Trends in biomarkers, biotic indices, and fish population size revealed contrasting long-term effects of recycled water on the ecological status of a Mediterranean river.

Recycled water is important for maintaining river flow in semi-arid regions. However, it has ecological risk, as suggested by comparison of habitat and white and red blood cell count in two wild fish species (Barbus meridionalis and Squalius laietanus) before and after an input of recycled water in Ripoll River (NE Spain) in 2009. Due to the lack of normal ranges for blood variables in wild fish, we surveyed seasonally the same river reaches in 2013 to test if blood alterations from 2009 compromised the viability of the fish populations. By examining other indicators of river health in baseline and polluted sites (fish abundance, mass-length relationships, and community indices in fish, diatoms and invertebrates), we tested for the superior utility of blood tests in biomonitoring. The comparison of water quality and scores of diatoms and invertebrate indices between polluted and reference sites showed that polluted sites improved from 2009 to 2013. The abundance of B. meridionalis also increased in polluted sites, but that of S. laietanus declined in 2013 compared to 2009. These results contrast with results of blood analyses in 2009, which suggested that B. meridionalis was more seriously affected by pollution than S. laietanus. The fish index did not reveal the risk of recycled water to fish health, whereas fish mass-length relationships suggested that S. laietanus individuals in 2013 had a better body condition in polluted than in reference sites. Given that the two fish species had opposite results in reference sites, and that the physical habitat was more suitable for B. meridionalis in polluted sites in 2013 than was for S. laietanus, trends in population size are not only explained by pollution. The role of phenology is suggested by peaks in blood disorders during the breeding season. However, more long-term studies combining indicators of river health at the individual and community scales are needed to fully assess the ecological risk of recycled water in this river. These studies will also help to develop blood tests as reliable health indicators in wild fish populations.

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.

Metal bioaccumulation in the Mediterranean barbel (Barbus meridionalis) in a Mediterranean river receiving effluents from urban and industrial wastewater treatment plants.

Although sewage treatment plants (STPs) play a crucial role in maintaining the water quality and flow of Mediterranean rivers, particularly during drought periods, few studies have addressed their impact on aquatic fauna. Here we analyzed the role of STPs as a source of metals in the Ripoll River, a heavily urbanized and industrialized watercourse with a long history of anthropogenic disturbance. For this purpose, we measured iron, mercury, cadmium, zinc, lead, nickel and copper accumulation in the liver and muscle of the Mediterranean barbel, Barbus meridionalis and also the concentrations of these metals in the river water. Industrial and urban sewage treatment plants are source of metals in Ripoll River but the former mainly increases Zn and Ni values. Significant differences in metal bioaccumulation between reference and polluted sites were detected. Nevertheless, there was only a significant positive relationship between bioaccumulation of Cu and Hg, and their concentration in water. In addition, the lead concentration in fish was not clearly associated with the presence of STPs. On the basis of morphometric parameters, the hepato-somatic index was the only one denoting significant differences between polluted and references sites. Given that fish are key elements in food webs, recreational fishing is practice in this area and that river water is used for agricultural purposes, we recommend long-term studies to analyze the impact of metal pollution in this river.

Patterns of species richness, abundance and individual-size distributions in native stream-fish assemblages invaded by exotic and translocated fishes.

Predicting the impacts of species introductions long has attracted the attention of ecologists yet there still is limited insight into how impacts on native assemblages vary with the degree of shared evolutionary context. Here, we used data from 535 stream-fish surveys from 15 catchments in north-eastern Spain (99,700 km2) to explore whether the relative effects on native fishes differ between fish introductions from two different ecoregions (i.e., evolutionary contexts), namely, catchments within Iberian Peninsula (i.e., 'translocated species') and catchments beyond Iberian Peninsula (i.e., 'exotic fishes'). We used hierarchical Bayesian models to relate taxon richness, abundance, and the individual-size distributions (ISDs) of native fishes to the presence, abundance, and weighted trophic level (TL) of translocated and exotic fishes, conditional on geographic and habitat covariates. Environmental covariates dominated the percentage of explained variance (≥ 65%) for all responses. Translocated fishes accounted for more of the explained variance than did exotic fishes for ISDs and abundance, but not for native fish species richness. The presence of translocated fishes was associated with lower abundance and richness of native fishes, with individuals being smaller in the presence of translocated fishes of higher TL. The presence of exotic fishes was associated with a greater abundance and richness of native fishes, with individuals generally being larger in the presence of exotic fishes. Our study suggests that translocated fishes could be as problematic as exotic fishes when angling and water transfers among catchments to deal with climate change may increase the establishment of translocated fishes. We also discuss the difficulties of using fish body size as species-blind, transferable assemblage-level trait in fish monitoring.

