Exploring the presence, genomic traits, and pathogenic potential of extended-spectrum ß-lactamase Escherichia coli in freshwater, wastewater, and hospital effluents.

AIMS: The purpose of this work was to study extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) in freshwaters, hospital effluents, and wastewaters during two sampling campaigns in 2021. METHODS AND RESULTS: Water sampling was performed at 24 stations in the Ourthe watershed in Belgium. A total of 644 ESBL (n = 642) and AmpC (n = 2) E. coli strains were isolated. Disk-diffusion assays were performed following the EUCAST's recommendations. All strains were tested for the presence of blaCTX-M-1, blaCTX-M-2, and blaCTX-M-9 gene groups by PCR. Genes belonging to blaCTX-M-1 and blaCTX-M-9 groups were detected, respectively, in 73.6% and 14.9% of the strains. No blaCTX-M-2 group's gene was found. A subset of strains (n = 40) was selected for whole genome sequencing. Escherichia coli serotype O18: H7 ST 1463 was predominant (n = 14) in the sequenced strains and showed pathogenicity in the Galleria mellonella larvae model. ß-lactamase genes identified were blaCTX-M (n = 21), with blaCTX-M-15 mostly represented (n = 15), as well as blaTEM (n = 11), blaOXA (n = 7), blaSHV (n = 9), and carbapenemase (CP) genes were observed in several strains-blaKPC-3 (n = 19), blaNDM-1 (n = 1), blaVIM-1 (n = 2), and blaOXA-244 (n = 2)-even from freshwaters. CONCLUSIONS: ESBL-EC are widely distributed in the aquatic environment in Belgium and contain a variety of ESBL and CP genes.

Thermal tolerance does not explain the altitudinal segregation of lowland and alpine aquatic insects.

Elevation gradients provide powerful study systems for examining the influence of environmental filters in shaping species assemblages. High-mountain habitats host specific high-elevation assemblages, often comprising specialist species adapted to endure pronounced abiotic stress, while such harsh conditions prevent lowland species from colonizing or establishing. While thermal tolerance may drive the altitudinal segregation of ectotherms, its role in structuring aquatic insect communities remains poorly explored. This study investigates the role of thermal physiology in shaping the current distribution of high-mountain diving beetles from the Sierra Nevada Iberian mountain range and closely related lowland species. Cold tolerance of five species from each altitudinal zone was measured estimating the supercooling point (SCP), lower lethal temperature (LLT) and tolerance to ice enclosure, while heat tolerance was assessed from the heat coma temperature (HCT). Alpine species exhibited wider fundamental thermal niches than lowland species, likely associated with the broader range of climatic conditions in high-mountain areas. Cold tolerance did not seem to prevent lowland species from colonizing higher elevations, as most studied species were moderately freeze-tolerant. Therefore, fundamental thermal niches seem not to fully explain species segregation along elevation gradients, suggesting that other thermal tolerance traits, environmental factors, and biotic interactions may also play important roles.

Main drivers of dragonflies and damselflies (Insecta; Odonata) metacommunities in streams inside protected areas in the Brazilian Amazon.

The evaluation of environmental and spatial influence in freshwater systems is crucial for the conservation of aquatic diversity. So, we evaluated communities of Odonata in streams inside and outside sustainable use areas in the Brazilian western Amazon. We predicted that these streams would differ regarding habitat integrity and species α and ß diversity. We also predict that environmental and spatial variables will be important for both suborders, but with more substantial effects on Zygoptera species, considering their nature of forest-specialist. The study was conducted in 35 streams, 19 inside and 16 outside sustainable use areas. The streams outside presented high species richness, abundance, and number of exclusive forest-specialist species from Zygoptera and higher scores of habitat integrity. In contrast, one sustainable use area presented the lowest values of these metrics. Besides, we found that environmental and spatial variables were significantly associated to Zygoptera species composition, but not with Anisoptera, which can be explained by their cosmopolitan nature. Our results indicated that an interplay between environmental and spatial processes determines the structure of the metacommunities of Zygoptera. The less effective dispersal rates and narrow ecological tolerance of Zygoptera species make them more influenced by local conditions and dispersal limitation, and more sensible to habitat modifications. We highlight the importance of improving the local management of the sustainable use areas by environmental agencies, mainly on areas that are losing their capacity to maintain the aquatic fauna, and implementation of social policies toward traditional people.

