Current water quality guidelines across North America and Europe do not protect lakes from salinization.

Human-induced salinization caused by the use of road deicing salts, agricultural practices, mining operations, and climate change is a major threat to the biodiversity and functioning of freshwater ecosystems. Yet, it is unclear if freshwater ecosystems are protected from salinization by current water quality guidelines. Leveraging an experimental network of land-based and in-lake mesocosms across North America and Europe, we tested how salinization-indicated as elevated chloride (Cl-) concentration-will affect lake food webs and if two of the lowest Cl- thresholds found globally are sufficient to protect these food webs. Our results indicated that salinization will cause substantial zooplankton mortality at the lowest Cl- thresholds established in Canada (120 mg Cl-/L) and the United States (230 mg Cl-/L) and throughout Europe where Cl- thresholds are generally higher. For instance, at 73% of our study sites, Cl- concentrations that caused a ≥50% reduction in cladoceran abundance were at or below Cl- thresholds in Canada, in the United States, and throughout Europe. Similar trends occurred for copepod and rotifer zooplankton. The loss of zooplankton triggered a cascading effect causing an increase in phytoplankton biomass at 47% of study sites. Such changes in lake food webs could alter nutrient cycling and water clarity and trigger declines in fish production. Current Cl- thresholds across North America and Europe clearly do not adequately protect lake food webs. Water quality guidelines should be developed where they do not exist, and there is an urgent need to reassess existing guidelines to protect lake ecosystems from human-induced salinization.

Biofilm phosphorus uptake capacity as a tool for the assessment of pollutant effects in river ecosystems.

Biofilms are a key component in the nutrient removal from the water column. However, nutrient uptake by biofilms may be hampered by the occurrence of pollutants or other stressors. This study aimed: (i) to investigate the biofilm phosphorus (P) uptake capacity as a relevant process for the maintenance of fluvial water quality and (ii) to explore the sensitivity of this process to different chemical and environmental stressors. We conducted chamber experiments to test for the relevance of biofilm P uptake capacity (PUC) as a tool to detect effects of pollutants on river self-depuration. PUC was calculated by measuring P temporal decay after performing controlled P-spikes in chambers with biofilm-colonized tiles. Four different experiments were conducted to evaluate the response of PUC to: (a) several river waters from increasing polluted sites; (b) the effect of the bactericide triclosan (TCS); (c) the combined effect of TCS and grazers; and (d) the effect of TCS after a drought episode that affected the biofilms. These experiments showed that biofilms decreased their PUC along the pollution gradient. The biofilm PUC was significantly reduced after receiving high TCS concentrations, though lower TCS concentrations also affected the biofilm when this was submitted to grazing pressure. PUC decrease was induced by flow interruption which further enhanced the TCS negative effects. Overall, PUC was sensitive to the effects of pollutants like TCS as well as to the action of biological (grazing) and environmental (drought) factors. The study also showed that multiple stressors enhance the negative effects of pollutants on the PUC of biofilms. Our study values the use of biofilms' PUC as a sensitive ecological-based tool to assess the effects of chemicals on freshwater communities and their derived functioning in river ecosystems.

Stream drying drives microbial ammonia oxidation and first-flush nitrate export.

Stream microbial communities and associated processes are influenced by environmental fluctuations that may ultimately dictate nutrient export. Discharge fluctuations caused by intermittent stream flow are increasing worldwide in response to global change. We examined the impact of flow cessation and drying on in-stream nitrogen cycling. We determined archaeal (AOA) and bacterial ammonia oxidizer (AOB) abundance and ammonia oxidation activity in surface and deep sediments from different sites along the Fuirosos stream (Spain) subjected to contrasting hydrological conditions (i.e., running water, isolated pools, and dry streambeds). AOA were more abundant than AOB, with no major changes across hydrological conditions or sediment layers. However, ammonia oxidation activity and sediment nitrate content increased with the degree of stream drying, especially in surface sediments. Upscaling of our results shows that ammonia oxidation in dry streambeds can contribute considerably (~50%) to the high nitrate export typically observed in intermittent streams during first-flush events following flow reconnection. Our study illustrates how the dry channels of intermittent streams can be potential hotspots of ammonia oxidation. Consequently, shifts in the duration, spatial extent and severity of intermittent flow can play a decisive role in shaping nitrogen cycling and export along fluvial networks in response to global change.

