Ecological impact and recovery of a Mediterranean river after receiving the effluent from a textile dyeing industry.

The textile industry is one of the largest sectors globally, representing up to 20% of industrial water pollution. However, there is limited insight into how fluvial ecosystems respond and recover from this impact. From summer 2012 to spring 2013, we examined water quality and ecological status upstream and 1.5km downstream the input of a textile industry wastewater treatment plant (WWTP) in Ripoll River, NE Spain. The ecological status was determined via diversity measures and 10 biotic indices based on diatoms, macrophytes, macroinvertebrates and fish. Our results showed that the WWTP severely deteriorated water quality and biological communities at the discharge site, but that they improved at 1.5km downstream. Severity also varied across taxa and seasons, being fish the most affected taxa and spring the season with the best ecological status. The strong correlation amongst water quality variables and many biotic indices across taxa indicated that this is a chronic pollution event affecting multiple trophic levels. Thus, this study suggests that there is an urgent need to invest in wastewater treatment in this industry to preserve the ecological integrity of Ripoll River and especially its fish fauna. Likewise, it illustrates the diagnostic power of biotic indices based on diatoms, macroinvertebrates and fish, as driven by the European Water Framework Directive.

The Andean Biotic Index (ABI): revised tolerance to pollution values for macroinvertebrate families and index performance evaluation.

Score-based biotic indices are widely used to evaluate the water quality of streams and rivers. Few adaptations of these indices have been done for South America because there is a lack of knowledge on macroinvertebrate taxonomy, distribution and tolerance to pollution in the region. Several areas in the Andes are densely populated and there is need for methods to assess the impact of increasing human pressures on aquatic ecosystems. Considering the unique ecological and geographical features of the Andes, macroinvertebrate indices used in other regions must be adapted with caution. Here we present a review of the literature on macroinvertebrate distribution and tolerance to pollution in Andean areas above 2,000 masl. Using these data, we propose an Andean Biotic Index (ABI), which is based on the BMWP index. In general, ABI includes fewer macroinvertebrate families than in other regions of the world where the BMWP index has been applied because altitude restricts the distribution of several families. Our review shows that in the high Andes, the tolerance of several macroinvertebrate families to pollution differs from those reported in other areas. We tested the ABI index in two basins in Ecuador and Peru, and compared it to other BMWP adaptations using the reference condition approach. The ABI index is extremely useful for detecting the general impairment of rivers but class quality boundaries should be defined independently for each basin because reference conditions may be different. The ABI is widely used in Ecuador and Peru, with high correlations with land-use pressures in several studies. The ABI index is an integral part of the new multimetric index designed for high Andean streams (IMEERA).

Taxonomic accuracy and complementarity between bulk and eDNA metabarcoding provides an alternative to morphology for biological assessment of freshwater macroinvertebrates.

Determining biological status of freshwater ecosystems is critical for ensuring ecosystem health and maintaining associated services to such ecosystems. Freshwater macroinvertebrates respond predictably to environmental disturbances and are widely used in biomonitoring programs. However, many freshwater species are difficult to capture and sort from debris or substrate and morphological identification is challenging, especially larval stages, damaged specimens, or hyperdiverse groups such as Diptera. The advent of high throughput sequencing technologies has enhanced DNA barcoding tools to automatise species identification for whole communities, as metabarcoding is increasingly used to monitor biodiversity. However, recent comparisons have revealed little congruence between morphological and molecular-based identifications. Using broad range universal primers for DNA barcode marker cox1, we compare community composition captured between morphological and molecular-based approaches from different sources - tissue-based (bulk benthic and bulk drift samples) and environmental DNA (eDNA, filtered water) metabarcoding - for samples collected along a gradient of anthropogenic disturbances. For comparability, metabarcoding taxonomic assignments were filtered by taxa included in the standardised national biological metric IBMWP. At the family level, bulk benthic metabarcoding showed the highest congruence with morphology, and the most abundant taxa were captured by all techniques. Richness captured by morphology and bulk benthic metabarcoding decreased along the gradient, whereas richness recorded by eDNA remained constant and increased downstream when sequencing bulk drift. Estimates of biological metrics were higher using molecular than morphological identification. At species level, diversity captured by bulk benthic samples were higher than the other techniques. Importantly, bulk benthic and eDNA metabarcoding captured different and complementary portions of the community - benthic versus water column, respectively - and their combined use is recommended. While bulk benthic metabarcoding can likely replace morphology using similar benthic biological indices, water eDNA will require new metrics because this technique sequences a different portion of the community.