Individual body mass and length dataset for over 12,000 fish from Iberian streams.

We provide a unique fish individual body size dataset collected from our own sampling and public sources in north-eastern Spain. The dataset includes individual body size measures (fork length and mass) of 12,288 individuals of 24 fish species within 10 families collected at 118 locations in large rivers and small streams. Fish were caught by one-pass electrofishing following European standard protocols. The fish dataset has information on the local instream conditions including climatic variables (i.e., temperature and precipitation), topography (i.e., altitude), nutrient concentration (i.e., total phosphorus and nitrates), and the IMPRESS values (a measure of cumulative human impacts in lotic ecosystems). The potential uses of this new fish dataset are manifold, including developing size-based indices to further estimate the ecological status of freshwater ecosystems, allometric models, and analysis of variation in body size structure along environmental gradients.

Ecologically relevant biomarkers reveal that chronic effects of nitrate depend on sex and life stage in the invasive fish Gambusia holbrooki.

Agricultural intensification and shifts in precipitation regimes due to global climate change are expected to increase nutrient concentrations in aquatic ecosystems. However, the direct effects of nutrients widely present in wastewaters, such as nitrate, are poorly studied. Here, we use multiple indicators of fish health to experimentally test the effects of three ecologically relevant nitrate concentrations (<10, 50 and 250 mg NO3-/l) on wild-collected mosquitofish (Gambusia holbrooki), a species widely introduced for mosquito biocontrol in often eutrophic waters. Overall, biomarkers (histopathology, feeding assays, growth and caloric content and stable isotopes as indicators of energy content) did not detect overt signs of serious disease in juveniles, males or females of mosquitofish. However, males reduced food intake at the highest nitrate concentration compared to the controls and females. Similarly, juveniles reduced energy reserves without significant changes in growth or food intake. Calorimetry was positively associated with the number of perivisceral fat cells in juveniles, and the growth rate of females was negatively associated with δ15N signature in muscle. This study shows that females are more tolerant to nitrate than males and juveniles and illustrates the advantages of combing short- and long-term biomarkers in environmental risk assessment, including when testing for the adequacy of legal thresholds for pollutants.

Managing small, highly prolific invasive aquatic species: Exploring an ecosystem approach for the eastern mosquitofish (Gambusia holbrooki).

Invasive exotic species threaten native biodiversity worldwide and their management is on the agenda of an increasing number of countries. We explored the potential of an ecosystem approach for the natural control of Gambusia holbrooki, which is among the most pernicious and widely distributed fish invaders. Individual-based linear mixed models were used to identify the ecosystem factors (conspecific density, environment and piscivorous birds) that most influenced life-history variation in male and female G. holbrooki (N = 654). All traits (body condition, growth, length, gonad weight, offspring size and number, real and potential fertility) were associated with at least one ecosystem factor from the 18 water bodies surveyed in north-eastern Spain. Models for female reproductive traits had the highest fit (R2 = 0.89) and those for body condition in both sexes the lowest (0.12). The life history of G. holbrooki was mostly affected by its density; increasing offspring number at the expense of offspring size at the sites with the highest fish density. Weaker effects on G. holbrooki life history were observed for the abundance of piscivorous birds and water-quality conditions, including turbidity and nutrient concentrations. Although effects were not consistent between traits, outputs supported that G. holbrooki has a wide tolerance to changes in water quality. Therefore, actions based solely on environmental changes within the range tested probably will fail in reducing the proliferation of G. holbrooki, especially if its body condition improved at the most naturalised sites. Overall, this study suggests that the management of G. holbrooki using ecologically sound treatments is likely to be very difficult in stagnant waters. Preventing new introductions and direct removal once established are the most appropriate actions for the management of this small, highly prolific fish invader.

