Carbamazepine, venlafaxine, tramadol, and their main metabolites: Toxicological effects on zebrafish embryos and larvae.

Pharmaceutical compounds and their metabolites are found in natural and wastewater. However, investigation of their toxic effects on aquatic animals has been neglected, especially for metabolites. This work investigated the effects of the main metabolites of carbamazepine, venlafaxine and tramadol. Zebrafish embryos were exposed (0.1-100 µg/L) for 168hpf exposures to each metabolite (carbamazepine-10,11-epoxide, 10,11-dihydrocarbamazepine, O-desmethylvenlafaxine, N-desmethylvenlafaxine, O-desmethyltramadol, N-desmethyltramadol) or the parental compound. A concentration-response relationship was found for the effects of some embryonic malformations. Carbamazepine-10,11-epoxide, O-desmethylvenlafaxine and tramadol elicited the highest malformation rates. All compounds significantly decreased larvae responses on a sensorimotor assay compared to controls. Altered expression was found for most of the 32 tested genes. In particular, abcc1, abcc2, abcg2a, nrf2, pparg and raraa were found to be affected by all three drug groups. For each group, the modelled expression patterns showed differences in expression between parental compounds and metabolites. Potential biomarkers of exposure were identified for the venlafaxine and carbamazepine groups. These results are worrying, indicating that such contamination in aquatic systems may put natural populations at significant risk. Furthermore, metabolites represent a real risk that needs more scrutinising by the scientific community.

Evaluation of the Potential of Marine Algae Extracts as a Source of Functional Ingredients Using Zebrafish as Animal Model for Aquaculture.

Research on immunotherapeutic agents has become a focus for the treatment of fish diseases. The ability of algae to produce secondary metabolites of potential interest as immunotherapeutics has been documented. The present research intended to assess antiviral and antibacterial activities of macro- and microalgae extracts against viral and bacterial pathogens and explore their immunomodulatory potential using zebrafish (Danio rerio) larvae as a model organism. The cytotoxicity and antiviral activity of eight methanolic and ethanolic extracts from two macroalgae (Fucus vesiculosus, Ulva rigida) and two microalgae (Nannochloropsis gaditana, Chlorella sp.) were analyzed in established fish cell lines. Six extracts were selected to evaluate antibacterial activity by disk diffusion and growth inhibition assays. The three most promising extracts were characterized in terms of fatty acid composition, incorporated at 1% into a plant-based diet, and evaluated their effect on zebrafish immune response and intestinal morphology in a short-term feeding trial. All extracts exhibited in vitro antiviral activity against viral hemorrhagic septicemia and/or infectious pancreatic necrosis viruses. Methanolic extracts from F. vesiculosus and U. rigida were richer in saturated fatty acids and exhibited in vitro antibacterial action against several bacteria. Most promising results were obtained in vivo with F. vesiculosus methanol extract, which exerted an anti-inflammatory action when incorporated alone into diets and induced pro-inflammatory cytokine expression, when combined with the other extracts. Moreover, dietary inclusion of the extracts improved intestinal morphology. In summary, the results obtained in this study support the potential of algae as natural sources of bioactive compounds for the aquaculture industry.

Effect of ultrasonic activation on the reduction of bacteria and endotoxins in root canals: a randomized clinical trial.

