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In the last decades, knowledge about the biology of the Amazon river dolphin Inia geoffrensis has increased considerably. Accordingly, we present a review of the published literature on this river dolphin, with a focus on topics with the most advancements and those that are important to the species conservation. We also present new information, including emerging knowledge on the other species of Inia. We summarize the current knowledge of the biology of Inia, covering aspects of taxonomy, distribution, life history and reproduction, genetics, anatomy and physiology, health, behavior, ecology, demographic parameters, and population trends. We also highlight the main anthropogenic threats affecting the species and the resulting conservation efforts.

We tracked thirteen giant otters from three different groups in the Balbina Hydroelectric Reservoir. One of them was a transient animal, while the others were living in groups of five and seven individuals. Travelling was the most frequent behaviour, both for the transient otter (>40%) and group otters (46%). Diurnal resting was the longest-lasting behaviour. Most giant otter behaviour in the reservoir showed little difference when compared with the behaviour of giant otters living in nondammed areas, indicating a significant degree of resilience of this species. However, otters in the reservoir spent more time travelling, probably because their territory sizes were much greater than the territories of giant otters in nondammed areas. The frequency of fishing was similar to what has been recorded elsewhere, suggesting that current fish density and availability in the reservoir is similar to what is observed in nondammed areas. Nevertheless, otters in Balbina spent more time fishing than those in nondammed areas, which alternatively could suggest that fish density in the reservoir was low and could be compensated by increasing the time spent fishing. The high frequency of intraspecific kleptoparasitism observed in this study (62%) is noteworthy, although virtually undocumented for this species. This is the first study of giant otter behaviour in a hydroelectric reservoir that can be used for the conservation of this endangered species in man-made lakes.

Studies on the microbiota of freshwater cetaceans are scarce and may provide important data on animal and environmental health. This study aimed to evaluate the antimicrobial susceptibility of Gram-negative bacteria recovered from two populations of free-ranging Amazon river dolphins (Inia geoffrensis). Twenty-one animals were captured and released, 13 from Negro River and 8 from Tapajos River, Brazil. Swab samples were obtained from the oral cavity, blowhole, genital opening and rectum and were cultured on MacConkey agar. Isolates were biochemically identified, and antimicrobial susceptibility was assessed by disk diffusion method. Overall, 132 isolates were recovered, of which 71 were recovered from animals from Negro River and 61 from Tapajos River. The most commonly recovered bacterial species were Enterobacter cloacae, Morganella morganii, Klebsiella pneumoniae and Pseudomonas aeruginosa. Overall, 51.6% (63/122) of the isolates were not-susceptible (intermediate resistance and resistance), of which 28/122 (22.9%) were resistant to at least one antimicrobial. Cephalothin, cefuroxime and cefepime were the drugs to which more resistant and intermediate results were observed (P

Body length is an important parameter in morphological, ecological and behavioral studies of a species and contributes to the understanding of the body condition of individuals. This parameter is essential for conservation and management strategies by informing studies evaluating growth rates, physical maturity and classification of individuals into age groups, promoting better accuracy for the biological parameters of the species. The aim of this study was to identify predictors of body length for Amazonian manatees (Trichechus inunguis) using metric characters of osteological materials. Eleven linear measurements of skull, jaw, scapulae and humeri were collected from 41 Amazonian manatee skeletons of different age classes (calf, juvenile and adult). Data were analyzed by simple linear regression. The condylobasal length was the best predictor of body length for the species (R = 0.943), however, all bones evaluated showed at least one measurement with the capacity to predict body size (R2 > 0.9). Results of this study are useful for inferring body length of Amazonian manatees using bones deposited in museums and biological collections, expanding the informative potential of these materials. RESUMO O comprimento corporal é um importante parâmetro em estudos morfológicos, ecológicos e comportamentais de uma espécie e contribui para o entendimento da condição corporal de um indivíduo. Este parâmetro é essencial para estratégias de conservação e manejo, informando estudos que avaliam taxas de crescimento, maturidade física e classificação dos indivíduos em grupos de idade, promovendo uma melhor precisão aos parâmetros biológicos de uma espécie. O objetivo desse trabalho foi identificar preditores de comprimento corporal para o peixe-boi da Amazônia (Trichechus inunguis) utilizando caracteres métricos de material osteológico. Onze medidas lineares do crânio, mandíbula, escápula e úmero foram coletadas de 41 esqueletos de peixe-boi da Amazônia de diferentes classes de idade (filhote, juvenil e adulto). Os dados foram analisados por regressão linear simples. O comprimento côndilobasal foi o melhor preditor de comprimento corporal para a espécie (R = 0,943), entretanto, todos os ossos avaliados mostraram pelo menos um caractere com capacidade de predizer o comprimento corporal (R2 > 0,9). Os resultados deste estudo são úteis para inferir o comprimento corporal de peixes-boi da Amazônia a partir de ossos depositados em museus e coleções biológicas, expandindo o potencial informativo destes materiais.

P-glycoprotein (P-gp) induction and/or activation have been proposed as therapeutic strategies in intoxication scenarios, by reducing the intestinal absorption of xenobiotics, including drugs. Oxygenated xanthones (OXs) have been described as P-gp modulators and, therefore, the main goals of this study were to: (a) investigate the potential modulatory effect of six OXs on P-gp expression and activity in SW480 cells; (b) validate these cells for the screening/identification of P-gp inducers/activators; (c) explore the potential OXs-mediated protective effects against the cytotoxicity of mitoxantrone (MTX), a toxic P-gp substrate. Four OXs (OX2, OX4, OX5, and OX6) increased P-gp expression 24 h after exposure. However, a lack of correlation between P-gp expression and activity was observed for OX1 and OX4. In addition, after a short incubation with the P-gp fluorescent substrate, rhodamine 123, all the studied OXs, except OX3, efficient and immediately increased P-gp activity, suggesting their potential as P-gp activators. Despite these results, OXs failed to afford protection against MTX-induced cytotoxicity. Docking simulations performed in a human P-gp model revealed that the lack of protection may be explained by the different binding locations of OXs and MTX within the P-gp drug-binding pocket. In conclusion, the in vitro results confirmed OXs potential for P-gp induction and/or activation and suggested SW480 cells as a suitable in vitro model for these studies. However, P-gp activation did not protected SW480 cells against MTX cytotoxicity. In silico studies suggested the different binding locations as a limiting step in the P-gp-mediated efflux of its substrates under P-gp activation.

Oxidative stress, triggered by UV radiation, is one of the major causes of free radical-associated disorders, such as skin cancer. The application of natural compounds (NCs) with antioxidant effects can attenuate free radicals’ accumulation and, therefore, provide a strategy for skin care and cancer prevention. In this work, three natural compounds, naringenin, nordihydroguaiaretic acid (NDGA), and kaempferol, were encapsulated into nanostructured lipid carriers (NLCs) aiming for the development of a formulation for cutaneous application with antioxidant properties. For the experiments, different formulation parameters were evaluated to optimize the NLCs that showed a diameter around 200 nm, which is an adequate particle size for incorporation in cosmetics. Transmission electron microscopy (TEM) analysis confirmed the NLCs’ typical spherical morphology. Encapsulation efficiency (EE) and loading capacity (LC) values revealed an effective production process, with EEs over 90% and LCs near the maximum value. The developed NLCs revealed a prolonged in vitro release of the natural compounds. The NLCs were stable under storage conditions, maintaining their psychochemical characteristics for 30 days. Additionally, they did not show any physical instability in accelerated stability studies, which also suggests long-term stability. Finally, the NCs antioxidant activity was evaluated. Interestingly, the NDGA and kaempferol mixture provided an antioxidant synergic effect. The NLC formulations’ cytotoxicity was tested in vitro in immortalized human keratinocytes (HaCaT). In addition, putative antioxidant effects of the developed NLC formulations against tert-butyl hydroperoxide (t-BHP)-induced oxidative stress were studied, and the NDGA-loaded NLC was revealed to be the one with the most protective effect. Therefore, we concluded that the naringenin, NDGA, and kaempferol incorporation into NLCs constitutes a promising strategy to increase their bioavailability and delivery to the skin.