Major determinants of the occurrence of a globally invasive parasite in riverine fish over large-scale environmental gradients.

The increased rate of outbreaks of infectious diseases in ecosystems is a dramatic consequence of global change, particularly when outbreaks affect important resources such as freshwater fish. However, the links between disease-inducing epizootics and widespread human impacts, including nutrient pollution and high water conductivity, in freshwater organisms are largely unexplored. We used data from extensive surveys in northeastern Spain (99,700 km2, 15 river catchments, n = 530 sites) to explore the environmental factors that singly, or in combination, are likely to influence the occurrence of the invasive parasite, Lernaea cyprinacea, after accounting for host fish characteristics. Smaller fish, lower altitudes, higher water conductivity and nutrient pollution were associated with higher probabilities of infection in 19 endemic and widely distributed fish species. We found no evidence that interactive effects among riverine stressors related to water and physical habitat quality better explained the probability of occurrence of L. cyprinacea in fish than did additive-stressor combinations. Nutrient pollution and high water conductivity were two of the major factors contributing to the increased occurrence of L. cyprinacea. Therefore, the improvement of wastewater treatment processes and agricultural practices probably would help to reduce the occurrence of this parasite among native fish.

Water-quality impacts in semi-arid regions: can natural 'green filters' mitigate adverse effects on fish assemblages?

The effective aridity in riparian areas is increasing from climate change and from human water consumption, which exacerbates the impacts of effluents from wastewater-treatment plants and from catchment run-off in rivers. The potential of natural riparian areas to act as 'green filters' has long been recognized, but the possible ecological benefits of natural riparian areas over large-scale environmental gradients on fish have not been explored in detail. Using an extensive data-set from northeastern Spain (99,700 km2, 15 catchments, 530 sites), ours is the first study to ask whether natural riparian vegetation can mitigate the effects of pollution on fish in rivers experiencing water scarcity. We used multimodel inference to explore the additive and interactive effects of riparian vegetation with nutrient pollution and water conductivity, which are among the world's worst river stressors, on multiple fish guilds, including widely distributed species and highly invasive alien fish species. Most models (54%) supported the additive effects of water-quality factors on fish, after having accounted for the influence of geography and hydrological alterations. Although many fewer models (7%) included riparian vegetation as an important predictor, riparian vegetation modulated the forms of the associations between fish and pollution. The relationship of nutrient pollution with native and alien fish richness changed from negative to positive with greater riparian structure or species richness. However, we found the opposite effect for the mean body size of sedentary fish, and only positive additive effects of riparian richness for the probability of occurrence of pelagic fish. Ammonium and nitrite concentrations adversely affected fish in these rivers up to 10 years after the enforcement of the implementation of the Water Framework Directive by the European Union. High conductivity also much affects fish, having negatives associations with migratory, pelagic, invertivorous and native fish, and positive associations with sedentary, benthic, omnivorous and alien fish. Therefore, the current status of natural riparian areas is unlikely to fully mitigate water-quality impacts on fish. The conservation of freshwater resources in semi-arid regions, such as north-eastern Spain, requires improved waste-water treatments and better agriculture practices.

Environmental correlates of food-chain length, mean trophic level and trophic level variance in invaded riverine fish assemblages.

Examining how the trophic structure of biotic assemblages is affected by human impacts, such as habitat degradation and the introduction of alien species, is important for understanding the consequences of such impacts on ecosystem functioning. We used general linear mixed models and hierarchical partitioning analyses of variance to examine for the first time the applicability of three hypotheses (ecosystem-size, productivity and disturbance) for explaining food-chain length (FCL) in invaded fish assemblages. We used Fishbase trophic level (TL) estimates for 16 native and 18 alien fish species in an extensive riverine system in north-eastern Spain (99,700 km2, 15 catchments, 530 sites). The FCL of assemblages ranged from 2.7 to 4.42. Ecosystem size-related variables (Strahler stream order, physical habitat diversity) and human-disturbance (conductivity) made the largest contribution to the explained variance in the FCL model after accounting for spatial confounding factors and collinearity among predictors. Within-assemblage TL also was positively associated with Strahler stream order, suggesting that large rivers have the highest trophic diversity. High conductivity was negatively associated with FCL, as did with the mean TL of fish assemblages. However, an inverse association was found between mean TL and Strahler stream order, possibly because the presence of fish species of high TL may be offset by larger numbers of alien species of lower TL in large rivers. Given that there may be trophic replacements among native and alien species, this inference needs to be addressed with detailed trophic studies. However, reducing water conductivity by improved wastewater treatment and better agricultural practices probably would help to conserve the fish species on the apices of aquatic food-webs.