AIM: This randomized clinical trial aimed to compare the effectiveness of ultrasonic activation with that of nonactivated irrigation on the removal of bacteria and endotoxin from root canals. METHODOLOGY: Fifty patients with necrotic pulps and asymptomatic apical periodontitis were randomly allocated into two groups according to the final irrigation protocol after root canal preparation: Group UI - ultrasonic irrigation (n = 25) and Group NI - needle irrigation (n = 25). The root canals were medicated with calcium hydroxide for 14 days. Microbiological sampling was performed before (S1) and after the root canal preparation (S2), after the irrigation protocols (S3) and after the removal of the intracanal medication (S4). Total bacteria counts were determined by qPCR and the endotoxin levels by the limulus amebocyte lysate assay. Intragroup analyses were performed using the Wilcoxon test for related samples, whereas intergroup analyses were performed using the Mann-Whitney U-test (P < 0.05). RESULTS: All S1 samples were positive for bacteria, with median numbers of 1.49 × 106 and 8.55 × 105 bacterial cells for the UI and NI groups, respectively. This number significantly decreased in S2 samples (UI: 1.41 × 104 ; NI: 3.53 × 104 ; both with P < 0.001). After final irrigation protocols, there was a significant decrease in bacterial load from S2 to S3 samples in both groups (UI: 4.29 × 103 ; NI: 1.08 × 104 ; P < 0.01). Intergroup analysis revealed a significant difference between irrigation methods regarding bacterial counts in S3 samples (P < 0.05). In contrast, no significant differences were observed between groups for endotoxin levels (P > 0.05). CONCLUSIONS: Ultrasonic activation was more effective than nonactivated irrigation for reducing the number of bacteria but not the endotoxin levels in root canals of teeth with apical periodontitis.

Morfologia do coração e dos vasos da base do pinguim-de-magalhães (Spheniscus magellanicus) / Morphology of heart and vessels of the cardiac base in Magellanic penguin (Spheniscus magellanicus)

O pinguim-de-magalhães é uma ave marinha de porte médio, de origem do hemisfério sul, com grandes colônias próximas à Patagônia. Em certas épocas do ano, alguns exemplares aparecem no litoral brasileiro, devido ao desvio de rotas de caça, e alguns indivíduos não conseguem retornar por debilidades na saúde. Foram utilizados 34 exemplares de Spheniscus magellanicus que vieram a óbito no litoral do estado de Espírito Santo. O presente estudo analisou a morfologia de câmaras e paredes cardíacas, valvas e artérias da base. Os fragmentos dessas regiões foram analisados histologicamente com coloração hematoxilina-eosina (HE) e Tricrômico de Gomori (TG), além da coloração Picrosirius Red (PSR) sob luz polarizada, visando observar, principalmente, a composição do tipo de colágeno existente em cada região descrita. Entre os 34 exemplares, nenhum apresentou discrepância em relação a sua morfologia. A tipificação do colágeno dessas regiões pelas colorações TG e PSR sob luz polarizada demonstrou a presença do colágeno tipo I em maior evidência que o tipo III, encontrada na maioria das estruturas, o que atribuiu a aparência avermelhada intensa a quase todas elas. Pode-se concluir que a anatomia cardíaca do pinguim-de-magalhães é semelhante à de outras aves, com predominância do colágeno do tipo I.(AU)

Magellanic penguin is medium-sized seabird originated from southern hemisphere with colonies near Patagonia. At certain times of the year in Brazilian coast, a few penguins lose their hunting routes and can´t return because they are very sick. Thirty-four penguins died in Espírito Santo´s coast. This study analyzed the cardiac morphology and morph metric of heart chambers and walls, valves, and arteries of the cardiac base. These parts were analysed and stained by Hematoxilin and eosin and Gomori´s trichrome. Mainly targeting the collagen´s composition in each described part the Picru-sirius Red´s stain under polarized light was used. Among thirty-four penguin hearts, none presented discrepancy in morphology, they were all very similar. The characterization of collagen by Picrusirius Red stain highlighted type 1 collagen in comparison to type 3 collagen in most structures, giving a more reddish appearance in almost of them. In conclusion, the cardiac anatomy of the Magellanic Penguin is similar to that of other birds, with a predominance of type I collagen.(AU)

Variation of age and total length in Sotalia guianensis (Van Bénéden, 1864) (Cetacea, Delphinidae), on the coast of Espírito Santo state, Brazil / Variação etária e de comprimento total de Sotalia guianensis (Van Bénéden, 1864) (Cetacea, Delphinidae), no Litoral do Espírito Santo, Brasil

Abstract Variations of age and total length of Sotalia guianensis from the state of Espírito Santo, Brazil, were evaluated. Specimens were found stranded. Age and total length of 44 Guiana dolphins were assessed based on tooth analysis. Age varied between 0.5 year and 33 years (mean = 8.23 years). Most specimens were between zero and 6 years old (47%). Total length varied from 119 cm to 198 cm, with mean of 172.52 cm. Asymptotic length was reached at 185 cm and approximately 5-6 years of age. Mean total length and age were higher than in other regions of the distribution range of the species. Nevertheless, more studies have to be carried out to evaluate the morphological variations in S. guianensis populations in the study area and Brazil.

