FMR1 allelic complexity in premutation carriers provides no evidence for a correlation with age at amenorrhea.

BACKGROUND: Premutations in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene, defined as between 55 and 200 CGGs, have been implicated in fragile X-associated primary ovarian insufficiency (FXPOI). Only 20% of female premutation carriers develop early ovulatory dysfunction, the reason for this incomplete penetrance is unknown. This study validated the mathematical model in premutation alleles, after assigning each allele a score representing allelic complexity. Subsequently, allelic scores were used to investigate the impact of allele complexity on age at amenorrhea for 58 premutation cases (116 alleles) previously published. METHODS: The allelic score was determined using a formula previously described by our group. The impact of each allelic score on age at amenorrhea was analyzed using Pearson's test and a contour plot generated to visualize the effect. RESULTS: Correlation of allelic score revealed two distinct complexity behaviors in premutation alleles. No significant correlation was observed between the allelic score of premutation alleles and age at amenorrhea. The same lack of significant correlation was observed regarding normal-sized alleles, despite a nearly significant trend. CONCLUSIONS: Our results suggest that the use of allelic scores combination have the potential to explain female infertility, namely the development of FXPOI, or ovarian dysfunction, despite the lack of correlation with age at amenorrhea. Such a finding is of great clinical significance for early identification of females at risk of ovulatory dysfunction, enhancement of fertility preservation techniques, and increasing the probability for a successful pregnancy in females with premutations. Additional investigation is necessary to validate this hypothesis.

Comparison of toxicity of silver nanomaterials and silver nitrate on developing zebrafish embryos: Bioavailability, osmoregulatory and oxidative stress.

The mechanisms of toxicity of engineered nanomaterials (ENMs) to the early life stages of freshwater fish, and the relative hazard compared to dissolved metals, is only partially understood. In the present study, zebrafish (Danio rerio) embryos were exposed to lethal concentrations of silver nitrate (AgNO3) or silver (Ag) ENMs (primary size 42.5 ± 10.2 nm). The 96 h-LC50 for AgNO3 was 32.8 ± 0.72 µg Ag L-1 (mean ± 95% CI) compared to 6.5 ± 0.4 mg L-1 of the whole material for Ag ENMs; with the ENMs being orders of magnitude less toxic than the metal salt. The EC50 for hatching success was 30.5 ± 1.4 µg Ag L-1 and 6.04 ± 0.4 mg L-1 for AgNO3 and Ag ENMs, respectively. Further sub-lethal exposures were performed with the estimated LC10 concentrations for both AgNO3 or Ag ENMs over 96 h, where about 3.7% of the total Ag as AgNO3 was internalised, as measured by Ag accumulation in the dechorionated embryos. However, for the ENM exposures, nearly all (99.8%) of the total Ag was associated with chorion; indicating the chorion as an effective barrier to protect the embryo in the short term. Calcium (Ca2+) and sodium (Na+) depletion was induced in embryos by both forms of Ag, but hyponatremia was more pronounced in the nano form. Total glutathione (tGSH) levels declined in embryos exposed to both Ag forms, but a superior depletion occurred with the nano form. Nevertheless, oxidative stress was mild as superoxide dismutase (SOD) activity stayed uniform and the sodium pump (Na+/K+-ATPase) activity had no appreciable inhibition compared to the control. In conclusion, AgNO3 was more toxic to the early life stage zebrafish than the Ag ENMs, still differences were found in the exposure and toxic mechanisms of both Ag forms.

Do Freshwater and Marine Bivalves Differ in Their Response to Wildfire Ash? Effects on the Antioxidant Defense System and Metal Body Burden.

