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.

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.

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.

Characterization of fungal communities in house dust samples collected from central Portugal-a preliminary survey.

House dust is a repository and concentrator of many chemical and biological agents including fungi. Considering that dust acts as a long-term reservoir of airborne fungi and that cumulative exposure is more relevant to potential health problems than single-day or short-term exposure, characterization of fungal communities in dust samples is of paramount importance. In the present study, the fungal composition of Portuguese house dust samples was determined. A total of 28 samples were obtained from vacuum cleaner deposits from households located in central Portugal. DNA was extracted from dust samples and fungal communities were analyzed using a culture-independent polymerase chain reaction (PCR)- denaturing gradient gel electrophoresis (DGGE) approach. Cultural analyses were also performed in order to identify the viable fungi species present in selected samples. Fungal diversity, reported as the number of operational taxonomic units (OTU), varied between 9 and 56 OTU. This analysis of viable fungi showed that Aspergillus was the most abundant genus, followed by Penicillium, Mucor, and Rhizomucor. Trichoderma, Chrysosporium, Fusarium, Rhizopus, and Stachybotrys were found in a limited number of houses. Our results demonstrated that dust is, in fact, home for a diverse and heterogeneous fungal community and that some of the species found are known allergic agents with severe negative impacts on human health.

Lethal and sub lethal effects of the biocide chlorhexidine on aquatic organisms.

Chlorhexidine is among the most used biocides in Europe, however its toxicity to aquatic organisms is scarcely known. The main objective of this study was to assess the lethal and sub lethal effects of chlorhexidine digluconate (ChD) on four aquatic model organisms: the bacteria Vibrio fischeri, the algae Pseudokirchneriella subcapitata, the crustacean Daphnia magna and the embryos of the fish Danio rerio. ChD was very toxic to algae and crustaceans, with a 72 h-EC50 of 62.2 µg/l and a 48 h-EC50 of 45.0 µg/l, respectively. Toxicity to fish embryos and the bacteria was lower, with a 96 h-EC50 of 804.0 µg/l and a 15 min-EC50 of 1,694.0 µg/l, respectively. Concerning sub lethal effects on D. magna (feeding inhibition) a 6 h-EC50 of 503.7 µg/l was obtained. In fish, ChD caused developmental abnormalities, namely alterations in the amniotic fluid (48 h-EC20 of 753.6 µg/l) and early hatching. Moreover, enzymatic biomarkers on fish embryos showed an induction of cholinesterase activity in all ChD tested concentrations (80-900 µg/l). The catalase activity was also induced at the highest concentration tested (900 µg/l) whereas no changes were observed for glutathione-S-transferase and lactate dehydrogenase activities. The toxicity of ChD to the algae and crustacean raises concerns about its potential effects in aquatic food webs, since these organisms are in the base of trophic chains, and highlights the need for further studies on ChD toxicity to aquatic organisms.

Mercury, pets' and hair: baseline survey of a priority environmental pollutant using a noninvasive matrix in man's best friend.

Pet cats and dogs have been successfully used as indicators of environmental pollution by a great variety of chemicals, including metals. However, information on mercury (a well know priority environmental pollutant) concentrations in household pets tissues and/or organs is scarce. Thus, in the present work we quantified total mercury (Hg(Total)) in blood and hair samples from twenty-six household dogs. The obtained results disclose relatively low levels of total mercury in the surveyed dogs, with values ranging from 0.16 to 12.38 ng g(-1) in blood; and from 24.16 to 826.30 ng g(-1) in hair. Mercury concentrations were independent of gender, age and diet type. A highly significant positive correlation was established between total mercury in blood and hair, validating the latter as a surrogate, non-invasive matrix for mercury exposure evaluation. Additionally, the obtained blood to hair ratio (200) is similar to the one described for humans reinforcing the suitability of dogs as sentinels. Overall, the determination of total mercury levels in dogs' hair samples proved to be a good screening method for the estimation of mercury burden in this species. We propose the quantification of Hg(Total) in hair as a screening method for sentinels like household pets to be performed in routine veterinary visits.

