Enhancement of Per- and Polyfluoroalkyl Substances (PFAS) quantification on surface waters from marinas in the Douro River, Portugal.

PFAS, known as "forever" compounds, are prevalent in various environments, including soils and aquatic systems, due to extensive usage. Surface waters in several European countries, especially marinas and ports with high boat traffic, require further study as potential contamination sources. Reliable methods for the extraction and quantification of these emergent compounds are essential. This study aimed to improve an existent solid phase extraction method to analyse marinas and ports' surface waters with variable salinities (2, 9 and 17 PSU). The objectives were to: 1) optimise the solid phase extraction method, considering matrix salinity effects and cross-contaminations, 2) validate the extraction and quantification method of 18 EPA 537.1 PFAS in estuarine surface waters, using the Ultra-High Performance Liquid Chromatography - Quadrupole Time - Of - Flight - Tandem Mass spectrometry, and 3) apply the optimised method for PFAS quantification in three Portuguese marinas. All ICH criteria were successfully validated considering 9 PSU. Limits of quantification ranged from 117.80 ng/L to 385 ng/L, except for PFHpA (645.85 ng/L). PFAS levels (PFOA, HFPO-DA, PFBS, PFHxS and PFOS) were relatively low, reaching a maximum of 0.32 ng/L only for the PFOA. In Freixo marina, total average concentrations were slightly higher (∑PFAS= 1.02 ng/L) when compared to the ones found in Cais da Ribeira Port (∑PFAS= 0.94 ng/L) and Afurada marina (∑PFAS= 0.81 ng/L). PFOS concentrations are below the limit values set by the Environmental Quality Standards (36000 ng/L of PFOS for inland surface water, respectively), similar to other Portuguese river studies. This study enabled the development of a precise and reliable extraction and quantification method to quantify PFAS in estuarine surface waters, particularly from marinas. This method can be readily applied to analyse PFAS in other estuarine samples.

Immunological and oxidative stress responses of the bivalve Scrobicularia plana to distinct patterns of heatwaves.

As a consequence of global warming, extreme events, such as marine heatwaves (MHW), have been increasing in frequency and intensity with negative effects on aquatic organisms. This innovative study evaluated for the first time, the immunological and physiological response of the estuarine edible bivalve Scrobicularia plana to different heatwaves, with distinct duration and recovery periods. So, extensive immune (total haemocyte count - THC, haemocyte viability, phagocytosis rate, respiratory oxidative burst of haemocytes, total protein, protease activity, nitric oxide and bactericidal activity of plasma) and oxidative stress (lipid peroxidation - LPO, superoxide dismutase - SOD, catalase - CAT and glutathione-S-Transferase - GST) analyses were performed in an experimental study that tested the impact of heatwaves during 25 days. The survival and condition of S. plana were not affected by the exposure to the extreme events. However, our data suggested that longer heatwaves with shorter recovery periods can be more challenging for the species, since THC and phagocytic activity were most affected under the temperature increase conditions. Regarding the oxidative status, the species increased its SOD activity while MDA production slightly declined to the increase of temperature, protecting the organism from cellular damage. These results indicate that S. plana has a great capacity to adapt to environmental temperature changes, however, the expected higher frequency/duration of heatwaves with climate change trends can cause some debility of the species face to other stressors, which can compromise its success in the future.

Lecanicillium aphanocladii: snake venom phospholipases A2 and proteases as tools to prospect enzymatic inhibitors.

Natural enzyme inhibitors have been widely described in literature because of its pharmacological and cosmetic applications. Fungi found in caves represent a promising source of bioactive substances that are still little explored scientifically. Thus, the present work evaluated the presence of enzymatic modulators in a filtrate obtained from the cultivation of the cave fungus Lecanicillium aphanocladii (Family: Cordycipitaceae). Snake venoms from Bothrops alternatus and Bothrops atrox were used as an enzymatic source for the induction of the phospholipase, proteolytic, thrombolytic, cytotoxic and coagulant activities. Compounds present in the fungal filtrate inhibited 50, 23·8, 26·6, 50·9 and 52·5% of the proteolytic, phospholipase, haemolytic, thrombolytic and coagulant activities respectively. The filtrate was not cytotoxic on erythrocytes, but induced partial dissolution of thrombi. Fungal enzyme inhibitors that have low or no toxicity and can be obtained on a large scale and at low cost are relevant in the medical-scientific context. Therefore, the inhibition of phospholipases A2 and proteases observed in the present work highlights the potential of fungal metabolites for the development of drugs that can be used in the treatment of haemostasis and inflammation-related disorders. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, secondary metabolites synthesized by Lecanicillium aphanocladii, a fungus isolated from caves, demonstrated modulating action on proteases and phospholipases A2 present in snake venoms of the Bothrops genus, widely used as tools for the study of pathophysiology processes related to haemostasis and inflammation. The results suggest the possibility of future applications for these metabolites in the development of pharmaceuticals of medical-scientific interest.