Wildfires constitute a source of contamination to both freshwater and marine ecosystems. This study aimed to compare the antioxidant defense response of the freshwater clam Corbicula fluminea and the marine cockle (Cerastoderma edule) to wildfire ash exposure and the concomitant metal body burden. Organisms were exposed to different concentrations (0%, 12.5%, 25%, 50%, and 100%) of aqueous extracts of Eucalypt ash (AEAs) from a moderate-to-high severity wildfire. The activity of various enzymes, as well as lipid peroxidation, protein content, and metal body burden, were determined after 96 h of exposure. A significant increase in the protein content of soft tissues was observed for C. edule at AEA concentrations ≥ 25%, unlike for C. fluminea. Similarly, significant effects on lipid peroxidation were observed for cockles, but not for clams. For both species, a significant effect in the total glutathione peroxidase activity was observed at AEA concentrations ≥ 25%. Relative to the control, AEAs-exposed clams showed higher Cd content, whereas AEAs-exposed cockles showed higher Cu content, thus exhibiting different responses to the exposure to wildfire ash. The susceptibility of bivalves to ashes, at environmentally relevant concentrations, raises concern about the effects of post-fire runoff to bivalve species.

Do sediment-bound nickel and lead affect chironomids life-history? Toxicity assessment under environmentally relevant conditions.

Metal pollution in aquatic ecosystems translates into increased concentrations of sediment-bound metals, representing a risk for benthic species. This risk might be enhanced in soft and moderately hard waters, world widely distributed, due to the protective role of hardness against metal toxicity. As lead (Pb) and nickel (Ni) are amongst the more abundant metals in aquatic systems, and since their combined effects to benthic species have been overlooked, in this study we aimed to investigate the life-cycle toxicity of Pb and Ni (using spiked sediment) to the benthic species Chironomus riparius, considering both single and mixture exposures, in moderately hard water. Environmentally relevant concentrations of each metal were used (25 and 75 mg kg-1, based on a scenario of pollution by runoff waters from burnt forests), following a full factorial design. Effects of the mixture with the highest metal concentrations (Pb 75 mg kg-1 dw + Ni 75 mg kg-1 dw) were also assessed in the second generation. In the first generation, exposure to Pb increased emergence and the weight of males, and decreased time to emergence of both males and females. Conversely, exposure to Ni delayed female emergence and decreased the weight of imagoes. Summarizing, Pb affected more endpoints but showed an apparent positive effect, whereas Ni affected less endpoints but exhibited adverse effects. Reproduction was not affected by these metals. In the second generation, the mixture Pb 75 mg kg-1 + Ni 75 mg kg-1 dw delayed emergence and reduced the emerged female fraction and their weight. These results highlight that Pb and Ni can alter the structure of C. riparius populations at environmentally relevant concentrations, which signals potential repercussions in the dynamics and functioning of freshwater ecosystems under these contamination scenarios. The findings of the present study are relevant not only for metal-polluted environments, in general, but also for fire-affected ecosystems.

Use of the FMR1 Gene Methylation Status to Assess the X-Chromosome Inactivation Pattern: A Stepwise Analysis.

X-chromosome inactivation (XCI) is a developmental process to compensate the imbalance in the dosage of X-chromosomal genes in females. A skewing of the XCI pattern may suggest a carrier status for an X-linked disease or explain the presence of a severe phenotype. In these cases, it is important to determine the XCI pattern, conventionally using the gold standard Human Androgen-Receptor Assay (HUMARA), based on the analysis of the methylation status at a polymorphic CAG region in the first exon of the human androgen receptor gene (AR). The aim of this study was to evaluate whether the methylation status of the fragile mental retardation protein translational regulator gene (FMR1) can provide an XCI pattern similar to that obtained by HUMARA. A set of 48 female carriers of FMR1 gene normal-sized alleles was examined using two assays: HUMARA and a FMR1 methylation PCR (mPCR). Ranges were defined to establish the XCI pattern using the methylation pattern of the FMR1 gene by mPCR. Overall, a 77% concordance of the XCI patterns was obtained between the two assays, which led us to propose a set of key points and a stepwise analysis towards obtaining an accurate result for the XCI pattern and to minimize the underlying pitfalls.

Development and Validation of a Mathematical Model to Predict the Complexity of FMR1 Allele Combinations.

