Portuguese Freshwaters Research Articles

Statistical Inferences Applying Non-Parametric Data on Cyanobacterial Investigations: Contributions to Water Quality and New Trends under Global Changes on Portuguese Freshwater Ecosystems.

Cyanobacteria are a bloom-forming ancient group of photosynthetic prokaryotes. A rise in temperature is a major contributor to its massive proliferation, namely on freshwater ecosystems, with social and economic impacts. Thus, reliable and cost-effective tools can permit the fast surveillance and assessment of temperature effects on potentially toxic cyanobacteria distribution and impacts. The occurrence of three potentially toxic cyanobacteria species was assessed on seven sampling points across three sampling years. Moreover, the association between the occurrence of those cyanobacteria species with climate change events was addressed. Here, we combined molecular and statistical methods to study the impacts of temperature on the occurrence of three globally occurring cyanotoxin-producing cyanobacteria species—Microcystis aeruginosa (microcystins), Raphidiopsis raciborskii (cylindrospermopsins and saxitoxins) and Planktothrix agardhii (microcystins and saxitoxins). Samples were collected on seven European temperate freshwater systems located on the North and Centre regions of Portugal, across three distinct sampling years with distinct ranges of air temperature. Data support that M. aeruginosa is still a common inhabitant of Portuguese freshwater ecosystems and a new trend was found on R. raciborskii recent invasion and establishment on the colder north ecosystems of Portugal. Additionally, the highest frequency of detection of both cyanobacteria was associated with warmer years. P. agardhii also revealed a new trend, being reported for the first time on North and Centre Regions of Portugal, however with no statistical relation with air temperature, demonstrating a higher ecological fitness. Distinct profiles of the statistical analysis on the three tested cyanobacteria species contribute to deepen the studies on other species as well as of our analyzed species on a global level. This assessment may help to anticipate possible repercussions on water quality and public health due to most probable alterations on cyanotoxins profile given the ecological fitness established among air temperature and PCR detection of potentially toxic cyanobacteria.

Review on Cyanobacterial Studies in Portugal: Current Impacts and Research Needs

Cyanobacteria have long been associated with harmful effects on humans, animals and aquatic biota. Cyanotoxins are their most toxic metabolite. This review summarizes the current research, impacts and future needs in cyanobacterial studies undertaken in Portugal, the southernmost country of Europe, and with a recent multiplication of cyanotoxicity due to climate change events. Microcystins are still the most prevalent, studied and the only regulated cyanotoxins in Portuguese freshwater systems much like most European countries. With the development of some tools, particularly in molecular studies, the recent discovery of cylindrospermopsins, anatoxins and saxitoxins, both genes and toxins, in North and Center ecosystems of our country highlight current impacts that overall communities are facing with increased risks of exposure and uptake to cyanotoxins. Research needs encompass the expansion of studies at all aspects due to the uprising of these cyanotoxins and reinforces the urgent need of increasing the frequency of surveillance to achieve tangible effects of cyanotoxins in Portugal to ultimately implement regulations on cylindrospermopsins, anatoxins and saxitoxins worldwide.

Evaluating the range expansion of recreational non-native fishes in Portuguese freshwaters using scientific and citizen science data

Evaluating the range expansion of recreational non-native fishes in Portuguese freshwaters using scientific and citizen science data

Sequential recovery of C-phycocyanin and chlorophylls from Anabaena cylindrica

Cyanobacteria are attracting the attention worldwide as a reliable and sustainable feedstock for the production of biofuels, food colorants and biochemically active compounds. Anabaena cylindrica (A. cylindrica) is a cyanobacterium with a significant widespread occurrence in Portuguese freshwaters, with C-phycocyanin and chlorophylls, compounds with a high market value, being abundantly produced by the species. This work presents the development of a sequential downstream process to obtain C-phycocyanin and chlorophyll from A. cylindrica. The C-phycocyanin extraction is carried using Na-phosphate (20 mM, pH 7) buffer, while aqueous solutions of surface-active compounds (250 mM), as well as ethanol, were screened for the extraction of the chlorophylls. After selecting the best solvents to recover both pigments from A. cylindrica, the operational conditions (solid-liquid ratio, time of extraction and temperature) were optimized, allowing the recovery of around 90% and 55% of the total content of C-phycocyanin and chlorophylls, respectively. Finally, dextran/copolymer-based ABS were applied to promote the purification of C-phycocyanin from the residual chlorophylls and other contaminant proteins, leading to an increase in the C-phycocyanin purity of 4-fold, without compromising its photostability.

