The herbicide glyphosate is the most widely used pesticide worldwide. Glyphosate and its major metabolite, aminomethylphosphonic acid (AMPA), have been frequently found in water samples. The widely used methods for determining these compounds are expensive and environmentally unfriendly due to reagent consumption for derivatization. Another possibility is the use of classic ion chromatography, albeit with low sensitivity and subject to interferences. Therefore, this study aimed to develop a method to directly and simultaneously determine glyphosate, AMPA, and common inorganic anions in water samples using gradient capillary ion chromatography without sample pre-treatment and derivatization. The proposed method was validated, presenting adequate linearity for glyphosate and AMPA with a determination coefficient (r2) > 0.998. Recoveries ranged from 94 to 105% and 79 to 113% for glyphosate and AMPA, respectively, with a relative standard deviation < 10%. The practical method limits of detection and quantification for both glyphosate and AMPA were 7.5 and 25 µg L 1, respectively. The method presented satisfactory results for the anions fluoride, chloride, bromide, nitrite, nitrate, phosphate, and sulfate, with limits of detection ranging from 7.5 to 200 µg L-1. Application of the method in water samples proved simple, efficient, and cost-effective, enabling the monitoring of these analytes in different water matrices.

ABSTRACT: The soybean looper (SBL), Chrysodeixis includens (Walker, [1858]) (Lepidoptera: Noctuidae), is a soybean and cotton pest in South America countries. Field-evolved resistance of SBL to inhibitors of chitin biosynthesis has been reported in Brazil; however, this mode of action is still widely used against SBL. On this basis, we conducted laboratory bioassays to investigate if adjuvants (Nimbus®, TA 35®, Break-Thru® S 240, and Rizospray Extremo®) added to the teflubenzuron spray increase the mortality of SBL strains (resistant, heterozygous, and susceptible to chitin biosynthesis inhibitors). Using chromatography analysis, we also evaluated the amount of teflubenzuron on soybean leaves when applied alone or in combination with adjuvants. In laboratory bioassays, the biological activity of teflubenzuron increased against the susceptible SBL strain when adjuvants were added. In contrast, no relevant effects of adjuvants added to the teflubenzuron spray against heterozygous and resistant SBL larvae were detected. In leaf bioassays, even leaves from the upper third part of the plants containing a significantly higher amount of teflubenzuron (3.4 mg/kg vs 1.7 and 0.6 mg/kg); the mortality of SBL strains was similar when teflubenzuron was applied alone or in mixture with adjuvants. Our findings indicated that adjuvants added to teflubenzuron spray do not provide a substantial increase in the mortality of SBL strains resistant to chitin biosynthesis inhibitors. Therefore, it is necessary to reduce the use of this mode-of-action insecticide against SBL and to give preference to other insecticides or control tactic.

RESUMO: A lagarta falsa-medideira, Chrysodeixis includens (Walker, [1858]) (Lepidoptera: Noctuidae), é uma praga da soja e do algodão nos países da América do Sul. A resistência de C. includens a inibidores da biossíntese de quitina tem sido relatada no Brasil. Entretanto, esse modo de ação ainda é amplamente utilizado para controle de C. includens. Com base nisso, conduzimos bioensaios em laboratório para investigar se adjuvantes (Nimbus®, TA 35®, Break-Thru® S 240 e Rizospray Extremo®) adicionados à calda inseticida de teflubenzuron aumentam a mortalidade de linhagens de C. includens (resistentes, heterozigotos e suscetíveis a inibidores da biossíntese de quitina). Usando análise cromatográfica, também avaliamos a quantidade de teflubenzuron em folhas de soja quando aplicado isolado ou em combinação com adjuvantes. Em bioensaios de laboratório, a atividade biológica do teflubenzuron aumentou para a linhagem suscetível quando os adjuvantes foram adicionados à calda inseticida. Em contraste, nenhum efeito relevante de adjuvantes adicionados ao teflubenzuron foi detectado para os heterozigotos e resistentes. Em bioensaios de folhas, mesmo naquelas do terço superior das plantas, as quais apresentaram uma maior deposição de teflubenzuron (3,4 mg/kg vs 1,7 e 0,6 mg/kg); a mortalidade das linhagens de C. includens foi semelhante quando o teflubenzuron foi aplicado isolado ou com adjuvantes. Nossos resultados indicam que os adjuvantes adicionados ao teflubenzuron não fornecem um aumento substancial na mortalidade de linhagens de C. includens resistentes aos inibidores da biossíntese de quitina. Portanto, é necessário reduzir o uso desse modo de ação para o manejo de C. includens e dar preferência a outros inseticidas ou tática de controle.