Small-scale heterogeneity of microbial N uptake in streams and its implications at the ecosystem level.

Large-scale factors associated with the environmental context of streams can explain a notable amount of variability in patterns of stream N cycling at the reach scale. However, when environmental factors fail to accurately predict stream responses at the reach level, focusing on emergent properties from small-scale heterogeneity in N cycling rates may help understand observed patterns in stream N cycling. To address how small-scale heterogeneity may contribute to shape patterns in whole-reach N uptake, we examined the drivers and variation in microbial N uptake at small spatial scales in two stream reaches with different environmental constraints (i.e., riparian canopy). Our experimental design was based on two ¹5N additions combined with a hierarchical sampling design from reach to microhabitat scales. Regardless of the degree of canopy cover, small-scale heterogeneity of microbial N uptake ranged by three orders of magnitude, and was characterized by a low abundance of highly active microhabitats (i.e., hot spots). The presence of those hot spots of N uptake resulted in a nonlinear spatial distribution of microbial N uptake rates within the streambed, especially in the case of epilithon assemblages. Small-scale heterogeneity in N uptake and turnover rates at the microhabitat scale was primarily driven by power relationships between N cycling rates and stream water velocity. Overall, fine benthic organic matter (FBOM) assemblages responded clearly to changes in the degree of canopy cover, overwhelming small-scale heterogeneity in its N uptake rates, and suggesting that FBOM contribution to whole-reach N uptake was principally imposed by environmental constraints from larger scales. In contrast, N uptake rates by epilithon showed no significant response to different environmental influences, but identical local drivers and spatial variation in each study reach. Therefore, contribution of epilithon assemblages to whole-reach N uptake was mainly associated with emerging properties from small-scale heterogeneity at lower spatial scales.

Exposure and recovery: The effect of different dilution factors of treated and untreated metal mining effluent on freshwater biofilm function and structure.

Abandoned mines generate effluents rich in heavy metals, and these contaminants are released uncontrolled into the nearby aquatic ecosystems, causing severe pollution. However, no real solution exists, leaving a legacy of global pollution. In this study, the efficiency of the treatment technologies in reducing the ecological impacts of mining effluents to freshwater ecosystems with different dilution capacities was tested using biofilm communities as biological indicators. The functional and structural recovery capacity of biofilm communities after 21 days of exposure was assessed. With this aim, we sampled aquatic biofilms from a pristine stream and exposed them to treated (T) and untreated (U) metal mining effluent from Frongoch abandoned mine (Mid Wales, UK). Additionally, we simulated two different flow conditions for the receiving stream: high dilution (HD) and low dilution (LD). After exposure, the artificial streams were filled with artificial water for 14 days to assess the biofilm recovery. Unexposed biofilm served as control for biofilm responses (functional and structural) measured throughout time. During the exposure, short term effects on biofilm functioning (photosynthetic efficiency, nutrient uptake) were observed in T-LD, U-HD, and U-LD, whereas long term effects (community composition, chl-a, and diatom metrics) were observed on the structure of all biofilms exposed to the treated and untreated mining effluent. On the other hand, metal accumulation occurred in biofilms exposed to the mining effluents. However, a functional recovery was observed for all treatments, except in the U-LD in which biofilm structure did not present a significant recovery after the exposure period. The results presented here highlight the need to consider the dilution capacity of the receiving stream to assess the real efficiency of treatment technologies applied to mining effluents to mitigate the ecological impact on freshwater ecosystems.

Nature-based bioreactors: Tackling antibiotic resistance in urban wastewater treatment.

The overuse and misuse of antibiotics have accelerated the selection of antibiotic-resistant bacteria, significantly impacting human, animal, and environmental health. As aquatic environments are vulnerable to antibiotic resistance, suitable management practices should be adopted to tackle this phenomenon. Here we show an effective, nature-based solution for reducing antibiotic resistance from actual wastewater. We utilize a bioreactor that relies on benthic (biofilms) and planktonic microbial communities to treat secondary effluent from a small urban wastewater treatment plant (<10,000 population equivalent). This treated effluent is eventually released into the local aquatic ecosystem. We observe high removal efficiency for genes that provide resistance to commonly used antibiotic families, as well as for mobile genetic elements that could potentially aid in their spread. Importantly, we notice a buildup of sulfonamide (sul1 and sul2) and tetracycline (tet(C), tet(G), and tetR) resistance genes specifically in biofilms. This advancement marks the initial step in considering this bioreactor as a nature-based, cost-effective tertiary treatment option for small UWWTPs facing antibiotic resistance challenges.