Observed and projected functional reorganization of riverine fish assemblages from global change.

Climate and land-use/land-cover change ("global change") are restructuring biodiversity, globally. Broadly, environmental conditions are expected to become warmer, potentially drier (particularly in arid regions), and more anthropogenically developed in the future, with spatiotemporally complex effects on ecological communities. We used functional traits to inform Chesapeake Bay Watershed fish responses to future climate and land-use scenarios (2030, 2060, and 2090). We modeled the future habitat suitability of focal species representative of key trait axes (substrate, flow, temperature, reproduction, and trophic) and used functional and phylogenetic metrics to assess variable assemblage responses across physiographic regions and habitat sizes (headwaters through large rivers). Our focal species analysis projected future habitat suitability gains for carnivorous species with preferences for warm water, pool habitats, and fine or vegetated substrates. At the assemblage level, models projected decreasing habitat suitability for cold-water, rheophilic, and lithophilic individuals but increasing suitability for carnivores in the future across all regions. Projected responses of functional and phylogenetic diversity and redundancy differed among regions. Lowland regions were projected to become less functionally and phylogenetically diverse and more redundant while upland regions (and smaller habitat sizes) were projected to become more diverse and less redundant. Next, we assessed how these model-projected assemblage changes 2005-2030 related to observed time-series trends (1999-2016). Halfway through the initial projecting period (2005-2030), we found observed trends broadly followed modeled patterns of increasing proportions of carnivorous and lithophilic individuals in lowland regions but showed opposing patterns for functional and phylogenetic metrics. Leveraging observed and predicted analyses simultaneously helps elucidate the instances and causes of discrepancies between model predictions and ongoing observed changes. Collectively, results highlight the complexity of global change impacts across broad landscapes that likely relate to differences in assemblages' intrinsic sensitivities and external exposure to stressors.

Eco-physiological Responses of Aquatic Fungi to Three Global Change Stressors Highlight the Importance of Intraspecific Trait Variability.

Anthropogenic change at a global scale is affecting life on Earth with impacts on freshwaters. Aquatic hyphomycetes are fungi that drive organic matter decomposition in freshwaters and mediate energy transfer to higher trophic levels. Intraspecific trait variability affects ecological processes and can account for species adaptations to environmental change. To ascertain how aquatic hyphomycetes respond to global change related stressors, we selected 20 strains (7 species), based on their co-occurrence in streams and phylogenetic relatedness. We measured fungal growth rates at different temperatures (7 levels), nutrient concentrations (6 levels) and medium moisture (6 levels). Our results indicate that all stressors affected fungal growth, and responses to nutrient enrichment and moisture were strain specific. Fungal responses to the stressors were not explained by their phylogenetic relatedness. In the absence of stressors, interspecific diversity best explained the variance in fungal traits, while the increase in the stress gradient increased the importance of intraspecific diversity.

A global agenda for advancing freshwater biodiversity research.

Global freshwater biodiversity is declining dramatically, and meeting the challenges of this crisis requires bold goals and the mobilisation of substantial resources. While the reasons are varied, investments in both research and conservation of freshwater biodiversity lag far behind those in the terrestrial and marine realms. Inspired by a global consultation, we identify 15 pressing priority needs, grouped into five research areas, in an effort to support informed stewardship of freshwater biodiversity. The proposed agenda aims to advance freshwater biodiversity research globally as a critical step in improving coordinated actions towards its sustainable management and conservation.

Production dynamics reveal hidden overharvest of inland recreational fisheries.

Recreational fisheries are valued at $190B globally and constitute the predominant way in which people use wild fish stocks in developed countries, with inland systems contributing the main fraction of recreational fisheries. Although inland recreational fisheries are thought to be highly resilient and self-regulating, the rapid pace of environmental change is increasing the vulnerability of these fisheries to overharvest and collapse. Here we directly evaluate angler harvest relative to the biomass production of individual stocks for a major inland recreational fishery. Using an extensive 28-y dataset of the walleye (Sander vitreus) fisheries in northern Wisconsin, United States, we compare empirical biomass harvest (Y) and calculated production (P) and biomass (B) for 390 lake year combinations. Production overharvest occurs when harvest exceeds production in that year. Biomass and biomass turnover (P/B) declined by ∼30 and ∼20%, respectively, over time, while biomass harvest did not change, causing overharvest to increase. Our analysis revealed that ∼40% of populations were production-overharvested, a rate >10× higher than estimates based on population thresholds often used by fisheries managers. Our study highlights the need to adapt harvest to changes in production due to environmental change.