Histopathological Evaluation of Combined Impacts of the Synthetic Progestin Levonorgestrel and Temperature on the Female Zebrafish Maturation Using a Semi-quantitative Grading Analysis-Is it Enough?

Pharmaceuticals contamination (e.g., synthetic progestins), and global climate change, represent two of the most stressful factors affecting aquatic species. To our knowledge, there is huge gap of data regarding the combined effects of both stressors on vertebrates' reproduction. Thus, it is crucial to implement rapid screenings of measurable histopathological alterations in fish gonads. For that, we propose: (1) an evaluation of the combined effects of progestin (levonorgestrel) and temperature on maturation of zebrafish female gonads, using a semi-quantitative method (i.e., grading) and (2) testing the robustness of the grading analysis comparatively to a quantitative method (i.e., stereology). Grading analysis showed a decrease on maturation stage of ovaries exposed to both stressors. Although grading is less robust than stereological analysis, it is recommended for a preliminary approach, since it gives a correct idea on trends and it is fast and cost-effective. For a detailed histological assessment, we recommend a stereological study.

Pollen limitation may be a common Allee effect in marine hydrophilous plants: implications for decline and recovery in seagrasses.

Pollen limitation may be an important factor in accelerated decline of sparse or fragmented populations. Little is known whether hydrophilous plants (pollen transport by water) suffer from an Allee effect due to pollen limitation or not. Hydrophilous pollination is a typical trait of marine angiosperms or seagrasses. Although seagrass flowers usually have high pollen production, floral densities are highly variable. We evaluated pollen limitation for intertidal populations of the seagrass Zostera noltei in The Netherlands and found a significant positive relation between flowering spathe density and fruit-set, which was suboptimal at <1200 flowering spathes m(-2) (corresponding to <600 reproductive shoots m(-2)). A fragmented population had ≈35 % lower fruit-set at similar reproductive density than a continuous population. 75 % of all European populations studied over a large latitudinal gradient had flowering spathe densities below that required for optimal fruit-set, particularly in Southern countries. Literature review of the reproductive output of hydrophilous pollinated plants revealed that seed- or fruit-set of marine hydrophilous plants is generally low, as compared to hydrophilous freshwater and wind-pollinated plants. We conclude that pollen limitation as found in Z. noltei may be a common Allee effect for seagrasses, potentially accelerating decline and impairing recovery even after environmental conditions have improved substantially.

Diversity and genetic parameter estimates for yield and its components in Jatropha curcas L.

Jatropha curcas L. is one of the most promising oilseeds for biodiesel and biokerosene production, but few basic studies or breeding programs have been conducted for the species. We estimated genetic parameters and diversity based on 10 yield traits in 77 half-sib progenies of J. curcas after 52 months in the field, and evaluated correlations between them and the oil content of the seeds. The mean grain yield per plant was 377.9 g (ranging from 169.8 to 772.1 g) and the mean oil content was 36.2% (ranging from 30 to 39.6%). Moderate estimates of heritability at the mean progeny level were obtained for the length of the fruit (84.7%), length (69.1%) and width (68.2%) of the seed, and grain yield per plant (62.2%). Oil content was only positively and significantly correlated with 100-seed weight. Our study revealed a range of possible crosses to be investigated in J. curcas. Progeny production should be evaluated over several crop seasons for the accurate selection of the best progenies.

In vitro assessment of the genotoxic and cytotoxic effects of boiled juice (tucupi) from Manihot esculenta Crantz roots.