The recently released Farm to Fork Strategy sets, for the first time ever, pesticide reduction goals at the EU level: 50% reduction in overall use and risk of chemical pesticides and 50% use reduction of more hazardous pesticides. However, little guidance is provided to achieve these targets. In this study, we compiled the characteristics and recommended application rates of 230 EU approved, synthetic, open-field use active substances and explored the potential of eight pesticide reduction scenarios (defined based on application rates, pesticide type, persistence, and hazard) to achieve the reduction goals. Our approach revealed that all 230 substances are potentially harmful to humans or ecosystems, and that only severe pesticide use restrictions such as full conversion to organic farming or allowing only low hazard substances will result in 50% reductions. Our results emphasis the need of an EC action plan on how to achieve and maintain the aimed reduction levels.

Diazepam is commonly used in the management of epileptic seizures, although it has limitations that can be overcome by using formulations that are easier to administer and capable of directing the drug to the brain. In this field, it has been reported that the use of nanostructured lipid carriers (NLC) via intranasal (or via nose-to-brain) promotes the targeting of drugs to the brain, improving the effectiveness of therapy. The aim of this work was to optimize two diazepam-loaded NLC formulations for nose-to-brain delivery, one with positive surface charge and one with negative surface charge. The quality by design (QbD) approach was used to design the experiments, where the quality target product profile (QTPP), the risk assessment and the critical quality attributes (CQAs) were defined to ensure safety, efficacy and quality of the final formulations. The experiments started with the optimization of critical material attributes (CMAs), related to the ratios of lipids and emulsifiers, followed by the selection of critical process parameters (CPPs), related to the production methods of the diazepam-loaded NLC formulation (ultrasound technique and high-pressure homogenization - HPH). Afterwards, the positive surface charge of the diazepam-loaded NLC was optimized. Finally, the biocompatibility with human neuronal cells of the formulation with a negative surface charge and of the formulation with a positive surface charge was evaluated. The results of the optimization of the CMAs showed that the ratios of lipids and emulsifiers more adequate were 6.7:2.9 and 4.2:0.3 (% w,w), respectively. Regarding the CPPs, HPH was considered the most suitable production method, resulting in an optimized diazepam-loaded NLC formulation (F1C15) with negative surface charge, showing particle size of 69.59 ± 0.22 nm, polydispersity index (PDI) of 0.19 ± 0.00, zeta potential (ZP) of −23.50 ± 0.24 mV and encapsulation efficiency (EE) of 96.60 ± 0.03 %. The optimized diazepam-loaded NLC formulation (F2A8) with positive surface charge had particle size of 124.40 ± 0.84 nm, PDI of 0.17 ± 0.01, ZP of 32.60 ± 1.13 mV and EE of 95.76 ± 0.24 %. In addition, the incorporation of diazepam in NLC resulted in a sustained release of the drug. No significant changes in particle size, PDI, ZP and EE were observed for the formulation F1C15, after 3 months of storage, whereas for formulation F2A8, particle size increased significantly. Biocompatibility studies showed that the formulation F2A8 was more cytotoxic than the formulation F1C15. Thereby, we conclude that the formulation F1C15 is more suitable for targeting the brain, when compared with the formulation F2A8. From the results of these studies, it can be confirmed that the QbD approach is an adequate and central tool to optimize NLC formulations.

We assessed the feeding habits of the Amazonian manatee inhabiting blackwater (Negro River) and clearwater (Tapajos River) igapos (floodplains) using δ13C and δ15N of dentin and bone collagen from different ontogenetic classes (nursing calves, juveniles, and adults). Within an individual, the dentine δ13C and δ15N values did not vary with tooth position. Bones were more depleted in 13C and 15N compared to teeth, and the δ13C and δ15N in bone differed among classes. Food sources had δ13C values typical of algae, plants of C3 or C4 photosynthetic pathways. Mixing models showed that lactating females (inferred by isotopic values from nursing calves) had higher proportional consumption of C4 plants, while, for other adults, C3 plants were more frequent in the diet of manatees from both rivers. Juveniles had a mixed diet of C3 pants and C4 plants. We hypothesize the C4 plants signal of calves results from female movements to the floodplains of nearby whitewater rivers (varzeas), where C4 plants are abundant. Individuals from Tapajos may be more resident, as C3 plants and C4 plants are available during the flood pulse. Results demonstrate that manatee diets vary with ontogenetic classes and water typology. Preservation of habitats (igapo/varzea) is therefore required for the survival of Amazonian manatees.

Herpesvirus (HV) infections in cetaceans are frequently associated with skin and mucosal lesions. Although HV infections have been reported worldwide, their occurrence in southern Atlantic marine mammals is still poorly understood. We tested skin, oral and genital mucosal beta-actin PCR-positive samples from 109 free-ranging Brazilian cetaceans using a universal herpesvirus DNA polymerase PCR. Herpesvirus-positive skin samples from a Guiana dolphin (Sotalia guianensis), a dwarf sperm whale (Kogia sima), a Bolivian river dolphin (Inia boliviensis), and a lingual sample from an Atlantic spotted dolphin (Stenella frontalis) were histologically evaluated. Additional tissue samples from these animals were also PCR-positive for HV, including a novel sequence obtained from the dwarf sperm whale's stomach and mesenteric lymph node. Four novel HV species were detected in the Guiana dolphin (one), the dwarf sperm whale (two) and the Bolivian river dolphin (one). The cutaneous lesions (marked, focally extensive, chronic proliferative dermatitis) of the Guiana dolphin and the Bolivian river dolphin were similar to previous HV reports in cetaceans, despite the absence of intranuclear inclusion bodies. This is the largest HV survey in South American cetaceans and the first detection of HV infection in riverine dolphins worldwide.

Pesticide use is a major foundation of the agricultural intensification observed over the last few decades. As a result, soil contamination by pesticide residues has become an issue of increasing concern due to some pesticides' high soil persistence and toxicity to non-target species. In this study, the distribution of 76 pesticide residues was evaluated in 317 agricultural topsoil samples from across the European Union. The soils were collected in 2015 and originated from 11 EU Member States and 6 main cropping systems. Over 80% of the tested soils contained pesticide residues (25% of samples had 1 residue, 58% of samples had mixtures of two or more residues), in a total of 166 different pesticide combinations. Glyphosate and its metabolite AMPA, DDTs (DDT and its metabolites) and the broad-spectrum fungicides boscalid, epoxiconazole and tebuconazole were the compounds most frequently found in soil samples and the compounds found at the highest concentrations. These compounds occasionally exceeded their predicted environmental concentrations in soil but were below the respective toxic endpoints for standard in-soil organisms. Maximum individual pesticide content assessed in a soil sample was 2.05 mg kg−1 while maximum total pesticide content was 2.87 mg kg−1. This study reveals that the presence of mixtures of pesticide residues in soils are the rule rather than the exception, indicating that environmental risk assessment procedures should be adapted accordingly to minimize related risks to soil life and beyond. This information can be used to implement monitoring programs for pesticide residues in soil and to trigger toxicity assessments of mixtures of pesticide residues on a wider range of soil species in order to perform more comprehensive and accurate risk assessments.