Biogeochemical functional responses to flow rate in a low order stream: implications for water quality monitoring.

In running waters, the concentration of components that define water quality can be subjected to ample fluctuations quantitatively linked to flow rate. If not properly considered, such variability may hinder assessment of the evolution of water quality, of the effects of management actions, and ultimately the understanding of processes driving water quality. The functional response to flow rate was characterized for multiple biogeochemical variables in a pristine, low order stream. Variability of responses spanned between a factor of 2 and > 34, and in all cases were associated to flow rate according to one of three patterns: positive asymptotic (for variables: seston, suspended particles, total nutrients, dissolved and particulated organic matter, dissolved inorganic nitrogen), negative asymptotic (conductivity and dissolved reactive silicon), and humped (dissolved inorganic phosphorous). Building on those results, a rationale is presented for an unambiguous, cost-effective approach to water quality evaluation in running systems with predominantly diffuse sources.

Litter Quality Is a Stronger Driver than Temperature of Early Microbial Decomposition in Oligotrophic Streams: a Microcosm Study.

Litter decomposition is an ecological process of key importance for forest headwater stream functioning, with repercussions for the global carbon cycle. The process is directly and indirectly mediated by microbial decomposers, mostly aquatic hyphomycetes, and influenced by environmental and biological factors such as water temperature and litter quality. These two factors are forecasted to change globally within the next few decades, in ways that may have contrasting effects on microbial-induced litter decomposition: while warming is expected to enhance microbial performance, the reduction in litter quality due to increased atmospheric carbon dioxide and community composition alteration may have the opposite outcome. We explored this issue through a microcosm experiment focused on early microbial-mediated litter decomposition under stream oligotrophic conditions, by simultaneously manipulating water temperature (10 °C and 15 °C) and litter quality (12 broadleaf plant species classified into 4 categories based on initial concentrations of nitrogen and tannins). We assessed potential changes in microbial-mediated litter decomposition and the performance of fungal decomposers (i.e., microbial respiration, biomass accrual, and sporulation rate) and species richness. We found stronger effects of litter quality, which enhanced the performance of microbial decomposers and decomposition rates, than temperature, which barely influenced any of the studied variables. Our results suggest that poorer litter quality associated with global change will have a major repercussion on stream ecosystem functioning.

A Shift from Snow to Rain in Midlatitude Japan Increases Fresh Submarine Groundwater Discharge and Doubled Inorganic Carbon Flux over 20 Years.

Fresh submarine groundwater discharge (FSGD), a well-established land-to-sea pathway for water and materials, has increased (∼30%) along the midlatitude coast of Japan with the shift from snowfall to rainfall since the 1980s. The terrestrial-derived FSGD in this area has a residence time of 10-20 years, and its material flux may respond quickly to the effects of recent warming. In this study, FSGD collected directly from the seafloor (∼10 m water depth) during two different periods (2017-2021 and 2001-2003) was observed using the same consistent method to evaluate the warming-related effects on FSGD material fluxes. In the FSGD system, dissolved constituents and the isotopic composition decreased by 10-30% and residence time by one-third compared to the previous data, even though homogeneous water quality was maintained among the FSGD-connected aquifers for each period. Most concerning is that these changes resulted in a drop in pH and an increase in FSGD-derived carbon flux. Total nutrient fluxes from land have roughly halved, resulting in lower primary productivity and an estimated doubling of excess dissolved inorganic carbon in coastal waters. Our findings present direct evidence of the sensitivity of carbon flux to climate change and the urgency for carbon-related FSGD research worldwide.

Plasticity of thermal performance curves in a narrow range endemic water beetle.