The population of Pará (a state in Brazil) has a very characteristic food culture, as a majority of the carbohydrates consumed are obtained from cassava (Manihot esculenta Crantz) derivatives. Tucupi is the boiled juice of cassava roots that plays a major role in the culinary footprint of Pará. Before boiling, this juice is known as manipueira and contains linamarin, a toxic glycoside that can decompose to hydrogen cyanide. In this study, the cytotoxic and genotoxic effects of tucupi on cultured human lymphocytes were assessed using the comet assay and detection of apoptosis and necrosis by differential fluorescent staining with acridine orange-ethidium bromide. Tucupi concentrations (v/v) were determined using the methylthiazole tetrazolium biochemical test. Concentrations of tucupi that presented no genotoxic effects (2, 4, 8, and 16%) were used in our experiments. The results showed that under our study conditions, tucupi exerted no genotoxic effects; however, cytotoxic effects were observed with cell death mainly induced by necrosis. These effects may be related to the presence of hydrogen cyanide in the juice.

Daily zero-reporting for suspect Ebola using short message service (SMS) in Guinea-Bissau.

OBJECTIVE: Intensified surveillance will be vital in the elimination phase to verify Ebola-free status and mitigate potential reemergence of the disease in West Africa. Zero-reporting from high-risk districts is a key strategy for surveillance. Our objective was to implement a pilot investigation to assess the feasibility of using short message service (SMS) texting for daily reporting of Ebola cases under investigation (CUI) in Guinea-Bissau in the context of an ongoing emergency-response training program known as Surveillance Training for Ebola Preparedness (STEP). STUDY DESIGN: Prospective cohort (pilot investigation) METHODS: The reporting period for the SMS pilot was January 24-March 24, 2015. STEP was conducted for two sequential groups during January 19-March 27, 2015 in Bissau, Guinea-Bissau. Training on SMS daily reporting occurred over one hour during the first week of didactic training of each group. Fourteen participants (nine from the first group and five from the second), including one surveillance officer from each of the 13 regions in Guinea-Bissau and one from the national laboratory, were selected as reporters, receiving a simple cell phone for sending SMS indicating the number of CUI for Ebola. The WHO suspect Ebola case definition was used initially and then modified on day 32 of the pilot. The text message was sent to the WiFi-connected smartphone at the Instituto Nacional Saúde Pública (INASA). The smartphone utilised an SMS-gateway application (Ushahidi SMSsync Android App) to upload the data to the Magpi cloud application. RESULTS: The average daily reporting from the first group was 7.7 of 9 (86%) and for the second group was 4.1 of 5 (82%). For the two groups combined, the reporting rate was 85%. Among the 14 reporters the median reporting rate was 85% (range 36%-100%). No cases meeting the definition for an Ebola CUI were reported during the 60 days. CONCLUSIONS: Real-time, SMS-based, daily zero-reporting can be implemented in a rapid, simple way in a low resource country. We believe that the high compliance rates were due to the simplicity and familiarity of SMS and heightened sensitivity that resulted from STEP to the importance of zero-reporting in the midst of an Ebola epidemic in neighbouring countries. This model could be useful for rapid scale-up and implementation of alert systems in other outbreaks and public health emergencies.

Remineralizing agents: effects on acid-softened enamel.

This study sought to evaluate whether remineralizing toothpastes can protect acid-softened enamel against further erosive episodes. Fifty enamel slabs of bovine teeth with preformed erosion-like lesions were randomly assigned to 1 control and 4 experimental groups (n = 10): group 1, nanohydroxyapatite (nanoHAp) dentifrice; group 2, arginine and calcium carbonate (CaCO3) dentifrice; group 3, potassium nitrate (KNO3) and high-fluoride (F) availability dentifrice; group 4, ordinary fluoridated dentifrice (OFD); and group 5, control (deionized water). Initial hardness measurements were taken after the different treatments were applied. Statistically significant mineral gains of 8.0% and 10.0% were exhibited in groups 1 and 4, respectively. Groups 2 and 3 showed mineral gains of 4.5% and 2.1%, respectively; these were not statistically significant. Group 5 showed mineral loss (-11.8%). A 1-way analysis of variance showed no statistically significant differences in the mean microhardness values among groups. However, there are indications that the nanoHAp and OFD toothpastes may decrease erosive lesions after treatment, while the arginine + CaCO3 and KNO3 + F pastes may prevent the progression of erosive lesions.