Abstract Most conventional sewage treatment systems are not able to fully remove micropollutants found in sewage. Thus, the simultaneous degradation of 11 pharmaceuticals identified in the effluent of a sewage treatment plant in Southern Brazil was herein investigated through advanced oxidation processes based on ozonation, ultraviolet radiation and hydrogen peroxide. For detection, samples were prepared through solid-phase extraction and pharmaceuticals were identified through ultra-high performance liquid chromatography tandem mass spectrometry. Active ingredients such as ciprofloxacin, oxytetracycline, paracetamol, sulfamethoxazole and trimethoprim had their concentrations increased for degradation analysis purposes. Trials were carried out on a bench at room temperature and neutral pH, with aliquots collected at 7.5 and 15 minutes. Two ozone doses (0.5 and 0.9 mg per mg of dissolved organic carbon), and combinations of the lowest ozone dose with photolysis (254 nm) and with 25 mg.L−1 of hydrogen peroxide were evaluated. Pharmaceuticals mineralization efficiency was assessed in a total organic carbon analyzer. The process combining ozone, hydrogen peroxide and ultraviolet radiation was the most efficient in the degradation of all pharmaceuticals detected in this study, since it enabled reducing oxytetracycline by 89.32%, caffeine by 96.79%, trimethoprim by 97.40%, ciprofloxacin by 97.75%, sulfamethoxazole by 99.79%, paracetamol by 99.96%, and clindamycin, ofloxacin, sulfadiazine, sulfathiazole and tylosin by 100%. This process also recorded the highest mineralization rate (60.52%), fact that confirmed the potential to decrease persistent pharmaceuticals found in conventional sewage treatment systems.

Resumo A maioria dos sistemas convencionais de tratamento de esgoto não é capaz de remover totalmente os micropoluentes presentes no esgoto. Assim, a degradação simultânea de 11 fármacos, detectados no efluente de uma estação de tratamento de esgoto no Sul do Brasil, foi investigada por meio de processos oxidativos avançados baseados em ozonização, radiação ultravioleta e peróxido de hidrogênio. Para detecção, as amostras foram preparadas por extração em fase sólida e os fármacos foram identificados por cromatografia líquida de ultra eficiência acoplada à espectrometria de massa em tandem. Ingredientes ativos como ciprofloxacina, oxitetraciclina, paracetamol, sulfametoxazol e trimetoprima tiveram suas concentrações aumentadas para análise de degradação. Os ensaios foram realizados em bancada a temperatura ambiente e pH neutro, com alíquotas coletadas aos 7,5 e 15 minutos. Duas doses de ozônio (0,5 e 0,9 mg por mg de carbono orgânico dissolvido) e combinações da menor dose de ozônio com fotólise (254 nm) e com 25 mg.L−1 de peróxido de hidrogênio foram avaliadas. A eficiência da mineralização dos fármacos foi verificada em um analisador de carbono orgânico total. O processo combinando de ozônio, peróxido de hidrogênio e radiação ultravioleta foi o mais eficiente na degradação de todos os fármacos detectados neste estudo, pois permitiu reduzir a oxitetraciclina em 89,32%, a cafeína em 96,79%, o trimetoprim em 97,40%, a ciprofloxacina em 97,75%, o sulfametoxazol em 99,79%, o paracetamol em 99,96% e a clindamicina, a ofloxacina, a sulfadiazina, o sulfatiazol e a tilosina em 100%. Esse processo também registrou a maior taxa de mineralização (60,52%), confirmando o potencial de redução de fármacos persistentes encontrados em sistemas convencionais de tratamento de esgoto.

