Antimicrobial resistance (AMR) represents an escalating global health challenge, primarily driven by the extensive use of antibiotics in agriculture and medicine. Environmental compartments like soil, water, and biofilms contribute to the spread and persistence of resistance genes. Biofilms and wildlife microbiota are recognized as reservoirs for antibiotic-resistant bacteria and antibiotic resistance genes (ARGs) in aquatic ecosystems. The ecological interactions between biofilms and wildlife gut microbiota, under antibiotic selective pressure, and their implication regarding the dissemination of antimicrobial resistance in aquatic ecosystems is poorly understood. This study examined the impact of sub-inhibitory concentrations of sulfamethoxazole, ciprofloxacin and trimethoprim (10 μg. L-1) on the resistome in a Biofilm-grazer system, using a river biofilm and Xenopus laevis larvae over a 12-day period. The results indicated that antibiotic pressure resulted in notable variations in microbial composition in both biofilms and gut microbiota. Additionally, comparison of bacterial community composition between gut and biofilm compartments indicated increased microbial exchange between these environments, with stronger effects observed under antibiotic exposure. An increase of the resistome was observed only in biofilm especially under antibiotics exposure. Variations in the grazer's bacterial communities appeared to attenuate the increase of ARGs within the gut. However, grazing activity concomitantly enhanced the resistome abundance and diversity within the biofilm. This study presents an original perspective on the understanding of impact of sub-inhibitory antibiotic concentration on ecosystems though a microcosm experiment, highlighting the necessity for environmental monitoring and further studies across diverse and complex ecological settings to mitigate antibiotic resistance dissemination.

Copper (Cu) is extensively used in agriculture, yet its environmental accumulation raises significant ecotoxicological concerns. Nano-formulations have been developed as alternatives allowing to decrease Cu application rates, but their effects on non-target species remain poorly understood, particularly in aquatic ecosystems. This study evaluated the ecotoxic potential of the commercial copper-based nanopesticide, Kocide 3000® in comparison with the conventional formulation Kocide 2000®, using the freshwater gastropod Planorbarius corneus as a model species. A copper salt was also included as a control to specifically assess copper- related toxicity. Juvenile snails were submitted to a 48-hour acute toxicity test. Egg masses (embryos) were exposed to a range of copper concentrations (10-500 µg Cu/L) for 12 days until hatching. Growth, developmental progression, heart rate, and shell size were assessed as multiple endpoints. A recovery assay was conducted to investigate whether the impact of an early exposure was reversible. All three formulations induced measurable adverse developmental effects with increasing copper concentrations. Across all endpoints, nano and conventional formulations induced comparable effects, suggesting minimal nanomaterial-specific impacts and indicating that copper load, primarily drives toxicity under these exposure conditions. Interestingly, recovery potential differed between the copper salt and the commercial formulations since only CuSO₄-exposed embryos showed a return to a developmental state comparable to unexposed individuals after transfer to clean water. This finding highlights the influence of product composition in persistence and recovery dynamics. These results emphasize the importance of evaluating complete pesticide formulations, not just active ingredients in the hazard assessment of agrochemicals.