Resumo Foram avaliadas variações de comprimento total e etárias de Sotalia guianensis provenientes do estado do Espírito Santo. Os espécimes foram encontrados encalhados. Idade e comprimento total de 44 S. guianensis foram avaliados com base na análise dentária. A idade variou entre 0,5 e 33 anos (média = 8,23 anos). A maioria dos exemplares tinha entre zero e seis anos de idade (47%). O comprimento total variou de 119 a 198 centímetros, com média de 172,52 centímetros. O comprimento assintótico foi atingido com 185 cm, aproximadamente entre 5-6 anos de idade. O comprimento total médio e a idade foram maiores que outras regiões de distribuição da espécie. No entanto, mais estudos devem ser realizados para avaliar as variações morfológicas em populações de S. guianensis na região e no Brasil.

Variation of age and total length in Sotalia guianensis (Van Bénéden, 1864) (Cetacea, Delphinidae), on the coast of Espírito Santo state, Brazil.

Variations of age and total length of Sotalia guianensis from the state of Espírito Santo, Brazil, were evaluated. Specimens were found stranded. Age and total length of 44 Guiana dolphins were assessed based on tooth analysis. Age varied between 0.5 year and 33 years (mean = 8.23 years). Most specimens were between zero and 6 years old (47%). Total length varied from 119 cm to 198 cm, with mean of 172.52 cm. Asymptotic length was reached at 185 cm and approximately 5-6 years of age. Mean total length and age were higher than in other regions of the distribution range of the species. Nevertheless, more studies have to be carried out to evaluate the morphological variations in S. guianensis populations in the study area and Brazil.

Single Low-Dose Ionizing Radiation Induces Genotoxicity in Adult Zebrafish and its Non-Irradiated Progeny.

This study investigated to what extent a single exposure to low doses of ionizing radiation can induce genotoxic damage in irradiated adult zebrafish (Danio rerio) and its non-irradiated F1 progeny. Four groups of adult zebrafish were irradiated with a single dose of X-rays at 0 (control), 100, 500 and 1000 mGy, respectively, and couples of each group were allowed to reproduce following irradiation. Blood of parental fish and whole-body offspring were analysed by the comet assay for detection of DNA damage. The level of DNA damage in irradiated parental fish increased in a radiation dose-dependent manner at day 1 post-irradiation, but returned to the control level thereafter. The level of DNA damage in the progeny was directly correlated with the parental irradiation dose. Results highlight the genotoxic risk of a single exposure to low-dose ionizing radiation in irradiated individuals and also in its non-irradiated progeny.

Effects of dietary exposure to herbicide and of the nutritive quality of contaminated food on the reproductive output of Daphnia magna.

Risk assessment of pesticides has been based on direct toxic effects on aquatic organisms. Indirect effects data are taken into account but with limitations, as it is frequently difficult to predict their real impacts in the ecosystems. In this context the main aim of this work was to assess how the exposure to the herbicide pendimethalin (Prowl(®)), under environmentally relevant concentrations, may compromise the nutritional composition of food for a relevant group of primary consumers of freshwater food webs-the daphnids, thus affecting their reproduction performance and subsequently the long-term sustainability of active populations of this grazer. Therefore, Daphnia magna individuals were chronically exposed in a clean medium to a control diet (NCF - i.e., non-contaminated green algae Raphidocelis subcapitata) and to a contaminated diet (CF - i.e., the same monoalgal culture grown in a medium enriched with pendimethalin in a concentration equivalent to the EC20 for growth inhibition of algae), during which reproductive endpoints were assessed. The algae were analysed for protein, carbohydrate and fatty acid content. The chemical composition of R. subcapitata in the CF revealed a slight decrease on total fatty acid levels, with a particular decrease of essential ω9 monounsaturated fatty acids. In contrast, the protein content was high in the CF. D. magna exposed to CF experienced a 16% reduction in reproduction, measured as the total number of offspring produced per female. Additionally, an internal pendimethalin body burden of 4.226µgg(-1) was accumulated by daphnids fed with CF. Hence, although it is difficult to discriminate the contribution of the pesticide (as a toxic agent transferred through the food web) from that of the food with a poor quality-compromised by the same pesticide, there are no doubts that, under environmentally relevant concentrations of pesticides, both pathways may compromise the populations of freshwater grazers in the long term, with consequences in the control of the primary productivity of these systems.