ABSTRACT. The study aimed to determine the fatty acid profile, omegas and lipid quality indeces in commercial cuts of pirarucu (Arapaima gigas) of the slaughter class 11.1 to 14.0 kg. Sample collections were carried out in two fish processing industries located in Rondônia state, Brazil. The experimental design was completely randomized, with processing performed in triplicate. Data were submitted to ANOVA to assess differences between commercial cuts in chemical compositions. If ANOVA appeared statistically significant (α=0.05), the averages were compared by Tukey's test. In the composition of fatty acids, there was a difference (p <0.05) between cuts. Commercial cuts that expressed the highest percentages of SFAs tail fillet 51.2%, of MUFAs fillet mignon 39.8% and of PUFAs deboned cut 20.7%. The indeces prescribed for lipid quality, ∑PUFAs/∑SFAs, ∑PUFAs (n-6/∑n-3), AI, TI and HH, indicate that commercial cuts have lipid quality. Deboned is the cut with the highest PUFA fatty acid content, with the highest values of Omega 3, 6, 7 and n-9 being also expressed. Nutritional information is important for the processes of conservation and processing, development of new products on the market, as well as guiding the form of preparation, thus providing commercial security for different market niches.

To preserve human health, persistent organic pollutants (POPs) and current-use pesticides (CUPs) should be monitored in fatty tissues, including breast milk. Therefore, this study aimed to optimize sample preparation conditions using a 32 factorial design for the determination of POPs and CUPs by gas chromatography coupled to mass spectrometry with negative chemical ionization (GC-NCI-MS). The method was validated for 57 POP and CUP compounds using hexane:acetone for extraction and clean-up by dispersive solid phase extraction (d-SPE) with Florisil®. The matrix effect was compensated by extracted analytical calibration. Method validation showed satisfactory results with limits of detection of 3 to 13 ng g-1 of fat. The method presented adequate accuracy (recoveries from 72 to 117%) and precision (relative standard deviation (RSD) ≤ 18%) and was applied to breast milk samples from Rio Grande do Sul State, southern Brazil, wherein all samples contained at least one compound. With principal component analysis, it was possible to associate the pesticides detected with the city of origin of the samples and the number of pregnancies of nursing mothers. Additionally, the analytical method was effective for the determination of trace levels of POPs and CUPs in breast milk and can be applied in biomonitoring studies.

ABSTRACT This study aimed to determine if there are any differences in the attributes and composition of the phytoplankton and epiphyton communities between organic (OF) and conventional (CF) rice fields. We also strove to identify if there were any variations in these communities by comparing samples taken from two different periods (12 and 35 days) after the application of the herbicide clomazone and penoxsulam in CF. The farms are located in the Pampa Biome, Southern Brazil. Phytoplankton samples from the subsurface water and epiphyton samples from the rice stems were analyzed using the Utermöhl method. The CF and OF had distinct environmental conditions (pH, conductivity, and turbidity values), and the residual concentration of the herbicides decreased over time. There were no significant differences in epiphyton and phytoplankton density, or in phytoplankton richness, between the rice fields; only the epiphyton richness and taxonomic composition showed differences between the rice fields. Cyanobacteria and Chlorophyceae comprised a large proportion of the epiphytic density in CF and OF, respectively. However, Bacillariophyceae and Chlorophyceae had greater phytoplankton densities in CF and OF, respectively. The taxonomic composition of communities should be considered an effective tool to show the differences between the two cultivation systems.

RESUMO: O presente trabalho tem por finalidade descrever e discutir os resultados do experimento realizado em vacas prenhes que foram alimentadas com 25kg/dia de bagaço de maçã. Experimentos foram conduzidos em duas etapas, a primeira no ano de 2015, de setembro a dezembro onde foram utilizadas 16 vacas prenhes da raça holandês. Estas foram divididas em quatro grupos: Grupo 0, Controle (5 vacas); Grupo I, 1 mês gestação (4 vacas); Grupo II, 3 meses gestação (4 vacas); Grupo III, 6 meses gestação (3 vacas). A segunda etapa foi realizada em abril de 2016 a fevereiro de 2017. Foram utilizadas 12 vacas prenhes da raça holandês, divididas em três grupos: Grupo 0, Controle (6 vacas); Grupo I, 1 mês gestação (3 vacas); Grupo II, 3 meses gestação (3 vacas). Todas as vacas receberam bagaço de maçã na dose de 25kg/dia/vaca. Para o primeiro experimento, uma vaca do Grupo III pariu uma bezerra, com ausência completa das vértebras coccígeas e cauda, encurvamento leve dos membros posteriores, escoliose na coluna torácica e dificuldade de locomoção. Decorridos 30 dias do nascimento, manifestou diarreia e pouco desenvolvimento, sendo eutanasiada para necropsia. Na macroscopia, havia malformações na coluna torácica, articulação coxofemoral e no aparelho urogenital. Em relação ao segundo experimento uma vaca do Grupo I pariu uma bezerra com membros pélvicos e torácicos, curvos e com articulações consideravelmente grossas e “achinelamento” de cascos. Na microscopia do fêmur foi observado na placa epifisária, zona hipertrófica desorganizada, irregular e zona de crescimento escassa. Na zona esponjosa primária observou-se trabéculas curtas e pouco mineralizadas. Amostras do bagaço de maçã utilizado na experimentação foram congeladas e enviadas para avaliação de resíduos agrotóxicos, onde foi encontrado resultado positivo para o fungicida carbendazim.