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.

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.

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.

Statistical approach to prenatal zygosity assessment following a decade of molecular aneuploidy screening.

In twin pregnancy studies, molecular genetic techniques have rarely been used to determine zygosity, despite their known precision and accuracy. The present work aimed to assess the power of discrimination in zygosity assessment, using a set of microsatellite markers that were routinely used for aneuploidy screening by multiplex-PCR in a prenatal context. Rapid aneuploidy screening using a group of 20 microsatellite markers (STRs) located on chromosomes 13, 18, 21 and X has been performed in our lab for over 10 years, with a total of approximately 1,500 samples studied to date. A retrospective analysis of the 257 prenatal samples from multiple pregnancies was carried out. A subset of 14 cases presenting theoretical monozygosity were re-evaluated by the use of biostatistics tools accessed via the ZygProb website. Further monozygosity determination relative to dizygosity was calculated, given an estimated overall error value of 0.093%. The results show that monozygosity had been correctly determined in all our previously studied twins. This work demonstrates that accurate zygosity assessment can be achieved with the same STRs applied in aneuploidy screening with a high power of discrimination and a matching probability of over 99.999999%.

A feeding inhibition based prediction of the toxic effect of dissolved metal mixtures upon Echinogammarus marinus (Crustacea: Amphipoda) at field relevant concentrations across a latitudinal gradient.

Risk assessment of metals in the environment is performed mainly with toxicity evaluations on single metals, which is largely inadequate since these substances occur in mixtures. The development of models predicting combined toxic effects on the basis of the concentration-response relationships of individual compounds has emerged as an answer. In the present study, metal effects on post-exposure anorexia (the concept of FdC(50)--concentration causing 50% of feeding inhibition--is implemented) in Echinogammarus marinus, a widely distributed gammarid amphipod, were assessed and compared with modelled ones obtained through the application of the concentration addition (CA) model, which represents a reasonable worst-case scenario for the risk assessment of metal mixtures. Data were validated using in situ experiments performed along a latitudinal gradient (Iceland, Scotland and Portugal) aiming at establishing a geographic profile of autochthonous population susceptibilities to metals. For all of the metals studied concentrations in the water column at exposure sites were in good agreement with feeding inhibition levels. Models gave low to relatively high percentage agreement between predictions and experimental data. Boreal populations demonstrated higher susceptibility to single metals, but not to mixture exposures. Meridional populations denoted lower susceptibilities with higher FdC(50).

Cholinesterase and glutathione-S-transferase activities in freshwater invertebrates as biomarkers to assess pesticide contamination.

Studies investigating the use of biomarkers in pesticide risk assessment have greatly increased in recent years; however, issues concerning the ecological meaning of enzymatic responses have proved controversial. Ideally a good biomarker response should be modulated by the environmental contaminants alone and demonstrate a predictable behavior towards certain types of toxins. As these premises are rarely observed, the present study aims to outline research that has contributed to an understanding of the behavior of two widely used biomarkers, cholinesterase and glutathione-S-transferase, describing environmental and biotic factors that affect their response in freshwater invertebrates. Studies were performed in the main classes of aquatic invertebrates with these biomarkers and conclusions were reached concerning their behavior towards the main classes of pesticides. Links between biomarker responses and conventional endpoints were evaluated so that ecological relevance could be attributed to enzymatic responses. Toxicity of mixtures was investigated, and cases of synergism and antagonism were pointed out as factors changing the expected toxicity of aquatic systems and leading to misinterpretations of biomarker responses. Finally, the use of biomarkers as a tool for biomonitoring and in situ assays was investigated, with discussion of advantages and disadvantages of their use.

Direct and indirect effects of the fungicide Carbendazim in tropical freshwater microcosms.

