Evaluation of the potential of translocated common cockle for ecological risk assessment studies: bioaccumulation and biomarkers test

Thesis submitted to the Faculdade de Ciências e Tecnologia to obtain the Master’s degree in Environmental Engineering, profile in Ecological EngineeringSediment–bound contamination is a major concern factor in estuaries and other confined coastal water bodies, frequently subjected to anthropogenic sources of pollution. In order to investigate the effects and responses of the common cockle (Cerastoderma edule, L. 1558, Bivalvia: Cardiidae) to sediment contaminants and to assess the species’ potential as an indicator organism, the bivalve was subjected to a laboratorial translocation assay with sediments collected from distinct sites of the Sado Estuary (Portugal). Cockles were collected from a mariculture site of the Sado estuary (Portugal), herewith identified as site A, and exposed through 28–day, semi–static laboratorial essays, to sediments collected from three other sites (B, C and D) of the estuary that revealed different levels of metals, organic contaminants and physico–chemical properties and that ranged from globally unimpacted to moderately impacted levels when compared to available sediment quality guidelines. The animals were surveyed for bioaccumulation of metals (Ni, Cu, Zn, As, Cd and Pb) and organic contaminants (PAHs, PCBs and DDTs). Two sets of potential biomarkers were employed to assess toxicity: whole–body metallothionein (MT) induction and digestive gland histopathology. The bioaccumulation factor (BAF) and the biota-to-soil accumulation factor(BSAF) were estimated as ecological indices of exposure to metals and organic compounds. Significant positive correlations between BSAF and MT were found for PHAs, and between each factor (BSAF and BAF) and MT were found for Cd. Histopathological alterations were found in cockles exposed to all sediments where they were translocated. The digestive gland integrity was found to be especially compromised in cockles from sediment B and C and at day 28 from sediment A. Results allowed concluding that C. edule responds to sediment–bound contamination and is capable to regulate and eliminate both types of contaminants and might, therefore, be suitable for biomonitoring. Still, the sediment contamination levels do not explain the variation in bioaccumulation and MT levels, which may result from the moderate contaminant concentrations found in sediments and, more importantly, from yet unexplained xenobiotic interaction effects

Seagrasses benefit from mild anthropogenic nutrient additions

Seagrasses are declining globally, in large part due to increased anthropogenic coastal nutrient loads that enhance smothering by macroalgae, attenuate light, and are toxic when in excessive concentrations of inorganic nitrogen and phosphorus. However, as sanitation is improved many seagrass meadows have been observed to recover, with a few studies suggesting that they may even benefit from mild anthropogenic nutrient additions. Monitoring seagrass demography and health has faced difficulties in establishing the adequate variables and metrics. Such uncertainty in the methods has caused uncertainty of the significance of results presented and compromised extrapolations to other seasons, areas, or species. One solution has come from within the plant self-thinning theories. During the 1980s, an interspecific boundary line (IBL) was determined as the upper limit of the combination of plant density and above-ground biomass for any stand on Earth, setting their maximum possible efficiency in space occupation. Recently, two meta-analyses to determine specific IBLs for algae and for seagrasses have been performed. The recently updated seagrass dataset comprises 5,052 observations from 78 studies on 18 species. These IBLs opened new perspectives for monitoring: the observed distance of a stand to the respective IBL (i.e., each stand's relative efficiency of space occupation) was demonstrated to be a valuable indicator of a population's health. Thus, this metric can be used to determine the impact of nutrients and pollutants on algae and seagrass populations. Furthermore, because the IBLs are common to all species, they may be used to compare all species from any location worldwide. This novel approach showed that Halodule wrightii, Halodule beaudettei, Halophila baillonii, Zostera marina, and Zostera noltei meadows benefit from anthropogenic additions of nitrogen and phosphorus, as long as these additions are moderate. In fact, the healthier Z. noltei meadows in Portugal (and among the healthiest meadows worldwide) were the ones exposed to effluents from wastewater treatment plants (WWTP) and a food factory. We conclude that those effluents are providing water with enough quality and that their optimal management should coordinate the technological solutions of the WWTP with the natural potential of seagrass meadows as water purifiers and biomass producers.info:eu-repo/semantics/publishedVersio