Considering that pesticides have been used in Europe for over 70 years, a system for monitoring pesticide residues in EU soils and their effects on soil health is long overdue. In an attempt to address this problem, we tested 340 EU agricultural topsoil samples for multiple pesticide residues. These samples originated from 4 representative EU case study sites (CSS), which covered 3 countries and four of the main EU crops: vegetable and orange production in Spain (S-V and S-O, respectively), grape production in Portugal (P-G), and potato production in the Netherlands (N-P). Soil samples were collected between 2015 and 2018 after harvest or before the start of the growing season, depending on the CSS. Conventional and organic farming results were compared in S-V, S-O and N-P. Soils from conventional farms presented mostly mixtures of pesticide residues, with a maximum of 16 residues/sample. Soils from organic farms had significantly fewer residues, with a maximum of 5 residues/sample. The residues with the highest frequency of detection and the highest content in soil were herbicides: glyphosate and its main metabolite AMPA (P-G, N-P, S-O), and pendimethalin (S-V). Total residue content in soil reached values of 0.8 mg kg-1 for S-V, 2 mg kg-1 for S-O and N-P, and 12 mg kg-1 for P-G. Organic soils presented 70-90% lower residue concentrations than the corresponding conventional soils. There is a severe knowledge gap concerning the effects of the accumulated and complex mixtures of pesticide residues found in soil on soil biota and soil health. Safety benchmarks should be defined and introduced into (soil) legislation as soon as possible. Soil remediation techniques should be developed to keep the levels of pesticide residues below such benchmarks. Furthermore, the process of transitioning to organic farming should take into consideration the residue mixtures and their residence time in soil.

Current farm systems rely on the use of Plant Protection Products (PPP) to secure high productivity and control threats to the quality of the crops. However, PPP use may have considerable impacts on human health and the environment. A study protocol is presented aiming to determine the occurrence and levels of PPP residues in plants (crops), animals (livestock), humans and other non-target species (ecosystem representatives) for exposure modelling and impact assessment. To achieve this, we designed a cross-sectional study to compare conventional and organic farm systems across Europe. Environmental and biological samples were/are being/will be collected during the 2021 growing season, at 10 case study sites in Europe covering a range of climate zones and crops. An additional study site in Argentina will inform the impact of PPP use on growing soybean which is an important European protein-source in animal feed. We will study the impact of PPP mixtures using an integrated risk assessment methodology. The fate of PPP in environmental media (soil, water and air) and in the homes of farmers will be monitored. This will be complemented by biomonitoring to estimate PPP uptake by humans and farm animals (cow, goat, sheep and chicken), and by collection of samples from non-target species (earthworms, fish, aquatic and terrestrial macroinvertebrates, bats, and farm cats). We will use data on PPP residues in environmental and biological matrices to estimate exposures by modelling. These exposure estimates together with health and toxicity data will be used to predict the impact of PPP use on environment, plant, animal and human health. The outcome of this study will then be integrated with socio-economic information leading to an overall assessment used to identify transition pathways towards more sustainable plant protection and inform decision makers, practitioners and other stakeholders regarding farming practices and land use policy. EEA Balcarce Fil: Silva, Vera. Wageningen University and Research. Soil Physics and Land Management Group; Países Bajos. Fil: Alaoui, Abdallah. University of Bern. Institute of Geography. Centre for Development and Environment; Suiza. Fil: Schlünssen, Vivi. Aarhus University. Department of Public Health; Dinamarca. National Research Centre for the Working Environment; Dinamarca. Fil: Vested, Anne. Aarhus University. Department of Public Health; Dinamarca. Fil: Graumans, Martien. Radboud Institute for Health Sciences; Países Bajos. Fil: Van Dael, Maurice. Radboud Institute for Health Sciences; Países Bajos. Fil: Trevisan, Marco. Università Cattolica del Sacro Cuore. Department for Sustainable Food Process; Italia. Fil: Suciu, Nicoleta. Università Cattolica del Sacro Cuore. Department for Sustainable Food Process; Italia. Fil: Mol, Hans. Wageningen University and Research. Wageningen Food Safety Research; Países Bajos. Fil: Beekmann, Karsten. Wageningen University and Research. Wageningen Food Safety Research; Países Bajos. Fil: Figueiredo, Daniel. Utrecht University. Institute for Risk Assessment Sciences; Países Bajos. Fil: Harkes, Paula. Wageningen University and Research. Soil Physics and Land Management Group; Países Bajos. Fil: Hofman, Jakub. Masaryk University. Faculty of Science. Research Centre for Toxic Compounds in the Environment; Republica Checa. Fil: Kandeler, Ellen. University of Hohenheim. Institute of Soil Science and Land Evaluation; Alemania. Fil: Abrantes, Nelson. University of Aveiro. Centre for Environmental and Marine Studies and Department of Environment and Planning; Portugal. Fil: Campos, Isabel. University of Aveiro. Centre for Environmental and Marine Studies and Department of Environment and Planning; Portugal. Fil: Martínez, María Ángeles. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España. Fil: Pereira, Joana Luísa. Centre for Environmental and Marine Studies and Department of Environment and Planning; Portugal. Fil: Goossens, Dirk. Wageningen University and Research. Soil Physics and Land Management Group. Países Bajos. KU Leuven Department of Earth and Environmental Sciences; Belgica. Fil: Gandrass, Juergen. Institute of Coastal Environmental Chemistry; Alemania. Fil: Debler, Freya. Institute of Coastal Environmental Chemistry; Alemania. Fil: Huerta Lwanga, Esperanza. Wageningen University and Research. Soil Physics and Land Management Group; Países Bajos. Fil: Jonker, Marlot. Dutch Mammal Society; Países Bajos. Fil: Van Langevelde, Frank. Wageningen University and Research. Wildlife Ecology and Conservation Group; Países Bajos. Fil: Sorensen, Martin. Aarhus University. Department of Animal Science; Dinamarca. Fil: Wells, Jerry. Wageningen University and Research. Host-Microbe Interactomics, Animal Sciences Group; Países Bajos. Fil: Boekhorst, Jos. Wageningen University and Research. Host-Microbe Interactomics, Animal Sciences Group; Países Bajos. Fil: Huss, Anke. Utrecht University. Institute for Risk Assessment Sciences; Países Bajos. Fil: Mandrioli, Daniele. Ramazzini Institute. Cesare Maltoni Cancer Research Center; Italia. Fil: Sgargi, Daria. Ramazzini Institute. Cesare Maltoni Cancer Research Center; Italia. Fil: Nathanail, Paul. Land Quality Management; Reino Unido. Fil: Nathanail, Judith. Land Quality Management; Reino Unido. Fil: Tamm, Lucius. Research Institute of Organic Agriculture; Suiza. Fil: Fantke, Peter. Technical University of Denmark. Department of Technology, Management and Economics; Dinamarca. Fil: Mark, Jennifer. Research Institute of Organic Agriculture; Suiza. Fil: Grovermann, Christian. Research Institute of Organic Agriculture; Suiza. Fil: Larsen, Ana Frelih. Ecologic Institute; Alemania. Fil: Herb, Irina. Ecologic Institute; Alemania. Fil: Chivers, Charlotte Anne. University of Gloucestershire. Countryside and Community Research Institute; Reino Unido. Fil: Mills, Jane. University of Gloucestershire. Countryside and Community Research Institute; Reino Unido. Fil: Alcon, Francisco. Universidad Politécnica de Cartagena; España. Fil: Contreras, Josefina. Universidad Politécnica de Cartagena; España. Fil: Baldi, Isabelle. Bordeaux University. INSERM U1219; Francia. Fil: Pasković, Igor. Institute of Agriculture and Tourism, Department of Agriculture and Nutrition; Croacia. Fil: Matjaz, Glavan. University of Ljubljana. Biotechnical Faculty; Eslovenia. Fil: Norgaard, Trine. Aarhus University. Department of Agroecology; Dinamarca. Fil: Aparicio, Virginia Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Fil: Ritsema, Coen. Wageningen University and Research. Soil Physics and Land Management Group; Países Bajos. Fil: Geissen, Violette. University and Research. Soil Physics and Land Management Group; Países Bajos. Fil: Scheepers, Paul. Radboud Institute for Health Sciences; Países Bajos.

