The potential contribution of aquatic wildlife to antibiotic resistance dissemination in freshwater ecosystems: A review.

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.

Environmental and Health Impacts of Graphene and Other Two-Dimensional Materials: A Graphene Flagship Perspective.

Two-dimensional (2D) materials have attracted tremendous interest ever since the isolation of atomically thin sheets of graphene in 2004 due to the specific and versatile properties of these materials. However, the increasing production and use of 2D materials necessitate a thorough evaluation of the potential impact on human health and the environment. Furthermore, harmonized test protocols are needed with which to assess the safety of 2D materials. The Graphene Flagship project (2013-2023), funded by the European Commission, addressed the identification of the possible hazard of graphene-based materials as well as emerging 2D materials including transition metal dichalcogenides, hexagonal boron nitride, and others. Additionally, so-called green chemistry approaches were explored to achieve the goal of a safe and sustainable production and use of this fascinating family of nanomaterials. The present review provides a compact survey of the findings and the lessons learned in the Graphene Flagship.

Graphene oxide worsens copper-mediated embryo-larval toxicity in the pacific oyster while reduced graphene oxide mitigates the effects.

Due to their properties, graphene-based nanomaterials (GBMs) are triggering a great interest leading to an increase of their global production and use in new applications. As a consequence, their release into the environment is expected to increase in the next years. When considering the current knowledge in the evaluation of GBMs ecotoxic potential, studies aiming to evaluate the hazard associated to these nanomaterials towards marine species and particularly considering potential interactions with other environmental pollutants such as metals are scarce. Here we evaluated the embryotoxic potential of GBMs, which include graphene oxide (GO) and its reduced form (rGO), both individually and in combination with copper (Cu) as a referent toxicant, towards early life stages of the Pacific oyster through the use of a standardized method (NF ISO 17244). We found that following exposure to Cu, dose-dependent decrease in the proportion of normal larvae was recorded with an Effective Concentration leading to the occurrence of 50% of abnormal larvae (EC50) of 13.85 ± 1.21 µg/L. Interestingly, the presence of GO at a non-toxic dose of 0.1 mg/L decreased the Cu EC50 to 12.04 ± 0.85 µg/L while it increased to 15.91 ± 1.57 µg/L in presence of rGO. Based on the measurement of copper adsorption, the obtained results suggest that GO enhances Cu bioavailability, potentially modifying its toxic pathways, while rGO mitigates Cu toxicity by decreasing its bioavailability. This research underscores the need to characterize the risk associated to GBMs interactions with other aquatic contaminants and supports the adoption of a safer-by-design strategy using rGO in marine environments. This would contribute to minimize the potential adverse effects on aquatic species and to reduce the risk for economic activities associated to coastal environments.

Gut microbiota impairment following graphene oxide exposure is associated to physiological alterations in Xenopus laevis tadpoles.

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.

Exposure of Midge Larvae (Chironomus riparius) to Graphene Oxide Leads to Development Alterations.

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.

Ecotoxicity of Heteroaggregates of Polystyrene Nanospheres in Chironomidae and Amphibian.

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.

Graphene oxide and reduced graphene oxide promote the effects of exogenous T3 thyroid hormone in the amphibian Xenopus laevis.

The interest for graphene-based nanomaterials (GBMs) is growing worldwide as their properties allow the development of new innovative applications. In parallel, concerns are increasing about their potential adverse effects on the environment are increasing. The available data concerning the potential risk associated to exposure of aquatic organisms to these GBMs are still limited and little is known regarding their endocrine disruption potential. In the present study, the endocrine disruption potential of graphene oxide (GO) and reduced graphene oxide (rGO) was assessed using a T3-induced amphibian metamorphosis assay. The results indicated that GBMs potentiate the effects of exogenous T3 with a more marked effect of GO compared to rGO. T3 quantifications in the exposure media indicated adsorption of the hormone on GBMs, increasing its bioavailability for organisms because GBMs are accumulated in the gut and the gills of these amphibians. This study highlights that the tested GBMs do not disrupt the thyroid pathway in amphibians but indicates that adsorption properties of these nanomaterials may increase the bioavailability and the toxicity of other pollutants.

Graphene-Based Nanomaterials Modulate Internal Biofilm Interactions and Microbial Diversity.

Graphene-based nanomaterials (GBMs), such as graphene oxide (GO) and reduced graphene oxide (rGO), possess unique properties triggering high expectations for the development of new technological applications and are forecasted to be produced at industrial-scale. This raises the question of potential adverse outcomes on living organisms and especially toward microorganisms constituting the basis of the trophic chain in ecosystems. However, investigations on GBMs toxicity were performed on various microorganisms using single species that are helpful to determine toxicity mechanisms but fail to predict the consequences of the observed effects at a larger organization scale. Thus, this study focuses on the ecotoxicological assessment of GO and rGO toward a biofilm composed of the diatom Nitzschia palea associated to a bacterial consortium. After 48 and 144 h of exposure to these GBMs at 0, 0.1, 1, and 10 mg.L-1, their effects on the diatom physiology, the structure, and the metabolism of bacterial communities were measured through the use of flow cytometry, 16S amplicon sequencing, and Biolog ecoplates, respectively. The exposure to both of these GBMs stimulated the diatom growth. Besides, GO exerted strong bacterial growth inhibition as from 1 mg.L-1, influenced the taxonomic composition of diatom-associated bacterial consortium, and increased transiently the bacterial activity related to carbon cycling, with weak toxicity toward the diatom. On the contrary, rGO was shown to exert a weaker toxicity toward the bacterial consortium, whereas it influenced more strongly the diatom physiology. When compared to the results from the literature using single species tests, our study suggests that diatoms benefited from diatom-bacteria interactions and that the biofilm was able to maintain or recover its carbon-related metabolic activities when exposed to GBMs.