Diet of European Catfish in a Newly Invaded Region

Biological invasions are considered to be one of the leading causes of biodiversity loss worldwide. Freshwater ecosystems are amongst the most biodiverse, containing about 50% of the world’s known fish species but, ironically, are the most invaded ecosystems. In Portuguese freshwaters, there are currently 20 identified non-native fishes, arriving at an unprecedented rate of a new species every two years. A recent non-native is the European catfish Silurus glanis, a top predator native to Central and Eastern Europe, that arrived at the Lower Tagus (Portugal) circa 2006. This study compares the dietary habits of this top predator in two different habitats of the Lower Tagus in order to evaluate its potential impact. The stomach contents of 96 individuals were analyzed, 61 from the lotic environment and 35 from the lentic systems. A total of 66 stomachs contained prey items, 38 from the lotic and 28 from the lentic. Diet varied considerably between habitats, with shrimp and crayfish being the most abundant prey items in the lentic systems and shrimp and fish the most abundant prey items in the lotic section. Tendency to piscivory was observed in large-sized individuals. Overall, the findings in this study provide the first evidence of the potential impacts of European catfish through predation on the fish communities.

Risk Levels of Toxic Cyanobacteria in Portuguese Recreational Freshwaters.

Portuguese freshwater reservoirs are important socio-economic resources, namely for recreational use. National legislation concerning bathing waters does not include mandatory levels or guidelines for cyanobacteria and cyanotoxins. This is an issue of concern since cyanotoxin-based evidence is insufficient to change the law, and the collection of scientific evidence has been hampered by the lack of regulatory levels for cyanotoxins in bathing waters. In this work, we evaluate the profile of cyanobacteria and microcystins (MC) in eight freshwater reservoirs from the center of Portugal, used for bathing/recreation, in order to determine the risk levels concerning toxic cyanobacteria occurrence. Three of the reservoirs did not pose a risk of MC contamination. However, two reservoirs presented a high risk in 7% of the samples according to the World Health Organization (WHO) guidelines for MC in bathing waters (above 20 µg/L). In the remaining three reservoirs, the risk concerning microcystins occurrence was low. However, they exhibited recurrent blooms and persistent contamination with MC up to 4 µg/L. Thus, the risk of exposure to MC and potential acute and/or chronic health outcomes should not be disregarded in these reservoirs. These results contribute to characterize the cyanobacterial blooms profile and to map the risk of toxic cyanobacteria and microcystins occurrence in Portuguese inland waters.

Low Genetic Diversity and High Invasion Success of Corbicula fluminea (Bivalvia, Corbiculidae) (Müller, 1774) in Portugal.

The Asian clam, Corbicula fluminea, is an invasive alien species (IAS) originally from Asia that has spread worldwide causing major ecological and economic impacts in aquatic ecosystems. Here, we evaluated C. fluminea genetic (using COI mtDNA, CYTb mtDNA and 18S rDNA gene markers), morphometric and sperm morphology variation in Portuguese freshwater ecosystems. The COI marker revealed a single haplotype, which belongs to the Asian FW5 invasive lineage, suggesting a common origin for all the 13 Portuguese C. fluminea populations analysed. Morphometric analyses showed differences between the populations colonizing the North (with the exception of the Lima River) and the Centre/South ecosystems. The sperm morphology examination revealed the presence of biflagellate sperm, a distinctive character of the invasive androgenetic lineages. The low genetic variability of the Portuguese C. fluminea populations and the pattern of sperm morphology have been illuminating for understanding the demographic history of this invasive species. We hypothesize that these populations were derived from a unique introductory event of a Corbicula fluminea FW5 invasive androgenic lineage in the Tejo River, which subsequently dispersed to other Portuguese freshwater ecosystems. The C. fluminea asexual reproductive mode may have assisted these populations to become highly invasive despite the low genetic diversity.