Graphene oxide (GO) and reduced graphene oxide (rGO) are carbon-based nanomaterials increasingly used in industrial applications, yet their environmental fate remains poorly understood. Once released into aquatic ecosystems, their interactions with biological systems can alter their physicochemical properties, with implications for their bioavailability and potential toxicity. This study investigates the digestive biotransformation of GO and rGO following ingestion by Xenopus laevis larvae, an established aquatic model organism. After 24 hours of exposure, feces containing the digested nanomaterials (dGO and drGO) were collected and analysed using transmission electron microscopy (TEM), Raman spectroscopy, and infrared spectroscopy (FTIR) to assess morphological, structural, and chemical changes. TEM imaging revealed particle alterations, edge erosion, and agglomeration in the digested samples. Raman analysis showed shifts in D* and D '' band positions and decreased defect-related intensity ratios, consistent with partial reduction of GO and rGO. IR spectroscopy confirmed a substantial loss of oxygenated functional groups, with a marked decrease in the oxygen-to-carbon signal ratio in dGO. Notably, carbonyl groups were more strongly reduced than C-O functional groups (epoxy, hydroxyl, alkoxy), suggesting preferential degradation at the sheet edges. rGO, being less oxidized initially, appeared less affected. In parallel, their toxic potential was assessed by measuring cellular viability of bacteria (Escherichia coli) and mammalian cell lines (IEC-6 and TR146) exposed to the materials. The results demonstrated that digested GO exhibited lower toxicity towards IEC-6 cells, while retaining antibacterial activity at low concentrations. However, antibacterial effects are lost at higher doses, likely due to agglomeration and reduced bioavailability, whereas rGO and drGO exhibited minimal toxicity across all conditions. These findings highlight the transformative role of digestive processes on graphene-based nanomaterials and underscore the need to consider such biotransformations in environmental risk assessments.

The omnipresence of organic micropollutants (MPs) in wastewater treatment plants and water bodies persists as an unresolved issue due to the partial efficiency of current conventional remediation technologies. In order to efficiently remove a wide range of MPs from wastewater, a hybrid process consisting of a Moving Bed Biofilm Reactor (MBBR) coupled with two membrane processes is designed for this study. This work aims to eliminate 19 MPs of different physicochemical properties from two different natures of non-conventional wastewater (i.e., domestic and hospital). The main particularity of these effluents is their low carbon content, resulting in C/N ratio ranging between 0.9 and 5.4. The 19 MPs are selected due to their occurrence and frequency of emergence on the European Union Watch List and inventory lists from French water agencies, as well as the feasibility of existing extraction protocols and analytical instruments to analyse them. The hybrid process notably the MBBR demonstrates excellent elimination efficiency of sCOD (>75 %) and relatively high ammonium-N removals (>65 %) regardless of the wastewater nature. Besides, the integrated system with the presence of a nanofiltration (NF) unit is efficient in eliminating most of the targeted MPs (>85 %) including recalcitrant compounds such as carbamazepine. The elimination of ketoprofen in the biological system has also significantly improved (5.9-50.3 %) thanks to the recirculation of NF concentrate in the bioreactors. Promising results from the lab-scale system highlight the importance and interest of implementing an integrated process to enhance the elimination of MPs and toxicity from wastewater.

Antimicrobial resistance (AMR) is a growing global health threat, with environmental compartments such as soil, water, and biofilms playing key roles in the dissemination and persistence of resistance genes. In this study, we explored the dynamics of AMR within a controlled Biofilm-Grazer system using Xenopus laevis larvae and biofilms A and B collected from two distinct rivers in Occitanie, France (collected respectively upstream from the large city of Toulouse and downstream from a wastewater treatment plant). The objective of the study was to investigate bacterial interactions between gut microbiota and biofilms, as well as the modulation of ARG (Antibiotic Resistance Gene) abundance and diversity over a 12-day period. Results showed a decrease of resistome in Xenopus gut microbiota feeding on both biofilms compared to feeding on commercial feed. In addition, an increase of resistome of Biofilm B compared to Biofilm A was observed. Procruste analysis and Pearson's correlations revealed a link between bacterial communities changes and ARG abundances in biofilms. Rhodobacter genus could be an ARG host shared between compartments. Furthermore, bacterial immigration predominantly occurred from the gut to the biofilms, with both biofilms acting as reservoirs for ARGs. Notably, Biofilm B, collected from a more polluted river, demonstrated a higher relative abundance of aac3-IVa resistance genes in the gut microbiota of larvae, compared to Biofilm A and a higher immigration rate from biofilm to gut. These findings highlight the complexity of interactions between biofilm communities and the gut microbiota, which may influence AMR dissemination.

