toxicity of chlorine dioxide and chlorite to larval and adult rainbow trout was investigated in 96-hour to 20-day laboratory exposures evaluating the wide range spectrum of biological indices under semi-static conditions. Results and Discussion Median lethal concentration (96-hour LC50) values derived from the tests were: 2.2 mg/l for larvae; 8.3 mg/l for adult fish and 20-day LC50 for larvae was 1.6 mg/l of chlorine dioxide, respectively. Chlorite was found to be from 48 to 18 times less acutely toxic to larvae and adult fish, correspondingly. Both chemical compounds induced similar toxic effects in rainbow trout larvae during chronic tests (they affected cardio-respiratory and growth parameters), but chlorine dioxide had a higher toxic potency than chlorite. A significant decrease in the heart rate and respiration frequency of larvae was established. However, within an increase in exposure duration recovery of cardio-respiratory responses was seen to have occurred in larvae exposed to chlorite. Meanwhile, in larvae exposed to chlorine dioxide, a significant decrease in cardio-respiratory responses remained during all 20-day chronic bioassays. Chlorine dioxide also more strongly affected growth parameters of rainbow trout larvae at much lower test concentrations. Decreased rate of yolk-sack resorption occurred only in the tests with chlorine dioxide. Conclusions Maximum-Acceptable-Toxicant-Concentration (MATC) of 0.21 mg/l for chlorine dioxide and of 3.3 mg/l for chlorite to fish was derived from chronic tests based on the most sensitive parameter of rainbow trout larvae (growth rate). According to substance toxicity classification accepted for Lithuanian inland waters, chlorine dioxide and chlorite can be referred to substances of \moderate\ toxicity to fish. Recommendations and Outlook Due to its very reactive nature, chlorine dioxide is rapidly (in a few hours) reduced to chlorite, which is persistent also as a biocide but 16 times less toxic to fish, according to MATC. Therefore, it is much more likely that fish will be exposed to chlorite than to chlorine dioxide in natural waters. Presently accepted, the Maximum-Permitted-Concentration of total residual chlorine (TRC) in waste-water discharging into receiving waters is 0.6 mg/l. If this requirement will not be exceeded, it is unlikely that fish would be exposed to lethal or even to sublethal concentrations of chlorine dioxide or chlorite. Furthermore, chlorine dioxide does not generate toxic nitrogenous (chloramines) or carcinogenic organic residuals (trihalomethanes). All these properties make chlorine dioxide a more promising biocide than chlorine.     

Chronic effects of Cd on reproduction of Polypedilum nubifer (Chironomidae) through water and food

Chronic effects of Cd on reproduction of Polypedilum nubifer (Chironomidae) were investigated by Cd-exposure from the egg stage using a flow-through aquarium. No significant effect of Cd on reproduction was observed in the midge larvae which had been exposed to 10 or 20 microg Cd litre(-1). Emergence of the Cd-exposed larvae peaked several days before that of the control, although growth was impaired in 1st and/or 2nd instars. The percentage emergence success decreased to about 46% of the control at 40 microg Cd litre(-1). However, other reproductive processes (adult sex ratio, oviposition success and egg hatchability) were not impaired. The emergence success decreased to less than 3% of the control at 80 microg Cd litre(-1). The emergence success of midge larvae which had been fed Cd-contaminated food with 220 or 1800 microg Cd g(-1) decreased to nearly 60% of the control. However, other reproductive processes were not impaired. Emergence success of larvae exposed to food containing 22 microg Cd g(-1) was unaffected.     

Identification of 2,6-diisopropylnaphthalene metabolites in carp part 1. In vivo experiment

The metabolism of diisopropylnaphthalene (DIPN) in fish was studied from an ecotoxicological viewpoint to clarify the environmental fate of DIPN, a solvent of carbonless paper which is widely used as a substitute for PCB. This study identified the following metabolites in carp by in vivo experiment; 2-(1-hydroxy-1-methyl) ethyl-6-isopropylnaphthalene, 2-(1-methyl-2-hydroxyethyl)-6-isopropyl-naphthalene, 2,6-(1-hydroxy-1-methyl) ethyl-naphthalene, 2-(1-hydroxy-1-hydroxymethyl) ethyl-6-isopropylnaphthalene and α-[2-(6-isopropylnaphthyl)] propionic acid. Identification was based on UV, IR, MS, and NMR analyses. The metabolism of 2,6-DIPN in carp was concluded to mainly through oxidation of the isopropyl chain.     