Considering that pesticides have been used in Europe for over 70 years, a system for monitoring pesticide residues in EU soils and their effects on soil health is long overdue. In an attempt to address this problem, we tested 340 EU agricultural topsoil samples for multiple pesticide residues. These samples originated from 4 representative EU case study sites (CSS), which covered 3 countries and four of the main EU crops: vegetable and orange production in Spain (S–V and S–O, respectively), grape production in Portugal (P-G), and potato production in the Netherlands (N–P). Soil samples were collected between 2015 and 2018 after harvest or before the start of the growing season, depending on the CSS. Conventional and organic farming results were compared in S–V, S–O and N–P. Soils from conventional farms presented mostly mixtures of pesticide residues, with a maximum of 16 residues/sample. Soils from organic farms had significantly fewer residues, with a maximum of 5 residues/sample. The residues with the highest frequency of detection and the highest content in soil were herbicides: glyphosate and its main metabolite AMPA (P-G, N–P, S–O), and pendimethalin (S–V). Total residue content in soil reached values of 0.8 mg kg−1 for S–V, 2 mg kg−1 for S–O and N–P, and 12 mg kg−1 for P-G. Organic soils presented 70–90% lower residue concentrations than the corresponding conventional soils. There is a severe knowledge gap concerning the effects of the accumulated and complex mixtures of pesticide residues found in soil on soil biota and soil health. Safety benchmarks should be defined and introduced into (soil) legislation as soon as possible. Furthermore, the process of transitioning to organic farming should take into consideration the residue mixtures at the conversion time and their residence time in soil.

Introduction: P-glycoprotein (P-gp) is an important efflux pump responsible for the extruding of many endogenous and exogenous substances out of the cells. P-gp can be modulated by different molecules – including xanthone derivatives – to surpass the multidrug resistance (MDR) phenomenon through P-gp inhibition, or to serve as an antidotal strategy in intoxication scenarios through P-gp induction/activation. Areas covered: This review provides a perspective on P-gp modulators, with particular focus on xanthonic derivatives, highlighting their ability to modulate P-gp expression and/or activity, and the potential impact of these effects on the pharmacokinetics, pharmacodynamics and toxicity of P-gp substrates. Expert opinion: Xanthones, of natural or synthetic origin, are able to modulate P-gp, interfering with its protein synthesis or with its mechanism of action, by decreasing or increasing its efflux capacity. These modulatory effects make the xanthonic scaffold a promising source of new derivatives with therapeutic potential. However, the mechanisms beyond the xanthones-mediated P-gp modulation and the chemical characteristics that make them more potent P-gp inhibitors or inducers/activators are still understudied. Furthermore, a new window of opportunity exists in the neuropathologies field, where xanthonic derivatives with potential to modulate P-gp should be further explored to optimize the prevention/treatment of brain pathologies.

This study aims to design and characterize Nanostructured lipid carriers (NLC) and Nanostructured lipid carrier-based hydrogels with Passiflora edulis seeds oil, a by-product from Madeira Island food industry. NLC were prepared by the ultrasonication technique, using passion fruit seeds oil as a liquid lipid and glyceryl distearate as a solid lipid. These NLC were then gelled with Poly (acrylic acid). Long-term stability studies were conducted with NLC and NLC-based hydrogels stored for 12 months. The following tests were performed: morphology, encapsulation efficiency, particle size analysis, polydispersity index analysis, zeta potential, pH measurement, color analysis, viscosity studies, texture analysis, in vitro occlusion test, ex vivo skin penetration study, tyrosinase inhibition activity, in vitro skin permeation experiments and in vitro cytotoxicity studies. The developed NLC had spherical shape and narrow particle sizes distribution with mean sizes in the range of 150 nm and PDI below 0.3, Zeta potential values around −30 mV and high Encapsulation efficiency. The tyrosinase inhibitory activity and skin retention of the nanoparticles was superior to that of the non-encapsulated oil. The developed formulations did not show cytotoxicity towards HaCat cells and presented suitable viscosity and texture properties for skin application, proving to be good candidates as depigmenting agent.

Progesterone (PRG) plays a crucial role in the female reproductive system, being the vaginal route the most adequate for its administration, as this drug has an extensive hepatic first pass effect. Nonetheless, vaginal PRG dosage forms originate immediate drug release and requires repeated administrations, which is unpleasant. Thereby, it is necessary to develop alternative delivery systems for prolonged vaginal release of PRG. The objective of this work was the development of pessaries for the prolonged vaginal delivery of PRG. Studies began with the preparation of an aqueous dispersion of PRG-loaded NLC (NLC_PRG), followed by the evaluation of its biocompatibility in human immortalized keratinocytes (HaCat cells), using three different methods (neutral red uptake, resazurin reduction and sulforhodamine B assays). Finally, the NLC_PRG was incorporated into pessaries, which were further characterized according to the European Pharmacopoeia to assess their suitability to prolong PRG release through the vaginal route. The results showed that, after preparation, 90% of the NLC_PRG had sizes equal or lower than 315.60 ± 0.01 nm, and an EE of 96.42 ± 0.00%. All the assays used to assess the biocompatibility of NLC_PRG showed the absence of cytotoxicity towards HaCaT cells for concentrations up to 10 μg/mL. In all cytotoxicity assays, a cytotoxic effect was only observed for concentrations equal or higher than 25 μg/mL, which provides high confidence in the obtained results. The outcomes of this study suggest the suitability of using pessaries containing PRG-loaded NLC for sustained drug release, which is an innovative therapeutic strategy and constitutes a promising alternative for the vaginal use of PRG. However, further ex vivo and in vivo studies are needed to fully clarify the pharmacokinetic and toxicological profile before reaching the clinical use.