From biomarkers to community composition: Negative effects of UV/chlorine-treated reclaimed urban wastewater on freshwater biota.

The use of urban wastewater reclaimed water has recently increased across the globe to restore stream environmental flows and mitigate the effects of water scarcity. Reclaimed water is disinfected using different treatments, but their effects into the receiving rivers are little studied. Physiological bioassays and biomarkers can detect sub-lethal effects on target species, but do not provide information on changes in community structure. In contrast, official monitoring programs use community structure information but often at coarse taxonomic resolution level that may fail to detect species level impacts. Here, we combined commonly used biomonitoring approaches from organism physiology to community species composition to scan a broad range of effects of disinfection of reclaimed water by UV-light only and both UV/chlorine on the biota. We (1) performed bioassays in one laboratory species (water flea Daphnia magna) and measured biomarkers in two wild species (caddisfly Hydropsyche exocellata and the barbel Luciobarbus graellsii), (2) calculated standard indices of biotic quality (IBQ) for diatoms, benthic macroinvertebrates, and fishes, and (3) analysed community species composition of eukaryotes determined by Cytochrome Oxidase C subunit I (cox1) metabarcoding. Only the UV/chlorine treatment caused significant changes in feeding rates of D. magna and reduced antioxidant defenses, increased anaerobic metabolism and altered the levels of lipid peroxidiation in H. exocellata. However, inputs of reclaimed water were significantly associated with a greater proportion of circulating neutrophils and LG-PAS cells in L. graellsii. Despite IBQ did not discriminate between the two water treatments, metabarcoding data detected community composition changes upon exposure to UV/chlorine reclaimed water. Overall, despite the effects of UV/chlorine-treated water were transient, our study suggests that UV-light treated is less harmful for freshwater biota than UV/chlorine-treated reclaimed water, but those effects depend of the organizational level.

What happens when salinization meets eutrophication? A test using stream microcosms.

Nutrient and salt pollution often co-occur in rivers and streams due to human activities (e.g., agriculture, urbanization). Thus, understanding the interactive effects of nutrients and salinity on freshwater ecosystems is critical for environmental management. We experimentally assessed the interactive effects of nutrient and salt pollution on stream microcosms using biofilm and macroinvertebrates as model systems. Six treatments were performed in triplicate: control (C: N-NH4+ = 0.05; P- PO43- = 0.037; Cl- = 33.5 mg L-1), intermediate nutrient (IN: N-NH4+ = 0.4; P- PO43- = 0.271; Cl- = 33. 5 mg L-1), high nutrient (HN: N-NH4+ = 0.84; P- PO43- = 0.80; Cl- = 33.5 mg L-1), salt (S: N-NH4+ = 0.05; P- PO43- = 0.037; Cl- = 3000 mg L-1), salt with intermediate nutrient (SIN: N-NH4+ = 0.4; P- PO43- = 0.27; Cl- = 3000 mg L-1) and salt with high nutrient (SHN: N-NH4+ = 0.84; P- PO43- = 0.80; Cl- = 3000 mg L-1). After 14 days of exposure, biofilm chlorophyll-a increased across all treatments, with cyanobacteria replacing diatoms and green algae. Treatments with no added nutrients (C and S) had more P uptake capacity than the rest. The indicator species analysis showed 8 significant taxa, with Orthocladius (Orthocladius) gr. Wetterensis and Virganytarsus significantly associated with the salinity treatment. Overall, salt pollution led to a very strong decline in macroinvertebrate richness and diversity. However, salt toxicity seemed to be ameliorated by nutrient addition. Finally, both structural equation models and biotic-abiotic interaction networks showed that complex biological interactions could be modulating the response of the biological communities to our treatments. Thus, our study calls for species-level assessments of salt and nutrient effects on river ecosystems and advocates for better management of co-occurring pollutants.

DNA Barcode of symbiotic chironomids: Findings in the genus Symbiocladius (Diptera: Chironomidae).