The polymorphic trinucleotide repetitive region in the FMR1 gene 5'UTR contains AGG interspersions, particularly in normal-sized alleles (CGG < 45). In this range repetitive stretches are typically interrupted once or twice, although alleles without or with three or more AGG interspersions can also be observed. AGG interspersions together with the total length of the repetitive region confer stability and hinder expansion to pathogenic ranges: either premutation (55 < CGG < 200) or full mutation (CGG > 200). The AGG interspersions have long been identified as one of the most important features of FMR1 repeat stability, being particularly important to determine expansion risk estimates in female premutation carriers. We sought to compute the combined AGG interspersion numbers and patterns, aiming to define FMR1 repetitive tract complexity combinations. A mathematical model, the first to compute this cumulative effect, was developed and validated using data from 131 young and healthy females. Plotting of their allelic complexity enabled the identification of two statistically distinct groups - equivalent and dissimilar allelic combinations. The outcome, a numerical parameter designated allelic score, depicts the repeat substructure of each allele, measuring the allelic complexity of the FMR1 gene including the AGGs burden, thus allowing new behavioral scrutiny of normal-sized alleles in females.

Establishing causal links between aquatic biodiversity and ecosystem functioning: Status and research needs.

Understanding how changes in biodiversity affects ecosystem functioning is imperative in allowing Ecosystem-Based Management (EBM), especially when addressing global change and environmental degradation. Research into the link between biodiversity and ecosystem functioning (BEF) has indeed increased considerably over the past decades. BEF research has focussed on terrestrial ecosystems and aquatic ecosystems have received considerably less attention. Due to differences in phylogenetic diversity, ecological processes and reported BEF relationships, however, it may at least be questionable whether BEF relationships are exchangeable between these ecosystems (i.e. terrestrial and aquatic). The aim of the present paper was therefore to pinpoint key areas and bottlenecks in establishing BEF relationships for aquatic ecosystems (freshwater, transitional, and marine). To this end, the available literature with special emphasis on the last 10 years was assessed to evaluate: i) reported mechanisms and shapes of aquatic BEF relationships; ii) to what extent BEF relations are interchangeable or ecosystem-specific; and iii) contemporary gaps and needs in aquatic BEF research. Based on our analysis, it may be concluded that despite considerable progress in BEF research over the past decades, several bottlenecks still need to be tackled, namely incorporating the multitude of functions supported by ecosystems, functional distinctiveness of rare species, multitrophic interactions and spatial-temporal scales, before BEF relationships can be used in ecosystem-based management.

Risk to the supply of ecosystem services across aquatic ecosystems.

The capacity of ecosystems to supply ecosystem services is decreasing. Sustaining this supply requires an understanding of the links between the impacts of pressures introduced by human activities and how this can lead to changes in the supply of services. Here, we apply a novel approach, assessing 'risk to ecosystem service supply' (RESS), across a range of aquatic ecosystems in seven case studies. We link aggregate impact risk from human activities on ecosystem components, with a relative score of their potential to supply services. The greatest RESS is found where an ecosystem component with a high potential to supply services is subject to high impact risk. In this context, we explore variability in RESS across 99 types of aquatic ecosystem component from 11 realms, ranging from oceanic to wetlands. We explore some causes of variability in the RESS observed, including assessment area, Gross Domestic Product (GDP) and population density. We found that Lakes, Rivers, Inlets and Coastal realms had some of the highest RESS, though this was highly dependent on location. We found a positive relationship between impact risk and service supply potential, indicating the ecosystem components we rely on most for services, are also those most at risk. However, variability in this relationship indicates that protecting the supply of ecosystem services alone will not protect all parts of the ecosystem at high risk. Broad socio-economic factors explained some of the variability found in RESS. For example, RESS was positively associated with GDP and artificial and agricultural land use in most realms, highlighting the need to achieve balance between increasing GDP and sustaining ecosystem health and human wellbeing more broadly. This approach can be used for sustainable management of ecosystem service use, to highlight the ecosystem components most critical to supplying services, and those most at risk.

Ecosystem-based management planning across aquatic realms at the Ria de Aveiro Natura 2000 territory.