The growing use of nanomaterials, including few-layer graphene (FLG) under various forms such as inks, raise concerns about their potential ecological impacts. This study evaluates the ecotoxicity on the aquatic compartment of a commercial ink composed of FLG stabilized with sodium deoxycholate (SDC) at a concentration of 1 g/L, through single-species bioassays and microcosm approaches on various aquatic organisms. The algae Raphidocelis subcapitata, the crustacean Daphnia magna, and the amphibian larvae Xenopus laevis were exposed to 0; 0.1; 10 and 50 mg/L of ink or SDC alone. The microcosm study was conducted with the ink or SDC at 0.1 mg/L, focusing on biofilm (diatoms and bacterial consortium) integrity and life-history traits of the dipteran Chironomus riparius. Results showed that although the ink exhibited weak toxicity in single-species bioassays or in microcosms, SDC alone caused significant toxic effects, depending on the biological model and concentration. Cellular alterations were observed in algae exposed to 0.1 mg/L of SDC, while acute toxicity occurred at concentrations over 50 mg/L in daphnia. Growth inhibition was observed in amphibian larvae at 0.1 and 1 mg/L without genotoxic effect. Microcosm study revealed slight changes in biofilm diversity and inhibition of chironomid growth by SDC (0.1 mg/L). This study highlights the importance of comprehensive ecological risk assessments for graphene-based materials and associated co-contaminants, emphasizing the environmental risks posed by SDC. The findings demonstrate the value of realistic approaches to evaluate the hazards of these chemicals and suggest that their release into the aquatic environments could pose significant ecological threats.

Copper-based plant protection products (PPPs) are widely used in agriculture to control fungal and bacterial diseases. However, concerns regarding copper accumulation in the environment have led to restrictions on its use. Copper-based nano-formulations have been proposed as an alternative, offering improved efficiency and reduced application rates. However, their environmental risks remain poorly understood, particularly their effects on aquatic ecosystems. This study evaluates the ecotoxicity of conventional and nano-formulated copper pesticides using Chironomus riparius as a holobiont model, considering both host life history traits and microbiota responses. Larvae were exposed to environmentally relevant concentrations (5, 50, and 500 µg Cu/L) of conventional and nano-formulated products under controlled laboratory conditions. Exposure to the highest concentration resulted in 100 % mortality. Following exposure to 50 µg Cu/L, sublethal effects were observed, including alteration of the larval microbiota composition, apparently shifting towards a copper-tolerant profile following exposure to the conventional formulation. Despite the distinct shifts in bacterial taxa between conventional and nano-formulated treatments, similar decreased adult emergences are noticed following exposure to both formulations. Despite comparable toxicity profiles, nano-formulations resulted in higher copper bioaccumulation in emerging adults, raising concerns about trophic transfer and ecosystem impacts. Our findings highlight the need for a holistic approach integrating host-microbiota interactions in ecotoxicological assessments. The comparable toxicity of nano-formulated and conventional copper pesticides, coupled with increased bioaccumulation potential, questions the assumption that nano-formulations represent a safer alternative. These results emphasize the importance of comprehensive risk assessments to ensure sustainable agricultural practices while minimizing environmental harm.

Antibiotic resistance (AR) is one of the major health threats of our time. The presence of antibiotics in the environment and their continuous release from sewage treatment plants, chemical manufacturing plants and animal husbandry, agriculture and aquaculture, result in constant selection pressure on microbial organisms. This presence leads to the emergence, mobilization, horizontal gene transfer and a selection of antibiotic resistance genes, resistant bacteria and mobile genetic elements. Under these circumstances, aquatic wildlife is impacted in all compartments, including freshwater organisms with partially impermeable microbiota.In this narrative review, recent advancements in terms of occurrence of antibiotics and antibiotic resistance genes in sewage treatment plant effluents source compared to freshwater have been examined, occurrence of antibiotic resistance in wildlife, as well as experiments on antibiotic exposure. Based on this current state of knowledge, we propose the hypothesis that freshwater aquatic wildlife may play a crucial role in the dissemination of antibiotic resistance within the environment. Specifically, we suggest that organisms with high bacterial density tissues, which are partially isolated from the external environment, such as fishes and amphibians, could potentially be reservoirs and amplifiers of antibiotic resistance in the environment, potentially favoring the increase of the abundance of antibiotic resistance genes and resistant bacteria. Potential avenues for further research (trophic transfer, innovative exposure experiment) and action (biodiversity eco-engineering) are finally proposed.

