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This work assesses the effects of river regulation on the diversity of different instream and riparian biological communities along a relieve gradient of disturbance in regulated rivers. Two case studies in Portugal were used, with different river regulation typology (downstream of run-of-river and reservoir dams), where regulated and free-flowing river stretches were surveyed for riparian vegetation, macrophytes, bryophytes, macroalgae, diatoms and macroinvertebrates. The assessment of the regulation effects on biological communities was approached by both biological and functional diversity analysis. Results of this investigation endorse river regulation as a major factor differentiating fluvial biological communities through an artificial environmental filtering that governs species assemblages by accentuating species traits related to river regulation tolerance. Communities' response to regulation gradient seem to be similar and insensitive to river regulation typology. Biological communities respond to this regulation gradient with different sensibilities and rates of response, with riparian vegetation and macroinvertebrates being the most responsive to river regulation and its gradient. Richness appears to be the best indicator for general fluvial ecological quality facing river regulation. Nevertheless, there are high correlations between the biological and functional diversity indices of different biological groups, which denotes biological connections indicative of a cascade of effects leading to an indirect influence of river regulation even on non-responsive facets of communities' biological and functional diversities. These results highlight the necessary holistic perspective of the fluvial system when assessing the effects of river regulation and the proposal of restoration measures. published

Review The biological assessment of rivers i.e., their assessment through use of aquatic assemblages, integrates the effects of multiple-stressors on these systems over time and is essential to evaluate ecosystem condition and establish recovery measures. It has been undertaken in many countries since the 1990s, but not globally. And where national or multi-national monitoring networks have gathered large amounts of data, the poor water body classifications have not necessarily resulted in the rehabilitation of rivers. Thus, here we aimed to identify major gaps in the biological assessment and rehabilitation of rivers worldwide by focusing on the best examples in Asia, Europe, Oceania, and North, Central, and South America. Our study showed that it is not possible so far to draw a world map of the ecological quality of rivers. Biological assessment of rivers and streams is only implemented officially nation-wide and regularly in the European Union, Japan, Republic of Korea, South Africa, and the USA. In Australia, Canada, China, New Zealand, and Singapore it has been implemented officially at the state/province level (in some cases using common protocols) or in major catchments or even only once at the national level to define reference conditions (Australia). In other cases, biological monitoring is driven by a specific problem, impact assessments, water licenses, or the need to rehabilitate a river or a river section (as in Brazil, South Korea, China, Canada, Japan, Australia). In some countries monitoring programs have only been explored by research teams mostly at the catchment or local level (e.g., Brazil, Mexico, Chile, China, India, Malaysia, Thailand, Vietnam) or implemented by citizen science groups (e.g., Southern Africa, Gambia, East Africa, Australia, Brazil, Canada). The existing large-extent assessments show a striking loss of biodiversity in the last 2–3 decades in Japanese and New Zealand rivers (e.g., 42% and 70% of fish species threatened or endangered, respectively). A poor condition (below Good condition) exists in 25% of South Korean rivers, half of the European water bodies, and 44% of USA rivers, while in Australia 30% of the reaches sampled were significantly impaired in 2006. Regarding river rehabilitation, the greatest implementation has occurred in North America, Australia, Northern Europe, Japan, Singapore, and the Republic of Korea. Most rehabilitation measures have been related to improving water quality and river connectivity for fish or the improvement of riparian vegetation. The limited extent of most rehabilitation measures (i.e., not considering the entire catchment) often constrains the improvement of biological condition. Yet, many rehabilitation projects also lack pre-and/or post-monitoring of ecological condition, which prevents assessing the success and shortcomings of the recovery measures. Economic constraints are the most cited limitation for implementing monitoring programs and rehabilitation actions, followed by technical limitations, limited knowledge of the fauna and flora and their life-history traits (especially in Africa, South America and Mexico), and poor awareness by decision-makers. On the other hand, citizen involvement is recognized as key to the success and sustainability of rehabilitation projects. Thus, establishing rehabilitation needs, defining clear goals, tracking progress towards achieving them, and involving local populations and stakeholders are key recommendations for rehabilitation projects (Table 1). Large-extent and long-term monitoring programs are also essential to provide a realistic overview of the condition of rivers worldwide. Soon, the use of DNA biological samples and eDNA to investigate aquatic diversity could contribute to reducing costs and thus increase monitoring efforts and a more complete assessment of biodiversity. Finally, we propose developing transcontinental teams to elaborate and improve technical guidelines for implementing biological monitoring programs and river rehabilitation and establishing common financial and technical frameworks for managing international catchments. We also recommend providing such expert teams through the United Nations Environment Program to aid the extension of biomonitoring, bioassessment, and river rehabilitation knowledge globally info:eu-repo/semantics/publishedVersion