Antioxidant and anti-inflammatory extracts from sea cucumbers and tunicates induce a pro-osteogenic effect in Zebrafish Larvae

Bone metabolic disorders such as osteoporosis are characterized by the loss of mineral from the bone tissue leading to its structural weakening and increased susceptibility to fractures. A growing body of evidence suggests that inflammation and oxidative stress play an important role in the pathophysiological processes involved in the rise of these conditions. As the currently available therapeutic strategies are often characterized by toxic effects associated with their long-term use, natural antioxidants and anti-inflammatory compounds such as polyphenols promise to be a valuable alternative for the prevention and treatment of these disorders. In this scope, the marine environment is becoming an important source of bioactive compounds with potential pharmacological applications. Here, we explored the bioactive potential of three species of holothurians (Echinodermata) and four species of tunicates (Chordata) as sources of antioxidant and anti-inflammatory compounds with a particular focus on polyphenolic substances. Hydroethanolic and aqueous extracts were obtained from animals' biomass and screened for their content of polyphenols and their antioxidant and anti-inflammatory properties. Hydroethanolic fractions of three species of tunicates displayed high polyphenolic content associated with strong antioxidant potential and anti-inflammatory activity. Extracts were thereafter tested for their capacity to promote bone formation and mineralization by applying an assay that uses the developing operculum of zebrafish (Danio rerio) to assess the osteogenic activity of compounds. The same three hydroethanolic fractions from tunicates were characterized by a strong in vivo osteogenic activity, which positively correlated with their anti-inflammatory potential as measured by COX-2 inhibition. This study highlights the therapeutic potential of polyphenol-rich hydroethanolic extracts obtained from three species of tunicates as a substrate for the development of novel drugs for the treatment of bone disorders correlated to oxidative stress and inflammatory processes.info:eu-repo/semantics/publishedVersio

Molecular biodiversity and advanced monitoring tools for macrobenthic communities of Portuguese coastal ecosystems