Thermal history can plastically alter the response of ectotherms to temperature, and thermal performance curves (TPCs) are powerful tools for exploring how organismal-level performance varies with temperature. Plasticity in TPCs may be favoured in thermally variable habitats, where it can result in fitness benefits. However, thermal physiology remains insufficiently studied for freshwater insects despite freshwater biodiversity being at great risk under global change. Here, we assess how acclimation at either summer or winter average temperatures changes TPCs for locomotion activity and metabolism in Enochrus jesusarribasi (Hydrophilidae), a water beetle endemic to shallow saline streams in SE Spain. This beetle is a bimodal gas exchanger and so we also assessed how aerial and aquatic gas exchange varied across temperatures for both acclimation treatments. Responses of locomotory TPCs to thermal acclimation were relatively weak, but high temperature acclimated beetles tended to exhibit higher maximum locomotor activity and reduced TPC breadth than those acclimated at lower temperature. High temperature acclimation increased the thermal sensitivity of metabolic rates, contrary to the response generally found in aquatic organisms. Higher metabolic rates upon high temperature acclimation were achieved by increasing aerial, rather than aquatic oxygen uptake. Such plastic respiratory behaviour likely contributed to enhanced locomotor performance at temperatures around the optimum and thermal plasticity could thus be an important component in the response of aquatic insects to climate change. However, high temperature acclimation appeared to be detrimental for locomotion in subsequent exposure at upper sublethal temperatures, suggesting that this narrow range endemic may be vulnerable to future climate warming. This study demonstrates that TPCs are context-specific, differing with performance metric as well as thermal history. Such context dependency must be considered when using TPCs to predict organismal responses to climate change.

Heavy metal toxicity in the water column and benthic sediments of a degraded tropical stream.

Agriculture and other anthropogenic activities on riparian corridors pose a great ecotoxicological risk to freshwater ecosystem and human health. Using the atomic absorption spectroscopy, concentrations of heavy metals (Cd, Cu, Fe, Zn, Pb and As) in the water column and benthic sediments of a degraded tropical stream (Opa Stream, Ile-Ife, Nigeria), were assessed with a view to determining the stream's health status. Three metals (Cu, Pb, and As) showed significant positive correlations between concentrations in the water column and the benthic sediments. All but one heavy metal (i.e. Zn) had reached disturbing concentrations in the stream's water column and exceeded their recommended limits in surface freshwaters. The geo-accumulation index (Igeo) and contamination factor (CF) indicated that the stream was moderately contaminated with Cd (0

The contamination of inland waters by microplastic fibres under different anthropogenic pressure: Preliminary study in Central Europe (Poland).

The contamination of freshwater ecosystems with microfibres has not yet been studied in Poland. We analysed samples from a river and three lakes located in central and northeastern Poland. A significantly higher number of fibres were reported in the river, which runs through large cities, compared with the lake situated within the Landscape Park. Fibres smaller than 1.0 mm dominated, especially in the river where they constituted 39% of all fibres detected. We found more microplastics (⩽ 4930 fibres·m-3) by using a mesh size of 20 µm compared with other studies of inland waters. The use of Raman spectroscopy allowed us to identify conventional plastic polymers: polyethylene terephthalate, polyester and polyurethane. We estimated that up to 25 g of microplastic in the form of fibres might be in the lake water under the surface. We found microplastic fibres in Majcz Lake situated within the Masurian Landscape Park. This suggests that microfibres are carried by the wind and rain and enter freshwater isolated from sewage outlets. By using the control sample and an air-test of microfibres in the laboratory, we observed that there is a high probability of contamination with microplastic in the field samples (up to 30% of all fibres detected). The contamination risk noted from the samples cannot be ignored; this could be particularly important for analysis of microplastic in remote freshwater ecosystems.

Dangerous relationships: biases in freshwater bioassessment based on observed to expected ratios.

The ecological assessment of freshwaters is currently primarily based on biological communities and the reference condition approach (RCA). In the RCA, the communities in streams and lakes disturbed by humans are compared with communities in reference conditions with no or minimal anthropogenic influence. The currently favored rationale is using selected community metrics for which the expected values (E) for each site are typically estimated from environmental variables using a predictive model based on the reference data. The proportional differences between the observed values (O) and E are then derived, and the decision rules for status assessment are based on fixed (typically 10th or 25th) percentiles of the O/E ratios among reference sites. Based on mathematical formulations, illustrations by simulated data and real case studies representing such an assessment approach, we demonstrate that the use of a common quantile of O/E ratios will, under certain conditions, cause severe bias in decision making even if the predictive model would be unbiased. This is because the variance of O/E under these conditions, which seem to be quite common among the published applications, varies systematically with E. We propose a correction method for the bias and compare the novel approach to the conventional one in our case studies, with data from both reference and impacted sites. The results highlight a conceptual issue of employing ratios in the status assessment. In some cases using the absolute deviations instead provides a simple solution for the bias identified and might also be more ecologically relevant and defensible.