The European Water Framework Directive (2000/60/EC) includes biological assessment of water bodies that has been implemented for many years. Indicator organisms such as diatoms respond to geological and hydrological features of rivers by modifying their structure. Therefore, when implementing the WFD, it was necessary to establish type-specific reference conditions to be able to measure the deviations of sampled communities due to anthropogenic impact.HTS-related eDNA metabarcoding has been developed to complement or even replace traditional approaches for its rapid, low-cost and highly accurate identification of communities for assessment of rivers’ ecological status (e.g. Mortágua et al., 2019; Pérez-Burillo et al. 2020) and proved to provide even more in-depth information about biological elements. The use of this information without assignment to species is being addressed once it eliminates the limiting factor of the reference database incompleteness and may provide new ecological information (e.g. Feio et al., 2020; Rivera et al., 2020). Since WFD requires the establishment of reference conditions for each water body type, for eDNA methods’ implementation it will be essential to review, confirm or reformulate, and perhaps create new typologies. Hereupon, the aim of this study is to analyze diatom communities from different typologies of Portuguese rivers resulting from DNA metabarcoding data and compare it with current typology system. To do so, we will verify the consistency of biological groups included in each type, validate the molecular data, analyze the correspondence of OTU/ISU/ESV to environmental characteristics of rivers. A total of 154 sampling sites were selected from central Portugal and northern Portugal in 2017 and 2019. The biofilm was collected for morphological identification and DNA sequencing of diatoms. Reference sites were selected for 4 river types (mountain, littoral, small and medium-large northern rivers) based on a set of pressure information (water quality, hydromorphology, land use and riparian zones). Diatom inventories were obtained from molecular and morphological analysis. DNA sequences were treated using Mothur software which processed two bioinformatic strategies in order to obtain the final ISU and OTU tables, while ESVs were treated with DADA2 package from R. Identification and counting of diatom valves took place under the light microscope concerning the morphological approach. We expect results to validate the molecular data for each typology either when assigning to species or not, and to understand whether it is necessary to establish new typologies for future use of the molecular approach in ecological assessment of rivers. Directive, W. F. (2000). Water Framework Directive. Journal reference OJL, 327, 1-73. Feio, M. J., Serra, S. R., Mortágua, A., Bouchez, A., Rimet, F., Vasselon, V., & Almeida, S. F. P. (2020). A taxonomy-free approach based on machine learning to assess the quality of rivers with diatoms. Science of the Total Environment, 722, 137900. https://doi.org/10.1016/j.scitotenv.2020.137900 Mortágua, A., Vasselon, V., Oliveira, R., Elias, C., Chardon, C., Bouchez, A., ... & Almeida, S. F. P. (2019). Applicability of DNA metabarcoding approach in the bioassessment of Portuguese rivers using diatoms. Ecological indicators, 106, 105470. https://doi.org/10.1016/j.ecolind.2019.105470 Pérez-Burillo, J., Trobajo, R., Vasselon, V., Rimet, F., Bouchez, A., & Mann, D. G. (2020). Evaluation and sensitivity analysis of diatom DNA metabarcoding for WFD bioassessment of Mediterranean rivers. Science of the Total Environment, 727, 138445. https://doi.org/10.1016/j.scitotenv.2020.138445 Rivera, S. F., Vasselon, V., Bouchez, A., & Rimet, F. (2020). Diatom metabarcoding applied to large scale monitoring networks: Optimization of bioinformatics strategies using Mothur software. Ecological indicators, 109, 105775. https://doi.org/10.1016/j.ecolind.2019.105775