ABSTRACT: This study aimed to describe and discuss the results of an experiment carried out in two stages with pregnant cows fed 25kg/apple pomace/day. The first stage involved 16 pregnant Holstein Friesian cows divided into four groups: Group 0 - Control (5 cows); Group I - 1 month-gestation (4 cows); Group II - 3 month-gestation (4 cows); Group III - 6 month-gestation (3 cows) and was performed from September to December 2015. The second stage comprised 12 pregnant Holstein Friesian cows divided into three groups: Group 0 - Control (6 cows), Group I - 1 month-gestation (3 cows), and Group II - 3 month-gestation (3 cows) and was conducted from April 2016 to February 2017. All study animals received apple pomace at a dose of 25kg/day. As for the first experiment stage, a cow in Group III bred a calf with complete absence of the coccygeal vertebrae and tail, slight bending of the hind limbs, scoliosis in the thoracic spine, and limited mobility. At 30 days, it presented with diarrhea and underdevelopment, and was euthanized for necropsy. At gross examination, malformations were observed in the thoracic spine, coxofemural joint, and genitourinary tract. Regarding the second experiment stage, a cow in Group I gave birth to a calf with curved pelvic and thoracic limbs with thick joints and flattening hooves. Microscopic examination of the femur showed disorganized, irregular hypertrophic zone and scarce growth zone, in addition to primary spongy zone with short, slightly mineralized trabeculae. Samples of the apple pomace used in this study were frozen and sent for laboratory evaluation of pesticide residues, which showed a positive result for the fungicide carbendazim.

A simple, fast and efficient method was developed for determination of six synthetic dyes (Tartrazine, Sunset Yellow, Amaranth, Ponceau 4R, Indigo Carmine and Brilliant Blue) in sports drinks using dispersive solid-phase extraction (d-SPE) with the polymeric sorbent Oasis® HLB. Sample preparation using d-SPE permitted to simplify the procedure and use less sorbent in comparison with SPE. The use of a syringe for the elution step make this easier and avoid the use of a manifold. High performance liquid chromatography with UV-Vis detection was employed for quantification. Recovery results, evaluated at 0.5; 1.0 and 1.5 mg L-1, were between 76 and 108% with relative standard deviation < 18%. The method limit of quantification was 0.5 mg L-1. The developed method was applied to the analysis of commercial sports drinks and the results indicated that all studied samples presented dye levels in conformity with the Brazilian legislation.

A fast and sensitive method using high performance liquid chromatography-fluorescence detection (HPLC-FD) and ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was developed and validated for the determination of avermectins residues in ovine muscle samples. QuEChERS (quick, easy, cheap, effective, rugged and safe) sample preparation based on acetonitrile extraction followed by partitioning with NaCl and Na2SO4 and dispersive solid-phase extraction (d-SPE) clean-up with C18 was applied. Na2SO4 was used instead of MgSO4 due to lower amounts of co-extractives in the final extract. The procedure was validated according to the Commission Decision 2002/657/EC. The method showed determination coefficients (r2) higher than 0.99, recoveries between 93.2 and 124.3% for spike levels between 0.5 and 2.0 times the maximum residues limit (MRL) values. The repeatability and intermediate precision RSD values ranged from 1 to 19%. Decision limits (CCα) and detection capabilities (CCβ) ranged from 10.7 to 59.4 µg kg-1 and 11.4 to 68.8 µg kg-1, respectively. Method performance was successfully evaluated by analyzing real samples and proficiency test with a z-score in the range of ±1.