Pesticides are heavily used in agriculture to reduce crop losses due to pests, weeds and pathogens. The intensive and long-term use of pesticides raises major health and environmental concerns since a substantial part of applied pesticides does not reach the target and is distributed into the environment instead. Exposure data (i.e. data on occurrence and levels of different pesticide residues), a pre-requisite to perform comprehensive and cumulative pesticide risk assessments, are scarce and fragmented, especially for soil. As analysing all EU soils for pesticide residues is not realistic, the contamination status of EU soils has to be inferred. Given pesticide use data limitations, the representative uses of the active substances (a.s.) allowed in the EU market, which cover different application schemes and recommended application rates, can be used as a proxy to estimate the type and amount of pesticide residues in EU soils. These representative uses are also considered in the calculation of predicted environmental concentrations in soil (PECs), the closest to a soil quality indicator when it comes to residues in soils of currently used pesticides. Although both pesticide representative uses and PECs are publicly available, this information is presented for individual a.s., in respective EU dossiers, which up to now were never compiled into a database and explored as such. Therefore, our study provides the first predictions on the total pesticide content in EU soils, calculated for 8 different crops (i.e. cereals, maize, root crops, non-permanent industrial crops, permanent crops, grapes, dry pulses-vegetables-flowers, and temporary grassland), 3 EU regions (i.e. Northern, Central and Southern Europe), and 2 pesticide use scenarios (i.e. all pesticides applied and no herbicides used). Such predictions are integrated into the Soil Quality Mobile App SQAPP, a recently launched and freely available tool that integrates existing soil quality information (covering both soil properties and soil threats) and provides tailored recommendations to improve soil quality. Furthermore, we present soil quality thresholds for all these crop-region-use combinations in terms of the number of active substances and the total pesticide content expected in soil. Our results indicate a much higher variety of products allowed in cereals than in other crops, yet the highest pesticide load is expected in dry pulses-vegetables-flowers and in grapes. At the most heavy use scenario (i.e. all allowed substances are applied, at the same time, and at the worst recommended use patterns), pesticide input can exceed 1,200 kg a.s. ha-1 year-1 (dry pulses-vegetables-flowers). Pesticide input is expected to be the highest in Southern Europe, and the lowest in Northern Europe or Central Europe, depending on crop type. Predicted pesticide levels in soil were in line with application data, with the highest contents in dry pulses-vegetables-flowers, and in Southern Europe. Predictions-based thresholds resulted in very low soil protection, especially when compared to measured data in literature, and measurement-based thresholds. Finally, our results reinforce the need of monitoring and surveillance programs for pesticide residues in soil, proper risk evaluation procedures for mixtures, as well as the need to establish threshold methodologies for pesticides.

Viticulture, which plays a major role in the economy of several EU countries, is highly dependent of pesticides. In the São Lourenço experimental catchment, located in an important wine-growing region in Portugal, vineyards are the dominant land cover. Previous studies in this catchment showed the presence of different pesticide residues in surface water and erosion rates in vineyards of up to 30 Mg ha-1 y-1. However, information on the presence of pesticide residues in topsoils and water-eroded sediments are lacking, not just in this catchment but in general. To address these knowledge gaps, we conducted two soil sampling campaigns (fall 2015, fall 2016) and a field runoff-erosion experiment (winter 2015). We sampled 3 vineyards in the first campaign and 9 in the second, both covering different soil types (humic cambisols, calcic cambisols and chromic luvisols). Soil samples were collected within and between the vine rows, at the top, middle and bottom of the slope, and at two soil depths (0-2 and 15 cm). The runoff-erosion experiment involved 9 plots of ~12 m2; pesticide residues were monitored in topsoil and eroded sediments at six occasions after important rainfall events (>50 mm). 47 prioritized pesticide residues were analysed in 162 soil and 108 sediment samples. The levels of residues in soil were compared with their predicted environmental concentrations (PECs), calculated according to European Food Safety Authority (EFSA) pesticide application recommendations and to local farmers records. All soil samples contained pesticide residues, dimethomorph, glyphosate and its metabolite AMPA being the most frequent detected compounds. Measured levels of the different residues in soil occasionally exceeded predictions. Pesticide levels seemed related to slope position, generally being higher at the bottom than at the top or middle of the slopes, but not to the position within or between vine rows. Soil depth played a noticeable role in pesticide levels, with 80% lower concentrations of glyphosate and tebuconazole at 15 cm than 0-2 cm depth. For dieldrin (the only long-banned pesticide detected), the concentration was higher at 15 than 0-2 cm depth. Sediments had fewer compounds than soils, but the frequencies with which they were detected were similarly high. Pesticide levels were significantly higher in sediments than soils, especially in the case of the finest sediments (

The two Sotalia species (the marine S. guianensis and the freshwater S. fluviatilis) have only recently been recognized, and both face several conservation challenges. We investigated the existence of hybridization between the two species in their possible area of sympatry in the Amazon Estuary, in northern Brazil. A fast and cheap PCR-RFLP diagnostic method using nuclear DNA was developed to discriminate between the two species, while allowing the detection of hybrids. All samples that could be identified (N = 51) were identified as S. guianensis, and no hybrids were detected. Our results, coupled with previous mitochondrial data, suggest that S. fluviatilis is not present in the Amazon delta. Thus, sympatry with S. guianensis, if it does occur, may be restricted to upstream areas of the Amazon River.

A maioria dos filhotes de Trichechus inunguis órfãos que chegam aos centros de pesquisa e reabilitação é lactante. Essa condição dificulta o processo de reabilitação, sendo necessário oferecer uma dieta láctea artificial adequada. Com o intuito de incrementar o teor de aminoácidos no sucedâneo lácteo buscou-se dentre os alimentos habitualmente amazônicos um ingrediente capaz de fornecer os nutrientes necessários para potencializar o sucedâneo utilizado, sendo facilmente adquiro, apresentar alto valor biológico, rápida manipulação, perecibilidade reduzida e baixo custo de obtenção. Dentre os alimentos que preenchem esses pré-requisitos, destaca-se a castanha-do-Brasil. Avaliou-se neste estudo a viabilidade nutricional em termos de aminoácidos da Bertholletia excelsa, para a possível suplementação no sucedâneo lácteo fornecido aos filhotes de peixe-boi da Amazônia em cativeiro. As amêndoas foram descascadas, maceradas e em seguida foi pesado 27 mg, digerido em HCl 6 M, aquecido a 150 oC por 90 minutos e analisado por Cromatografia Líquida de Alta Eficiência por troca iônica. Os resultados demonstraram uma concentração elevada de aminoácidos não essenciais, dentre eles arginina e ácido glutâmico + glutamina. Os aminoácidos em menor concentração foram prolina, alanina e tirosina. Dentre os aminoácidos essenciais, a valina e a leucina apresentaram as maiores concentrações, entretanto os aminoácidos menos abundantes foram à histidina, isoleucina. Os demais aminoácidos apresentaram teores equivalentes na análise da composição em aminoácidos da castanha. Concluiu-se nesse trabalho que a castanha do Brasil é uma fonte potencial de enriquecimento nutricional na dieta láctea de filhotes de T.inunguis em cativeiro.

This work relied on the use microcosms to evaluate the individual and the combined effects of different levels of copper sulfate (0.0, 0.013, 0.064 and 0.318 mg Cu L−1) – a fungicide commonly exceeding allowable thresholds in agricultural areas – and a range of water temperature increase scenarios (15, 20 and 25 °C) on freshwater species belonging to different functional groups. Hence, the growth inhibition of primary producers (the microalgae Raphidocelis subcapitata and the macrophyte Lemna minor), as well as the survival and feeding behavior of a shredder species (the Trichoptera Schizopelex sp.) were evaluated. The results revealed that copper was toxic to primary producers growth, as well as shredders growth and survival, being the growth of L. minor particularly affected. Higher water temperatures had generally enhanced the growth of primary producers under non-contaminated (microalgae and macrophytes) or low-contaminated (macrophytes) conditions. Despite the tendency for a more pronounced toxicity of copper under increasing water temperatures, a significant interaction between the two factors was only observed for microalgae. Since the test organisms represent relevant functional groups for sustaining freshwater systems functions, the present results may raise some concerns on the impacts caused by possible future climate change scenarios in aquatic habitats chronically exposed to the frequent or intensive use of the fungicide copper sulfate.