Genetic characterization of Microcystis aeruginosa isolates from Portuguese freshwater systems.

Cyanobacteria are microorganisms that pose a serious threat to the aquatic waterways through the production of dense blooms under eutrophic conditions and the release of toxic secondary metabolites-cyanotoxins. Within cyanobacteria, the colonial planktonic Microcystis aeruginosa is widely distributed in both fresh and brackish aquatic environments throughout the world being frequently observed in the Portuguese water systems. Apart from the well-established distribution of M. aeruginosa in Portugal, knowledge of its genetic diversity and population structure is unknown. Therefore, in this study twenty-seven strains were obtained from the North, Centre and South regions of Portugal and were subjected to extensive phylogenetic analyses using simultaneously four distinct genetic markers (16S rRNA, 16S-23S ITS, DNA gyrase subunit ß and cell division protein (ftsZ)) encompassing in total 2834bp. With this work we characterized the phylogenetic relationship among the Portuguese strains, with the southern strains showing higher genetic structure relatively to the North and Centre strains. A total of fifteen genotypes were determined for M. aeruginosa in Portuguese water systems revealing a high genetic diversity. This is also the first study to report geographic variation on the population structure of the Portuguese M. aeruginosa.

Are insect repellents toxic to freshwater insects? A case study using caddisflies exposed to DEET

Stream ecosystems face ever-increasing pressures by the presence of emergent contaminants, such as, personal care products. N, N-diethyl-3-methylbenzamide (DEET) is a synthetic insect repellent that is being found in surface waters environments in concentrations up to 33.4 μg/L. Information concerning DEET's toxicity in the aquatic environment is still limited and focused only on its acute effects on model species. Our main objective was to assess the effects of DEET exposure to a caddisfly non-target species using sub-lethal endpoints. For that, we chose Sericostoma vittatum, an important shredder in Portuguese freshwaters that has been already used in different ecotoxicological assays. Besides acute tests, S. vittatum were exposed during 6 days to a gradient of DEET concentrations (8, 18 and 40.5 mg/L) to assess effects on feeding behaviour and biochemical responses, such as, lipid peroxidation levels (LPO), catalase and acetylcholinesterase (AChE) activities, and also assess effects on energy reserves and consumption. Acute tests revealed a 48 h-LC50 of 80.12 mg/L and DEET exposure caused feeding inhibition with a LOEC of 36.80 mg/L. Concerning the biochemical responses, DEET caused no effects in LPO nor on catalase activity. A non-significant decrease in AChE activity was observed. Regarding energetic reserves, exposure to DEET caused a significant reduction in S. vittatum carbohydrates levels. These results add important information for the risk assessment of insect repellents in the aquatic environment and suggest that reported environmental concentrations of DEET are not toxic to non-target freshwater insects.

The impact of some microcystins on the growth of heterotrophic bacteria from Portuguese freshwater reservoirs

The impact of some microcystins on the growth of heterotrophic bacteria from Portuguese freshwater reservoirs Microcystins (MCs) are hepatotoxins that are abundantly produced by cyanobacteria. Studies have shown that these toxins affect many multicellular organisms that inhabit aquatic ecosystems; however, their impact on bacteria that co-occur with freshwater cyanobacteria is still unclear. In this study, the impact of the three most common variants of MCs (MCLR, MCRR, and MCYR) on the growth of heterotrophic bacteria isolated from four Portuguese reservoirs was evaluated. Some isolates were derived from freshwater sources where blooms of cyanobacteria that often contain microcystin-producing strains are frequently observed and from a reservoir where these phenomena do not occur. Morphological and molecular characterisation of the bacterial isolates was performed, and these bacteria were exposed to three different concentrations of each MC variant. The effect of MCs on bacterial growth was then evaluated. This study showed that MCLR, MCRR and MCYR can reduce the growth of some heterotrophic bacteria isolated from freshwater sources. To our knowledge, this is the first study in which the impact of several variants of MCs was evaluated in a diverse group of freshwater heterotrophic bacteria.