An easy and efficient sample preparation method was developed for the determination of natamycin residues in wine samples by solvent extraction and ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) analysis. After choosing acetonitrile as extraction solvent, an experimental design was carried out with different amounts of C18 and primary secondary amine (PSA) sorbents in order to optimize the clean-up step. Validation results were satisfactory, with recoveries from 77 to 95% and relative standard deviation (RSD) lower than 10% for the spike levels 5, 10 and 20 µg L-1. Method limit of detection (LOD) and quantification (LOQ) were 1.5 and 5.0 µg L-1, respectively. The validated method proved to be an excellent analytical tool, with a total analysis time of 3 min. The method was applied in 10 Brazilian wine samples and no residues of natamycin were detected.

ABSTRACT The objective of this study was to verify the richness and density of aquatic benthic macroinvertebrates after exposure to fungicides and insecticides of the rice paddy fields. In the crop seasons of 2012/13 and 2013/14, field experiments were performed, which consisted of single-dose applications of the fungicides trifloxystrobin + tebuconazole and tricyclazole, and the insecticides lambda-cyhalothrin + thiamethoxam and diflubenzuron, in 10 m2 experimental plots, over rice plants in the R3 stage. Control plots with and without rice plants were maintained in order to simulate a natural environment. Soil samples were collected during rice cultivation for assessment of the macroinvertebrate fauna. Chemical-physical parameters assessed in the experiments included temperature, pH and oxygen dissolved in the water and pesticide persistence in the water and in the soil. The application of a single dose of the pesticides and fungicides in the recommended period does not cause significant negative effects over the richness and density of the macroinvertebrates. Tebuconazole, tricyclazole and thiamethoxam showed high persistence in the irrigation water of rice paddy fields. Thus, the doses and number of applications of these products in crops should be carefully handled in order to avoid contamination of the environment.

RESUMO. O estudo objetivou analisar resíduos de fungicidas e inseticidas nos grãos de arroz submetidos a diferentes formas de processamento. O trabalho foi realizado no campo durante três safras agrícolas com a aplicação de fungicidas e inseticidas sobre a parte aérea das plantas de arroz. Foi realizado um tratamento com a aplicação dos fungicidas azoxystrobin, difenoconazole, propiconazole, tebuconazole, trifloxystrobin no estádio de desenvolvimento R2, e outro tratamento com os mesmos fungicidas com duas aplicações nos estádios R2 e R4 em parcelas individuais. Um tratamento com inseticida consistiu na aplicação de cypermethrin, lambda-cyhalothrin, permethrin, thiamethoxam no estádio R2, e outro tratamento na aplicação de permethrin com intervalo de cinco dias entre as aplicações a partir do estádio de desenvolvimento R4 até um dia antes da colheita. Foram analisados resíduos de fungicida e inseticida nos grãos polidos e integral, cru, cozido e parboilizado, e na casca de arroz, totalizando 1.458 amostras. As amostras foram analisadas por cromatografia em fase gasosa com detecção por captura de elétrons (GC-ECD) usando QuEChERS modificados como método de extração. Resíduos de fungicidas e inseticidas não foram detectados nos grãos de arroz. No entanto, resíduos de azoxystrobin e cypermethrin foram detectados na casca de arroz.

ABSTRACT. The objective of this study was to analyse residues of fungicides and insecticides in rice grains that were subjected to different forms of processing. Field work was conducted during three crop seasons, and fungicides and insecticides were applied at different crop growth stages on the aerial portion of the rice plants. Azoxystrobin, difenoconazole, propiconazole, tebuconazole, and trifloxystrobin fungicides were sprayed only once at the R2 growth stage or twice at the R2 and R4 growth stages; cypermethrin, lambda-cyhalothrin, permethrin, and thiamethoxam insecticides were sprayed at the R2 growth stage; and permethrin was sprayed at 5-day intervals from the R4 growth stage up to one day prior to harvest. Pesticide residues were analysed in uncooked, cooked, parboiled, polished and brown rice grains as well as rice hulls during the three crop seasons, for a total of 1,458 samples. The samples were analysed by gas chromatography with electron capture detection (GC-ECD) using modified QuEChERS as the extraction method. No fungicide or insecticide residues were detected in rice grain samples; however, azoxystrobin and cypermethrin residues were detected in rice hull samples.