Movements and dispersal of brown trout (Salmo trutta Linnaeus, 1758) in Mediterranean streams: influence of habitat and biotic factors.

Dispersal is a critical determinant of animal distribution and population dynamics, and is essential information for management planning. We studied the movement patterns and the influence of habitat and biotic factors on Mediterranean brown trout (Salmo trutta) by mark-recapture methods in three headwater streams of the Ebro Basin (NE Iberian Peninsula). Fish were sampled by electrofishing on five occasions over 18-24 months and movements of over 3,000 individually tagged trout (age 1+ onwards) were recorded. Most of the tagged fish exhibited limited movement and were recaptured within 100 m from the initial capture section. Small seasonal differences in the movement pattern were observed, but in two of the streams, displacement distances increased prior the spawning period in autumn. The frequency distributions of dispersal distances were highly leptokurtic and skewed to the right and fitted well to a two-group exponential model, thus trout populations were composed of mobile and stationary individuals, the latter being the predominant component in the populations (71.1-87.5% of individuals). The mean dispersal distances, for fish captured at least in three sampling events, ranged 20.7-45.4 m for the stationary group and 229.4-540.5 m for the mobile group. Moving brown trout were larger than non-moving individuals and exhibited higher growth rates in two of the streams. Habitat features were not consistently linked to movement rates, but there were some interaction effects between stream and habitat characteristics such as depth, cover and water velocity.

Functional diversity measures revealed impacts of non-native species and habitat degradation on species-poor freshwater fish assemblages.

Trait-based ecology has been developed for decades to infer ecosystem responses to stressors based on the functional structure of communities, yet its value in species-poor systems is largely unknown. Here, we used an extensive dataset in a Spanish region highly prone to non-native fish invasions (15 catchments, N=389 sites) to assess for the first time how species-poor communities respond to large-scale environmental gradients using a taxonomic and functional trait-based approach in riverine fish. We examined total species richness and three functional trait-based indices available when many sites have ≤3 species (specialization, FSpe; originality, FOri and entropy, FEnt). We assessed the responses of these taxonomic and functional indices along gradients of altitude, water pollution, physical habitat degradation and non-native fish biomass. Whilst species richness was relatively sensitive to spatial effects, functional diversity indices were responsive across natural and anthropogenic gradients. All four diversity measures declined with altitude but this decline was modulated by physical habitat degradation (richness, FSpe and FEnt) and the non-native:total fish biomass ratio (FSpe and FOri) in ways that varied between indices. Furthermore, FSpe and FOri were significantly correlated with Total Nitrogen. Non-native fish were a major component of the taxonomic and functional structure of fish communities, raising concerns about potential misdiagnosis between invaded and environmentally-degraded river reaches. Such misdiagnosis was evident in a regional fish index widely used in official monitoring programs. We recommend the application of FSpe and FOri to extensive datasets from monitoring programs in order to generate valuable cross-system information about the impacts of non-native species and habitat degradation, even in species-poor systems. Scoring non-native species apart from habitat degradation in the indices used to determine ecosystem health is essential to develop better management strategies.

The presence of non-native species is not associated with native fish sensitivity to water pollution in greatly hydrologically altered rivers.