Craspedacusta sowerbii Lankester, 1880 in southern Portugal

Craspedacusta sowerbii Lankester, 1880 is reported for the first time from one of the largest Portuguese hydrographic basins. Three specimens collected in October 2012 represent the third record from Portugal and the first on the Sado hydrographic basin (south Portugal). We provide evidence on the range extension of C. sowerbii within Portuguese freshwater habitats, and outline the current lack of knowledge on the distribution in the southwest Iberian Peninsula.

Morphological, biochemical and molecular characterization of Anabaena, Aphanizomenon and Nostoc strains (Cyanobacteria, Nostocales) isolated from Portuguese freshwater habitats

Studies of cyanobacterial nostocacean taxa are important to the global scientific community, mainly because a significant number of beneficial strains that belong to the order Nostocales fix atmospheric nitrogen, thus contributing to the fertility of agricultural soils worldwide, while others behave as nuisance microorganisms in aquatic ecosystems due to their involvement in toxic bloom events. However, in spite of their ecological importance and environmental concerns, their identification and taxonomy are still problematic and doubtful, often being based on current morphological and physiological studies, which generate confusing classification systems and usually vary under different conditions. Therefore, the present research aimed to investigate through a polyphasic approach differences in morphological, biochemical and genotypic features of three nostocacean cyanobacterial strains isolated from central-western Portuguese shallow freshwater bodies. Morphometric, genetic (16S rRNA, nifH and hetR fragments) and biochemical (fatty acid methyl ester; FAME profiles) data were used to characterize the strains. Morphological analysis and sequencing of 16S rRNA fragments showed that the strains belonged to Anabaena cylindrica (UTAD_A212), Aphanizomenon gracile (UADFA16) and Nostoc muscorum (UTAD_N213) species. These strains showed clear distinct morphological and genetic features, allowing easy allocation to their respective genera. The same happened by using partial sequences of hetR and nifH genes, in spite of the scarcity of deposited sequences. Biochemical characterization showed that the FAME profiles obtained were consistent with both morphological and molecular analyses. It was suggested that the ratio of monounsaturated to polyunsaturated FAMEs, together with the unsaturation index, could be used as genus-specific chemotaxonomic biomarkers.

Production of anatoxin-a by cyanobacterial strains isolated from Portuguese fresh water systems

The occurrence of anatoxin-a in several freshwater systems in Portugal and its production by Portuguese cyanobacterial strains, after cultivation in laboratory, were studied. Surface water samples from 9 water bodies, for recreational and human consumption usage, were surveyed for anatoxin-a presence and for obtaining cultures of pure cyanobacterial strains. Anatoxin-a analysis was performed by high performance liquid chromatography (HPLC) with fluorescence detection (FLD) followed by Mass Spectrometry (MS) confirmation. No anatoxin-a was detected in all the natural water samples (limit of detection (LOD) = 25 ng l(-1)) but among the 22 isolated cyanobacterial strains, 13 could produce anatoxin-a in laboratory conditions (LOD = 3 ng g(-1) dw). This proportion of anatoxin-a producing strains (59.1%) in laboratory is discussed considering the hypothesis that anatoxin-a is a more frequent metabolite in cyanobacteria than it was thought before and making its occurrence in Portuguese freshwaters almost certain. Therefore, health and ecological risks caused by anatoxin-a in Portugal, should be seriously considered.