Chironomidae of symbiotic habits have been recorded in different parts of the world, among commensals and parasites. There are different genera reported at the moment, however questions such as the origin of commensal or parasitic life, which occurred first or what are their benefits remain debatable. In order to contribute with information to elucidate the above mentioned issues, the present study reports the finding of immature stages of Symbiocladius (Acletus) wygodzinskyi Roback, 1965 in the Churup stream located in the Andes Cordillera (Peru), living on nymphs of Leptophlebiidae (Ephemeroptera). We present a morphological description of immature stages of this species and for the first time the sequence of COX1 gene S. (A.) wygodzinskyi. The genetic result also supports differences between the morphospecies of Symbiocladius (Symbiocladius) rhithrogenae Zavrel, 1924 and S. (A.) wygodzinskyi in 23%.

Life history and secondary production of Anomalocosmoecusilliesi Marlier, 1962 (Trichoptera, Limnephilidae) in a small stream in the northern Ecuadorian Paramo.

Life history of benthic faunas of tropical high-altitude cold environments are poorly studied. Here, monthly larval and adult data are presented for Anomalocosmoecusilliesi at Saltana Stream in Ecuador. In cold conditions throughout the year (6 °C), this species showed an asynchronous and continuous production. Larval density showed two peaks in August and April. All five larval instars were present in most months. Using the size-frequency method an annual rate of secondary production per biomass of 4.8 was calculated. The measured biomass was 785 mg/m2.

Genetic Variability of Polypedilum (Diptera: Chironomidae) from Southwest Ecuador.

Chironomids show a wide distribution and can occupy several habitats due to their high adaptive capacity in different freshwater environments. The genus Polypedilum is found along a wide elevational and environmental gradient in the neotropics, and its genetic variability could help to elucidate factors determining its distribution and tolerance to the environmental changes of different species or populations. This study examines the genetic variability of Polypedilum in an important biogeographic area that acts as a geographical barrier of biodiversity at the border of the Choco and Tumbes biomes. We identified five Polypedilum morphotypes using classic taxonomic methods. We examined 68 Polypedilum individuals from eight sampling sites in El Oro Province, Ecuador, analyzing the putative molecular species using the cytochrome c oxidase subunit 1 (CO1) mitochondrial gene fragment. Then, we calculated molecular diversity indices, Haplotype diversity (Hd), and θs and θπ estimators. Seven Polypedilum OTUs were determined from which a high molecular diversity was registered. A CCA was conducted to understand the population composition in relation to environmental characteristics. Results indicated that dissolved oxygen and temperature are the main environmental factors affecting Polypedilum distribution across elevational gradients and between basins.

Evaluation of side-effects of glyphosate mediated control of giant reed (Arundo donax) on the structure and function of a nearby Mediterranean river ecosystem.

The aim of this study was to evaluate the effect of the application of the herbicide Herbolex (Aragonesas Agro, S.A., Madrid, Spain) to control giant reed (Arundo donax), which has glyphosate as active ingredient, on the structure and function of a nearby river ecosystem. Specifically, we assessed glyphosate environmental fate in the surrounding water and its effects on transplanted Daphnia magna, field collected caddisfly (Hydropsyche exocellata) and on benthic macroinvertebrate structure assemblages. Investigations were conducted in the industrialized and urbanized Mediterranean river Llobregat (NE Spain) before and after a terrestrial spray of glyphosate. Four locations were selected to include an upstream site and three affected ones. Measured glyphosate levels in river water following herbicide application were quite high (20-60 microg/l) with peak values of 137 microg/l after three days. After 12 days of its application, leaching of glyphosate from sprayed riverbanks was quite high in pore water (20-85 microg/l) but not in the river. Closely linked with the measured poor habitat and water physico-chemical conditions, macroinvertebrate communities were dominated by taxa tolerant to pollution and herbicide application did not affect the abundance or number of taxa in any location. Nevertheless, significant specific toxic effects on transplanted D. magna and field collected H. exocellata were observed. Effects included D. magna feeding inhibition and oxidative stress related responses such as increased antioxidant enzyme activities related with the metabolism of glutathione and increased levels of lipid peroxidation. These results emphasize the importance of combined chemical, ecological and specific biological responses to identify ecological effects of pesticides in the field.

Agricultural impacts on streams near Nitrate Vulnerable Zones: A case study in the Ebro basin, Northern Spain.