Methylmercury Biogeochemistry in Freshwater Ecosystems: A Review Focusing on DOM and Photodemethylation.

Mercury contamination is a growing concern for freshwater food webs in ecosystems without point sources of mercury. Methylmercury (MeHg) is of particular concern, as this is the form of mercury that crosses the blood-brain barrier and is neurotoxic to organisms. Wetlands and benthic sediments have high organic content and low oxygen availability. Anaerobic bacteria drive the metabolic function in these ecosystems and subsequently can methylate mercury. The bioavailability of MeHg is controlled by physicochemical characteristics such as pH, binding affinities, and dissolved organic matter (DOM). Similarly, photodemethylation is influenced by similar characteristics and thereby the two processes should be studied in tandem. The degradation of MeHg through photochemistry is an effective destruction mechanism in freshwater lakes. This review will highlight the uncertainties and known effects of DOM on subsequent photoreactions that lead to the occurrence of mercury photodemethylation and reduction in mercury bioavailability in freshwater ecosystems.

Integrating within-catchment and interbasin connectivity in riverine and nonriverine freshwater conservation planning in the North China Plain.

Freshwater ecosystems encompass all inland water bodies, in which riverine and nonriverine freshwaters are linked through hydrological connectivity within a catchment. However, riverine and nonriverine freshwaters have often been assessed separately and their interdependence and connection has not been considered appropriately in prevailing freshwater conservation planning. To address the representation and persistence of freshwater ecosystems in conservation assessment, we integrated riverine and nonriverine freshwater wetlands as broad-scale conservation surrogates and incorporated longitudinal, lateral and vertical connectivity rules in a conservation planning for the freshwater wetlands in the North China Plain (NCP). We also considered interbasin connectivity by incorporating conservation features of key transferring nodes of the South-to-North Water Diversion Project (SNWD) in the NCP to safeguard their unique ecosystem services of regulating interbasin freshwater. Three scenarios (i.e., 2D, 3D and interbasin scenario) were developed by incorporating different multiple conservation targets, and their spatial priorities and cost-efficiency in freshwater conservation were compared. We applied systematic conservation framework and modified Marxan to accommodate these multidirectional and interbasin connectivity targets in our freshwater conservation assessment. The results indicated that the existing conservation system covered approximately 20% of the freshwater wetlands in the NCP, and there were still considerable conservation gaps that need to be filled. The optimal scenarios could substantially improve the representation, complementarity and persistence for the conservation of freshwater ecosystems, but would not significantly increased overall costs. The framework developed by our research has the potential to facilitate further application of systematic methods in freshwater conservation and rehabilitation planning at multiple scales.

Molecular phylogeny of the highly diversified catfish subfamily Loricariinae (Siluriformes, Loricariidae) reveals incongruences with morphological classification.

The Loricariinae belong to the Neotropical mailed catfish family Loricariidae, the most species-rich catfish family. Among loricariids, members of the Loricariinae are united by a long and flattened caudal peduncle and the absence of an adipose fin. Despite numerous studies of the Loricariidae, there is no comprehensive phylogeny of this morphologically highly diversified subfamily. To fill this gap, we present a molecular phylogeny of this group, including 350 representatives, based on the analysis of mitochondrial and nuclear genes (8426 positions). The resulting phylogeny indicates that Loricariinae are distributed into two sister tribes: Harttiini and Loricariini. The Harttiini tribe, as classically defined, constitutes a paraphyletic assemblage and is here restricted to the three genera Harttia, Cteniloricaria, and Harttiella. Two subtribes are distinguished within Loricariini: Farlowellina and Loricariina. Within Farlowellina, the nominal genus formed a paraphyletic group, as did Sturisoma and Sturisomatichthys. Within Loricariina, Loricaria, Crossoloricaria, and Apistoloricaria are also paraphyletic. To solve these issues, and given the lack of clear morphological diagnostic features, we propose here to synonymize several genera (Quiritixys with Harttia; East Andean members of Crossoloricaria, and Apistoloricaria with Rhadinoloricaria; Ixinandria, Hemiloricaria, Fonchiiichthys, and Leliella with Rineloricaria), to restrict others (Crossoloricaria, and Sturisomatichthys to the West Andean members, and Sturisoma to the East Andean species), and to revalidate the genus Proloricaria.