Graphene based nanomaterials (GBMs) have been drawing the attention of the scientific communities these past years. As their market is increasing every year, their potential environmental and health risk are to be assessed. As microbial communities represent the basis of every ecosystem, it is essential to evaluate the risks of GBMs on these communities. The effects of GBMs on bacteria has been widely studied, while the effects on phototrophic species are less represented. In this study, the diatom Nitzschia palea was exposed to graphene oxide (GO) and reduced graphene oxide (rGO), at 0.1, 1 and 10 mg.L-1. rGO had no effect on the diatom while GO induced an increase in growth, indicating the importance of the oxidation rate in the observed effect. Further results showed that shading effect of GO was countered by increased chlorophyll contents and by the accumulation of GO into the biofilm. This “sticking” mechanism increased the proximity between the biofilm and the GO, which might favour interactions between GO and the biofilm. These interactions led to the increase of defects and the reduction of GO. In addition, increased heterotrophic activity was suggested. To our knowledge, no diatom before Nitzschia palea has been shown to enhance its growth in presence of a GBM, but also capable to modify it. These results demonstrate the potential of GBMs, and in particular those with higher oxidation rates, to disrupt the contribution of the diatom Nitzschia palea to carbon cycling, which could have broader consequences at ecosystem scale.

The increase in industrial production of multi-walled carbon nanotubes (MWCNTs) raises concerns about their potential adverse effects associated to environmental releases, especially in aquatic environments where they are likely to accumulate. This study focuses on the environmental impact of MWCNTs, specifically on a benthic freshwater diatom (Nitzschia linearis), which plays a major role in the primary production of water bodies. The obtained results indicate that exposure to MWCNTs in the presence of natural organic matter (NOM) inhibits diatom's growth in a dose-dependent manner after 72 h of exposure. Interestingly, the photosystem II quantum yield (PSIIQY) in diatoms remains unaffected even after exposure to MWCNTs at 10 mg/L. After 48 h of exposure, MWCNTs are found to bind preferentially to extracellular polymeric substances (EPS) produced by diatoms, which could decrease their toxicity by limiting their interaction with this organism. However, measurement of genotoxicity and teratogenicity in diatoms exposed to MWCNTs revealed that the exposure to MWCNTs increased the occurrence of cells with micronuclei and abnormal frustules. Microscopy analyses including two-photon excitation microscopy (TPEM) revealed the internalization of MWCNTs. Investigations of the diatom's frustule structure using Scanning electron microscopy (SEM) indicated that the presence of pore structures constitutes a pathway allowing MWCNTs uptake. The presence in the diatom's cytoplasm of MWCNTs might possibly induce disturbances of the cellular components, leading to the observed genotoxic and teratogenic effects. In view of previous studies, this work underscores the need for further studies on the interaction between nanomaterials and different diatom species, given the species-specific nature of the interactions.

The behavior and removal of roxithromycin (ROX), oxytetracycline (OTC), chlortetracycline (CTC), and enrofloxacin (ENR) were investigated during the steady state of sludge anaerobic digestion (AD) in semi-continuous mode (37 °C). Sludge was spiked at realistic concentrations (50 μg/L of each antibiotic) and then used to feed the bioreactor for 80 days. Antibiotics were extracted from the substrate and digested sludge samples by accelerated solvent extraction (ASE). Accurate determination of antibiotics was obtained by the standard addition method (SAM) associated with the liquid chromatography-tandem mass spectrometry (LC–MS/MS). The presence of antibiotics at a concentration of 2.5 μg/g TS had no inhibitory effects on methane (CH4) production, total and volatile solids (TS and VS) removal as well as chemical oxygen demand (COD) removal. During the steady-state, antibiotics were removed significantly by 50, 100, and 59