There is a risk of 'ecological surprises' if multiple potentially interacting stressors are managed individually, which is a question attracting significant current interest. Habitat degradation and species introductions are major threats to global biodiversity, and riverine fish are among the most threatened taxa in the world. Our interest was whether the presence of non-native species can affect native fish sensitivity to water quality deterioration in a large region in northeastern Spain (99,700km2, 15 catchments, 530 sites). We used a 'base model' with geographical and hydro-morphological variables, which are the major shaping factors in rivers. We tested whether water pollution, non-native species, or their interaction provided an improved understanding of patterns of distributions and health measurements of the twelve most common native species. There was little evidence that variation in native species abundance, where they occurred, the presence of diseases and changes in mean fish length or body condition was affected by water deterioration, the presence of non-native species, or their interaction. The disease rate and occurrence of native species might be affected, to a minor degree, by water quality changes and the presence of non-native species. Environmental conditions between sites with and without non-native fish differed in the condition of riparian areas and in water quality. Based on presence-absence data and changes in abundances through weighted average equations we also derived potential safe levels of salinization, nutrient pollution, and pH for the native fish. Overall, additive effects of stressors prevail over interactions, and the restoration of natural hydro-morphology in rivers is likely to be the most effective management approach to improving the prospects for the native fish fauna.

Fine-scale determinants of conservation value of river reaches in a hotspot of native and non-native species diversity.

Global freshwater biodiversity is declining at unprecedented rates while non-native species are expanding. Examining diversity patterns across variable river conditions can help develop better management strategies. However, many indicators can be used to determine the conservartion value of aquatic communities, and little is known of how well they correlate to each other in making diagnostics, including when testing for the efficacy of protected areas. Using an extensive data set (99,700km2, n=530 sites) across protected and unprotected river reaches in 15 catchments of NE Spain, we examine correlations among 20 indicators of conservation value of fish communities, including the benefits they provide to birds and threatened mammals and mussels. Our results showed that total native fish abundance or richness correlated reasonably well with many native indicators. However, the lack of a strong congruence led modelling techniques to identify different river attributes for each indicator of conservation value. Overall, tributaries were identified as native fish refugees, and nutrient pollution, salinization, low water velocity and poor habitat structure as major threats to the native biota. We also found that protected areas offered limited coverage to major components of biodiversity, including rarity, threat and host-parasite relationships, even though values of non-native indicators were notably reduced. In conclusion, restoring natural hydrological regimes and water chemical status is a priority to stem freshwater biodiversity loss in this region. A complementary action can be the protection of tributaries, but more studies examining multiple components of diversity are necessary to fully test their potential as fluvial reserves in Mediterranean climate areas.

Ecological relevance of biomarkers in monitoring studies of macro-invertebrates and fish in Mediterranean rivers.

Mediterranean rivers are probably one of the most singular and endangered ecosystems worldwide due to the presence of many endemic species and a long history of anthropogenic impacts. Besides a conservation value per se, biodiversity is related to the services that ecosystems provide to society and the ability of these to cope with stressors, including climate change. Using macro-invertebrates and fish as sentinel organisms, this overview presents a synthesis of the state of the art in the application of biomarkers (stress and enzymatic responses, endocrine disruptors, trophic tracers, energy and bile metabolites, genotoxic indicators, histopathological and behavioural alterations, and genetic and cutting edge omic markers) to determine the causes and effects of anthropogenic stressors on the biodiversity of European Mediterranean rivers. We also discuss how a careful selection of sentinel species according to their ecological traits and the food-web structure of Mediterranean rivers could increase the ecological relevance of biomarker responses. Further, we provide suggestions to better harmonise ecological realism with experimental design in biomarker studies, including statistical analyses, which may also deliver a more comprehensible message to managers and policy makers. By keeping on the safe side the health status of populations of multiple-species in a community, we advocate to increase the resilience of fluvial ecosystems to face present and forecasted stressors. In conclusion, this review provides evidence that multi-biomarker approaches detect early signs of impairment in populations, and supports their incorporation in the standardised procedures of the Water Frame Work Directive to better appraise the status of European water bodies.

Metal concentration in water, sediment and four fish species from Lake Titicaca reveals a large-scale environmental concern.