The spermatozoa from seven adult Amazon river dolphins (Inia geoffrensis, CETACEA: INIIDAE) were analyzed by light and electron microscopy. The spermatozoa showed an elongated ellipsoid shaped head and a long tail with a well distinguishable midpiece. The head spermatozoa have a smooth surface like other odontocetes examined, with the exception of the Delphinidae family. The mean dimensions of the spermatozoa were within the range already reported for other cetaceans. The spermatozoa midpiece, as in other cetaceans, showed a random pattern of mitochondria, different from that described for other mammals. Further studies of sperm morphology of a wider spectrum of cetacean families could help to better understand the reproductive biology of these animals and the intergeneric and intrageneric relationships among them, as well as, among other mammals. Anat Rec, 300:1519-1523, 2017. © 2017 Wiley Periodicals, Inc.

The number of wildfires has markedly increased in Mediterranean Europe, including in Portugal. Wildfires are environmentally concerning, not only due to the loss of biodiversity and forest area, but also as a consequence of environmental contamination by specific compounds including metals and polycyclic aromatic compounds (PAHs). These contaminants, mostly bound to ashes, can reach downstream water bodies, namely through surface runoff, being ultimately dispersed by vast areas and contacting with aquatic biota. Being toxicologically noteworthy, the potential toxic outcomes of the input of such chemicals across the aquatic compartment must be characterized. In this context, the present study used a biomarker-based approach to find early-warning signals of toxicity triggered by the exposure of the mosquitofish, Gambusia holbrooki, to affected aqueous runoff and stream water samples collected from a forest burnt area. The chemical analysis revealed concerning levels of metals and polycyclic aromatic hydrocarbons in both runoff and stream water samples. Biological responses elicited by the collected samples showed the occurrence of pro-oxidative modifications, specifically driven by enzymatic forms involved in the metabolism of glutathione. Despite these effects, no further signs of involvement of metals and PAHs were elicited in terms of neurotoxicity. The overall set of data implicates chemicals resulting from wildfires in clear deleterious effects in exposed fish. published

P-glycoprotein (P-gp) plays a crucial role in the protection of susceptible organs, by significantly decreasing the absorption/distribution of harmful xenobiotics and, consequently, their toxicity. Therefore, P-gp has been proposed as a potential antidotal pathway, when activated and/or induced. Knowing that xanthones are known to interact with P-gp, the main goal was to study P-gp induction or/and activation by six new oxygenated xanthones (OX 1-6). Furthermore, the potential protection of Caco-2 cells against paraquat cytotoxicity was also assessed. The most promising compound was further tested for its ability to increase P-gp activity ex vivo, using everted intestinal sacs from adult Wistar-Han rats. The oxygenated xanthones interacted with P-gp in vitro, increasing P-gp expression and/or activity 24 h after exposure. Additionally, after a short-incubation period, several xanthones were identified as P-gp activators, as they immediately increased P-gp activity. Moreover, some xanthones decreased PQ cytotoxicity towards Caco-2 cells, an effect prevented under P-gp inhibition. Ex vivo, a significant increase in P-gp activity was observed in the presence of OX6, which was selectively blocked by a model P-gp inhibitor, zosuquidar, confirming the in vitro results. Docking simulations between a validated P-gp model and the tested xanthones predicted these interactions, and these compounds also fitted onto previously described P-gp induction and activation pharmacophores. In conclusion, the in vitro, ex vivo, and in silico results suggest the potential of some of the oxygenated xanthones in the modulation of P-gp, disclosing new perspectives in the therapeutics of intoxications by P-gp substrates.

4-Oxoquinoline derivatives have been often used in drug discovery programs due to their pharmacological properties. Inspired on chromone and 4-oxoquinoline chemical structure similarity, a small series of quinoline-based compounds was obtained and screened, for the first time, toward human monoamine oxidases isoforms. The data showed the N-(3,4-dichlorophenyl)-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide 10 was the most potent and selective MAO-B inhibitor (IC50 = 5.30 ± 0.74 nM and SI: ≥1887). The data analysis showed that prototropic tautomerism markedly influences the biological activity. The unequivocal characterisation of the quinoline tautomers was performed to understand the attained data. To our knowledge, there have been no prior reports on the characterisation of quinolone tautomers by 2D NMR techniques, namely by 1H–15N HSQC and 1H–15N HMBC, which are proposed as expedite tools for medicinal chemistry campaigns. Computational studies on enzyme-ligand complexes, obtained after MM-GBSA calculations and molecular dynamics simulations, supported the experimental data.

The potential value of protected areas for the conservation of cetaceans is widely recognized; however, few evaluation methods exist to assess their effectiveness. In this study, a modeling approach based on long-term mark-recapture/resight data was used to assess the effectiveness of a Brazilian reserve in protecting endangered Amazon River dolphins or boto (Inia geoffrensis), a species killed for use as fish bait. We built an annual discrete-time model with subdivisions based on hydrological periods and age classes. It included transition probabilities in and out of the reserve that were estimated utilizing multi-state mark-recapture models. To evaluate five reserve configurations, we re-estimated the transition probabilities to represent changes in the reserve boundaries. Model predictions showed that four scenarios, including one representing the existing boundaries, would be insufficient to protect the local boto population (no = 528) and a steep decline in abundance would occur in the next 50 years (0 ≤ n50 ≤ 108). However, one reserve configuration, encompassing both flooded forests and adjacent river habitats, and including beaches and channel/floodplain entrances, resulted in a nearly stable population in the same time frame (n50 = 515). These findings suggest that, with careful design and efficient management, protected areas could be an effective conservation tool for boto populations that exhibit site fidelity to Amazonian floodplains. With appropriate mark-recapture/resight data, the modeling framework employed could be extended to evaluate and design protected areas for populations of other species in other systems.

Wildfires, increasingly frequent and severe in the Mediterranean countries, can cause serious impacts on ecosystems. As a diffuse source of contaminants – namely inorganic elements (metals, metalloids and non-metallic elements) and polycyclic aromatic hydrocarbons (PAHs) – post-fire runoff may cause toxic effects on the aquatic biota. Despite the recognition of these likely impacts, this issue has been still largely neglected. This study explores the impacts of ash from wildfires on the freshwater clam Corbicula fluminea and the ability of this filter feeder to concentrate the contaminants associated with ash in post-fire runoff. The bio-concentration potential of the clam was assessed in a 7-day filtration experiment, using aqueous extract of ashes (AEA) to represent post-fire runoff. Despite the noticeable mortality of the clams exposed to the AEA (∼30% higher in AEA than in control after 7 days of exposure), there was a clear reduction on the concentration of almost all pyrolytic substances in the water linked to bio-filtration and bio-concentration activity. Along 7 days the clams operated decreases of more than 70% for some chemicals (Cu, Mn and Zn) while others were no longer detected in the water at the end of the experiment (Cr, NAP and PHE). The removal from water was accompanied by contaminants’ accumulation in clams’ soft tissues and, less extensively, in shells. Lower pollutant loads of filtered AEA translated into decreased toxicity of the matrix towards standard aquatic species (microalgae, macrophytes and cladocerans) confirming an improvement as well in the biological quality of the water through bio-filtration. Although post-fire loads can compromise C. fluminea survival, this study evidenced the potential of this invasive species to contribute to water quality improvement.

Deliberate killing for use as bait in a regional catfish (Calophysus macropterus) fishery is the primary threat affecting the survival of the Amazon river dolphin, or boto (Inia geoffrensis). Establishing and improving freshwater protected areas has been suggested as a possible course of action to protect the species. However, the ecology of the boto is poorly understood and more information is needed on the species’ habitat use and movement patterns to ensure that spatial protection initiatives meet conservation needs. In this study, mark-recapture/resight data collected in and near the Mamiraua Sustainable Development Reserve, a varzea in the Brazilian Amazon, was used to examine transition probabilities of botos between various habitat types. Our findings suggest that it is imperative that spatial protection initiatives focused on the boto take into account the importance of varzea habitat and bay systems for individuals in early life stages. Moreover, because botos frequently occupy the main rivers, particularly during the low water period, protecting areas of the main river waters adjacent to varzeas could aid in the protection of hunted populations.