This study aims to select fungi isolates to reduce olive mill wastewater (OMWW) chemical oxygen demand (COD) and phenolic compounds (PC), as well as their genotoxicity effect. Treatment with mold, isolated by an innovative technique using phenolic compound-selective media, showed a reduction rate of about 4% for COD and 2% for PC during one month of incubation without optimization of the treatment conditions. Whereas this percentage reached 98% and 96% for COD and PC, respectively, after only 12 days of treatment, when the C:N ratio was adjusted to 30 by adding urea as a nitrogen source at 150 rpm agitation speed. Genetic sequence homology of the most efficient mold isolate showed 100% similarity to Penicillium chrysogenum. High-performance liquid chromatography analysis of phenolic extracts of untreated OMWW showed the presence of five compounds—hydroxytyrosol at 1.22 g.L−1, tyrosol at 0.05 g.L−1, caffeic acid at 0.16 g.L−1, p-coumaric acid at 0.05 g.L−1 and oleuropein at 0.04 g.L−1—that were eliminated during the degradation process at 88.82%. Genotoxicity, assessed by the Vicia-faba root cell, showed a significant decrease in micronucleus frequency of about 96% after fungal treatment. These results confirm the positive role of fungal treatment of OMWW to eliminate genotoxicity and their ability to improve the agronomic potential.

Tests using algae and/or cyanobacteria, invertebrates (crustaceans) and fish form the basic elements of an ecotoxicological assessment in a number of regulations, in particular for classification of a substance as hazardous or not to the aquatic environment according to the Globally Harmonised System of Classification and Labelling of Chemicals (GHS-CLP) (GHS, 2022) and the REACH regulation (Registration, Evaluation, Authorisation and Restriction of Chemicals, EC, 2006). Standardised test guidelines (TGs) of the Organisation for Economic Co-operation and Development (OECD) are available to address the regulatory relevant endpoints of growth inhibition in algae and cyanobacteria (TG 201), acute toxicity to invertebrates (TG 202), and acute toxicity in fish (TG 203). Applying these existing OECD TGs for testing two dimensional (2D) graphene nanoforms may require more attention, additional considerations and/or adaptations of the protocols, because graphene materials are often problematic to test due to their unique attributes. In this review a critical analysis of all existing studies and approaches to testing used has been performed in order to comment on the current state of the science on testing and the overall ecotoxicity of 2D graphene materials. Focusing on the specific tests and available guidance's, a complete evaluation of aquatic toxicity testing for hazard classification of 2D graphene materials, as well as the use of alternative tests in an integrated approach to testing and assessment, has been made. This information is essential to ensure future assessments generate meaningful data that will fulfil regulatory requirements for the safe use of this "wonder" material.

Graphene-based nanomaterials such as graphene oxide (GO) possess unique properties triggering high expectations for the development of technological applications. Thus, GO is likely to be released in aquatic ecosystems. It is essential to evaluate its ecotoxicological potential to ensure a safe use of these nanomaterials. In amphibians, previous studies highlighted X. laevis tadpole growth inhibitions together with metabolic disturbances and genotoxic effects following GO exposure. As GO is known to exert bactericidal effects whereas the gut microbiota constitutes a compartment involved in host homeostasis regulation, it is important to determine if this microbial compartment constitutes a toxicological pathway involved in known GO-induced host physiological impairments. This study investigates the potential link between gut microbial communities and host physiological alterations. For this purpose, X. laevis tadpoles were exposed during 12 days to GO. Growth rate was monitored every 2 days and genotoxicity was assessed through enumeration of micronucleated erythrocytes. Genomic DNA was also extracted from the whole intestine to quantify gut bacteria and to analyze the community composition. GO exposure led to a dose dependent growth inhibition and genotoxic effects were detected following exposure to low doses. A transient decrease of the total bacteria was noticed with a persistent shift in the gut microbiota structure in exposed animals. Genotoxic effects were associated to gut microbiota remodeling characterized by an increase of the relative abundance of Bacteroides fragilis. The growth inhibitory effects would be associated to a shift in the Firmicutes/Bacteroidetes ratio while metagenome inference suggested changes in metabolic pathways and upregulation of detoxification processes. This work indicates that the gut microbiota compartment is a biological compartment of interest as it is integrative of host physiological alterations and should be considered for ecotoxicological studies as structural or functional impairments could lead to later life host fitness loss.