Uranium uptake and depuration in the aquatic invertebrate Chironomus tentans

Evaluation of aqueous uranium (U) uptake and depuration in larvae of the midge Chironomus tentans were investigated in two separated experiments. First, a static-renewal experiment was performed with 10-d old C. tentans larvae exposed to 300 μg U/L. The animals steadily accumulated U (K_u = 20.3) approaching steady-state conditions (BAF = 56) in approximately 9-11 d. However, accumulated U was readily depurated (K_d = 0.36) with U tissue concentration decreasing rapidly within 3 d of the larvae being placed in clean water (t_1/2 = 1.9 d). Also, the growth of C. tentans larvae appeared to decrease after 6-11 d of U exposure, probably due to the reallocation of resources into U detoxification mechanisms. However, growth significantly increased once C. tentans were transferred to clean water. A separate short-term experiment was performed to evaluate the possible mechanism of U uptake in this invertebrate. Results suggested a passive mechanism of U uptake coupled with an active mechanism of U depuration but no details related to the type of mechanisms or pathway was investigated.     

Distribution and accumulation of elements (As, Cu, Fe, Hg, Mn, and Zn) in tissues of fish species from different trophic levels in the Danube River at the confluence with the Sava River (Serbia)

Pikeperch (Sander lucioperca), European catfish (Silurus glanis), common carp (Cyprinus carpio), and gobies (Neogobius gymnotrachelus, Neogobius melanostomus) were collected from the Danube River (Belgrade section), and samples of liver, muscle, or whole-body composites (in the case of gobies) were analyzed for As, Cu, Fe, Hg, Mn, and Zn with inductively coupled plasma optical spectrometry to find out if there was a correlation between accumulation of these elements in predatory and prey species, as well as in pairs of species with overlapping diets. Concentrations of all analyzed elements were either higher (Cu, Fe, Mn, Zn) in liver than in muscle, or equal (As, Hg), except for Hg in carp, which was higher in muscle. Mercury concentration in liver and muscle of predators (catfish, pikeperch) was significantly (<10^?4) higher than in prey fishes (carp and gobies). The results indicate that Hg concentration was biomagnified through the food chain. Concentrations of As, Fe, and Hg in carp liver and gobies whole-body composite were similar, but carp had significantly (<10^?4) higher values of Zn and Cu in liver. The regression analysis and trendline equations indicate that the concentrations of all tested elements, except for As in liver, and Mn and Fe in muscle, were similar in predatory fish (pikeperch and catfish), on one hand, and in prey fish (carp and gobies), on the other hand. Distinctly high Zn concentration in carp is very common in this species due to its physiology. Concentrations of Hg and Zn were higher than the maximum acceptable concentration due to the high pollution level in this section of the Danube River, accordingly posing a risk for the human consumption of these fish species.     

Bioconcentration of n-dodecane and its highly branched isomer 2,2,4,6,6-pentamethylheptane in fathead minnows

Petroleum products are complex mixtures of hydrocarbons. They are important as constituents of fuels and lubricants, and as key raw materials for the chemicals industry. Since there is a potential for accidental releases to the aquatic environment, bioaccumulation of higher hydrocarbons is of concern. Here, the bioconcentration behaviour of two representative hydrocarbons, the dodecane isomers n-dodecane and 2,2,4,6,6-pentamethylheptane (PMH), was investigated in fathead minnows at concentrations in water below their maximum aqueous solubility. The concentration of n-dodecane in fish did not exceed our method limit of detection of 60 μg/kg. In contrast, PMH could be quantified in fish. No significant increase in the ratio of PMH concentrations in fish to water could be detected indicating that an exposure time of 4–10 days is sufficient to approach steady-state. For n-dodecane the upper limit of the bioconcentration factor (BCF) is estimated by dividing the method limit of detection by the exposure concentration and a value of 240 l/kg is derived. For PMH the bioconcentration factor, estimated as the average fish/water concentration ratio during the steady-state part of the experiment, ranges between 880 and 3500 l/kg. The BCFs of both compounds are small compared to their hydrophobicity. Given that both linear and branched hydrocarbons are known to be biotransformed by fish, it appears that efficient metabolism of the test compounds in fathead minnows prevents bioaccumulation.     

Accumulation and partition of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) in the muscle and liver of fish

The present study monitored fish from sites contaminated for many years, and the data reflected the results of actual, long-term environmental exposure. The sum concentrations and TEQs of PCDD/F in the muscle of common carp (Cyprinus carpio (linnaeus)) and big head (Aristichthys nobilis) from the different site of a heavily polluted lake in China are decreased with decrease of PCDD/F concentration in sediment and water. Concentration in fish can be highly variable, but the concentration is comparable in muscle between different species of fish if the concentrations are normalized by lipid content. PCDD/F accumulate in certain tissues with the highest proportions found in the liver of fish. The concentrations and TEQs in livers were also decreased with concentrations of sediments and water. The concentrations of PCDD/F in muscle are significantly correlated with those in liver. BCFs of 2,3,7,8-substituted PCDD/F calculated directly from water were consistent with that calculated indirectly from sediment. PCDD/F concentrations in sediment are much higher than that in water, therefore the measurement is much easier and the results are more accurate. Estimated BCFs for superhydrophobic chemicals derived from sediment may also serve as acceptable surrogates for "true" bioaccumulation potential.