Wildfires can cause immediate and drastic impacts on the structure and functioning of ecosystems, and there has been an increasing interest in wildfire effects on water chemistry and aquatic biota. Wildfires are increasingly recognized as a diffuse source of contamination of aquatic ecosystems, through the production of deleterious pyrolytic substances and their subsequent transport, mostly attached to ashes. To study the deleterious effects of the ash-laden runoff from burnt areas on water quality, composite ash samples of ashes were collected immediately after a forest fire and then used to prepare aqueous extracts of ash (AEA). The AEA were analyzed with respect to a large group of chemical elements and the sixteen prioritized polycyclic aromatic hydrocarbons (PAHs). Ca, S, Mg, K and Na were found to be the principal elements in the AEA, while only two low molecular weight PAHs (phenanthrene and naphthalene) were present in quantifiable amounts. In parallel, an ecotoxicological screening of the AEA was performed with four standard aquatic species from different functional groups and trophic levels. The AEA was found to induce a statistically significant decrease in the growth of two primary producers – Pseudokirchneriella subcapitata and Lemna minor – and inhibited the luminescence of the bacteria Vibrio fischeri . By contrast, AEA did not produce a significant immobilization of Daphnia magna suggesting that short-term acute toxicity may be absent at higher trophic levels. Overall, the present results emphasize the role of wildfires as a potential source of diffuse contamination for downstream water bodies, compromising both chemical and ecological conditions. At the same time, this study highlights the need for further research into the complexity of the potentially deleterious ecological effects of wildfires on aquatic communities, with a particular focus on cascading effects along the trophic web.

ABC (ATP-binding cassette) transporters represent one of the largest and most diverse superfamily of proteins in living species, playing an important role in many biological processes such as cell homeostasis, cell signaling, drug metabolism and nutrient uptake. Moreover, using the energy generated from ATP hydrolysis, they mediate the efflux of endogenous and exogenous substrates from inside the cells, thereby reducing their intracellular accumulation. At present, 48 ABC transporters have been identified in humans, which were classified into 7 different subfamilies (A to G) according to their phylogenetic analysis. Nevertheless, the most studied members with importance in drug therapeutic efficacy and toxicity include P-glycoprotein (P-gp), a member of the ABCB subfamily, the multidrug-associated proteins (MPRs), members of the ABCC subfamily, and breast cancer resistance protein (BCRP), a member of the ABCG subfamily. They exhibit ubiquitous expression throughout the human body, with a special relevance in barrier tissues like the blood-brain barrier (BBB). At this level, they play a physiological function in tissue protection by reducing or limiting the brain accumulation of neurotoxins. Furthermore, dysfunction of ABC transporters, at expression and/or activity level, has been associated with many neurological diseases, including epilepsy, multiple sclerosis, Alzheimer's disease, and amyotrophic lateral sclerosis. Additionally, these transporters are strikingly associated with the pharmacoresistance to central nervous system (CNS) acting drugs, because they contribute to the decrease in drug bioavailability. This article reviews the signaling pathways that regulate the expression and activity of P-gp, BCRP and MRPs subfamilies of transporters, with particular attention at the BBB level, and their mis-regulation in neurological disorders.

Piperine has been associated with neuroprotective effects and monoamine oxidase (MAO) inhibition, thus being an attractive scaffold to develop new antiparkinsonian agents. Accordingly, we prepared a small library of piperine derivatives and screened the inhibitory activities towards human MAO isoforms (hMAO-A and hMAO-B). Structure-activity relationship (SAR) studies pointed out that the combination of α-cyano and benzyl ester groups increased both potency and selectivity towards hMAO-B. Kinetic experiments with compounds 7, 10 and 15 indicated a competitive hMAO-B inhibition mechanism. Compounds 15 and 16, at 10 μM, caused a small but significant decrease in P-gp efflux activity in Caco-2 cells. Compound 15 stands out as the most potent piperine-based hMAO-B inhibitor (IC50 = 47.4 nM), displaying favourable drug-like properties and a broad safety window. Compound 15 is thus a suitable candidate for lead optimization and the development of multitarget-directed ligands.

An adult male Amazonian manatee Trichechus inunguis under human care presented with 3 circular cutaneous lesions on the dorsal aspect of the rostrum and between the nostrils (plenum). Initially these lesions were superficial, hypopigmented, without warmth and non-painful. Microbiological cultures of skin swabs isolated Candida sp. and Pseudomonas aeruginosa, and topical treatment with antiseptic, antifungal, anti-inflammatory and antibiotic medication was instituted. This treatment strategy did not lead to any clinical improvement, and after 6 mo, the lesions progressed to a confluent abscess (5.0 × 3.0 cm) with increased temperature and obvious discomfort on palpation. An impression smear of a cutaneous biopsy was submitted for Ziehl-Neelsen staining and after detection of acid-fast bacilli, the cutaneous biopsy and a swab from the lesion were sent for histopathology, culture and sensitivity testing. After 5 d of incubation and through PCR-restriction analysis of the isolates, Mycobacterium fortuitum and M. abscessus were identified. Sensitivity testing indicated that the isolates were susceptible to ciprofloxacin and clarithromycin, and after draining of the lesion and administration of systemic antibiotic treatment, there was rapid clinical improvement. This report describes non-healing lesions in an aquatic animal and illustrates the importance of evaluating the presence of non-tuberculous mycobacteria, opportunistic pathogens which are ubiquitous in the aquatic environment, in protracted, non-responsive cases. We also highlight the importance of a correct diagnosis and treatment approach, and we review concerns that these bacteria are zoonotic agents and are frequently resistant to conventional antibiotics. © Inter-Research 2018.

We monitored the underwater behavior of botos (Inia geoffrensis) using stereo acoustic data loggers to observe their local habitat use and its diel changes at the Mamiraua Sustainable Development Reserve, Brazil. A-tags were set at five sites in three different habitat types: Lake (low current), Channel (middle current), and Junction (junction of two channels). The presence index during nighttime was significantly greater than during daytime in the Lake and Junction. Underwater movement was estimated from the changing pattern (trajectory) of the relative angle of the sound source from A-tags. A staying-type trajectory was dominant in the Lake, although the prevalence of moving-type trajectory increased at night. More than 80% of detected trajectories were the staying type in the Junction, while moving-type trajectories dominated in the Channel. The frequency of click trains was greatest in the Lake, followed by the Junction and Channels. The average interpulse interval, which reflects the mean target distance of echolocation, was shortest in the Lake, followed by the Junction and Channel. These results suggest that the botos used the Lake as their primary habitat for active behaviors like foraging, especially at night, and the Junction as their primary habitat for relatively inactive behaviors at night.