Development of a powdered activated carbon in bar adsorptive micro-extraction for the analysis of morphine and codeine in human urine.

In the present work, bar adsorptive microextraction using an activated carbon (AC) adsorbent phase followed by liquid desorption and high-performance liquid chromatography with diode array detection was developed to monitor morphine (MOR) and codeine (COD) in human urine. Under optimized experimental conditions, assays performed in aqueous media spiked at the 30.0 µg/L level yielded recoveries of 41.3 ± 1.3% for MOR and 38.4 ± 1.7% for COD, respectively. The textural and surface chemistry properties of the AC phase were also correlated with the analytical data for a better understanding of the overall enrichment process. The analytical performance showed good precision (relative standard deviation < 8.0%), suitable detection limits (0.90 and 0.06 µg/L for MOR and COD, respectively) and convenient linear dynamic ranges (r(2) > 0.991) from 10.0 to 330.0 µg/L. By using the standard addition methodology, the applications of this analytical approach to water and urine matrices allowed remarkable performance to monitor MOR and COD at the trace level. This new confirmatory method proved to be a suitable alternative to other sorptive micro-extraction methodologies in monitoring trace levels of opiate-related compounds, because it was easy to implement, reliable, sensitive and required a low sample volume.

Crescimento e desenvolvimento de boto-cinza (Sotalia guianensis) do litoral do Espírito Santo / Growth and development of estuarine dolphin (Sotalia guianensis) from Espírito Santo coast

Estudou-se o desenvolvimento do boto-cinza (Sotalia guianensis) do litoral do Espírito Santo e verificou-se se a análise das radiografias da aleta peitoral pode ser utilizada para tal. Trinta e sete botos passaram pela determinação do comprimento total (CT), análise do grau de fusionamento das vértebras e radiografia das aletas peitorais. O CT ao nascimento foi de 103,3cm e o de animais maduros fisicamente 187,5cm. Observou-se correlação entre o CT, as epífises distais do rádio e da ulna e correlação entre o grau de fusionamento vertebral e a epífise distal do rádio. Concluiu-se que a análise radiográfica das aletas peitorais é uma boa maneira de avaliar o grau de maturação física do boto.

The aim of this study is to analyze the development of the estuarine dolphin from the coast of Espírito Santo and verify that the analysis of X-rays of pectoral fin can be used for such. Thirty-seven Sotalia guianensis were used, determining the TL, examination of the degree of fusion of the vertebrae and X-ray of pectoral fins. At birth the TL is 103.3cm and in physically mature animals the TL is 187.5cm. The TL showed a significant correlation with the epiphyses of the distal radius and ulna and the degree of spinal fusion showed significant correlation with distal radius. It can be concluded that the X-ray analysis of the pectoral fins is a good way of assessing the physical maturation of these animals and can be used in routine procedures.

Cork-based activated carbons as supported adsorbent materials for trace level analysis of ibuprofen and clofibric acid in environmental and biological matrices.

