Fluvial Biofilms Research Articles

Antioxidant enzyme activities in biofilms as biomarker of Zn pollution in a natural system: An active bio-monitoring study

This study aimed to explore the use of antioxidant enzyme activities (AEA) and biofilm metal accumulation capacity in natural communities as effect-based indicator of metal exposure in fluvial systems. To achieve these objectives, an active biomonitoring using fluvial biofilm communities was performed during 5 weeks. Biofilm was colonized over artificial substrata in a non-polluted site. After 5 weeks, biofilms were translocated to four different sites with different metal pollution in the same stream. The evolution of environmental parameters as well as biofilm responses was analysed over time.Physicochemical parameters were different between sampling times as well as between the most polluted site and the less polluted ones, mainly due to Zn pollution. In contrast, AEA and metal accumulation in biofilms allowed us to discriminate the high and moderate metal pollution sites from the rest. Zn, the metal with the highest contribution to potential toxicity, presented a fast and high accumulation capacity in biofilms. According to the multivariate analysis, AEA showed different responses. While catalase (CAT) and ascorbate peroxidase (APX) variability was mainly attributed to environmental stress (pH, temperature and phosphate concentration), glutathione-S-transferase (GST) changes were related to metal pollution. Glutathione reductase (GR) and superoxide dismutase (SOD) responses were related to both stress factors.AEA and metal accumulation are proposed as sensitive effect-based field methods, to evaluate biofilm responses after acute metal exposure (e.g. an accidental spill) due to their capacity to respond after few hours, but also in routinely monitoring due to their persistent changes after few weeks of exposure. These tools could improve the Common Implementation Strategy (CIS) of the Water Framework Directive (WFD) as expert group request.

Headwaters are critical reservoirs of microbial diversity for fluvial networks

Streams and rivers form conspicuous networks on the Earth and are among nature's most effective integrators. Their dendritic structure reaches into the terrestrial landscape and accumulates water and sediment en route from abundant headwater streams to a single river mouth. The prevailing view over the last decades has been that biological diversity also accumulates downstream. Here, we show that this pattern does not hold for fluvial biofilms, which are the dominant mode of microbial life in streams and rivers and which fulfil critical ecosystem functions therein. Using 454 pyrosequencing on benthic biofilms from 114 streams, we found that microbial diversity decreased from headwaters downstream and especially at confluences. We suggest that the local environment and biotic interactions may modify the influence of metacommunity connectivity on local biofilm biodiversity throughout the network. In addition, there was a high degree of variability in species composition among headwater streams that could not be explained by geographical distance between catchments. This suggests that the dendritic nature of fluvial networks constrains the distributional patterns of microbial diversity similar to that of animals. Our observations highlight the contributions that headwaters make in the maintenance of microbial biodiversity in fluvial networks.

Effects of pesticides and pharmaceuticals on biofilms in a highly impacted river

We investigated the effects of pharmaceuticals and pesticides detected in a Mediterranean river, on fluvial biofilms by translocation experiments performed under controlled conditions. Water was sampled from three sites along a pollution gradient. Biofilms grown in mesocosms containing relatively clean water were translocated to heavily polluted water. Several biofilm descriptors were measured before and after translocations. Fifty-seven pharmaceuticals and sixteen pesticides compounds were detected in river waters. The translocation from less to more polluted site was the most effective. Autotrophic biomass and peptidase increased while phosphatase and photosynthetic efficiency decreased. Multivariate analysis revealed that analgesics and anti-inflammatories significantly affected biofilm responses. Ibuprofen and paracetamol were associated with negative effects on photosynthesis, and with the decrease of the green algae/cyanobacteria ratio, while diclofenac was associated with phosphatase activity. The effects of these emerging compounds on biofilms structure and function may cause important alterations in river ecosystem functioning.