International audience; Organic contaminants, and herbicides in particular, represent a risk for aquatic ecosystems. The primary target of herbicides are producers, the base of food webs, but frequently they end up far from the application point affecting non-target species. Its presence can work as sub-lethal stimulus, which sort the genetic and phenotypic differences within a species. Intraspecific variation allows adaptation to changes in the environment but also to new niches due to variations in species' sensitivity and biochemical response to a certain chemical. A better understanding of these variations can lead to the development of improved strategies for ecosystem protection. This research aimed to compare a sensitive and a tolerant strain of the freshwater diatom Nitzschia palea to atrazine. Strains were exposed to three concentrations within their tolerance range, during 96 h. The activity of the antioxidant enzymes superoxide dismutase, catalase, glutathione-S-transferases and glutathione peroxidases was determined. In addition, chlorophylls a and c, carotenoids, reduced glutathione, proteins and lipid peroxidation were quantified. Both strains displayed different strategies to deal with atrazine toxicity: while the sensitive strain decreased the oxidative stress, increasing the activity of antioxidant enzymes such as superoxide dismutase, the tolerant strain invested in conjugation pathways and carotenoids' maintenance.

Epub 2017 Jul 06; Diatoms are used as indicators of freshwater ecosystems integrity. Developing diatom-based tools to assess impact of herbicide pollution is expected by water managers. But, defining sensitivities of all species to multiple herbicides would be unattainable. The existence of a phylogenetic signal of herbicide sensitivity was shown among diatoms and should enable prediction of new species sensitivity. However, diatoms present a cryptic diversity that may lead to variation in their sensitivity to herbicides that would need to be taken into account. Using bioassays, the sensitivity to four herbicides (Atrazine, Terbutryn, Diuron, Isoproturon) was evaluated for 11 freshwater diatom taxa and intraspecific variability was assessed for two of them (Nitzschia palea and Achnanthidium spp.). Intraspecific variability of herbicide sensitivity was always smaller than interspecific variability, but intraspecific variability was more important in N. palea than in Achnanthidium spp. Indeed, one species showed no intraspecific phylogenetic signal (N. palea) whereas the other did (Achnanthidium spp.). On one hand, species boundaries are not set properly for Achnanthidium spp. which encompass several taxa. On the other hand, there is a higher phenotypic plasticity for N. palea. Finally, a phylogenetic signal of herbicide sensitivity was measured at the interspecific level, opening up prospects for setting up reliable biomonitoring tools based on sensitivity prediction, insofar as species boundaries are correctly defined.

Aljustrel mines were classified as having high environmental hazard due to their large tailings volume and high metal concentrations in waters and sediments. To assess acid mine drainage impacted systems whose environmental conditions change quickly, the use of biological indicators with short generation time such as diatoms is advantageous. This study combined geochemical and diatom data, whose results were highlighted in 3 groups: Group 1, with low pH (1.9-5.1) and high metal/metalloid (Al, As, Cd, Co, Cu, Fe, Mn, Ni, Pb, Zn; 0.65-1032 mg/L) and SO4 (405-39124 mg/L) concentrations. An acidophilic species, Pinnularia aljustrelica, was perfectly adapted to the adverse conditions; in contrast, teratological forms of Eunotia exigua were found, showing that metal toxicity affected this species. The low availability of metals/metalloids in sediments of this group indicates that metals/metalloids of the exchangeable fractions had been solubilized, which in fact enables metal/metalloid diatom uptake and consequently the occurrence of teratologies; Group 2, with sites of near neutral pH (5.0-6.8) and intermediate metal/metalloid (0.002-6 mg/L) and SO4 (302-2179 mg/L) concentrations; this enabled the existence of typical species of uncontaminated streams (Brachysira neglectissima, Achnanthidium minutissimum); Group 3, with samples from unimpacted sites, showing low metal/metalloid (0-0.8 mg/L) and SO4 (10-315 mg/L) concentrations, high pH (7.0-8.4) and Cl contents (10-2119 mg/L) and the presence of brackish to marine species (Entomoneis paludosa). For similar conditions of acidity, differences in diversity, abundance and teratologies of diatoms can be explained by the levels of metals/metalloids. (C) 2015 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

Investigations on organism ecology, biodiversity and biogeography often use large compiled datasets to extract information on species ecological preferences, which then can be used in environmental assessment. Freshwater benthic diatoms are commonly used in this context. However, it is important that the taxonomic information of the separate diatom datasets is compatible. At present, inconsistencies between diatom datasets, mainly due to differences and uncertainties in diatom identification, may misinform diatom taxon-specific ecological preferences, geographical distribution and water quality assessment. It is our opinion that these inconsistencies in diatom datasets can be reduced with quality assurance (QA), such as identification exercises. However, the results of these exercises must be well documented and well communicated; otherwise, gained knowledge may not spread inter-regionally or internationally. As a first step to reach greater consistency in QA/harmonization studies, this article (1) presents and compares information of existing diatom identification and counting QA from published and grey (non-peer reviewed) European literature to identify advantages and drawbacks of each approach; (2) summarizes taxa that can easily be misidentified according to European identification exercises; and (3) suggests a consistent design of identification exercises for diatom dataset QA.