In this contribution, powdered activated carbons (ACs) from cork waste were supported for bar adsorptive micro-extraction (BAµE), as novel adsorbent phases for the analysis of polar compounds. By combining this approach with liquid desorption followed by high performance liquid chromatography with diode array detection (BAµE(AC)-LD/HPLC-DAD), good analytical performance was achieved using clofibric acid (CLOF) and ibuprofen (IBU) model compounds in environmental and biological matrices. Assays performed on 30 mL water samples spiked at the 25.0 µg L(-1) level yielded recoveries around 80% for CLOF and 95% for IBU, under optimized experimental conditions. The ACs textural and surface chemistry properties were correlated with the results obtained. The analytical performance showed good precision (<15%), suitable detection limits (0.24 and 0.78 µg L(-1) for CLOF and IBU, respectively) and good linear dynamic ranges (r(2)>0.9922) from 1.0 to 600.0 µg L(-1). By using the standard addition methodology, the application of the present approach to environmental water and urine matrices allowed remarkable performance at the trace level. The proposed methodology proved to be a viable alternative for acidic pharmaceuticals analysis, showing to be easy to implement, reliable, sensitive and requiring low sample volume to monitor these priority compounds in environmental and biological matrices.

Designing novel hybrid materials by one-pot co-condensation: from hydrophobic mesoporous silica nanoparticles to superamphiphobic cotton textiles.

This work reports the synthesis and characterization of mesoporous silica nanoparticles (MSNs) functionalized with tridecafluorooctyltriethoxysilane (F13) and their in situ incorporation onto cotton textiles. The hybrid MSNs and the functional textiles were prepared by a one-pot co-condensation methodology between tetraethylorthosilicate (TEOS) and F13, with hexadecyltrimethylammonium chloride (CTAC) as the template and triethanolamine as the base. The influence of the F13 to TEOS molar ratio (1:10, 1:5 and 1:3) on the nanoparticle morphology, porosity, degree of functionalization, and hydro/oleophobic properties is discussed. The hybrid nanosilicas presented high colloidal stability and were spherical and monodispersed with average particle size of ∼45 nm. They also showed high surface areas, large pore volumes, and a wormhole-type mesoporous structure. The increase in the organosilane proportion during the co-condensation process led to a more radially branched wormhole-like mesoporosity, a decrease in the surface area, pore volume, and amount of surface silanol groups, and an enrichment of the surface with fluorocarbon moieties. These changes imparted hydrophobic and oleophobic properties to the materials, especially to that containing the highest F13 loading. Cotton textiles were coated with the F13-MSNs through an efficient and less time-consuming route. The combination between surface roughness and mesoporosity imparted by the MSNs, and the low surface energy provided by the organosilane resulted in superhydrophobic functional textiles. Moreover, the textile with the highest loading of fluorocarbon groups was superamphiphobic.

Powdered activated carbons as effective phases for bar adsorptive micro-extraction (BAµE) to monitor levels of triazinic herbicides in environmental water matrices.

Bar adsorptive micro-extraction using three powdered activated carbons (ACs) as adsorbent phases followed by liquid desorption and high performance liquid chromatography with diode array detection (BAµE(ACs)-LD/HPLC-DAD), was developed to monitor triazinic herbicides (atrazine, simazine and terbutylazine) in environmental water matrices. ACs used present apparent surface areas around 1000 m(2) g(-1) with an important mesoporous volume and distinct surface chemistry characteristics (pH(PZC) ranging from 6.5 to 10.4). The textural and surface chemistry properties of the ACs adsorbent phases were correlated with the analytical data for a better understanding of the overall enrichment process. Assays performed on 10 mL water samples spiked at the 10.0 µg L(-1) levels under optimized experimental conditions yielded recoveries around 100% for the three herbicides under study. The analytical performance showed good precision (RSD<15.0%), convenient detection limits (≈0.1 µg L(-1)) and suitable linearity (1.0-12.0 µg L(-1)) with good correlation coefficients (r(2)>0.9914). By using the standard addition method, the application of the present method on real water matrices, such as surface water and wastewater, allowed very good performances at the trace level. The proposed methodology proved to be a suitable sorptive extraction alternative for the analysis of priority pollutants with polar characteristics, showing to be easy to implement, reliable, sensitive and requiring a low sample volume to monitor triazinic compounds in water matrices.