RESUMO Foi conduzido um estudo para avaliar diferentes graus de hidratação e adição de fubá de milho (FMI) para a ensilagem do farelo de arroz integral (FAR) para conservação do produto. Foram utilizadas relações FAR: FMI 10:0; 9:1; 8:2; 7:3 e 6:4 e níveis de hidratação da mistura 10, 20, 30, 40 e 50% do peso inicial, perfazendo um esquema fatorial de 5×5 com quatro repetições. Paralelamente, foram fabricados quatro silos de características semelhantes aos demais com FAR sem a hidratação, denominados silos referência, e avaliada a peroxidação lipídica. Após a abertura (120 dias de ensilagem) foram avaliados o pH, acidez titulável (AT), índice de acidez (IA), índice de fungos (IF) e atividade de água (Aw). A temperatura (análise para verificar a estabilidade aeróbica) e o pH, pós abertura, foram determinados em 24, 48 e 120 horas após a abertura. Houve interações entre as variáveis para: pH na abertura, AT, IA e IF. Para a variável Aw não houve interação. A inclusão de 40% de FMI e 42,3% de água melhorou o pH da silagem na abertura, e a inclusão de 43,6% de água melhorou o pH 24 horas pós abertura. Após 48 horas de abertura as silagens estavam inapropriadas para o consumo, e a inclusão do milho acelerou a deterioração da massa ensilada após este período. Somente nos silos controles foi observado peroxidação lipídica. O uso do método de ensilagem para preservar a qualidade do FAR foi promissor como ferramenta para diminuir a peroxidação, independentemente do nível de hidratação. Para a confecção de silagem de farelo de arroz recomenda-se a inclusão de FMI de 40% e hidratação de 42,3% sobre o peso para otimizar o pH na abertura.

SUMMARY A study was carried out to evaluate the addition of cornmeal (FMI) at different hydration degrees to rice bran, as a strategy for its conservation by silage (FAR). Five relations of FAR and FMI were used: 10:0; 9:1; 8:2; 7:3 and 6:4, combined with five hydration levels: 10, 20, 30, 40 and 50% over initial weight, performing a 5×5 factorial design with four replications. In parallel, were prepared four similar silos only with FAR, without hydration (control silos). After 120 days of storage, the pH, titratable acidity, acidity index, fungi index, and water activity (Aw) were evaluated. The temperature (regarding to verify the aerobic stability) and the pH were determined at 24, 48 and 120 hour after opening. Interactions were observed for pH at opening time, titratable acidity, acid index and fungi index. No interactions were observed with Aw. The inclusion of 40% of cornmeal and 42.3% water improved the silage pH at opening and 43,6% improve the silage pH at 24 hours after opening. However, after 48 h of opening, all silages were inappropriate for consumption, the addition of cornmeal accelerated the deterioration of silage. Only on the control silos, the peroxidation process was observed. Then, the use of silage method for preserving the quality of rice bran was promising as a tool to reduce peroxidation independent of hydration level. We recommended for silage rice bran the inclusion of FMI 40% and 42.3% of water to optimize the pH in the opening.

An effective analytical method for pesticides multiresidue determination in grape samples was developed and validated. A modified quick, easy, cheap, effective, robust and safe (QuEChERS) method was used to extract the target compounds. Several sorbent materials were tested for the clean-up step using dispersive solid phase extraction (d-SPE) and Florisil® was selected. Samples extracts were evaporated before injection in the gas chromatography with mass spectrometry (GC-MS) system in order to improve detectability. Recoveries from blank samples spiked at 0.04, 0.3 and 1.0 mg kg−1 ranged from 95 to 102% with relative standard deviation (RSD) from 1.3 to 19.7%. Method limits of detection (LODm) and quantification (LOQm) ranged from 0.006 to 0.012 and 0.02 to 0.04 mg kg−1, respectively. The positive matrix effect caused an increase in the peak areas of all compounds and matrix matched calibration curve linearity (coefficient of determination, r2) was higher than 0.98 for all target analytes. The validated method was successfully applied for the determination of 19 pesticides in grape samples.