International audience; The EuropeanWater Framework Directive establishes a framework for the protection ofwater resources. However, common water management tools demand common understanding of assessment methods, so quality goals are equallymet. Intercalibration ofmethods ensures the comparability of biological elements across similar geographical areas. Many aspects can influence the outcome of intercalibration: data sampling, treatment methods, taxonomic reliability of databases, choice of metrics for ecological quality status classification, and criteria for selecting reference sites. This study describes the potentials and constraints of the intercalibration of indices using diatoms for assessment of Mediterranean rivers. Harmonisation of diatom taxonomy and nomenclature was based on a previous ring test which took place at the European level. Four diatom indices (Indice de Polluosensibilité Spécifique-IPS, Indice Biologique Diatomées-IBD 2007, Intercalibration Common Metric Italy-CMi and Slovenian Ecological Status assessment system) were intercalibrated using data from six European Mediterranean countries (Cyprus, France, Italy, Portugal, Slovenia and Spain). Boundaries between High/Good and Good/Moderate quality classes were harmonised by means of the Intercalibration Common Metric (ICM). Comparability between countries was assured through boundary bias and class agreement. The national boundaries were adjusted when they deviated more than a quarter of a class equivalent (0.25) from the globalmean. All national methods correlated well with the ICM, which was sensitive to water quality (negatively correlated to nutrients). Achnanthidium minutissimum sensu lato was the most discriminative species of Good ecological status class. Planothidium frequentissimum, Gomphonema parvulum and Nitzschia paleawere themost contributive toModerate ecological status class.Some taxa were discriminative for bothGood andModerate ecological status classes due to low indication and ecological discriminative power but also due to differences in taxonomy between countries. This intercalibration exercise allowed establishment of common water quality goals across Mediterranean Europe, which is substantiated with the ICM.

An indoor channel system was colonised with fluvial biofilms to study the chronic effects of high Fe and SO4 2- concentrations and acidic pH, the water chemistry in the surrounding streams of Aljustrel mining area (Alentejo, Portugal), and their contribution to community (in)tolerance to metal toxicity by short-term experiments with Cu and Zn. Biofilms were subjected to four different treatments during 8 weeks: high Fe and SO4 2- concentrations (1 mg Fe l-1+ 700 mg SO 4 2- l-1) and acidic pH, high Fe and SO4 2- at alkaline pH; lower Fe and SO4 2- at acidic pH: and lower Fe and SO4 2- concentrations at alkaline pH as negative control. During chronic exposure, acidic pH affected growth negatively, based on low values of algal biomass and the autotrophic index, high values of the antioxidant enzyme activities and low diversity diatom communities, dominated by acidophilic species (Pinnularia aljustrelica) in acidic treatments, being the effects more marked with high Fe and SO4 2-. Co-tolerance to metals (Cu and Zn) was also shown in biofilms from the acidic treatments, contrasting with the higher sensitivity observed in the alkaline treatments. We can conclude that the Aljustrel mining area acidic environment limits algal growth and exerts a strong selection pressure on the community composition which is in turn, more tolerant to metal exposure The authors are grateful to the Biology and Geosciences Departments of the University of Aveiro, Portugal, to the Fundaçao para a Ciência e Tecnologia, Portugal (grant number SFRH/BD/36137/2007) and to the University of Girona for their financial support by the project FLUVIALMULTISTRESS funded by the Spanish ‘‘Ministerio de Economı´a y competitividad’’ ref: CTM2009-14111-CO2-01