Public perception vs ecological quality status: Examining the ecological restoration of the Congost River's Environment.

This study examines the public's perceptions of the ecological restoration of the Congost River over the past thirty years, focusing on the period between 2010 and 2022. We conducted a survey of 112 river users across five key zones identified through a pilot study for their high pedestrian density, aiming to analyse how different sociodemographic groups perceive the river's ecological state. A structured questionnaire was distributed along both sides of the river to engage a diverse range of individuals typically utilizing the river environment. The collected data were analysed using regression models and Mann-Whitney U tests to assess differences between groups, with Bonferroni adjustments applied to control for multiple comparisons. The results reveal a broad increase in appreciation for the river since 2010, alongside measurable ecological improvements supported by scientific data. Despite these positive changes, a majority of surveyed users remain sceptical about the river's recovery, with less pronounced scepticism among older respondents, those with higher education, and environmental volunteers. These groups' perceptions align more closely with empirical evidence, highlighting the influence of sociodemographic factors on environmental awareness. Individuals living closer to natural settings and frequent river visitors were found to be more attuned to changes in the river's environment, particularly in aesthetic and sensory aspects. The study underscores the persistence of a perceptual gap between scientific assessments of ecological health and public sentiment, emphasizing the complex relationship between community perceptions and objective environmental indicators. These insights underline the complex relationship between community perceptions and objective environmental indicators, reflecting a broader trend in environmental awareness and the importance of factual communication in ecological issues.

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.

Dissipation of pesticides by stream biofilms is influenced by hydrological histories.

To evaluate the effects of hydrological variability on pesticide dissipation capacity by stream biofilms, we conducted a microcosm study. We exposed biofilms to short and frequent droughts (daily frequency), long and less frequent droughts (weekly frequency) and permanently immersed controls, prior to test their capacities to dissipate a cocktail of pesticides composed of tebuconazole, terbuthylazine, imidacloprid, glyphosate and its metabolite aminomethylphosphonic acid. A range of structural and functional descriptors of biofilms (algal and bacterial biomass, extracellular polymeric matrix (EPS) concentration, microbial respiration, phosphorus uptake and community-level physiological profiles) were measured to assess drought effects. In addition, various parameters were measured to characterise the dynamics of pesticide dissipation by biofilms in the different hydrological treatments (% dissipation, peak asymmetry, bioconcentration factor, among others). Results showed higher pesticide dissipation rates in biofilms exposed to short and frequent droughts, despite of their lower biomass and EPS concentration, compared to biofilms in immersed controls or exposed to long and less frequent droughts. High accumulation of hydrophobic pesticides (tebuconazole and terbuthylazine) was measured in biofilms despite the short exposure time (few minutes) in our open-flow microcosm approach. This research demonstrated the stream biofilms capacity to adsorb hydrophobic pesticides even in stressed drought environments.

Experimental assessment of salinization effects on freshwater zooplankton communities and their trophic interactions under eutrophic conditions.

Freshwater ecosystems are becoming saltier due to human activities. The effects of increased salinity can lead to cascading trophic interactions, affecting ecosystem functioning and energy transfer, through changes in community and size structure. These effects can be modulated by other environmental factors, such as nutrients. For example, communities developed under eutrophic conditions could be less sensitive to salinization due to cross-tolerance mechanisms. In this study, we used a mesocosm approach to assess the effects of a salinization gradient on the zooplankton community composition and size structure under eutrophic conditions and the cascading effects on algal communities. Our results showed that zooplankton biomass, size diversity and mean body size decreased with increased chloride concentration induced by salt addition. This change in the zooplankton community did not have cascading effects on phytoplankton. The phytoplankton biomass decreased after the chloride concentration threshold of 500 mg L-1 was reached, most likely due to direct toxic effects on the osmotic regulation and nutrient uptake processes of certain algae rather than as a response to community turnover or top-down control. Our study can help to put in place mitigation strategies for salinization and eutrophication, which often co-occur in freshwater ecosystems.