Ph. D. Thesis Molecular and Environmental Biology - Specialization on Evolution, Biodiversity and EcologyBenthic communities (BCs) are components of paramount importance for aquatic ecosystems function and therefore central elements in aquatic biodiversity monitoring programmes (e.g. European Union's Water Framework Directive – WFD). Current methodologies (e.g. morphological identification) are very time-consuming, technically difficult and frequently species-level data cannot be obtained. Molecular approaches such as DNA barcodes can greatly facilitate and speed up monitoring of BCs. DNA barcodes are also proficient in unravelling latent biodiversity and clarifying taxonomic ambiguities. The emergence of high throughput sequencing (HTS) technologies, combined with DNA barcodes, have the potential to overtake many of the limitations of the morphology-based method. In this thesis we use DNA barcodes to investigate the biodiversity of macrobenthic organisms of Portuguese coastal ecosystems and develop a high-throughput DNA metabarcoding approach for macrobenthic communities. To this purpose, a) we optimized the primer design in order to enhance the amplification success of DNA barcodes, b) compiled DNA barcodes for dominant groups and species and c) tested primer success in DNA metabarcoding of mock communities, and compared the effectiveness of morphology-based and DNA metabarcoding approaches in the inventory of wild estuarine macrobenthic communities. DNA barcodes were used to contribute to the enrichment of the global reference library with prominent species of the estuarine and marine benthic communities of Portugal, focusing on annelids, crustaceans and mollusks. This thesis contributed to generate DNA barcodes for macrobenthic morphospecies, which include 51 polychaetes, 43 amphipods and 34 gastropods. Evidence of hidden or cryptic species based on high intra-specific distances was found 6 polychaete, 7 amphipod and 3 gastropod morphospecies, corresponding to the detection of a possible total of 30 hidden species. To assess the ability of the HTS to recover DNA barcodes from all macrobenthic species present in a bulk community DNA extract, 3 phylogenetically diverse communities were assembled. Over 80% of the species of the tested communities were recovered. Subsequently, the efficiency of morphology-based identifications compared to metabarcoding-based approaches in the assessment of the species composition of macrobenthos was determined in four distinct sites of the Sado estuary, Portugal. The results indicate that the species richness would be underestimated if only morphological methods were used, since the metabarcoding approach was able to provide unambiguous species-level assignments for a much greater number of species, which are susceptible to scrutiny and verification in the future. We conclude that the DNA metabarcoding approach can perform equally or better than morphology based inventories, and that high-throughput biodiversity assessment of macrobenthic communities is attainable through this approach. Nevertheless, further refinement is still required to improve its performance and potential, as for example the improvement of the recovery rates through additional optimization of primers, or by testing alternative primer combinations. Finally, since the reference libraries are still incipient for marine invertebrates, the continuing completion will be decisive to fully materialize the potential of metabarcoding.This work was supported by FEDER through POFC-COMPETE by national funds from ‘Fundação para a Ciência e a Tecnologia (FCT)’ in the scope of the grant FCOMP-01-0124-FEDER-015429 and also by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) also funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI). Jorge Lobo was supported by a PhD fellowship (SFRH/BD/69750/2010) from FCT.As comunidades bentónicas são componentes de extrema importância para o funcionamento dos ecossistemas aquáticos e portanto, elementos centrais nos programas de monitoramentor da biodiversidade aquática (por exemplo, Directiva Quadro da Água da União Europeia – DQA). As metodologias actuais (identificação morfológica) são muito demoradas, tecnicamente difíceis e frequentemente não se podem obter dados até o nível da espécie. Abordagens moleculares, como o citocromo c oxidase I DNA barcodes, podem facilitar e acelerar em grande medida o monitoramento das comunidades bentónicas. DNA barcodes também são capazes de revelar a biodiversidade latente e esclarecer ambiguidades taxonómicas. O aparecimento de tecnologias de sequenciação de alto rendimento (HTS), combinado com DNA barcodes, têm o potencial para ultrapassar muitas das limitações encontradas nos métodos baseados na morfologia. Nesta tese, usamos DNA barcodes para investigar a biodiversidade de organismos macrobentónicos dos ecossistemas costerios portugueses e desenvolver uma abordagem de DNA metabarcoding de alto rendimento para as comunidades macrobetónicas. Para este propósito, a) optimizámos o desenho de primers de modo a aumentar o sucesso de amplificação de DNA barcodes, b) compilámos DNA barcodes para grupos e espécies dominantes e c) testámos o sucesso de primers em DNA metabarcoding de comunidades simuladas e, comparámos a eficiência de abordagens baseadas na morfologia com DNA metabarcoding de comunidades naturais de organismos macrobentónicos estuarinos. Foram utilizados DNA barcodes de modo a contribuir ao enriquecimento de uma biblioteca de referência global de espécies proeminentes das comunidades bentónicas estuarinas e marinas de Portugal, focada em crustáceos, anelídeos e moluscos. Esta tese contribuiu com DNA barcodes para morfoespécies macrobentónicas, entre as quais incluem-se 51 polychetas, 43 anfípodes e 34 gastrópodes. Devido aos grandes valores de distancias intraespecíficas, encontraram-se evidencias de espécies crípticas em 6 poliquetas, 7 anfípodes e 3 gastrópodes, correspondendo à possível detecção de um total de 30 espécies crípticas. Para avaliar a capacidade da HTS que tem de recuperar os DNA barcodes de todas as espécies macrobentónicas presentes em um extracto de DNA de uma amostra composta por toda a comunidade, foram criadas artificialmente 3 comunidades filogeneticamente diversas. Foi recuperado mais de 80 % das espécies das comunidades testadas. Posteriormente, foi comparada a eficiência de duas abordagens, uma baseada na identificação morfológica e outra baseada em metabarcoding, para determinar a composição de espécies macrobentónicas de 4 locais diferentes do estuário do Sado, Portugal. Os resultados indicam que a riqueza de espécies seria subestimada se usarmos apenas métodos morpfológicos, pois a abordagem de metabarcoding foi capaz de fornecer atribuições até o nível de espécie sem ambiguidade para um maior número de espécies, as quais são susceptíveis de escrutínio e verificação no futuro. Conclui-se que a abordagem de DNA metabarcoding pode realizar-se igualmente ou melhor do que os inventários baseados na morfologia, e que avaliação da biodiversidade de alto rendimento das comunidades macrobentónicas é possível através desta abordagem. No entanto, ainda é necessário um aperfeiçoamento para melhorar o seu desempenho e potencial, como por exemplo, melhorar as taxas de recuperação através de um optimização dos primers o testar combinações de primers alternativos. Finalmente, devido a que as bibliotecas de referencia ainda são incipientes para invertebrados marinhos, será decisivo que estas continuem a ser completadas para poder materializar plenamente o potencial do metabarcoding