Agricultural intensification during the last century has caused river degradation across Europe. From the wide range of stressors derived from agricultural activities that impact rivers, diffuse agricultural pollution has received most of the attention from managers and scientists. The aim of this study was to determine the main stressors exerted by intensive agriculture on streams around Nitrate Vulnerable Zones (NVZs), which are areas of land that drain into waters polluted by nitrates according to the European Nitrate Directive (91/676/EEC). The study area was located in the NW of La Rioja (Northern Spain), which has some of the highest nitrate concentrations within the Ebro basin. The relationships between 40 environmental variables and the taxonomic and functional characteristics of the macroinvertebrate assemblages (which are useful indicators of water quality) were analyzed in 11 stream reaches differentially affected by upstream agricultural activity. The streams affected by a greater percentage of agricultural land cover in the surrounding catchment had significantly higher nitrate concentrations than the remaining sites. However, hydromorphological alteration (i.e. channel simplification, riparian forest and habitat degradation), which is closely linked to agricultural practices, was the main factor affecting macroinvertebrate assemblages. We suggest that "good agricultural practices" should be implemented in streams affected by NVZs to reverse stream degradation, in concordance with the European Water Framework Directive (WFD).

Combined use of Daphnia magna in situ bioassays, biomarkers and biological indices to diagnose and identify environmental pressures on invertebrate communities in two Mediterranean urbanized and industrialized rivers (NE Spain).

Environmental factors affecting aquatic invertebrate communities were assessed using Daphnia magna in situ bioassays and biological indices based on community assemblages of benthic macroinvertebrates. Investigations were carried out in two heavily industrialized and urbanized river basins from the NE of Spain (Llobregat and Besós). Measures of energy consumption (i.e. algal grazing), and of specific biochemical responses (biomarkers) were conducted on individuals transplanted upstream and downstream from effluent discharges of sewage treatment plants. In both rivers there was a clear deterioration of the ecological water quality parameters and benthic communities towards downstream reaches. In all but one of the 19 locations studied, transplanted organisms were affected in at least one of the five measured responses. In three of them, significant effects were detected in most of the traits considered. Principal Component and Partial Least Square Projections to Latent Structures regression analyses indicated that the measured responses in D. magna in situ bioassays and those of macroinvertebrate assemblages were affected by distinct environmental factors. From up to 20 environmental variables considered, seven of them including habitat degradation, suspended solids, nitrogenous and conductivity related parameters affected macroinvertebrate assemblages. On the other hand, levels of organophosphorus compounds and polycyclic aromatic hydrocarbons were high enough to trigger the responses of D. magna in situ bioassays. These results emphasize the importance of combining biological indices with biomarkers and more generalized and ecologically relevant (grazing) in situ responses to identify ecological effects of effluent discharges from sewage treatment plants in surface waters.

Effects of Didymosphenia geminata massive growth on stream communities: Smaller organisms and simplified food web structure.

This study aims to contribute to the understanding of the impact of Didymosphenia geminata massive growths upon river ecosystem communities' composition and functioning. This is the first study to jointly consider the taxonomic composition and functional structure of diatom and macroinvertebrate assemblages in order to determine changes in community structure, and the food web alterations associated with this invasive alga. This study was carried out in the Lumbreras River (Ebro Basin, La Rioja, Northern Spain), which has been affected by a considerable massive growth of D. geminata since 2011. The study shows a profound alteration in both the river community composition and in the food web structure at the sites affected by the massive growth, which is primarily due to the alteration of the environmental conditions, thus demonstrating that D. geminata has an important role as an ecosystem engineer in the river. Thick filamentous mats impede the movement of large invertebrates-especially those that move and feed up on it-and favor small, opportunistic, herbivorous organisms, mainly chironomids, that are capable of moving between filaments and are aided by the absence of large trophic competitors and predators -prey release effect-. Only small predators, such as hydra, are capable of surviving in the new environment, as they are favored by the increase in chironomids, a source of food, and by the reduction in both their own predators and other midge predators -mesopredator release-. This change in the top-down control affects the diatom community, since chironomids may feed on large diatoms, increasing the proportion of small diatoms in the substrate. The survival of small and fast-growing pioneer diatoms is also favored by the mesh of filaments, which offers them a new habitat for colonization. Simultaneously, D. geminata causes a significant reduction in the number of diatoms with similar ecological requirements (those attached to the substrate). Overall, D. geminata creates a community dominated by small organisms that is clearly different from the existing communities in the same stream where there is an absence of massive growths.

Chironomus alchichica sp. n. (Diptera: Chironomidae) from Lake Alchichica, Mexico.