Hypersaline mining effluents affect the structure and function of stream biofilm.

The salinisation of freshwater ecosystems is a global environmental problem that threatens biodiversity, ecosystem functioning and human welfare. The aim of this study was to investigate the potential impact of a realistic salinity gradient on the structure and functioning of freshwater biofilms. The salinity gradient was based on the real ion concentration of a mining effluent from an abandoned mine in Germany. We exposed biofilm from a pristine stream to 5 increasing salinities (3 to 100 g L-1) under controlled conditions in artificial streams for 21 days. We evaluated its functional (photosynthetic efficiency, nutrient uptake, and microbial respiration) and structural responses (community composition, algal biomass and diatom, cyanobacteria and green algae metrics) over time. Then we compared their responses with an unexposed biofilm used as control. The functionality and structure of the biofilm exposed to the different salinities significantly decreased after short-term and long-term exposure, respectively. The community composition shifted to a new stable state where the most tolerant species increased their abundances. At the same time, we observed an increase in the community tolerance (measured as Pollution-Induced Community Tolerance) along the salinity gradient. This study provides relevant information on the salt threshold concentrations that can substantially damage algal cells (i.e., between 15 and 30 g L-1). The results provide new insights regarding the response and adaptation of stream biofilm to salinity and its potential implications at the ecosystem level.

Effects of an hypersaline effluent from an abandoned potash mine on freshwater biofilm and diatom communities.

Potash abandoned mines cause severe environmental damage to their bordering environment, with significant impacts on freshwater ecosystems mostly through uncontrolled discharge of hypersaline effluents. This study aimed to evaluate the ecological impact caused by a hypersaline effluent from an abandoned potash mine (Menteroda, Germany) on freshwater biofilms and, specifically, on diatom communities. Biofilm from a pristine stream was exposed under controlled conditions in microcosms to a mining effluent (ME), and its structural (algal biomass, community composition, diatom metrics) and functional (photosynthetic activity, nutrient uptake) responses were evaluated over time and compared with unexposed biofilms used as control. Biofilm exposed to ME showed drastic functional responses after one day of exposure, with a significant decrease in photosynthetic efficiency and nutrient uptake, that were recovered over time. Biofilm exposed to ME showed a progressive increase in diatom metrics (abundance, density and growth rate) over time, compared to the control. However, a significant decrease in diatom species diversity, richness and cell size was also observed in biofilm exposed to ME. This study revealed that the ME affected the biofilm causing short-term functional responses, which were recovered simultaneously with a drastic diatom community structure shift.

Driving Factors of Geosmin Appearance in a Mediterranean River Basin: The Ter River Case.

In recent decades, human activity coupled with climate change has led to a deterioration in the quality of surface freshwater. This has been related to an increase in the appearance of algal blooms, which can produce organic compounds that can be toxic or can affect the organoleptic characteristics of the water, such as its taste and odor. Among these latter compounds is geosmin, a metabolite produced by certain cyanobacteria that confers an earthy taste to water and which can be detected by humans at very low concentrations (nanogram per liter). The difficulty and cost of both monitoring the presence of this compound and its treatment is a problem for drinking water treatment companies, as the appearance of geosmin affects consumer confidence in the quality of the drinking water they supply. In this field study, the evaluation of four sampling sites with different physicochemical conditions located in the upper part of the Ter River basin, a Mediterranean river located in Catalonia (NE Spain), has been carried out, with the aim of identifying the main triggers of geosmin episodes. The results, obtained from 1 year of sampling, have made it possible to find out that: (i) land uses with a higher percentage of agricultural and industrial activity are related to high nutrient conditions in river water, (ii) these higher nutrient concentrations favor the development of benthic cyanobacteria, (iii) in late winter-early spring, when these cyanobacteria are subjected to both an imbalance of the dissolved inorganic nitrogen and soluble reactive phosphorus ratio, guided by a phosphorus concentration increase, and to cold-mild temperatures close to 10°C, they produce and release geosmin, and (iv) 1-2 weeks after cyanobacteria reach a high relative presence in the whole biofilm, an increase in geosmin concentration in water is observed, probably associated with the cyanobacteria detachment from cobbles and consequent cell lysis. These results could serve as a guide for drinking water treatment companies, indicating under what conditions they can expect the appearance of geosmin episodes and implement the appropriate treatment before it reaches consumers' tap.