Ria de Aveiro represents a coastal territory, in which its natural capital, mostly classified under a Natura 2000 network of protected areas, is of paramount importance for the regional and national economy, supporting harbour activities and maritime traffic, agriculture, commercial fisheries, aquaculture, manufacturing, tourism, sports and recreational activities. Current and foreseen changes connected to human activities, namely land and water uses and potential conflicts, in frame of environmental policies, sustainable economic development and human well-being require the implementation of ecosystem-based management (EBM) planning processes considering the connectivity across marine, transitional, freshwater, and terrestrial domains. The main objective is to elaborate on the co-development of the EBM planning process across the three water domains, all characterized by high biodiversity and by the wide range of services provided by ecosystems and their abiotic components, for the mitigation of impacts from the management plan under implementation. The approach used follows a stepwise procedure in frame of resilience principles, considering the analysis of the relationship between the social and ecological components and on how these can be connected through risk assessment and a spatial multi-criteria analysis based on the delivery of ecosystem services. Stakeholders' perception matched the ecosystem services provisioning risk assessment and supported the planning EBM response that consist in saltmarshes and seagrasses meadows restoration programs. Compliance of the proposed measures is achievable regarding policies (policy targets and policy instruments) and feasibility (scientific and technological knowledge and financial resources). The EBM response can support the Vouga estuary management plan and regional smart specialization (RIS3 Centro).

Participatory coastal management through elicitation of ecosystem service preferences and modelling driven by "coastal squeeze".

The Baixo Vouga Lagunar (BVL) is part of Ria de Aveiro coastal lagoon in Portugal, which is classified as a Special Protection Area under the European Habitats and Birds Directives. This part of the system, corresponding to the confluence of the Vouga River with the lagoon, is very important culturally and socioeconomically for the local communities, taking place several human activities, especially agriculture. To prevent salt water intrusion from the Ria de Aveiro into agriculture fields, a floodbank was initiated in the 90's. In frame of ongoing changes in Ria de Aveiro hydrodynamics, the existing floodbank will be now extended, introducing further changes in the ecological dynamics of the BVL and its adjacent area. As a consequence, the water level in the floodbank downstream side is expected to rise, increasing the submersion period in tidal wetlands, and leading to coastal squeeze. The aim of this study is to apply an ecosystem based-management approach to mitigate the impacts on biodiversity resulting from the management plan. To do so, we have modelled the implications of the changes in several hydrological and environmental variables on four saltmarsh species and habitats distribution, as well as on their associated ecosystem services, both upstream and downstream of the floodbank. The ecosystem services of interest were prioritized by stakeholders' elicitation, which were then used as an input to a spatial multi-criteria analysis aimed to find the best management actions to compensate for the unintended loss of biodiversity and ecosystem services in the BVL. According to our results, the main areas to be preserved in the BVL were the traditional agricultural mosaic fields; the freshwater courses and the subtidal estuarine channels. By combining ecology with the analysis of social preferences, this study shows how co-developed solutions can support adaptive management and the conservation of coastal ecosystems.

Exploring variability in environmental impact risk from human activities across aquatic ecosystems.

Aquatic ecosystems are under severe pressure. Human activities introduce an array of pressures that impact ecosystems and their components. In this study we focus on the aquatic domains of fresh, coastal and marine waters, including rivers, lakes and riparian habitats to transitional, coastal as well as shelf and oceanic habitats. In an environmental risk assessment approach, we identified impact chains that link 45 human activities through 31 pressures to 82 ecosystem components. In this linkage framework >22,000 activity-pressure-ecosystem component interactions were found across seven European case studies. We identified the environmental impact risk posed by each impact chain by first categorically weighting the interactions according to five criteria: spatial extent, dispersal potential, frequency of interaction, persistence of pressure and severity of the interaction, where extent, dispersal, frequency and persistence account for the exposure to risk (spatial and temporal), and the severity accounts for the consequence of the risk. After assigning a numerical score to each risk criterion, we came up with an overall environmental impact risk score for each impact chain. This risk score was analysed in terms of (1) the activities and pressures that introduce the greatest risk to European aquatic domains, and (2) the aquatic ecosystem components and realms that are at greatest risk from human activities. Activities related to energy production were relevant across the aquatic domains. Fishing was highly relevant in marine and environmental engineering in fresh waters. Chemical and physical pressures introduced the greatest risk to the aquatic realms. Ecosystem components that can be seen as ecotones between different ecosystems had high impact risk. We show how this information can be used in informing management on trade-offs in freshwater, coastal and marine resource use and aid decision-making.

Cost-effective restoration and conservation planning in Green and Blue Infrastructure designs. A case study on the Intercontinental Biosphere Reserve of the Mediterranean: Andalusia (Spain) - Morocco.