Seasonal changes in antioxidant enzyme activities of freshwater biofilms in a metal polluted Mediterranean stream

While seasonal variations in fluvial communities have been extensively investigated, effects of seasonality on community responses to environmental and/or chemical stress are poorly documented. The aim of this study was to describe antioxidant enzyme activity (AEA) variability in fluvial biofilms over an annual cycle, under multi-stress scenarios due to environmental variability (e.g., light intensity, water flow, and temperature) and metal pollution (Zn, Mn and Fe). The annual monitoring study was performed at three sites according to their water and biofilm metal concentrations. Metal concentration was affected by water flow due to dilution. Low flow led to higher dissolved Zn concentrations, and thus to higher Zn accumulation in the biofilm. Water temperature, light intensity and phosphate concentration were the environmental factors which determined the seasonality of biofilm responses, whereas dissolved Zn and Zn accumulation in biofilms were the parameters linked to sites and periods of highest metal pollution. Community algal succession, from diatoms in cold conditions to green algae in warm conditions, was clearer in the non metal-polluted site than in those metal-polluted, presumably due to the selection pressure exerted by metals. Most AEA were related with seasonal environmental variability at the sites with low or no-metal pollution, except glutathione-S-transferase (GST) which was related with Zn (dissolved and accumulated in biofilm) pollution occurring at the most polluted site. We can conclude that seasonal variations of community composition and function are masked by metal pollution. From this study we suggest the use of a multi-biomarker approach, including AEA and a set of biological and physicochemical parameters as an effect-based field tool to assess metal pollution.

Modelling the effects of dispersal mechanisms and hydrodynamic regimes upon the structure of microbial communities within fluvial biofilms

The spatial distribution of microbial taxa is determined primarily by physical and chemical environments and by dispersal. In a homogeneous landscape with limited dispersal, the similarity in abundance of taxa in samples declines with separation distance. We present a one-dimensional model for the spatial autocorrelation in abundances arising from immigration from some remote community and dispersal between environmentally similar landscape patches. Spatial correlation in taxa abundances were calculated from biofilms from the beds of two flumes which differed only in their bedform profiles; one flat and the other a periodic sawtooth shape. The hydraulic regime is approximately uniform over the flat bed, whereas the sawtooth induces fast flow over the peaks and recirculation in the troughs. On the flat bed, the correlation decline between samples was reproduced by a model using one biologically reasonable parameter. A decline was apparent in the other flume; however, a better fit was achieved when dispersal was not assumed constant everywhere. However, analysis of finer-resolution data for the heterogeneous flume suggested even this model did not adequately capture the community's complexity. We conclude that hydrodynamics are a strong driver of taxa-abundance patterns in stream biofilms. However, local adaptability must also be considered to build up a complete mechanistic model.

Light History Influences the Response of Fluvial Biofilms to Zn Exposure.

Fluvial biofilms are subject to multistress situations in natural ecosystems, such as the co-occurrence of light intensity changes and metal toxicity. However, studies simultaneously addressing both factors are rare. This study evaluated in microcosm conditions the relationship between short-term light intensity changes and Zn toxicity on fluvial biofilms with long-term photoacclimation to different light conditions. Biofilms that had long-term photoacclimation to 25μmol photons·m(-2) · s(-1) (low light [LL] biofilms), 100μmol photons· m(-2) ·s(-1) (medium light [ML] biofilms), and 500μmol photons·m(-2) ·s(-1) (high light [HL] biofilms) were characterized by different structural (Chlorophyll-a [Chl-a], total biomass-AFDW, EPS, algal groups, and diatom taxonomy) and physiological attributes (ETR-I curves and photosynthetic pigments). HL biofilms showed higher light saturation intensity and a higher production of xanthophylls than LL biofilms. In contrast, LL biofilms had many structural differences; a higher proportion of diatoms and lower AFDW and EPS contents than ML and HL biofilms. A clear effect of light intensity changes on Zn toxicity was also demonstrated. Zn toxicity was enhanced when a sudden increase in light intensity also occurred, mainly with LL biofilms, causing higher inhibition of both the Φ'PSII and the ΦPSII . A decoupling of NPQ from de-epoxidation reaction (DR) processes was also observed, indicating substantial damage to photoprotective mechanisms functioning in biofilms (i.e., xanthophyll cycle of diatoms) due to Zn toxicity. This study highlights the need to take into account environmental stress (e.g., light intensity changes) to better assess the environmental risks of chemicals (e.g., metals).