Morphological analysis of all developmental stages (except female), mitochondrial DNA sequences from cytochrome c oxidase subunit I (cox1) and cytological analysis of the polytene chromosomes were used to describe a new species of Chironomus found in the littoral and profundal zones of an endorheic, warm-monomictic lake in Mexico. Male imago is distinguished by the shape of superior volsella and by an antennal and bristle ratio lower than two. The pupa is characterized by the spur morphology of abdominal segment VIII. There is also a continuous row of hooklets on abdominal segment II. The larva is distinguished by a combination of antenna, mentum, mandible, and pecten epipharyngis characteristics, and abdominal ventral tubules. Molecular and cytological analysis supported the morphological differences found. The maximum likelihood tree obtained shows that Chironomus alchichica sp. n. clusters together with Chironomus decorus-group sp. 2 Butler et al. (1995) (bootstrap support = 92%), but genetic p-distances within C. alchichica sp. n. (0.004) were lower than the p-distances between other species of the decorus-group (C. decorus-group sp. 2, Chironomus bifurcatus Wülker et al., 2009 and Chironomus maturus Johannsen, 1908) confirming that it is a different species. The new species belongs to thummi cytocomplex, (decorus-group), with chromosome set- 2n = 8 and chromosome arm combinations: AB CD EF G. Karyologically, the species is closest to Chironomus riihimaekiensis Wülker (1973). This species has very compact salivary gland chromosomes with well heterochromatinized centromere regions in chromosomes AB CD G. Several fixed homozygous inversions distinguish arm A of the species from that of C. riihimaekiensis. Arm E differs from that of C. riihimaekiensis by simple fixed homozygous inversion. Some similarities in band sequences of this arm were found with species from the decorus-group as Chironomus blaylocki Wülker et al., 2009 and C. bifurcatus (decorus-group). The position of the key constrictions in chromosome G: Nucleolar organizer (NOR) and Balbiani rings (BRs) is similar to the species of decorus-group. C. alchichica sp. n. has been found in soft sediments rich in organic matter in well mineralized waters (where conductivity >10 mS cm-1) and with a high pH (≥9). The profundal zone is inhabited only during the mixing period, when dissolved oxygen is present.

TREHS: An open-access software tool for investigating and evaluating temporary river regimes as a first step for their ecological status assessment.

When the regime of a river is not perennial, there are four main difficulties with the use of hydrographs for assessing hydrological alteration: i) the main hydrological features relevant for biological communities are not quantitative (discharges) but qualitative (phases such as flowing water, stagnant pools or lack of surface water), ii) stream flow records do not inform on the temporal occurrence of stagnant pools, iii) as most of the temporary streams are ungauged, their regime has to be evaluated by alternative methods such as remote sensing or citizen science, and iv) the biological quality assessment of the ecological status of a temporary stream must follow a sampling schedule and references adapted to the flow- pool-dry regime. To overcome these challenges within an operational approach, the freely available software tool TREHS has been developed within the EU LIFE TRIVERS project. This software permits the input of information from flow simulations obtained with any rainfall-runoff model (to set an unimpacted reference stream regime) and compares this with the information obtained from flow gauging records (if available) and interviews with local people, as well as instantaneous observations by individuals and interpretation of ground-level or aerial photographs. Up to six metrics defining the permanence of water flow, the presence of stagnant pools and their temporal patterns of occurrence are used to determine natural and observed river regimes and to assess the degree of hydrological alteration. A new regime classification specifically designed for temporary rivers was developed using the metrics that measure the relative permanence of the three main phases: flow, disconnected pools and dry stream bed. Finally, the software characterizes the differences between the natural and actual regimes, diagnoses the hydrological status (degree of hydrological alteration), assesses the significance and robustness of the diagnosis and recommends the best periods for biological quality samplings.

Monitoring metal and metalloid bioaccumulation in Hydropsyche (Trichoptera, Hydropsychidae) to evaluate metal pollution in a mining river. Whole body versus tissue content.