Molecular identification, typing and traceability of cyanobacteria from freshwater reservoirs

In order to assess the potential of several molecular targets for the identification, typing and traceability of cyanobacteria in freshwater reservoirs, molecular techniques were applied to 118 cyanobacterial isolates mostly sourced from Portuguese freshwater reservoirs and representative of three orders of cyanobacteria: Chroococcales (54), Oscillatoriales (15) and Nostocales (49). The isolates were previously identified by morphological methods and subsequently characterized by composite hierarchical cluster analysis of STRR and LTRR (short and long tandemly repeated repetitive sequences) PCR fingerprinting profiles. Representative isolates were selected from each cluster and their molecular identification, at the species level, was obtained or confirmed by phylogenetic positioning using 16S rRNA gene and rpoC1 phylogenies. A highly congruent association was observed between STTR- and LTRR-based clusters and taxonomic affiliation, revealing the usefulness of such PCR fingerprinting profiles for the identification of cyanobacteria. Composite analysis of hierarchical clustering of M13 and ERIC PCR fingerprints also appeared suitable for strain typing and traceability within a reservoir, indicating its potential for use in cyanobacterial monitoring, as a quality management control. Based on Simpson (D) and Shannon-Wiener (J') indices a high diversity was observed within all species, with Planktothrix agardhii showing the lowest diversity values (D=0.83; J'=0.88) and Aphanizomenon flos-aquae the highest ones (D=J'=0.99). A diagnostic key based on 16S-ARDRA, ITS amplification and ITS-ARDRA for identification purposes is also presented.

Detection of Planktothrix rubescens (Cyanobacteria) associated with microcystin production in a freshwater reservoir

So far, the presence of microcystins in Portuguese freshwater resources has always been attributed to the bloom-forming cyanobacteria Microcystis aeruginosa. In 2005, however, microcystins were detected at the Beliche reservoir (Algarve, South Portugal), following the development of a bloom dominated by Planktothrix rubescens. The identity of the causative organism was confirmed by combining both morphological and 16S rRNA gene sequence analysis. Its ability to produce microcystins was confirmed by HPLC and MALDI-TOF MS. Unlike M. aeruginosa that usually accumulates near the water surface, P. rubescens found at the Beliche reservoir accumulated only at deep water levels. Being invisible from the surface, the occurrence of toxic P. rubescens in freshwater resources requires special attention when designing site inspection and sampling procedures for the correct risk assessment and management of cyanobacterial blooms in the field.

Ecological, anatomical and physiological traits of benthic macroinvertebrates: Their use on the health characterization of freshwater ecosystems

Ecological, anatomical, and physiological traits of benthic macroinvertebrates: their use on the health characterization of freshwater ecosystems After some studies made in Northern Portuguese freshwater ecosystems using the benthic macroinvertebrate communities for water quality evaluation, it has been possible to see that only the analysis of some characteristics allowed to distinguish different levels of environmental disturbance. The studies of some characteristics of the macroinvertebrates and the comparison with the results of the physico-chemical pa- rameters analysed show that the more disturbed the ecosystems had, the lower dissolved oxygen concentration and organisms with greater independence from this concentration. The objective of this work is to define groups of organisms, attending to some of their characteristics that are related to getting oxygen. The groups were defined through bibliographic information and from direct observation of their behaviour. The ability of these groups to distinguish different types of ecosystems was analysed by comparison of the obtained results on their distribution in a group of sampling sites located along gradients of contamination, with the results obtained by the calculation of biological indices, descriptive metric analyses of the community, and the water chemical quality. After this study, it is possible to say that the results obtained with the use of respiratory groups can be more informative than that obtained with the analysis of the physico-chemical parameters or other biological parameters.

Seasonal variation of phytoplankton and cyanobacteria composition and associated microcystins in six Portuguese freshwater reservoirs