Antioxidant enzyme activities as biomarkers of Zn pollution in fluvial biofilms

The potential of the antioxidant enzyme catalase (CAT) and ascorbate peroxidase (APX) as molecular biomarkers of Zn toxicity in freshwater biofilms has been explored in this study jointly with other classical functional and structural endpoints (photosynthetic parameters, algal group composition and bioaccumulation). Biofilms were colonized in an indoor microcosm system for 5 weeks and then exposed to Zn for 5 weeks. To evaluate Zn effects, biofilms were sampled 5 and 3 days before exposure, just before exposure (time 0), and after 6h, 1, 3, 7, 21 and 35 days of metal exposure. Most endpoints measured were affected by Zn exposure (320μgZnL−1) during both periods of exposure. APX was the only functional parameter responding after a few hours of Zn exposure, highlighting its use as an early toxicity biomarker. Structural changes began after 3 days of exposure, starting with a decrease in algal biomass and an increase in the OD 430:665 ratio. Structural changes in biofilm communities were observed after 1 week, leading to a shift from diatoms to cyanobacteria and green algae-dominated communities. CAT activity was thereafter enhanced (after three weeks of exposure) and attributed not only to a direct effect of Zn bioaccumulation but also to an indirect effect of the community composition changes driven by chronic metal exposure. It can be concluded that biofilm antioxidant enzyme activities may provide evidence of early stress caused by metal exposure and also provide information about the mechanism of community adaptation. This information can be of great interest to improve current tools used for risk assessment.

Enhanced co-tolerance and co-sensitivity from long-term metal exposures of heterotrophic and autotrophic components of fluvial biofilms

Understanding the interactive effects of multiple stressors on ecosystems has started to become a major concern. The aim of our study was therefore to evaluate the consequences of a long-term exposure to environmental concentrations of Cu, Zn and As on the pollution-induced community tolerance (PICT) of lotic biofilm communities in artificial indoor channels. Moreover, the specificity of the PICT was assessed by evaluating the positive and negative co-tolerance between these metals. Photosynthetic efficiency and substrate-induced respiration (SIR), targeting the autotrophic and heterotrophic communities respectively were used in short-term inhibition bioassays with Cu, Zn and As to assess sensitivities of pre-exposed biofilms to the metals tested. Diversity profiles of a phototrophic, eukaryotic and prokaryotic community in biofilms following the different treatments were determined and analyzed with principal component analysis. The results demonstrated that pre-exposure to metals induced structural shifts in the community and led to tolerance enhancements in the phototrophic and heterotrophic communities. On the other hand, whatever the functional parameter used (i.e. photosynthesis and SIR), communities exposed to Cu were more tolerant to Zn and vice versa. Furthermore, only phototrophic communities pre-exposed to As developed tolerance to Cu but not to Zn, whereas no co-tolerance between Cu and As was observed in the heterotrophic communities. Finally, phototrophic and heterotrophic communities exposed to Cu and Zn became more sensitive to As, reflecting a negative co-tolerance between these metals. Overall, our findings support the fact that although the mode of action of the different metals is an important driver for the structure and thus the tolerance of the communities, it appears that the detoxification modes are the most important factors for the occurrence of positive or negative co-tolerance.

In situ spatio-temporal changes in pollution-induced community tolerance to zinc in autotrophic and heterotrophic biofilm communities