Effects of the interaction between nutrient concentration and DIN:SRP ratio on geosmin production by freshwater biofilms.

The global increase of cyanobacterial blooms occurrence has been associated with the presence of compounds that generate earthy and musty odor in freshwater systems, among which geosmin stands out. The lack of information on the factors associated to geosmin production by benthic organisms has driven the development of this study, whose main goal is to determine the effects of nutrient concentration and DIN:SRP ratio on geosmin formation and release. The experiment was performed in 18 microcosms under controlled conditions for 21 days, using a natural biofilm suspension from Ter river (NE, Spain) to promote biofilm settlement. Six treatments were set crossing three DIN:SRP ratios (A = 4:1, B = 16:1 and C = 64:1) with two nutrient concentrations (Low and High). After 7 days of experiment, geosmin was detected in biofilm, being higher under high nutrient concentration and low DIN:SRP ratio conditions. In this treatment, geosmin in biofilm reached its maximum concentration at day 16 (3.8 ± 0.9 ng/mg), decreasing at the end of the experiment (21d) due to cyanobacteria detachment and geosmin release into the water (136 ± 6 ng/L). Overall, this experimental study showed that high nutrient concentration and low DIN:SRP ratio favored the Oscillatoria genus development within biofilm communities, generating the optimal conditions for geosmin production. The interaction between these two factors was demonstrated to be a potential driver of benthic geosmin production and release, and should be monitored and controlled in rivers exploited for drinking water purposes.

Development of a Heavy Metal Sensing Boat for Automatic Analysis in Natural Waters Utilizing Anodic Stripping Voltammetry.

Determination of the levels of heavy metal ions would support assessment of sources and pathways of water pollution. However, traditional spatial assessment by manual sampling and off-site detection in the laboratory is expensive and time-consuming and requires trained personnel. Aiming to fill the gap between on-site automatic approaches and laboratory techniques, we developed an autonomous sensing boat for on-site heavy metal detection using square-wave anodic stripping voltammetry. A fluidic sensing system was developed to integrate into the boat as the critical sensing component and could detect ≤1 µg/L Pb, ≤6 µg/L Cu, and ≤71 µg/L Cd simultaneously in the laboratory. Once its integration was completed, the autonomous sensing boat was tested in the field, demonstrating its ability to distinguish the highest concentration of Pb in an effluent of a galena-enriched mine compared to those at other sites in the stream (Osor Stream, Girona, Spain).

Lifestyle preferences drive the structure and diversity of bacterial and archaeal communities in a small riverine reservoir.

Spatial heterogeneity along river networks is interrupted by dams, affecting the transport, processing, and storage of organic matter, as well as the distribution of biota. We here investigated the structure of planktonic (free-living, FL), particle-attached (PA) and sediment-associated (SD) bacterial and archaeal communities within a small reservoir. We combined targeted-amplicon sequencing of bacterial and archaeal 16S rRNA genes in the DNA and RNA community fractions from FL, PA and SD, followed by imputed functional metagenomics, in order to unveil differences in their potential metabolic capabilities within the reservoir (tail, mid, and dam sections) and lifestyles (FL, PA, SD). Both bacterial and archaeal communities were structured according to their life-style preferences rather than to their location in the reservoir. Bacterial communities were richer and more diverse when attached to particles or inhabiting the sediment, while Archaea showed an opposing trend. Differences between PA and FL bacterial communities were consistent at functional level, the PA community showing higher potential capacity to degrade complex carbohydrates, aromatic compounds, and proteinaceous materials. Our results stressed that particle-attached prokaryotes were phylogenetically and metabolically distinct from their free-living counterparts, and that performed as hotspots for organic matter processing within the small reservoir.

Water Flow and Light Availability Influence on Intracellular Geosmin Production in River Biofilms.