Uptake and depuration of PCB-153 in edible shrimp Palaemonetes varians and human health risk assessment.

A medium-term mesocosm exposure study was conducted to elucidate bioaccumulation and depuration of polychlorinated biphenyl congener 153 (PCB-153) in edible shrimp Palaemonetes varians. Over the 15-day exposure period, shrimp under different exposure concentrations exhibited a significant increase in PCB-153 concentration compared with control organisms. Distinct bioaccumulation patterns and uptake rates were observed depending on the exposure concentrations. For low PCB-153 exposure levels (0.25µgL(-1)), accumulation followed a saturation model, reaching an apparent steady state after fifteen days exposure. For intermediate (2.5µgL(-1)) and high PCB-153 levels (25µgL(-1)), accumulation was faster and linear. In addition, the bioaccumulation rate was not proportional to PCB-153 concentration, and the bioaccumulation was higher at intermediate exposure concentrations. Regarding the depuration phase, P. varians lost up to 30% of PCB-153 after 72h and levels continued slowly to decrease until the end of the 30-d experimental period. However, PCB-153 levels in shrimp did not reach background values, and those exposed to moderate and high PCB-153 concentrations presented contamination levels much higher than the regulatory limit for human food consumption (75ngg(-1) ww for Σ6 PCB).

Discrimination of common bean cultivars using multiplexed microsatellite markers.

Analysis of DNA polymorphisms allows for the genetic identification and precise discrimination of species with a narrow genetic base such as common bean. The primary objectives of the present study were to molecularly characterize commercial common bean varieties developed at various research institutions using microsatellite markers and to determine the degree of genetic diversity among the bean varieties analyzed. Fifty cultivars representing 12 grain classes and 64 genitors, i.e., accessions used to develop these cultivars, were characterized. Based on an analysis of 24 simple sequence repeats, the estimates for the average number of alleles and genetic diversity were 8.29 and 0.646, respectively. The combined probability of identity was estimated at 7.05 x 10(-17), indicating a high individual discriminatory power. Thirty-two percent of the cultivars exhibited heterogeneity for multiple loci that reflected either homozygosity for different alleles of a given locus in different individuals or heterozygosity for the locus. The average genetic diversity for the groups of cultivars and genitors was 0.605 and 0.660, respectively, with no genetic differentiation (FST) between these groups. Although similar estimates of expected heterozygosity were observed when the cultivars were grouped by release date, a greater number of private alleles was observed in the most recent cultivars. The genetic differentiation among cultivars originating from different institutions was not different from zero (FST = 0.01). The molecular profile database derived from these analyses may increase the statistical power of genetic estimates and may be incorporated into breeding programs for common bean. Furthermore, the profiles obtained for the different cultivars may be used as molecular descriptors to complement traditional descriptors used in distinctiveness, uniformity and stability tests, thereby improving the traceability of samples and their derivatives and helping to protect the intellectual property rights of breeders.

Potential distribution and cost estimation of the damage caused by Cryptotermes brevis (Isoptera: Kalotermitidae) in the Azores.

In the Azores archipelago, a significant proportion of buildings are infested with the urban exotic drywood termite Cryptotermes brevis (Walker), causing major economical and patrimonial losses. This work aims to understand the potential spread of this termite species in the Azores and estimate the costs for both treatment and reconstruction of infested buildings in the entire archipelago. A maximum entropy niche modeling process was used to determine the potential occurrence of the species on each island. Different scenarios were built using independent global and regional incidence and environmental data. Both projections reveal the same pattern. Generally, the probability of occurrence is higher near the coast line, where, in Azores, the majority of the towns and villages are located. We also predict that the infestation has potential to spread to yet unaffected towns and islands. It is estimated that the cost of treating all currently infested buildings in the archipelago is Euro 51 million, while reconstruction of the same buildings would rise the costs to Euro 175 million. We predict that the absence of a control strategy will cause a further expansion of the pest to more localities in the Azores. An estimate to future scenarios implies higher costs, with treatment and rebuilding values rising up to eight times the current values.

Application of a robust analytical method for quantifying progestins in environmental samples from three Portuguese Estuaries.