Although intensive mining activity and urban sewage discharge are major sources of metal inputs to Lake Titicaca, the risk posed by metal pollution to wildlife and human populations has been poorly studied. In this study we compared the concentrations of Cu, Zn, Cd, Hg, Pb, Co, and Fe in water, sediment, and two tissues (liver and muscle) of four fish species (Odontesthes bonariensis, Orestias luteus, Orestias agassii, and Trichomycterus rivulatus) across important fishery areas in Lake Titicaca. The concentration of Pb in water at the discharge sites of the main rivers and of most elements, with the exception of Co and Fe, in all fish collected in this study exceeded the safety thresholds established by international legislation. The highest metal concentrations were observed in benthopelagic species, and liver tissue was identified as the main depository for all metals with the exception of mercury. The metal bioaccumulation pattern in fish was weakly related to the metal concentrations in the environment with the exception of Hg at the most polluted location, partly explained by the different metabolic role of essential and non-essential elements and the influence of other factors such as species' ecology and individual traits in the bioaccumulation of most metals. As metal pollution extended across the study area and high metal concentrations were detected in all four fish species, we urge the authorities to enforce legislation for water and fish consumption and to evaluate the effects of metal pollution on fish health.

Metal concentrations and pathological responses of wild native fish exposed to sewage discharge in a Mediterranean river.

The requirements of the Water Framework Directive suggest the need for further research to test and develop sensitive tools that will allow freshwater managers to detect impacts on fish communities. Diagnostic refinement often encompasses the use of lethal diagnostic tools that are incompatible with the conservation of native ichthyofauna. Here we determine the metal concentration and the pathological response of Squalius laietanus exposed to sewage discharges in the Ripoll river (north-eastern Spain), and compare these findings with our previous studies on Barbus meridionalis using lethal and non-lethal diagnostic tools. Metals concentrations (Zn, Cu, Pb, Hg, Fe, Cd and Ni) were determined in liver and muscle. A complete blood cell profile (haematocrit, haemoglobin, differential leukocyte count, erythrocytic nuclear abnormalities, erythrocytes in division and the development stage of erythrocytes) was used as a non-lethal diagnostic tool to determine early warning signs of disease in these two fish species. As the reference range for these haematological variables is lacking, liver histology, calculation of body condition (CF) and organosomatic indices (HSI and GSI) were employed to support the findings of the blood analyses. Compared to our previous results on B. meridionalis, S. laietanus appeared to have fewer pathological responses than B. meridionalis under the environmental conditions measured and the fish size range examined in this study. Both species showed a similar bioaccumulation pattern, but B. meridionalis stored high Hg and Cu concentrations in muscle and liver, respectively. Hg, Cu and Pb concentrations in fish tissues exceeded the thresholds of European and Spanish legislation. Our findings pinpoint the potential suitability of the blood variables determined in the health diagnoses of these species. Further research will be necessary to establish the natural variability of these and other haematological variables to convert haematology into a sensitive and useful non-lethal diagnostic tool in wild fish populations.

Changes in non-specific biomarkers in the Mediterranean barbel (Barbus meridionalis) exposed to sewage effluents in a Mediterranean stream (Catalonia, NE Spain).

The effluents from sewage treatment plants strongly influence the water quality and flow of Mediterranean streams. These effluents play a crucial role in maintaining the aquatic communities of these ecosystems, particularly in the absence of natural flow resulting from climate constraints or intensive water use. To detect the ecological effects of these effluents, we used non-lethal biomarkers in Barbus meridionalis and traditional assessment protocols in the Ripoll River (NE of Spain). Our results demonstrate the utility of haematological parameters. In comparison with fish at reference sites, we detected a decrease in haematocrit and haemoglobin, neutrophilia, lymphopenia, monocytosis, a rise in the nucleo-cytoplasmatic ratio of erythrocytes and an increase in the frequency of abnormal, immature and senescent erythrocytes. Many haematological parameters correlated significantly with the environmental parameters measured. In addition to these haematological alterations, histopathological examination also revealed damage in fish livers but no impact was detected by the regional index of biotic integrity using fish as bioindicators. To the best of our knowledge, this study is the first to report this battery of haematological parameters as biomarkers on a freshwater fish in a Mediterranean stream. In a region in which more than 50% of native fish species are classified as endangered or vulnerable, there is an urgent need to find biomarkers that do not harm the animals.