Hydro-morphological alterations in water bodies caused by climate change and human activities affects the ecosystem functioning and generate important water quality problems. Some of these alterations can generate an increase in cyanobacterial blooms, which are associated with the appearance of bad taste and odorous compounds such as geosmin. The factors that trigger their production are still unclear, and this inability to predict geosmin episodes provokes economic problems for water supply companies. This study aims to evaluate the effects of water flow and light availability on biofilm development and intracellular geosmin formation. A mesocosm experiment was performed between February-April, 2019. The mesocosms were a set of 10 outdoor 3 m long flumes, with a continuous water supply from the Ter river (Catalonia, NE Spain). Two light intensities were established: natural light and light reduced to 80%, combined with five gradual water flows from 0.09 to 1.10 L/s. Water samples were taken to analyze nutrients, and biofilm samples, to analyze geosmin concentration, chlorophyll a and the community. Geosmin in biofilm was detected in those treatments in which Oscillatoria sp. appeared. The concentration of intracellular geosmin was higher at lower water flows (0.09 and 0.18 L/s), and the highest (2.12 mg/g) was found in the flume with the lowest water flow (0.09 L/s) and irradiation (20%). This flume was the one that presented a greater concentration of Oscillatoria sp. (21% of the community). It stands out that, when geosmin in biofilm was found, the dissolved inorganic nitrogen and soluble reactive phosphorus ratio decreased, from an average of 417:1 to 14:1. This was mainly due to an increase in inorganic phosphorus concentration generated by a change in the nutrient uptake capacity of the community's biofilm. The results obtained in this study indicated the potential implications for stream ecosystem management to control geosmin appearance. Likewise, they could be used as an early warning system, establishing that in times of drought, which lead to a general decrease in river water flow, the situation could be optimal for the appearance and development of geosmin producing cyanobacteria in low-flow areas near the river banks.

Antibiotic resistance along an urban river impacted by treated wastewaters.

Urban rivers are impacted ecosystems which may play an important role as reservoirs for antibiotic-resistant (AR) bacteria. The main objective of this study was to describe the prevalence of antibiotic resistance along a sewage-polluted urban river. Seven sites along the Zenne River (Belgium) were selected to study the prevalence of AR Escherichia coli and freshwater bacteria over a 1-year period. Culture-dependent methods were used to estimate E. coli and heterotrophic bacteria resistant to amoxicillin, sulfamethoxazole, nalidixic acid and tetracycline. The concentrations of these four antibiotics have been quantified in the studied river. The antibiotic resistance genes (ARGs), sul1, sul2, tetW, tetO, blaTEM and qnrS were also quantified in both particle-attached (PAB) and free-living (FLB) bacteria. Our results showed an effect of treated wastewaters release on the spread of antibiotic resistance along the river. Although an increase in the abundance of both AR E. coli and resistant heterotrophic bacteria was observed from upstream to downstream sites, the differences were only significant for AR E. coli. A significant positive regression was also found between AR E. coli and resistant heterotrophic bacteria. The concentration of ARGs increased from upstream to downstream sites for both particle-attached (PAB) and free-living bacteria (FLB). Particularly, a significant increase in the abundance of four among six ARGs analyzed was observed after crossing urban area. Although concentrations of tetracycline significantly correlated with tetracycline resistance genes, the antibiotic levels were likely too low to explain this correlation. The analysis of ARGs in different fractions revealed a significantly higher abundance in PAB compared to FLB for tetO and sul2 genes. This study demonstrated that urban activities may increase the spread of antibiotic resistance even in an already impacted river.

Occurrence and persistence of carbapenemases genes in hospital and wastewater treatment plants and propagation in the receiving river.

This study aims to investigate the prevalence of clinically relevant carbapenemases genes (blaKPC, blaNDM and blaOXA-48) in water samples collected over one-year period from hospital (H), raw and treated wastewater of two wastewater treatment plants (WWTPs) as well as along the Zenne River (Belgium). The genes were quantified in both particle-attached (PAB) and free-living (FLB) bacteria. Our results showed that absolute abundances were the highest in H waters. Although absolute abundances were significantly reduced in WWTP effluents, the relative abundance (normalized per 16S rRNA) was never lowered through wastewater treatment. Particularly, for the PAB the relative abundances were significantly higher in the effluents respect to the influents of both WWTPs for all the genes. The absolute abundances along the Zenne River increased from upstream to downstream, peaking after the release of WWTPs effluents, in both fractions. Our results demonstrated that blaKPC, blaNDM and blaOXA-48 are widely distributed in the Zenne as a consequence of chronic discharge from WWTPs. To conclude, the levels of carbapenemases genes are significantly lower than other genes conferring resistance to more widely used antibiotics (analyzed in previous studies carried out at the same sites), but could raise up to the levels of high prevalent resistance genes.