Ultrassonografia e histopatologia renal em cães / Renal ultrasonography and histopathology in dogs

The kidneys are the organs responsible for maintaining the hydro-electrolytic equilibrium of the organism. There are several diseases that can affect these organs, causing changes and may be one of the causes of death in clinical routine. The ultrasound is the main diagnostic method to detect changes in the kidneys because it is noninvasive and allow the assessment of the size and the renal parenchyma. The histopathology is essential for confirmation of the diagnosis, and it was performed and compared with the ultrasound examination, so the degree of compatibility between ultrasound and pathological diagnostic methods could be analyzed. Twenty-one dogs were used in the experiment. After the ultrasound, the animals were autopsied for removal of the kidneys. The kidneys were fixed in 10% formalin, processed and observed by light microscopy. The findings were 52% in ultrasound examination and 81% in histological examination. The index of compatibility between the two tests was 66%, which confirmed the literature data that describe the ultrasound examination as the best method of non-invasive diagnosis for renal disorders.

Effects of the microcystin profile of a cyanobacterial bloom on growth and toxin accumulation in common carp Cyprinus carpio larvae.

A 12 day growth trial was conducted to compare the effect of the variation in microcystins (MC) composition of two bloom samples of Microcystis aeruginosa on the growth performance and microcystin accumulation in common carp Cyprinus carpio larvae. Two M. aeruginosa natural bloom samples with different MC profiles were collected and larvae were exposed to cyanobacterial cells through their diet. Three diets, a basal control diet and two diets prepared from the basal diet plus the same toxins content (60 ng MC g(-1) diet) of each cyanobacterial bloom, were given at the same ration level to three groups of larvae during the experimental period. Larval mass and standard length from day 9 were significantly different between cyanobacterial treatments and in both cases lower than that of the control. The MC accumulation by larvae, inversely correlated with the growth performance, was also significantly different between cyanobacterial treatments (26.96 v. 17.32 ng g(-1) at the end of the experimental period). These results indicate that MC variants profile may have effects on the toxin uptake and toxicity. To date, this is the first laboratory study to show that fish accumulate MC depending on the toxin profile of the cyanobacterial bloom.

Disruption of zebrafish (Danio rerio) embryonic development after full life-cycle parental exposure to low levels of ethinylestradiol.

Exposure of fish to the synthetic estrogen ethinylestradiol (EE2) has been shown to induce a large set of deleterious effects. In addition to the negative impact of EE2 in reproductive endpoints, concern has recently increased on the potential effects of EE2 in fish embryonic development. Therefore, the present study aimed at examining the effects of EE2 on the full embryonic development of zebrafish in order to identify the actual phases where EE2 disrupts this process. Hence, zebrafish were exposed to environmentally relevant low levels of EE2, 0.5, 1 and 2ng/L (actual concentrations of 0.19, 0.24 and 1ng/L, respectively) from egg up to eight months of age (F(1)), and the survival as well as the occurrence of abnormalities in their offsprings (F(2)), per stage of embryonic development, was investigated. A thorough evaluation of reproductive endpoints and transcription of vtg1 gene in the parental generation (F(1)) at adulthood, was performed. No significant differences could be observed for the two lowest EE2 treatments, in comparison with controls, whereas vtg1 transcripts were significantly elevated (40-fold) in the 2ng/L EE2 treatment. In contrast to the findings in the F(1) generation,a significant concentration-dependent increase in egg mortality between 8 and 24hours post-fertilization (hpf) was observed for all EE2 treatments, when compared with controls. The screening of egg and embryo development showed a significant increase in the percentage of abnormalities at 8 hpf for the highest EE2 concentration, a fact that might explain the increased embryo mortality at the 24 hpf time-point observation. Taken together, these findings indicate that the two lowest tested EE2 concentations impact late gastrulation and/or early organogenesis, whereas exposure to 2ng/L EE2 also disrupts development in the blastula phase. After early organogenesis has been completed (24 hpf), no further mortality was observed. These results show that increased embryo mortality occurs at EE2 levels below those inducing reproductive impairment and vtg1 gene induction in the male parental generation, thus suggesting that EE2 may impact some fish populations at levels below those inducing an increase in vtg1 transcripts. Hence, these findings have important implications for environmental risk assessment, strongly supporting the inclusion of embryonic development studies in the screening of endocrine disruption in wild fish populations.