Pollution-induced community tolerance (PICT) uses increased tolerance in populations at contaminated sites as an indicator of contaminant effects. However, given the broad structural and functional complexity that characterizes biological communities, the acquisition of PICT could vary with (i) target community, (ii) intensity of toxicant exposure, (iii) the species succession stage, and (iv) the physicochemical characteristics of the studied site. To assess the spatio-temporal changes of zinc-induced tolerance in fluvial biofilm communities, we conducted an in situ study in Osor River (North-East Catalonia, Spain), which has zinc contamination. Biofilms were developed for 5 weeks in a non-metal-polluted site, and were then transferred to different sites in Osor River with different levels of zinc contamination. The spatio-temporal changes of biofilm PICT to zinc was determined using photosynthetic activity bioassays and respiration-induced aerobic bioassays at T(0), and at 1, 3 and 5 weeks of exposure. We also performed physicochemical characterization of the sites, taxonomic analysis of diatoms, bacterial and fungal diversity and profiled pigments of phototrophic communities. We used multivariate ordination to analyze results. In addition to natural species succession, the intensity of metal pollution exerted structural pressure by selecting the most metal-tolerant species, but differently depending on the type of biofilm. Zn-tolerance values indicated that exposure to high levels of zinc had effects that were similar to a longer exposure to lower levels of zinc.

Chl-a fluorescence parameters as biomarkers of metal toxicity in fluvial biofilms: an experimental study

A study was carried out to evaluate the sensitivity of different chlorophyll-a (chl-a) fluorescence parameters measured in freshwater biofilms as metal pollution biomarkers of short- and long-term metal exposures at environmentally realistic concentrations. A microcosm experiment was performed using indoor channels. Mature biofilms were exposed from hours to weeks to three different treatments: No-Metal, Zn (400 μg l−1); and Zn plus Cd (400 μg l−1 and 20 μg l−1, respectively). Metal concentration was based on a real case study: the Riou-Mort River (France). Biofilms exposed to Zn bioaccumulated similar Zn contents per dry weight to those exposed to the mixture (Zn plus Cd) causing a similar inhibition of the effective quantum yield $$(\Upphi_{\text{M}}^{\prime})$$ during the first hours of exposure. A reduction of the algal biomass, a shift in the community composition (a high reduction of diatoms), a reduction of the maximal quantum yield (ΦM) and a strong reduction of non-photochemical quenching (NPQ) were observed from day 14 until the end of the experiment (35 days). The results indicate that the effects of the metal mixture present in the Riou-Mort on biofilms could be attributed to Zn toxicity. The use of a set of chl-a fluorescence measurements, including photochemical and NPQ parameters, are recommended as a reliable biomarker tool box to evaluate both short- and long-term effects of metals on biofilms containing oxygenic photoautotrophs, suggesting its use in field applications.

Catalase in fluvial biofilms: a comparison between different extraction methods and example of application in a metal-polluted river

Antioxidant enzymes are involved in important processes of cell detoxification during oxidative stress and have, therefore, been used as biomarkers in algae. Nevertheless, their limited use in fluvial biofilms may be due to the complexity of such communities. Here, a comparison between different extraction methods was performed to obtain a reliable method for catalase extraction from fluvial biofilms. Homogenization followed by glass bead disruption appeared to be the best compromise for catalase extraction. This method was then applied to a field study in a metal-polluted stream (Riou Mort, France). The most polluted sites were characterized by a catalase activity 4-6 times lower than in the low-polluted site. Results of the comparison process and its application are promising for the use of catalase activity as an early warning biomarker of toxicity using biofilms in the laboratory and in the field.

Discharge and the response of biofilms to metal exposure in Mediterranean rivers

The expected response of fluvial biofilms to the environment and metal pollution prevailing under different discharge conditions was investigated. The relationship between inter-annual hydrological variability and metal concentration in water and sediments was explored in Mediterranean rivers (Catalonia, NE Spain) affected by low but chronic metal pollution, using monitoring data provided by the Catalan Water Agency (ACA). During the period investigated (2000–2006), metal pollution was characterized by low water concentrations and high concentrations in sediments. The most consistent pattern was observed for sediment cadmium (Cd) concentrations, showing a positive relationship with annual discharge, reaching values of environmental concern (above ecotoxicological benchmarks). A different pattern was observed for Cu, Zn, and As increasing with flow in some sites and decreasing in others. While Cd seems to proceed from diffuse sources being washed by surface runoff, Zn, Pb, and As may proceed from either diffuse or point-sources in the different river sites investigated. The relevance of diffuse metal pollution in the area of study indicates that polluted landfills runoff might be an important source of metals causing repetitive pulses of high metal concentration in the receiving water courses. The experimental results presented demonstrate that metal effects in fluvial biofilms may be accumulative, increasing the toxicity after repetitive pulse exposures. Since draughts and extreme rain events are expected to increase at higher latitudes due to global change, the sources of metal pollution, its final concentration and potential effects on the fluvial ecosystem may also change following the patterns expected for human-impacted Mediterranean rivers.