In the last years, progestins have raised special concerns for their documented negative effects on aquatic species, yet little is known about their environmental levels in surface waters and bioaccumulation in the trophic web. This study aimed to 1) adapt an extraction method for quantifying progestins in freeze-dried matrices, 2) validate the analytical procedure for three matrices: bivalve, polychaete, and crustacean, and 3) characterize levels of the four most prescribed synthetic progestins in key species across three Portuguese estuaries. Through the validated method, progestins were only quantifiable for the crustacean. Values were generally low, peaking with drospirenone values in Ria de Aveiro (1.33 ± 0.26 ng/g ww) and Tagus estuary (1.42 ± 0.55 ng/g ww), while Ria Formosa exhibited the lowest progestin concentrations (< 1 ng/g ww). This study enabled the development of a precise extraction and analytical method for quantifying steroid hormones in three distinct biological matrices.

Localization and symbiotic status of probiotics in the coral holobiont.

Corals establish symbiotic relationships with microorganisms, especially endosymbiotic photosynthetic algae. Although other microbes have been commonly detected in coral tissues, their identity and beneficial functions for their host are unclear. Here, we confirm the beneficial outcomes of the inoculation of bacteria selected as probiotics and use fluorescence in situ hybridization (FISH) to define their localization in the coral Pocillopora damicornis. Our results show the first evidence of the inherent presence of Halomonas sp. and Cobetia sp. in native coral tissues, even before their inoculation. Furthermore, the relative enrichment of these coral tissue-associated bacteria through their inoculation in corals correlates with health improvements, such as increases in photosynthetic potential, and productivity. Our study suggests the symbiotic status of Halomonas sp. and Cobetia sp. in corals by indicating their localization within coral gastrodermis and epidermis and correlating their increased relative abundance through active inoculation with beneficial outcomes for the holobiont. This knowledge is crucial to facilitate the screening and application of probiotics that may not be transient members of the coral microbiome. IMPORTANCE: Despite the promising results indicating the beneficial outcomes associated with the application of probiotics in corals and some scarce knowledge regarding the identity of bacterial cells found within the coral tissue, the correlation between these two aspects is still missing. This gap limits our understanding of the actual diversity of coral-associated bacteria and whether these symbionts are beneficial. Some researchers, for example, have been suggesting that probiotic screening should only focus on the very few known tissue-associated bacteria, such as Endozoicomonas sp., assuming that the currently tested probiotics are not tissue-associated. Here, we provide specific FISH probes for Halomonas sp. and Cobetia sp., expand our knowledge of the identity of coral-associated bacteria and confirm the probiotic status of the tested probiotics. The presence of these beneficial microorganisms for corals (BMCs) inside host tissues and gastric cavities also supports the notion that direct interactions with the host may underpin their probiotic role. This is a new breakthrough; these results argue against the possibility that the positive effects of BMCs are due to factors that are not related to a direct symbiotic interaction, for example, that the host simply feeds on inoculated bacteria or that the bacteria change the water quality.

Mercury bioaccumulation and the population dynamics of Mesopodopsis slabberi (Crustacea: Mysidacea) along a mercury contamination gradient.

The mercury bioaccumulation and population dynamics of the mysid Mesopodopsis slabberi was assessed along a mercury gradient in Ria de Aveiro (Portugal). M. slabberi is one of the most important mysid species in European temperate coastal shallow waters playing a key ecological role. Nevertheless, no references were found concerning the possible consequences of the Hg on the trophodynamics of these coastal ecosystems. M. slabberi showed a clear bioaccumulation along the Hg gradient and through life, with mature females reaching the highest concentrations. In terms of population structure, higher densities and biomasses of M. slabberi were assessed in the most contaminated areas contrarily to the least polluted areas. Despite the mercury accumulation in its tissues no strong negative effects on the structure and population dynamics of the species were observed. However, mysids might be important in the transfer of metals from the sediments and zooplankton to higher trophic levels such as fishes, most of them with commercial interest.

Molecular characterization of high performance inbred lines of Brazilian common beans.