Whole body metal concentration is easier to analyse than concentration in tissues, but is often not an accurate measure of the potential toxicity of the metal. We tested if whole body metal and metalloid concentrations in the caddisfly Hydropsyche (Trichoptera, Hydropsychidae), common in a mine-polluted river (Guadiamar River, SW Spain), were a useful measure to extrapolate environmental degradation. Our results confirmed that metal and metalloid accumulation by Hydropsyche strongly correlated with metal and metalloid concentration both in the water and the sediment, and also correlated with macroinvertebrate community parameters such as total richness and abundance, as well as the richness of some taxonomic groups (EPT and OCH). Seven elements were analysed, Zn, Cu, Pb, As, Cd, Tl and Sb; only As did not show significant correlations. In addition, after leaving live organisms in clean water for 24 or 48 h, we found that 24 h were sufficient to clean the gut content and that between 33% and 75% of the whole body metal content were concentrated in this organ. Although concentration in tissues also discriminated between control and affected stations, we found better results with whole body concentrations when evaluating environmental quality.

Top predator absence enhances leaf breakdown in an intermittent stream.

Current biodiversity loss is characterized by the extinction of top predators, but small-bodied freshwater fish are often overlooked in research and conservation management even when threatened because they usually lack commercial value. Therefore, the ecosystem impacts of their possible loss remain mostly unknown. We assessed whether the presence/absence of an endangered predatory fish (Barbus meridionalis (A. Risso, 1827)) in an intermittent stream affects leaf fungal biomass and leaf quality (i.e. leaf carbon:nitrogen ratio and leaf toughness), macroinvertebrate assemblages colonizing leaf packs, and leaf breakdown rates. We conducted a leaf bag experiment comparing a control reach with a population of B. meridionalis with an adjacent upstream fishless reach. In the fishless reach, leaf fungal biomass and microbially mediated breakdown rate were lower compared to the control reach. This was probably caused by the lack of the bottom-up stimulation through nutrient recycling by fish. Shredders and scrapers were found at higher abundance and biomass in the fishless compared to the control reach, and the whole macroinvertebrate community composition changed with fish absence. Consequently, macroinvertebrate mediated leaf breakdown was faster in the fishless than in the control reach, not only compensating for the lower microbially mediated leaf breakdown in the fishless reach, but accelerating the overall leaf breakdown rate. Our study contributes to understand the potential cascading effects produced by the extirpation of endangered small-bodied fish.

A trait-based approach reveals the feeding selectivity of a small endangered Mediterranean fish.

Functional traits are growing in popularity in modern ecology, but feeding studies remain primarily rooted in a taxonomic-based perspective. However, consumers do not have any reason to select their prey using a taxonomic criterion, and prey assemblages are variable in space and time, which makes taxon-based studies assemblage-specific. To illustrate the benefits of the trait-based approach to assessing food choice, we studied the feeding ecology of the endangered freshwater fish Barbus meridionalis. We hypothesized that B. meridionalis is a selective predator which food choice depends on several prey morphological and behavioral traits, and thus, its top-down pressure may lead to changes in the functional composition of in-stream macroinvertebrate communities. Feeding selectivity was inferred by comparing taxonomic and functional composition (13 traits) between ingested and free-living potential prey using the Jacob's electivity index. Our results showed that the fish diet was influenced by 10 of the 13 traits tested. Barbus meridionalis preferred prey with a potential size of 5-10 mm, with a medium-high drift tendency, and that drift during daylight. Potential prey with no body flexibility, conical shape, concealment traits (presence of nets and/or cases, or patterned coloration), and high aggregation tendency had a low predation risk. Similarly, surface swimmers and interstitial taxa were low vulnerable to predation. Feeding selectivity altered the functional composition of the macroinvertebrate communities. Fish absence favored taxa with weak aggregation tendency, weak flexibility, and a relatively large size (10-20 mm of potential size). Besides, predatory invertebrates may increase in fish absence. In conclusion, our study shows that the incorporation of the trait-based approach in diet studies is a promising avenue to improve our mechanistic understanding of predator-prey interactions and to help predict the ecological outcomes of predator invasions and extinctions.

Chloride and sulphate toxicity to Hydropsyche exocellata (Trichoptera, Hydropsychidae): Exploring intraspecific variation and sub-lethal endpoints.