Green and Blue Infrastructure (GBI) is a network designed and planned to deliver a wide range of ecosystem services and to protect biodiversity. Existing GBI designs lacked a systematic method to allocate restoration zones. This study proposes a novel approach for systematically selecting cost-effective areas for restoration on the basis of biodiversity, ecosystem services, and ecosystem condition to give an optimal spatial design of GBI. The approach was tested at a regional scale, in a transboundary setting encompassing the Intercontinental Biosphere Reserve of the Mediterranean in Andalusia (Spain) - Morocco (IBRM), across three aquatic ecosystems: freshwater, coastal and marine. We applied Marxan with Zones to stakeholder-defined scenarios of GBI in the IBRM. Specifically, we aimed to identify management zones within the GBl that addressed different conservation, restoration and exploitation objectives. Although almost all conservation targets were achieved, our results highlighted that the proportion of conservation features (i.e., biodiversity, ecosystem services) that would be compromised in the GBl, and the proportion of provisioning services that would be lost due to conservation (i.e., incidental representation) are potentially large, indicating that the probability of conflicts between conservation and exploitation goals in the area is high. The implementation of restoration zones improved connectivity across the GBI, and also achieved European and global policy targets. Our approach may help guide future applications of GBI to implement the flexible conservation management that aquatic environments require, considering many areas at different spatial scales, across multiple ecosystems, and in transboundary contexts.

Linking biodiversity to ecosystem services supply: Patterns across aquatic ecosystems.

Global initiatives have been increasingly focusing on mainstreaming the values of biodiversity and ecosystem services into decision-making at all levels. Due to the accelerated rate at which biodiversity is declining and its consequences for the functioning of ecosystems and subsequently, the services they provide, there is need to develop comprehensive assessments of the services and the benefits nature delivers to society. Based on expert evaluation, we identified relevant flow linkages in the supply-side of the socio-ecological system, i.e. from biodiversity to ecosystem services supply for eight case studies across European aquatic ecosystems covering freshwater, transitional, coastal and marine waters realms. Biological mediated services were considered, as well as those reliant on purely physical aspects of the ecosystem, i.e. abiotic outputs, since both have implications for spatial planning, management and decision-making. Due to the multidimensional nature of ecosystems and their biodiversity, our approach used ecosystem components such as habitats and biota as proxies for biodiversity and as the focal point for linkage identification. Statistical analysis revealed the importance of considering mobile biota in the spatial assessment of habitats. Contrary to literature evidences so far, our results showed significantly different and complementary ecosystem services supply patterns across the continuum of aquatic realms. The implemented score of ecosystem services supply has a high potential for integrated aquatic ecosystem service supply assessments in the context of ecosystem-based management.

Ecotoxicological assessment of the herbicide Winner Top and its active substances-are the other formulants truly inert?

Formulants used in Plant Protection Products (PPPs) to promote their efficiency are normally undisclosed in the PPP documentation, unless they bear a human health or environmental hazardous potential per se. PPP regulation also demands the assessment of putative interactions among formulants within each product recipe and consequent effects, but these results are often unavailable. Such a case is that of the herbicide Winner Top (Selectis®, Portugal), which we selected as a model commercial formulation in the present study specifically aiming at (i) characterising its aquatic toxicity towards sensitive eco-receptors (Raphidocelis subcapitata, Chlorella vulgaris, Lemna minor and Lemna gibba), as well as that of its active substances (a.s.) nicosulfuron and terbuthylazine; (ii) comparing the ecotoxicity among the commercial formulation, the corresponding mixture of its a.s. and this a.s.'s mixture increasingly enriched with the formulants. Single chemical testing revealed that terbuthylazine was the strongest microalgae growth inhibitor and nicosulfuron was the strongest macrophyte growth inhibitor. On the other hand, the commercial formulation was consistently less toxic than the corresponding mixture of the a.s., suggesting that Winner Top formulants (72.9% of the commercial formulation) interact with the a.s., promoting less than additive effects in the selected non-target species. Importantly, this environmentally protective effect of the formulation can be apparent. Because macrophytes share most physiological features with the weeds targeted by the studied herbicide, it is likely that increased application doses are required to reach desired efficacy levels with the consequent detrimental increase of PPP residues load in edge-of-field freshwater ecosystems.