Learning lessons and moving forward

Funding Information: This work was supported by the Directorate-General Environment of the European Commission (Grant Number 110661/2018/794607/SUB/ENV.C2 ). Fundação para a Ciência e a Tecnologia ( FCT , Portugal), through the strategic projects , and the project LA/P/0069/2020 granted to the Associate Laboratory ARNET - Aquatic Research Network. We are very grateful to all partners of the RAGES (Risk Based Approaches to Good Environmental Status; https://www.msfd.eu/rages/rages.html) consortium for discussions during the development of this work, particularly to Jean-Marc Brignon and Valentin Chapon for their contributions to discussions around the application of the ELECTRE II approach. We are also indebted to the RAGES partners as well as to invited experts that contributed to the NIS lists and to the HS exercise. Finally, we would like to thank two anonymous reviewers for their remarks, which improved the quality of this work. Publisher Copyright: © 2023 The AuthorsRisk-based Approaches (RBA) are increasingly playing an explicit and important role in a number of environmental regulations across Europe and globally. In this paper, we summarise a generic RBA developed for the Marine Strategy Framework Directive (MSFD) and its application to two descriptors of Good Environmental Status (GES) for marine waters, Non-Indigenous Species and Underwater Energy and Noise (Descriptors 2 and 11). We provide an overview of the findings and outcomes emerging from the application, which focus on identifying common advantages as well as common challenges encountered in the application of the RBA. Recommendations are then made, aimed at identifying potential solutions to the common problems, particularly in relation to data and expert-judgement approaches. Further recommendations address the development of governance structures to facilitate the uptake of risk-based approaches at the level of the MSFD common implementation strategy. Finally, some general and specific recommendations are made to effectively embed RBA and enhance regional cooperation for future implementation of the MSFD.publishersversionpublishe

Integrative Taxonomy Reveals That the Marine Brachyuran Crab Pyromaia tuberculata (Lockington, 1877) Reached Eastern Atlantic

Pyromaia tuberculata is native to the north-eastern Pacific Ocean and currently established in distant regions in the Pacific Ocean and southwest Atlantic. Outside its native range, this species has become established in organically polluted enclosed waters, such as bays. The Tagus estuary, with a broad shallow bay, is one of the largest estuaries in the west coast of Europe, located in western mainland Portugal, bordering the city of Lisbon. In this study, sediment samples were collected in the estuary between 2016 and 2017. Several adult specimens of P. tuberculata, including one ovigerous female, were morphologically and genetically identified, resulting in accurate identification of the species. The constant presence of adults over a 16-month sampling period suggests that the species has become established in the Tagus estuary. Moreover, their short life cycle, which allows for the production of at least two generations per year, with females reaching maturity within six months after settlement, favours population establishment. Despite being referred to as invasive, there are no records of adverse effects of P. tuberculata to the environment and socio-economy in regions outside its native range. However, due to its expanding ability, its inclusion in European monitoring programmes would indeed be desirable

On the Properties Evolution of Eco-Material Dedicated to Manufacturing Artificial Reef via 3D Printing: Long-Term Interactions of Cementitious Materials in the Marine Environment

his paper deals with the evolution monitoring of biomass colonization and mechanical properties of 3D printed eco-materials/mortars immersed in the sea. Measurements of tensile strength, compressive strength, and Young’s modulus were determined on samples deployed along the Atlantic coast of Europe, in France, United Kingdom, Spain, and Portugal. The samples were manufactured using 3D printing, where six mix designs with a low environmental impact binder were used. These mortars were based on geopolymer and cementitious binders (Cement CEM III), in which sand is replaced by three types of recycled sand, including glass, seashell, and limestone by 30%, 50%, and 100% respectively. The colonization of concrete samples by micro/macro-organisms and their durability were also evaluated after 1, 3, 6, 12, and 24 months of immersion. The results showed that both biomass colonization and mechanical properties were better with CEM III compared to geopolymer-based compositions. Therefore, the mixed design optimized according to mechanical properties show that the use of CEM III should be preferred over these geopolymer binders in 3D printed concrete for artificial reef application