Does Grazing Pressure Modify Diuron Toxicity in a Biofilm Community?

Herbicides affect the structure and functional parameters of fluvial biofilm. Diuron is toxic to primary producers and disrupts endocrine activity. Here, we studied the interaction between this toxicant and several biological compartments in a simple food chain composed of herbivores (the snail Physella [Costatella] acuta) and biofilm. We used indoor experimental channels to which Diuron was added at a realistic concentration (2 mug/L). Bacterial survival and chlorophyll-a and photosynthetic activity were analyzed in the biofilm. We monitored biomass, mortality, reproduction, and motility as end points in the freshwater snail P. acuta. Our results showed that bacterial survival and photosynthetic activity were sensitive to Diuron. Snails were not affected by the herbicide at the concentration tested. No significant interactions between the toxicant and grazers were observed on the biofilm. Reproductive traits, however, were slightly affected, indicating a possible endocrine disruption.

Fluvial biofilms: A pertinent tool to assess β-blockers toxicity

Among increasingly used pharmaceutical products, β-blockers have been commonly reported at low concentrations in rivers and littoral waters of Europe and North America. Little is known about the toxicity of these chemicals in freshwater ecosystems while their presence may lead to chronic pollution. Hence, in this study the acute toxicity of 3 β-blockers: metoprolol, propranolol and atenolol on fluvial biofilms was assessed by using several biomarkers. Some were indicative of potential alterations in biofilm algae (photosynthetic efficiency), and others in biofilm bacteria (peptidase activity, bacterial mortality). Propranolol was the most toxic β-blocker, mostly affecting the algal photosynthetic process. The exposure to 531 μg/L of propranolol caused 85% of inhibition of photosynthesis after 24 h. Metoprolol was particularly toxic for bacteria. Though estimated No-Effect Concentrations (NEC) were similar to environmental concentrations, higher concentrations of the toxic (503 μg/L metoprolol) caused an increase of 50% in bacterial mortality. Atenolol was the least toxic of the three tested β-blockers. Effects superior to 50% were only observed at very high concentration (707 mg/L). Higher toxicity of metoprolol and propranolol might be due to better absorption within biofilms of these two chemicals. Since β-blockers are mainly found in mixtures in rivers, their differential toxicity could have potential relevant consequences on the interactions between algae and bacteria within river biofilms.

Primary and complex stressors in polluted mediterranean rivers: Pesticide effects on biological communities

Summary We examined the presence of pesticides in the Llobregat river basin (Barcelona, Spain) and their effects on benthic biological communities (invertebrates and diatoms). The Llobregat river is one of Barcelona’s major drinking water resources. It has been highly polluted by industrial, agricultural, and urban wastewaters, and—as a typical Mediterranean river—is regularly subjected to periodic floods and droughts. Water scarcity periods result in reduced water flow and dilution capacity, increasing the potential environmental risk of pollutants. Seven sites were selected, where we analysed the occurrence of 22 pesticides (belonging to the classes of triazines, organophosphates, phenylureas, anilides, chloroacetanilides, acidic herbicides and thiocarbamates) in the water and sediment, and the benthic community structure. Biofilm samples were taken to measure several metrics related to both the algal and bacterial components of fluvial biofilms. Multivariate analyses revealed a potential relationship between triazine-type herbicides and the distribution of the diatom community, although no evidence of disruption in the invertebrate community distribution was found. Biofilm metrics were used as response variables rather than abundances of individual species to identify possible cause-effect relationships between pesticide pollution and biotic responses. Certain effects of organophosphates and phenylureas in both structural and functional aspects of the biofilm community were suggested, but the sensitivity of each metric to particular stressors must be assessed before we can confidently assign causality. Complemented with laboratory experiments, which are needed to confirm causality, this approach could be successfully incorporated into environmental risk assessments to better summarise biotic integrity and improve the ecological management.