International audience; The European Union (EU) Water Framework Directive depends, for effective implementation, on Member States (MSs) agreeing to a concept of the unimpacted “reference” state, which will then provide the “expected” value in Ecological Quality Ratio (EQR) calculations. Reference assemblages of organism groups will, in turn, vary, due to geological, hydrological, climatic, physicochemical and biological factors. Member States tackle this by establishing “types” which share common characteristics. However, for the purposes of ensuring consistent application, broad transboundary types were also established within five Geographical Intercalibration Groups (GIGs, referred to here as “regions”) as part of the EU’s intercalibration exercise. In this paper, we evaluate these types using river diatom assemblages and also provide reference threshold values for two common metrics used in pan-European diatom assessments. A database was assembled, representing 14 EU Member States from Ireland and Portugal in the West, to Estonia and Cyprus in the East, in order to explore biogeographical patterns in assemblages unaffected by anthropogenic pressures. Multivariate analyses were used to examine this pattern and its relationship with geographic, typological and abiotic parameters. After taxonomic harmonisation, NMDS ordination of samples indicated weak differences in assemblage composition among regions. ANOSIM analyses, in turn, indicated that MS was the best factor to group similar samples whereas alkalinity, recognised as the primary environmental variable structuring diatom communities, although significant, explained less variability in the dataset. This, we believe, reflects the importance of methodological factors other than taxonomy (e.g. counting protocol, sample season) that may be constant within a MS but which vary between MSs. When two diatom metrics, the TI and IPS, were applied to the data, differences in the distribution of the metric scores between MS were generally not statistically significant even though some differences between regions were apparent. A trend of increasing values of TI (decreasing values of IPS) was observed in the sequence: Nordic < Alpine < Mediterranean < Central-Baltic < Eastern Continental regions. Additionally, some differences were observed among types within the Mediterranean and Nordic regions, though not for other regions. The data used in this exercise provides us with a region and, in some cases, a type specific benchmark dataset against which national reference data can be compared.

International audience; In 2009, seventeen analysts participated in a pan-European diatom ring-test (intercalibration), analyzing nine samples from seven countries following the European standard EN 14407. The objective of this exercise was to agree on practical conventions on diatom identification to facilitate future intercalibration work and to assess the extent to which national differences in sample analysis (counting protocol and identification conventions) contribute to variability in EU-level comparisons of diatom-based methods. Differences in the reported taxa lists were large, but not a major source of variation in values of a common metric (the phytobenthos Intercalibration Common Metric, ICM). Therefore, every country can apply its own identification conventions for national assessments, and still be fairly confident that the ICM reflects the national classification of its streams. Part of the index variation was due to differences in counting protocols and care should be taken when handling broken valves, girdle views and small taxa. More work at both national and European level is needed to provide a harmonized way of using diatoms for ecological status assessments in the future.

Diatoms are widely used in stream bioassessment due to their broad distribution, extraordinary variability and the ability to integrate changes in water quality. The indices Specific Polluosensitivity Index (SPI), standardized Biological Diatom Index (BDI), European Economic Community Index (CEC) and Generic Diatom Index (GDI), originally developed in France, are often applied in Portugal to evaluate stream ecological quality based on diatom communities. Alternatively, predictive models resulting from the comparison between the communities of the study site and those of a set of reference sites representing undisturbed or the best available conditions of a given region have been proposed as valuable methods for evaluating the ecological status of streams. In the present study, we applied the four above-mentioned widely used diatom-based indices (SPI, BDI, CEC and GDI) and a predictive model (MoDi) to 54 sites located in central Portugal to assess the sensitivity of the five methods to a range of anthropogenic disturbances cumulatively affecting streams and represented by 27 variables (e.g., organic enrichment, changes in morphology of the channels, integrity of the riparian corridor, land use in the catchment). The results were analyzed comparatively through Spearman correlations, Boxplots and Stepwise Discriminant Analysis. This study confirmed the sensitivity of diatoms to organic and nutrient contamination (showed by the MoDi, BDI, CEC and SPI) and revealed the importance of suspended solids (through the MoDi, GDI, and SPI). The relevance of modifications in land use to diatoms was shown by all methods applied, except for the GDI. The MoDI also revealed the importance of changes in the structure and morphology of the reach and the channel, like the construction of artificial walls or embankments and connectivity; the BDI also related its assessments with the riparian zone integrity; and the SPI was not useful in detecting morphological pressures. The GDI produced the most divergent assessments and was less effective in revealing the anthropogenic disturbances. The use of the predictive model (MoDi) is therefore a good method for the assessment of streams in central Portugal because it expresses a great diversity of quantitative and qualitative changes in freshwater systems reflected in the structure (species richness and abundance) of diatom communities.