On the Properties Evolution of Eco-Material Dedicated to Manufacturing Artificial Reef via 3D Printing: Long-Term Interactions of Cementitious Materials in the Marine Environment

ABSTRACT: This paper deals with the evolution monitoring of biomass colonization and mechanical properties of 3D printed eco-materials/mortars immersed in the sea. Measurements of tensile strength, compressive strength, and Young’s modulus were determined on samples deployed along the Atlantic coast of Europe, in France, United Kingdom, Spain, and Portugal. The samples were manufactured using 3D printing, where six mix designs with a low environmental impact binder were used. These mortars were based on geopolymer and cementitious binders (Cement CEM III), in which sand is replaced by three types of recycled sand, including glass, seashell, and limestone by 30%, 50%, and 100% respectively. The colonization of concrete samples by micro/macro-organisms and their durability were also evaluated after 1, 3, 6, 12, and 24 months of immersion. The results showed that both biomass colonization and mechanical properties were better with CEM III compared to geopolymer-based compositions. Therefore, the mixed design optimized according to mechanical properties show that the use of CEM III should be preferred over these geopolymer binders in 3D printed concrete for artificial reef applications.Funding was provided by Interreg Atlantic area through the project EAPA_174/2016- 3DPARE-Artificial Reef 3D Printing for Atlantic area granted to the Faculty of Sciences of the University of Porto; Bournemouth University; ESITC—École Supérieure d’Ingénieurs des Travaux de la Construction de Caen; University of Cantabria; and IPMA—Instituto Português do Mar e da Atmosfera. Other supports were provided by MARE (FCT/MCTES–UIDB/04292/2020), by CIIMAR (FCT/MCTES–UIDB/04423/2020 and UIDP/04423/2020), and by the project LA/P/0069/2020 granted to the Associate Laboratory ARNET

Prophylaxis of postoperative endophthalmitis following cataract surgery: Results of the ESCRS multicenter study and identification of risk factors

Purpose: To identify risk factors and describe the effects of antibiotic prophylaxis on the incidence of postoperative endophthalmitis after cataract surgery based on analysis of the findings of the European Society of Cataract & Refractive Surgeons (ESCRS) multicenter study. Setting: Twenty-four ophthalmology units in Austria, Belgium, Germany, Italy, Poland, Portugal, Spain, Turkey, and the United Kingdom. Methods: A prospective randomized partially masked multicenter cataract surgery study recruited 16 603 patients. The study was based on a 2 × 2 factorial design, with intracameral cefuroxime and topical perioperative levofloxacin factors resulting in 4 treatment groups. The comparison of case and non-case data was performed using multivariable logistic regression analyses. Odds ratios (ORs) associated with treatment effects and other risk factors were estimated. Results: Twenty-nine patients presented with endophthalmitis, of whom 20 were classified as having proven infective endophthalmitis. The absence of an intracameral cefuroxime prophylactic regimen at 1 mg in 0.1 mL normal saline was associated with a 4.92-fold increase (95% confidence interval [CI], 1.87-12.9) in the risk for total postoperative endophthalmitis. In addition, the use of clear corneal incisions (CCIs) compared to scleral tunnels was associated with a 5.88-fold increase (95% CI, 1.34-25.9) in risk and the use of silicone intraocular lens (IOL) optic material compared to acrylic with a 3.13-fold increase (95% CI, 1.47-6.67). The presence of surgical complications increased the risk for total endophthalmitis 4.95-fold (95% CI, 1.68-14.6), and more experienced surgeons were more likely to be associated with endophthalmitis cases. When considering only proven infective endophthalmitis cases, the absence of cefuroxime and the use of silicone IOL optic material were significantly associated with an increased risk, and there was evidence that men were more predisposed to infection (OR, 2.70; 95% CI, 1.07-6.8). Conclusions: Use of intracameral cefuroxime at the end of surgery reduced the occurrence of postoperative endophthalmitis. Additional risk factors associated with endophthalmitis after cataract surgery included CCIs and the use of silicone IOLs. © 2007 ASCRS and ESCRS