Direct and indirect effects of the fungicide carbendazim on ecosystem structure and functioning were studied < or =8 weeks after application (nominal concentrations: 0, 3.3, 33, 100, and 1000 microg/L) to outdoor microcosms in Thailand. Direct effects on macroinvertebrates are discussed in detail in a separate article. The present article presents the effects on other end points and discusses the hypothesized ecologic effect chain. Negative treatment effects on the zooplankton community were only recorded for the highest carbendazim treatment (NOEC(community) = 100 microg/L). The rotifer Keratella tropica, cladocerans (Moina micrura, Ceriodaphnia cornuta, and Diaphanosoma sp.), and cyclopoid copepods were decreased or even eliminated at this treatment level. The decrease in zooplankton and macroinvertebrate abundances was accompanied by an increase in numbers of several tolerant invertebrates, presumably caused by a release from competition and predation. The death of sensitive invertebrates probably also led to an overall decreased grazing pressure because increased levels of chlorophyll-a and bloom of the floating macrophyte Wolffia sp. were noted. The increase in primary producers is discussed to be the probable cause of changes in physicochemical water conditions, eventually resulting in an anoxic water layer during the last 3 weeks of the experiment. This is likely to have resulted in decreased invertebrate abundances noted in that period. Furthermore, the decreased decomposition of Musa (banana) leaves observed 8 weeks after application is considered to be the indirect effect of a decreased microbial activity resulting from these anoxic water conditions, rather than a direct toxic effect of carbendazim.

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.

Comparison of fate and ecological effects of the herbicide linuron in freshwater model ecosystems between tropical and temperate regions.

This paper compares the fate and effects of linuron in an outdoor plankton-dominated microcosm study carried out in Thailand with those reported in temperate model ecosystem studies evaluating linuron and other photosynthesis-inhibiting herbicides. Lower linuron concentrations disappeared slightly faster from the water compartment compared to temperate conditions, which appears to be related with the experimental design rather than differences in climatic conditions. Sensitivity of primary producers and zooplankton were similar for the climatic regions, whereas effects on ecosystem functioning were less pronounced in tropical microcosms. Recovery potential of affected endpoints appears higher for tropical ecosystems compared to their temperate counterparts. These findings support the use of toxicity data generated in temperate countries in the tropics. Recommendations for the methodology of tropical model ecosystem experiments are discussed.

Influence of exposure scenario on pesticide toxicity in the midge Kiefferulus calligaster (Kieffer).

In aquatic environments, many factors determine the overall impact of a toxic event, constituting "exposure scenarios" that must be understood so that risk assessment strategies can be successfully applied. In this work, an experimental design was planned to investigate the influence of the organism age, duration of exposure and subsequent recovery period (after cessation of toxic exposure) on chlorpyrifos or carbendazim toxicity to Kiefferulus calligaster. The endpoints studied were cholinesterase (ChE) activity and glutathione S-transferase (GST) activity. The influence of age and exposure time and the existence of recovery depend on the chemical and the biomarkers tested, suggesting that no general rule can be applied when trying to assess overall toxicity of pesticides and that detailed knowledge has to be acquired on a case-to-case basis.

Ecological effects of the herbicide linuron in tropical freshwater microcosms.

Effects of a single application of the photosynthesis-inhibiting herbicide linuron (0, 15, 50, 150, and 500 microg/L) on the ecology of outdoor plankton-dominated microcosms were studied in Thailand. As a result of the decreased photosynthesis, DO and pH decreased while EC, alkalinity and nutrient concentrations increased. Chlorophytes belonging to the genera Scenedesmus, Coelastrum and Pediastrum were the most sensitive taxa, whereas several other chlorophytes, diatoms, and cryptophytes increased in abundance. Tolerant taxa appeared to be less digestable for several zooplankton taxa, which subsequently decreased in abundances. Chamaesiphon sp. (Cyanobacteria) was the most susceptible periphyton species. As a consequence of functional redundancy, effects of the herbicide on the chlorophyll-a content of periphyton and especially phytoplankton did not always reflect the effects noted on community level.

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.

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).