Evidence of Helicobacter spp. in freshwaters from Roraima Tepui, Guayana Shield, South America.

Helicobacter presence and viability in waters is not well characterized. The identification of natural reservoirs and infection sources may provide novel insights into its waterborne transmission. The goal of this study was to investigated the occurrence of Helicobacter spp. in natural freshwaters from Roraima Tepui, a little studied and unique ecosystem of the Guayana Shield. Freshwaters collected from two localities at Roraima Tepui were cultured in HP selective broth and agar for Helicobacter pylori and analysed by fluorescent in situ hybridization (FISH), specific PCR assays, 16S rRNA gene sequencing and phylogenetic analysis. The presence of other bacteria in freshwater enrichments was determined using clone library sequencing of the 16S rRNA gene and phylogenetic inferences. Helicobacter spp. were detected by semi-nested PCR and FISH in freshwater enrichments from both sites. Coccoid viable but nonculturable (VBNC) cells were evidenced using 16S rRNA gene Helicobacter species and H. pylori-specific probes. Partial 16S rRNA gene sequences of two HP enrichments showed high similarity to H. pylori and Helicobacter nemestrinae (99-100 %). Other bacteria such as Serratia, Aquitalea, Chromobacterium, Mycobacterium, Acinetobacter, Curvibacter and Dysgonomonas were also detected using complete 16S rRNA gene sequences, with Serratia, Aquitalea and Chromobacterium the most common genera (40.9, 18.2 and 15.2 %, respectively). This is the first time that Helicobacter spp. have been reported in freshwaters of a tepui ecosystem. Our results contribute to the current knowledge of these bacteria in the aquatic environment and expand their known/potential sites outside the human host.

Fossil-based comparative analyses reveal ancient marine ancestry erased by extinction in ray-finned fishes.

The marine-freshwater boundary is a major biodiversity gradient and few groups have colonised both systems successfully. Fishes have transitioned between habitats repeatedly, diversifying in rivers, lakes and oceans over evolutionary time. However, their history of habitat colonisation and diversification is unclear based on available fossil and phylogenetic data. We estimate ancestral habitats and diversification and transition rates using a large-scale phylogeny of extant fish taxa and one containing a massive number of extinct species. Extant-only phylogenetic analyses indicate freshwater ancestry, but inclusion of fossils reveal strong evidence of marine ancestry in lineages now restricted to freshwaters. Diversification and colonisation dynamics vary asymmetrically between habitats, as marine lineages colonise and flourish in rivers more frequently than the reverse. Our study highlights the importance of including fossils in comparative analyses, showing that freshwaters have played a role as refuges for ancient fish lineages, a signal erased by extinction in extant-only phylogenies.

Effects of heavy metals on ultrastructure and Hsp70 induction in Lemna minor L. exposed to water along the Sarno River, Italy.

The effects of freshwater pollution in the highly contaminated river Sarno (Campania, Southern Italy) have been evaluated using bags containing the aquatic plant Lemna minor (Lemnacee, Arales), in order to determine morpho-physiological modifications as a response to pollutants. The exposition of Lemna bags for 7 days on three different sites along the river path showed alterations in chloroplasts and vacuoles shape and organization. Moreover, some specimens were exposed in vitro at the same heavy metal (HM) concentrations measured in the polluted sites of the river, and compared with data from the bag experiment; to verify the dose and time dependent effects, samples were exposed to HM in vitro at concentrations ranging from 10(-6) to 10(-4)M up to 7 days. Transmission electron microscopy (TEM) observations on in vitro plants confirmed that ultrastructural alterations affected most of plastids and the shape of different subcellular structures, namely vacuoles; in in vitro stressed specimens, Heat Shock Proteins 70 (Hsp70) levels changed, in dependence of changing levels of HM measured in different sites along the river path. Thus L. minor exhibited a possible correlation between the levels of HM pollution and Hsp70 occurrence; interestingly, the data presented showed that copper specifically increased Hsp70 levels at concentrations detected in polluted river waters, whereas cadmium and lead did not; on the other side, the latter represent highly toxic elements when specimens were exposed to higher levels in vitro. The effects of specific elements in vitro are compared to those observed in bags exposed along the river path; thus results are examined in order to propose L. minor as an organism able to be utilized to monitor heavy metals pollution; the possibility of using Hsp70s as specific markers of HM pollution is discussed.