Effects of chronic copper exposure on fluvial systems: Linking structural and physiological changes of fluvial biofilms with the in-stream copper retention

Long-term metal exposure is known to be responsible for a large variety of structural and functional changes in periphyton communities which allow these communities to adapt to metal-polluted conditions. This study aimed to link the changes that chronic copper (Cu) exposure causes on the structure and physiology of fluvial biofilms with the efficiency of the river systems in retaining phosphate and Cu. The effects of a chronic Cu exposure on the structure, physiology and induction of Cu tolerance of the community were evaluated by comparing this community with a non-exposed one. Results showed that periphyton chronically exposed to Cu had lower algal biomass, higher proportion of green algae, lower proportion of brown algae, and higher EPS content per unit of biomass than the un-exposed community. In addition, the chronically-exposed community showed a Cu content (both total and intracellular Cu content) ten times higher than the un-exposed community. While in-stream phosphate retention was not markedly influenced by chronic Cu exposure; Cu retention was clearly reduced, as was shown by a reduction in Cu retention efficiency (Cu– S w) and demand (Cu–Vf). The chronically-exposed periphyton, in spite of having high intracellular Cu concentration, showed similar photosynthetic efficiency than the un-exposed community and showed a higher Cu tolerance. It indicated that this community was acclimatized to Cu exposure and that this acclimatization was probably linked to the ability to detoxify and immobilize metals. These observations suggest that the fate of Cu in fluvial ecosystems will be influenced by the exposure history of the system. The results from this study indicate that metals will travel longer distances in metal-polluted streams compared to pristine systems having effects on water quality farther downstream.

Measuring in-stream retention of copper by means of constant-rate additions

Human practices entail inputs of nutrients and toxicants such as heavy metals to the fluvial ecosystems. While nutrient dynamics in fluvial ecosystems have been widely studied for over three decades, dynamics of toxicants still remain unclear. In this investigation, the nutrient spiraling concept and associated methodologies to quantify nutrient retention in streams were applied to study copper (Cu) dynamics in streams. The present study aimed to quantify total dissolved Cu retention using a simplified system of indoor channels colonized with fluvial biofilms. Cu retention was studied at sub-toxic concentrations to avoid negative/lethal effects on biota. In addition, Cu retention was compared with retention estimates of a macronutrient, phosphate (PO 4 3−), which has been widely studied within the context of the nutrient spiraling concept. The methodology used allowed a successful quantification of Cu and PO 4 3− retention. The results showed higher retention efficiency for PO 4 3− than for Cu. The biofilm played a key role in retaining both solutes. Although retention efficiency for both solutes was higher in the experiments with colonized substrata compared to uncolonized substrata, we found a positive relationship between uptake rate and chlorophyll- a only for PO 4 3−. Finally, retention efficiency for both solutes was influenced by water discharge, showing lower retention efficiencies under higher flow conditions. These results suggest that the fate and toxic effects of copper on stream biota may be strongly influenced by the prevailing environmental conditions. Our results indicate that the experimental approach considered can provide new insights into the investigation of retention of toxic compounds in fluvial systems and their controlling mechanisms.

Use of multivariate analyses to investigate the contribution of metal pollution to diatom species composition: search for the most appropriate cases and explanatory variables

The aim of this study was to elucidate how the spatial scale and the set of variables included influence our ability to detect the effects of different types of pollution on the biota. Using variance partitioning analysis, we assessed the individual importance of a set of environmental factors (eutrophication and organic pollution) versus metal level pollution on the community structure of diatom assemblages at different spatial scales. At regional scale, environmental factors did not explain more of the variance compared to the watershed study. The results of the watershed scale field survey indicate that diatom community composition was influenced by low metal concentrations, but this pattern was only observed by the inclusion of biofilm metal concentration data. We recommend the analysis of metal traces in the water phase and the biota (fluvial biofilms) as well as the use of the Diffusive Gradient in Thin films (DGT) technique to characterize low metal level pollution in freshwater systems.