Mixture toxicity assisting the design of eco-friendlier plant protection products: a case-study using a commercial herbicide combining nicosulfuron and terbuthylazine.

The development of environmentally friendly plant protection products (PPPs), including pesticides, is a challenge nowadays. A commercial herbicide combining terbuthylazine and nicosulfuron as active substances (a.s.) was selected as a model PPP. The suitability of manipulating the ratio between a.s. towards alternative formulations with reduced impacts in a non-target indicator (Lemna minor) was assessed. The efficacy of such eco-friendlier a.s. ratios was then assessed using a target weed, Portulaca oleracea. Single and mixture toxicity testing with L. minor revealed an antagonistic joint action of the a.s., suggesting an environmentally protective effect of the combination compared to single dosing of a.s. The efficacy testing against the target weed of single and combined treatments of the a.s. showed that (i) the a.s. behave antagonistically throughout the whole P. oleracea response surface; (ii) there were no environmentally safe a.s. combinations ensuring target-efficacy; (iii) terbuthylazine alone was effective in controlling P. oleracea with no environmental hazardous potential, dosed at concentrations 10-fold lower than those involved in commercially recommended application doses. Overall, this case-study suggests that modelling tools widely used in the field of environmental risk assessment of PPPs may also have application in PPP design stages for a more efficient meeting of efficacy and environmental friendliness requirements.

Phytotoxicity of silver nanoparticles to Lemna minor: Surface coating and exposure period-related effects.

Silver nanoparticles (Ag NPs) exponential production raises concern about their environmental impact. The effects of Ag NPs to aquatic plants remain scarcely studied, especially in extended exposures. This paper aims to evaluate Ag NPs effects in Lemna minor at individual and sub-individual levels, focusing on three variables: Ag form (NPs versus ions - Ag+), NPs surface coating (citrate vs polyvinylpyrrolidone - PVP) and exposure period (7 vs 14days). Endpoints were assessed at individual level (specific growth rate, chlorosis incidence and number of fronds per colony) and sub-individual level (enzymatic activities of catalase (CAT), guaiacol peroxidase (GPx) and glutathione-S-transferase (GST)). Generally, plants exposed to all Ag forms underwent decays on growth rate and fronds per colony, and increases on chlorosis, GPX and GST, but no effects on CAT. The most sensitive endpoints were specific growth rate and GPx activity, showing significant effects down to 0.05mg/L for Ag NPs and 3µg/L for Ag+, after 14days. Ag+ showed higher toxicity with a 14d-EC50 of 0.0037mg Ag/L. Concerning surface coating, PVP-Ag NPs were more deleterious on growth rate and fronds per colony, whereas citrate-Ag NPs affected more the chlorosis incidence and GPx and GST activities. The exposure period significantly affected chlorosis: 14days triggered a chlorosis increase in Ag+-exposed plants and a decrease in Ag NPs-exposed plants when compared to 7days. Ag NPs induced an oxidative stress status in cells, thus ensuing upregulated enzymatic activity as a self-defense mechanism. Since Ag NPs dissolution might occur on a steady and continuous mode along time, and the average longevity of fronds, we propose longer exposures periods than the recommended by the OECD guideline. This approach would provide more relevant and holistic evidences on the overall response of freshwater plants to Ag NPs in an ecological relevant scenario.

The sugarcane herbicide ametryn induces oxidative stress and developmental abnormalities in zebrafish embryos.

Ametryn (AMT) is one of the most widely used herbicides in tropical sugarcane crops, the main culture of São Paulo State, Brazil. It is known as a diffuse pollutant, being found in surface water and sediment of water bodies adjacent to the crop fields. In the present study, the toxicity of AMT to zebrafish (Danio rerio) embryos was evaluated using developmental and biochemical endpoints. At the biochemical level, lactate dehydrogenase responded at the lowest concentration tested (4 µg L-1) indicating a high demand of energy required to cope with the stress condition. Antioxidant enzyme levels were changed at intermediate/high concentrations while oxidative damage (lipid peroxidation) was observed at the last concentration tested (10 mg L-1). This suggests that, like for other herbicides from the triazine group, oxidative stress is a major pathway of toxicity for AMT. Several developmental effects such as oedemas and tail deformities were also observed. The 96 h-EC50 values calculated for different developmental parameters were between 17 and 29 mg L-1. AMT also affected hatching (96 h-EC50 = 22.5 mg L-1) and positioning in the water column (96 h-EC50 = 13.2 mg L-1). In a previous work of the group, lethal toxicity of AMT showed to be much higher to adults than to embryos. However, in the present work, sublethal endpoints assessed suggest that important effects are observed at lower concentrations, improving the sensitivity of the embryo test. Moreover, in this work, sublethal effects were observed for concentrations in the same range as the ones found in the environment, and thus, given that this chemical is widely used in tropical fields, a refined evaluation of risk should be performed based on the monitoring of sublethal and long-term parameters and considering mixture scenarios.