The rivers and streams of the world are becoming saltier due to human activities. In spite of the potential damage that salt pollution can cause on freshwater ecosystems, this is an issue that is currently poorly managed. Here we explored intraspecific differences in the sensitivity of freshwater fauna to two major ions (Cl(-) and SO4(2-)) using the net-spinning caddisfly Hydropsyche exocellata Dufour 1841 (Trichoptera, Hydropsychidae) as a model organism. We exposed H. exocellata to saline solutions (reaching a conductivity of 2.5mScm(-1)) with Cl(-):SO4(2-) ratios similar to those occurring in effluents coming from the meat, mining and paper industries, which release dissolved salts to rivers and streams in Spain. We used two different populations, coming from low and high conductivity streams. To assess toxicity, we measured sub-lethal endpoints: locomotion, symmetry of the food-capturing nets and oxidative stress biomarkers. According to biomarkers and net building, the population historically exposed to lower conductivities (B10) showed higher levels of stress than the population historically exposed to higher conductivities (L102). However, the differences between populations were not strong. For example, net symmetry was lower in the B10 than in the L102 only 48h after treatment was applied, and biomarkers showed a variety of responses, with no discernable pattern. Also, treatment effects were rather weak, i.e. only some endpoints, and in most cases only in the B10 population, showed a significant response to treatment. The lack of consistent differences between populations and treatments could be related to the high salt tolerance of H. exocellata, since both populations were collected from streams with relatively high conductivities. The sub-lethal effects tested in this study can offer an interesting and promising tool to monitor freshwater salinization by combining physiological and behavioural bioindicators.

Can salinity trigger cascade effects on streams? A mesocosm approach.

Human activities have greatly increased the salt concentration of the world's rivers, and this might be amplified by water scarcity in the future. While the lethal effects of salinity have been documented for a wide variety of stream invertebrates, the sub-lethal effects (i.e. changes in biological condition without mortality) are not deeply understood yet. One important sub-lethal effect that has yet to be investigated is changes in predation efficiency, which could trigger cascade effects associated to the abundance of herbivorous invertebrates that control algae biomass. In this study we combined the use of biomarkers with community-level data in a stream mesocosm to evaluate the potential cascade effect of increased salinity on the trophic food web. Both predation and salt treatments had an effect on the aquatic invertebrate abundance, richness and community composition. The presence of predators had a clear cascade effect, it reduced herbivorous invertebrate abundance and richness leading to higher chlorophyll a concentrations. The salt treatment significantly reduced taxa richness, but only in the gravel bed. The predators were significantly stressed by salt addition, as shown by the different analyzed biomarkers. Concordantly, in the presence of predators, Tanytarsini registered higher abundances and chlorophyll a showed a lower concentration when salt was added. However, none of these changes was significant. Therefore, although salt addition significantly stressed Dina lineata, our results suggest that a longer exposure time is needed to fully capture cascading effects (e.g. a decrease in chlorophyll a due to a relaxation of predation on herbivorous invertebrates). We suggest that the potential cascade effects of salinization need to be evaluated when addressing the impacts of water scarcity (as caused by climate change and increasing water demand) on river ecosystems, since flow reductions will lead to higher salt concentrations.

Small but powerful: top predator local extinction affects ecosystem structure and function in an intermittent stream.

Top predator loss is a major global problem, with a current trend in biodiversity loss towards high trophic levels that modifies most ecosystems worldwide. Most research in this area is focused on large-bodied predators, despite the high extinction risk of small-bodied freshwater fish that often act as apex consumers. Consequently, it remains unknown if intermittent streams are affected by the consequences of top-predators' extirpations. The aim of our research was to determine how this global problem affects intermittent streams and, in particular, if the loss of a small-bodied top predator (1) leads to a 'mesopredator release', affects primary consumers and changes whole community structures, and (2) triggers a cascade effect modifying the ecosystem function. To address these questions, we studied the top-down effects of a small endangered fish species, Barbus meridionalis (the Mediterranean barbel), conducting an enclosure/exclosure mesocosm experiment in an intermittent stream where B. meridionalis became locally extinct following a wildfire. We found that top predator absence led to 'mesopredator release', and also to 'prey release' despite intraguild predation, which contrasts with traditional food web theory. In addition, B. meridionalis extirpation changed whole macroinvertebrate community composition and increased total macroinvertebrate density. Regarding ecosystem function, periphyton primary production decreased in apex consumer absence. In this study, the apex consumer was functionally irreplaceable; its local extinction led to the loss of an important functional role that resulted in major changes to the ecosystem's structure and function. This study evidences that intermittent streams can be affected by the consequences of apex consumers' extinctions, and that the loss of small-bodied top predators can lead to large ecosystem changes. We recommend the reintroduction of small-bodied apex consumers to systems where they have been extirpated, to restore ecosystem structure and function.