Toxic cyanobacteria are common in Portuguese freshwaters and are a cause of concern, given that exposure to subacute levels of cyanobacterial toxins through drinking and recreational water might have deleterious effects on human health. Since 1996 several laboratories have been involved in the screening of cyanotoxins in different freshwater bodies, some on a regularly basis but most on a sporadically basis. Here we present data on the phytoplankton communities and on microcystins production occurring in six freshwater reservoirs located in a dry region of south Portugal, from May to December 2005 and April to July 2006. Most of the reservoirs experienced cyanobacterial blooms of various intensities, following noticeable shifts in phytoplankton composition towards cyanobacterial dominance. The seasonal dominance of cyanobacteria during warmer periods was not a fi xed pattern since considerable amounts of cyanobacteria were also recorded during winter months in some reservoirs. Microcystins were detected in 23% of the 53 samples tested and some of them had concentrations higher than the WHO guideline of 1μg L-1 of microcystins, but their amounts not always reflected the encountered cyanobacteria biomass. Although Microcystis aeruginosa seems to be the major species responsible for microcystins production, other potentially toxigenic bloom-forming species were also found. These results reflect the irregular and unpredictable nature of cyanobacterial blooms in what respects to their occurrence, their composition, their intensity and persistency, as well as to their overall toxicity, strengthening the need of a systematic survey of freshwater resources for the correct risk assessment of cyanobacteria and associated toxins in natural environments.

Effects of Cadmium and Zinc on the feeding behaviour of two freshwater crustaceans: Atyaephyra desmarestii (Decapoda) and Echinogammarus meridionalis (Amphipoda)

Lethal and sublethal effects of Cadmium and Zinc on two freshwater crustaceans were investigated. The 96-h LC50 for Cadmium and Zinc were 42.20 microg l(-1) and 5.43 mg l(-1) for Atyaephyra desmarestii, and 36.17 microg l(-1) and 4.61 mg l(-1) for Echinogammarus meridionalis, respectively. An increase in metal concentrations at sublethal levels resulted in significant reductions of the feeding rate of both species. The LOECs in the chronic feeding assays for Zinc were 1.29 mg l(-1) for A. desmarestii and 0.4 mg l(-1) for E. meridionalis. For Cadmium the LOEC was 6.53 microgl(-1) for both species. The results suggest the potential use of these two crustacean species as test organisms in ecotoxicological tests. Feeding assays appear to be a rapid, cheap and effective tool to be used in biomonitoring studies of Portuguese freshwaters.

Invasibility Patterns of Knotgrass (Paspalum distichum) in Portuguese Riparian Habitats

Spatial patterns of the exotic riverine knotgrass (Paspalum distichum L.) were examined in Mediterranean river basins in Southwestern Iberia. The major goals of this study were to assess the degree of invasibility of riparian habitats by this species and to determine the influence of environmental factors and human-induced disturbances that this knotgrass has on both the landscape and the habitat scales. The present study demonstrates the ability of knotgrass to invade riparian habitats in Portuguese freshwater ecosystems. However, most of the spatial variation of the knotgrass cover seemed to be driven by local factors, such as fine sediment enrichment and the fragmentation of riparian woods, and by other anthropogenic interferences in relation to both the fluvial system and the surrounding landscape.

Molecular characterization of Cylindrospermopsis raciborskii strains isolated from Portuguese freshwaters

Cylindrospermopsis raciborskii is a toxic bloom forming cyanobacteria that is a common component of the phytoplankton assemblage in temperate freshwaters, as well as in temperate climates. This species is of major concern in public health, due to its known ability to produce toxins, including cylindrospermopsin and paralytic shellfish poisoning toxin (PSP). In this study, M13 PCR fingerprinting, ERIC PCR fingerprinting and amplification of the internal transcribed spacer (ITS) region were used to characterize nine cultured strains of C. raciborskii, sourced from several freshwater lakes and rivers in Portugal, and two other Australian. Strains belonging to other taxa including Microcystis aeruginosa, Aphanizomenon spp., Planktothrix agardhii and Oscillatoria neglecta were also analysed to evaluate the taxonomical potential of the fingerprinting methods. Data obtained from genomic fingerprinting were used to perform hierarchical cluster analysis and demonstrated ability to differentiate strains at intra-specific level. However, the high level of variability prevents their use as an identification tool. ITS amplification displayed intra-specific polymorphism both in number and length of the obtained amplicons, but revealed itself as a good method for strain clustering. The unsuccessful amplification of peptide synthetase (PS) and polyketide synthase (PKS) genes pointed to the inability of Portuguese C. raciborskii strains to produce cylindrospermopsin. HPLC analysis further confirmed this lack of toxicity, since negative results were obtained for cylindrospermopsin and PSP toxins.