The identification of germplasm genetic variability in breeding programs of the common bean (Phaseolus vulgaris) is essential for determining the potential of each combination of parent plants to obtain superior genotypes. The present study aimed to estimated the extent of genetic diversity in 172 lineages and cultivars of the common bean by integrating five tests of value for cultivation and use (VCU) that were conducted over the last eight years by the breeding program of Embrapa Arroz e Feijão in Brazil. Nine multilocus genotyping systems composed of 36 fluorescent microsatellite markers distributed across 11 different chromosomes of the common bean were used, of which 24 were polymorphic in all trials. One hundred and eighty-seven alleles were identified, with an average of 7.79 alleles per locus and an average gene diversity of 0.65. The combined probability of identity for all loci was 1.32 x 10(-16). Lineages that are more genetically divergent between the selection cycles were identified, allowing the breeding program to develop a crossbreed between elite genotypes with a low degree of genetic relatedness. HE values ranged from 0.31 to 0.63, with a large reduction in the genetic base over successive selection cycles. The test showed a significant degree of differentiation (FST = 0.159). Private alleles (26%) were identified and can be directly incorporated into the gene pool of cultivated germplasm, thereby contributing effectively to the expansion of genetic diversity in this bean-breeding program.

Identification and assessment of kefir yeast potential for sugar/ethanol-resistance.

Biochemical and molecular analysis was used for identification of different kefir yeasts species from Brazil, Canada and the United States of America. The sugar/ethanol-resistant activity of the yeasts was evaluated. Saccharomyces cerevisiae and Kluyveromyces marxianus had the highest growth rates, suggesting biotechnological applications possible for these strains.

Combined effects of climate change and environmentally relevant mixtures of endocrine disrupting compounds on the fitness and gonads' maturation dynamics of Nucella lapillus (Gastropoda).

Coastal areas are affected by multiple stressors like climate change and endocrine disruptors (EDCs). In the laboratory, we investigated the combined effects of increased temperature and EDCs (drospirenone and mercury) on the fitness and gonads' maturation dynamics of the marine gastropod Nucella lapillus for 21 days. Survival was negatively affected by all the stressors alone, while, in combination, a synergistic negative effect was observed. Both chemicals, as single factors, did not cause any effect on the maturation stage of ovaries and testis. However, in the presence of a higher temperature, it was clear a delay in the maturation stage of the ovaries, but not in the testis, suggesting a higher negative impact of the stressors in females than in males. In summary, drospirenone caused a low negative impact in aquatic species, like gastropods, but in combination with other EDCs and/or increased temperature can be a matter of concern.

Baseline progestins characterization in surface waters of three main Portuguese estuaries.

Synthetic progestins are micropollutants of special concern, due to their growing use in human and veterinary therapies and their risks to aquatic life. Currently, there is a lack of environmental information on these compounds, worldwide. The main objective of this work was to characterize the levels of the most consumed progestins in Portugal. For that, Ria de Aveiro, Tagus estuary and Ria Formosa were sampled in a temporal perspective to evaluate levels of drospirenone (DRO), desogestrel (DSG), gestodene (GST) and levonorgestrel (LNG). Drospirenone and desogestrel were the most abundant progestins. In the North of Portugal, DSG was the most abundant (Aveiro: 193.9 ng L-1 in summer), while DRO was more representative in the South (Tagus: 178.9 ng L-1; Formosa: 125.7 ng L-1) and also in summer. These spatial differences can be associated with the hydrodynamics of each estuarine system as well as the distinct population and tourist levels associated with each site.

Parasitological diagnosis in ornamental freshwater fish from different fish farmers of five Brazilian states.

This study aimed to search parasites in 333 ornamental fish from five Brazilian states (Ceará, Minas Gerais, São Paulo, Paraná and Santa Catarina). Fish were sent from eight farms located in the municipalities of Fortaleza, Patrocínio do Muriaé, São Francisco do Glória, Cascavel, Timbó, Iguape, Jacareí and Mairinque. All fish received anesthesia earlier to euthanasia procedures. After the search for parasites, it was verified that 70.6% (235/333) of fishes were infected by at least one type of parasite, being 12 types of parasites identified: monogeneans, digenean metacercariae, cestodes, nematodes, Lernaea cyprinacea, trichodinids, Piscinoodinium pillulare, Ichthyophthirius multifiliis, diplomonad flagellates, Ichthyobodo sp., Chilodonella sp., and Tetrahymena sp. The proportion of infected fishes among the farms is compared through statistical tests, besides, animal handling adopted in each farm is also discussed. The importance of ensuring fish health in order to make the ornamental freshwater fish industry economically viable and reduce losses in production is highlighted.