Teeth and otoliths are metabolically inert structures that preserve a chronology of chemical variations that may be related to the environmental histories experienced by each organism. Because of the natural decrease of barium (Ba) and increase of strontium (Sr) bioavail- ability in water with increasing salinity, these elements may be especially useful to track habitat use in aquatic organisms. Therefore, we tested whether the Ba/Ca and Sr/Ca ratios in the teeth of dolphins represent a salinity gradient. The main aim was to determine whether these elements can be used as a natural tag for different aquatic environments. Teeth from 2 freshwater dolphins (Inia geoffrensis and Sotalia fluviatilis) and 2 marine species (S. guianensis and Pontoporia blainvillei) from Brazil and Uruguay were analyzed using a Laser Ablation Inductively Coupled Plasma-Mass Spectrometer. Intensity ratios of 138 Ba/ 43 Ca and 86 Sr/ 43 Ca were measured along a line that covered all growth increments in the dentin from the second year of life onwards. Teeth from the freshwater species had mean Ba/Ca values tenfold higher than marine dolphins, confirm- ing the inverse relationship between salinity (and thus ambient Ba/Ca) and elemental ratios in teeth. Furthermore, Ba/Ca ratios could also differentiate dolphins from lower-salinity estuarine areas from those in areas with minimal freshwater discharge. No significant differences were found for Sr/Ca values. Results presented encouraging indications for the application of this tech- nique as a potential new tool for studying habitat use in aquatic mammals.

Until the recent construction of hydroelectric dams, a series of 18 rapids divided the upper and lower Madeira River, and these rapids were thought to separate two species of Amazonian freshwater dolphins (boto): Inia boliviensis (above) and I. geoffrensis (below). Some reports and articles, however, mention the occurrence of botos within the rapids region and that they occasionally cross the rapids, but without mentioning the species concerned. Based on our previous studies, it is likely that I. boliviensis occurs in the region of the rapids. To test this supposition, we sampled 18 individuals from this region, and collected mitochondrial (control region, cytochrome b and cytochrome oxidase I) and nuclear (10 microsatellite loci) DNA data, in order to test if there is connectivity between the dolphins that were found within the rapids region and dolphins collected upstream and downstream of the rapids, and investigate population structuring between these localities. All animals in our study were molecularly identified using three mitochondrial markers as belonging to the species I. boliviensis. Animals upstream of the Teotonio waterfall, the main and highest waterfall of the region, had nuclear genome of I. boliviensis, while most dolphins downstream of the waterfall had nuclear genome of I. geoffrensis. Inia boliviensis collected in the rapids region above the Teotonio waterfall belong to a management unit (MU) distinct from the I. boliviensis MU occupying Bolivian rivers. Downstream of Teotonio waterfall most dolphins are I. boliviensis/geoffrensis hybrids, with remaining individuals being migrant I. boliviensis. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114, 764–777.

The aims of this study were to validate an enzyme immunoassay (EIA) for the measurement of luteinizing hormone (LH) in urine samples of Amazonian manatees (Trichechus inunguis; Mammalia: Sirenia) and to monitor urinary LH and reproductive steroids during the ovarian cycle in this species. Urine samples were collected from two captive males following a hormonal challenge with a gonadotropin-releasing hormone (GnRH) analogue. The urinary LH results from hormonal challenge were compared with urinary androgens for the purpose of EIA validation. Furthermore, urine samples were collected daily, over a 12-wk period, from two captive adult females, for 2 consecutive yr. The urinary LH pattern from females was compared with the patterns of urinary progestagens and estrogen conjugates throughout the ovarian cycle. An LH peak was observed in both male Amazonian manatees after the hormonal challenge, occurring prior to or together with peak androgen levels. In the females, the ovarian cycle ranged from 40 to 48 days (mean of 43.7 days). Two distinct peaks of estrogen conjugates were observed across all cycles analyzed, and the urinary LH peaks observed were accompanied by peaks of urinary estrogen conjugates. The EIA was validated as a method for the quantification of urinary LH from Amazonian manatees, as it was able to detect variations in the levels of LH in urine samples. These results suggest that T. inunguis exhibits a peculiar hormonal pattern during the ovarian cycle. Therefore, further studies are desirable and necessary to clarify the relationship between this hormonal pattern and morphological changes, as well as mating behavior, in Amazonian manatee.

Approval for glyphosate-based herbicides in the European Union (EU) is under intense debate due to concern about their effects on the environment and human health. The occurrence of glyphosate residues in European water bodies is rather well documented whereas only few, fragmented and outdated information is available for European soils. We provide the first large-scale assessment of distribution (occurrence and concentrations) of glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) in EU agricultural topsoils, and estimate their potential spreading by wind and water erosion. Glyphosate and/or AMPA were present in 45% of the topsoils collected, originating from eleven countries and six crop systems, with a maximum concentration of 2 mg kg− 1. Several glyphosate and AMPA hotspots were identified across the EU. Soil loss rates (obtained from recently derived European maps) were used to estimate the potential export of glyphosate and AMPA by wind and water erosion. The estimated exports, result of a conceptually simple model, clearly indicate that particulate transport can contribute to human and environmental exposure to herbicide residues. Residue threshold values in soils are urgently needed to define potential risks for soil health and off site effects related to export by wind and water erosion.

Toothed whales have evolved to live in extremely different habitats and yet they all rely strongly on echolocation for finding and catching prey. Such biosonar based foraging involves distinct phases of searching for, approaching, and capturing prey, where echolocating animals gradually adjust sonar output to actively shape the flow of sensory information. Measuring those outputs in absolute levels requires hydrophone arrays centred on the biosonar beam axis, but this has never been done for wild toothed whales approaching and capturing prey. Rather, field studies make the assumption that toothed whales will adjust their biosonar in the same manner to arrays as they will when approaching prey. To test this assumption, we recorded wild botos (Inia geoffrensis) as they approached and captured dead fish tethered to a hydrophone in front of a star-shaped seven-hydrophone array. We demonstrate that botos gradually decrease interclick intervals and output levels during prey approaches, using stronger adjustment magnitudes than extrapolated from previous boto array data. Prey interceptions are characterised by high click rates, but although botos buzz during prey capture, they do so at lower click rates than marine toothed whales, resulting in a much more gradual transition from approach phase to buzzing. We also demonstrate for the first time that wild toothed whales broaden biosonar beamwidth when closing in on prey, as it is also seen in captive toothed whales and in bats, thus resulting in a larger ensonified volume around the prey, likely aiding prey tracking by decreasing the risk of prey evading ensonification.

Negative interactions between fishers and the Amazon River dolphin (Inia geoffrensis), or boto, have increased substantially in the last few decades. Herein, we investigate these interactions with focus on assessing fisher perceptions, attitudes, and behaviors toward botos. Moreover, we evaluate the effect that the Mamiraua Sustainable Development Reserve (MSDR) in the Brazilian Amazon, and related programs, has had on fisher attitudes and behaviors toward botos. The results suggest that interactions between fishers and botos, such as depredation and incidental entanglement, are frequent, and that the illegal harvest for botos, for use as bait, occurs in the majority of the study communities. However, the assessment revealed that most fishers have positive attitudes toward botos and that these attitudes have been influenced by participation in the MSDR activities such as research and ecotourism. Our results also highlight the importance of community-based enforcement in addressing the issue of boto harvesting. The MSDR programs have successfully promoted positive attitudes toward botos and have likely played a role in limiting boto mortality through behavioral controls, though the scope of influence of these programs has been restricted to a small geographical area. The current extent of these programs is insufficient to prevent the decline of the boto population; therefore, we suggest the MSDR model be used to improve and expand boto conservation efforts with communities in the region.

The mammaries from carcasses of two female Amazonian manatees were examined. Trichechus inunguis possesses two axillary mammaries beneath the pectoral fins, one on each side of the body. Each papilla mammae has a small hole on its apex--the ostium papillare. The mammaries are covered by a stratified squamous keratinized epithelium. The epithelium of the mammary ducts became thinner more deeply in the tissue and varied from stratified to simple cuboidal. There was no evidence of glandular activity or secretion into the ducts of the mammary glands.