Effects of cyanobacterial extracts containing anatoxin-a and of pure anatoxin-a on early developmental stages of carp.

This study compares the effects of pure anatoxin-a and cyanobacterial extracts of an anatoxin-a producing strain on early stages of development of carp. Carp eggs were exposed from 2:30 h to 4 days post-fertilization to different ecologically relevant concentrations of anatoxin-a, provided as pure toxin or contained in the cyanobacterial extracts. Data on time to mortality, mortality rate, time to hatching, hatching rate, skeletal malformations rate, and larval standard length were registered until 8 days post-fertilization. At any tested concentration of anatoxin-a, the pure toxin was almost harmless to carp early stages of development, contrarily to cell extracts that were highly toxic. Only an adverse effect on the larval length was found at the highest concentration of pure toxin, while increasing concentrations of cell extracts caused increasing adverse effects in all the analyzed parameters. Anatoxin-a producing cyanobacteria should be regarded as putative modulators of aquatic ecosystems communities.

[Surgical treatment of complete penile duplication]. / Tratamiento quirúrgico de duplicidad peneana completa.

Penile duplication is a rare anomaly with an incidence of 1 in 5,500,000. It is almost associated with other malformations like double bladder, presence of the cloaca, imperforate anus, duplication of the recto sigmoid and vertebral deformities. The authors present the surgical technique to resolve a rare case of complete penile duplication in a 4 years old child, without any other malformation.

Detection of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in effluents and sludge of a hospital sewage treatment plant.

AIMS: To detect ESBL (extended-spectrum beta-lactamase)-producing Klebsiella pneumoniae present in the effluents and sludge of a hospital sewage treatment plant, evaluating the treatment plant's potential to remove these micro-organisms. METHODS AND RESULTS: Twenty samples (crude sewage, UASB reactor effluent, filtered effluent and sludge) were collected in the period from May to December 2006, in order to analyse antimicrobial susceptibility and to check ESBL production, the disc-diffusion and the combined disc methods were used. Total and faecal coliform concentrations were also determined. ESBL-producing K. pneumoniae were detected in all samples analysed, representing 46 x 5% of the total strains isolated. Among the non-ESBL-producing strains, 26% were multiresistant and one strain resistant to eight of the nine antimicrobials tested was detected in the treated effluent. CONCLUSIONS: The hospital wastewater treatment plant did not show a satisfactory efficacy in removing pathogenic micro-organisms, allowing for the dissemination of multiresistant bacteria into the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The inefficacy of hospital wastewater treatment plants can result in routes of dissemination of multiresistant bacteria and their genes of resistance into the environment, thus contaminating water resources, and having serious negative impact on public health.

Genotoxic effects of binary mixtures of xenoandrogens (tributyltin, triphenyltin) and a xenoestrogen (ethinylestradiol) in a partial life-cycle test with Zebrafish (Danio rerio).

A partial life-cycle test with the model fish Danio rerio was performed in order to evaluate the genotoxic potential of binary mixtures of xenoandrogenic (tributyltin--TBT; triphenyltin--TPT) and an estrogenic compound (ethinylestradiol--EE2). Five days post-fertilisation larvae were diet-exposed to environmental relevant concentrations of TBT and TPT (25 ng/g-100 ng/g), and water-exposed to ethinylestradiol (3.5 ng/L) for a four-month period; binary mixtures of TBT plus EE2 and TPT plus EE2 were run in parallel. The erythrocytic nuclear abnormalities (ENA) assay in circulating erythrocytes was used to evaluate genotoxicity in the end of the four-month exposure period. A significant increase (p<0.05, Kruskall-Wallis non-parametric ANOVA) in ENA frequency, in comparison with control animals, was observed in those animals exposed to TBT and TPT (the highest doses only), and to EE2 and the binary mixtures, although neither synergistic nor additive effects of the tested compounds were evident. Overall, the results clearly indicate that chronic exposure to low levels of TBT, TPT, EE2 and binary mixtures of TBT plus EE2 and TPT plus EE2 are genotoxic to zebrafish, which may suggest that wild fish populations may be under increased DNA damage in areas contaminated by these endocrine disrupting chemicals.