The effects of Copper and Zinc on survival, growth and reproduction of the cladoceran Daphnia longispina: introducing new data in an "old" issue.

Metal contamination is still a major environmental issue due to their continuous deposition and persistence. In this work we intended to assess the impact that Copper (Cu) and Zinc (Zn) exert in life-history parameters of Daphnia longispina, a common cladoceran in freshwater environments. Thus, we studied the effects of Cu (20-300 µg/L) and Zn (500-4000 µg/L) on the survival, growth, reproduction, feeding rate and population growth rate of D. longispina. Though survival was only reduced for the highest concentration of each metal, other endpoints were strongly affected by lower concentrations. Growth was affected by both metals, especially in the period 0-7 d, being significant for Cu ≥ 40 µg/L and Zn ≥ 500 µg/L. Indeed, growth endpoints at day 7 (body length and growth rate) were equally or more sensitive than the corresponding endpoints at day 21. The size at first reproduction decreased (significant for Cu ≥ 40 µg/L and Zn ≥ 500 µg/L). Reproduction decreased, which was partially explained by the reduced growth, but additional effects were supported by a direct effect of metals on reproduction (based on the relationship body length-brood size). Reduced growth and reproduction are likely a food mediated effect due to feeding inhibition caused by the metals. Globally, the studied endpoints were affected by Cu and Zn differently, supporting a chemical-specificity of the effects, with Zn causing more pronounced effects than Cu. This work presents an innovative approach to the effects of Cu and Zn to D. longispina, giving a general and comprehensive overview of those effects.

Levels of TBT and other selected organotin compounds in duplicate diet samples.

Organotin compounds (OTs) are ubiquitous contaminants with a broad range of applications ranging from biocides and pesticides to catalysts for the production of polyurethane foams and silicones. The deleterious effects of some OTs (particularly tributyltin - TBT) upon wildlife and experimental animals are well documented and include endocrine disruption, immunotoxicity, neurotoxicity, genotoxicity and metabolic dysfunction in which obesity is included. However, virtually no data on the current human exposure levels is available. In order to bridge this gap, we quantified for the first time the levels of OTs in duplicate diet samples from members of the University of Aveiro in Portugal. OTs were detected in 32% of the 28 diet samples analyzed, at relatively low levels. TBT and monobutyltin were detected only in two samples and dibutyltin was detectable in three samples. Dioctyltin was quantified in four samples and monooctyltin in three samples. Phenyltins were below the detection limit in all the diet samples analyzed. Overall, for the vast majority of the samples (89%), the estimated daily intakes (EDI) of organotins through food were much lower than the established tolerable daily intakes (TDI). Hence, for the majority of the participants the risk associated with food ingestion is low.

Characterization of silver nanoparticles-alginate complexes by combined size separation and size measurement techniques.

The detection and quantification of nanoparticles is a complex issue due to the need to combine "classical" identification and quantification of the constituent material, with the accurate determination of the size of submicrometer objects, usually well below the optical diffraction limit. In this work, the authors show that one of the most used analytical methods for silver nanoparticles, asymmetric flow field-flow fractionation, can be strongly influenced by the presence of dissolved organic matter (such as alginate) and lead to potentially misleading results. The authors explain the anomalies in the separation process and show a very general way forward based on the combination of size separation and size measurement techniques. This combination of techniques results in more robust AF4-based methods for the sizing of silver nanoparticles in environmental conditions and could be generally applied to the sizing of nanoparticles in complex matrices.