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This study investigated the fate of antibiotics during composting and its relationship with organic matter fractionation. Sludge was spiked with roxithromycin (ROX), chlortetracycline (CTC), oxytetracycline (OTC), and ciprofloxacin (CIP), at 3 different levels. Accelerated solvent extraction (ASE) and liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) were used for the extraction and the quantification of antibiotics. Sludge composting effectively removed parent compounds (p < 0.01) such as ROX, CTC, and OTC (52–87, 69–95 %, and 100 % respectively), while CIP persisted in the final compost (p > 0.05). The thermophilic stage was responsible for ROX removal, and the maturation stage was more implicated in removing CTC and OTC. Chemical accessibility and 3D fluorescence showed that high level of antibiotics affected the behavior of organic fractions. ROX removal was more associated with decreasing the most accessible fractions. The removals of CTC and OTC was more associated with the depletion of the complex organic matter. The observed results were confirmed by PCA and dendrograms analysis that confirmed the relationship between antibiotics removal and the evolution of organic pools. On the other hand, germination test indicated that cress and turnip were more sensitive to high antibiotic concentration. These results have been explored for the first time and they are recommended for controlling antibiotic removal based on organic matter fractionation.

Due to their various properties as polymeric materials, plastics have been produced, used and ultimately discharged into the environment. Although some studies have shown their negative impacts on the marine environment, the effects of plastics on freshwater organisms are still poorly studied, while they could be widely in contact with this pollution. The current work aimed to better elucidate the impact and the toxicity mechanisms of two kinds of commercial functionalized nanoplastics, i.e., carboxylated polystyrene microspheres of, respectively, 350 and 50 nm (PS350 and PS50), and heteroaggregated PS50 with humic acid with an apparent size of 350 nm (PSHA), all used at environmental concentrations (0.1 to 100 µg L−1). For this purpose, two relevant biological and aquatic models—amphibian larvae, Xenopus laevis, and dipters, Chironomus riparius—were used under normalized exposure conditions. The acute, chronic, and genetic toxicity parameters were examined and discussed with regard to the fundamental characterization in media exposures and, especially, the aggregation state of the nanoplastics. The size of PS350 and PSHA remained similar in the Xenopus and Chironomus exposure media. Inversely, PS50 aggregated in both exposition media and finally appeared to be micrometric during the exposition tests. Interestingly, this work highlighted that PS350 has no significant effect on the tested species, while PS50 is the most prone to alter the growth of Xenopus but not of Chironomus. Finally, PSHA induced a significant genotoxicity in Xenopus.

Despite the fast-growing use and production of graphene-based nanomaterials (GBMs), data concerning their effects on freshwater benthic macroinvertebrates are scarce. This study aims to investigate the effects of graphene oxide (GO) on the midge Chironomus riparius. Mortality, growth inhibition, development delay and teratogenicity, assessed using mentum deformity analysis, were investigated after a 7-day static exposure of the first instar larvae under controlled conditions. The collected data indicated that the survival rate was not impacted by GO, whereas chronic toxicity following a dose-dependent response occurred. Larval growth was affected, leading to a significant reduction in larval length (from 4.4 to 10.1%) in individuals reaching the fourth instar at any of the tested concentrations (from 0.1 to 100 mg/L). However, exposure to GO is not associated with an increased occurrence of mouthpart deformities or seriousness in larvae. These results highlight the suitability of monitoring the larval development of C. riparius as a sensitive marker of GO toxicity. The potential ecological consequences of larval size decrease need to be considered for a complete characterization of the GO-related environmental risk.

This review discusses the interactions between graphene-based nanomaterials and microbial communities in different environmental compartments reconstituted in microcosms, from soil and freshwater to bioprocesses treating waste.