Heteroaggregates of Polystyrene Nanospheres and Organic Matter: Preparation, Characterization and Evaluation of Their Toxicity to Algae in Environmentally Relevant Conditions.

The environmental fate and behavior of nanoplastics (NPs) and their toxicity against aquatic organisms are under current investigation. In this work, relevant physicochemical characterizations were provided to analyze the ecotoxicological risk of NPs in the aquatic compartment. For this purpose, heteroaggregates of 50 nm polystyrene nanospheres and natural organic matter were prepared and characterized. The kinetic of aggregation was assimilated to a reaction-limited colloid aggregation mode and led to the formation of heteroaggregates in the range of 100-500 nm. Toxicities of these heteroaggregates and polystyrene nanospheres (50 and 350 nm) were assessed for a large range of concentrations using four benthic and one planktonic algal species, in regards to particle states in the media. Heteroaggregates and nanospheres were shown to be stable in the exposure media during the ecotoxity tests. The algal species exhibited very low sensitivity (growth and photosynthetic activity), with the noteworthy exception of the planktonic alga, whose growth increased by more than 150% with the heteroaggregates at 1 µg L-1. Despite the lack of a strong direct effect of the NPs, they may still impair the functioning of aquatic ecosystems by destabilizing the competitive interactions between species. Moreover, further work should assess the toxicity of NPs associated with other substances (adsorbed pollutants or additives) that could enhance the NP effects.

Ecotoxicological assessment of commercial boron nitride nanotubes toward Xenopus laevis tadpoles and host-associated gut microbiota.

Despite the growing interest for boron nitride nanotubes (BNNT) due to their unique properties, data on the evaluation of the environmental risk potential of this emerging engineered nanomaterial are currently lacking. Therefore, the ecotoxicity of a commercial form of BNNT (containing tubes, hexagonal-boron nitride, and boron) was assessed in vivo toward larvae of the amphibian Xenopus laevis. Following the exposure, multiple endpoints were measured in the tadpoles as well as in bacterial communities associated to the host gut. Exposure to BNNT led to boron accumulation in host tissues and was not associated to genotoxic effects. However, the growth of the tadpoles increased due to BNNT exposure. This parameter was associated to remodeling of gut microbiome, benefiting to taxa from the phylum Bacteroidetes. Changes in relative abundance of this phylum were positively correlated to larval growth. The obtained results support the finding that BNNT are biocompatible as indicated by the absence of toxic effect from the tested nanomaterials. In addition, byproducts, especially free boron present in the tested product, were overall beneficial for the metabolism of the tadpoles.

Gut microbiota of aquatic organisms: A key endpoint for ecotoxicological studies.

Gut microbial communities constitute a compartment of crucial importance in regulation of homeostasis of multiple host physiological functions as well as in resistance towards environmental pollutants. Many chemical contaminants were shown to constitute a major threat for gut bacteria. Changes in gut microbiome could lead to alteration of host health. The access to high-throughput sequencing platforms permitted a great expansion of this discipline in human health while data from ecotoxicological studies are scarce and particularly those related to aquatic pollution. The main purpose of this review is to summarize recent body of literature providing data obtained from microbial community surveys using high-throughput 16S rRNA sequencing technology applied to aquatic ecotoxicity. Effects of pesticides, PCBs, PBDEs, heavy metals, nanoparticles, PPCPs, microplastics and endocrine disruptors on gut microbial communities are presented and discussed. We pointed out difficulties and limits provided by actual methodologies. We also proposed ways to improve understanding of links between changes in gut bacterial communities and host fitness loss, along with further applications for this emerging discipline.

Thermal Reduction of Graphene Oxide Mitigates Its In Vivo Genotoxicity Toward Xenopus laevis Tadpoles.

The worldwide increase of graphene family materials raises the question of the potential consequences resulting from their release in the environment and future consequences on ecosystem health, especially in the aquatic environment in which they are likely to accumulate. Thus, there is a need to evaluate the biological and ecological risk but also to find innovative solutions leading to the production of safer materials. This work focuses on the evaluation of functional group-safety relationships regarding to graphene oxide (GO) in vivo genotoxic potential toward X. laevis tadpoles. For this purpose, thermal treatments in H2 atmosphere were applied to produce reduced graphene oxide (rGOs) with different surface group compositions. Analysis performed indicated that GO induced disturbances in erythrocyte cell cycle leading to accumulation of cells in G0/G1 phase. Significant genotoxicity due to oxidative stress was observed in larvae exposed to low GO concentration (0.1 mg.L-¹). Reduction of GO at 200 °C and 1000 °C produced a material that was no longer genotoxic at low concentrations. X-ray photoelectron spectroscopy (XPS) analysis indicated that epoxide groups may constitute a good candidate to explain the genotoxic potential of the most oxidized form of the material. Thermal reduction of GO may constitute an appropriate "safer-by-design" strategy for the development of a safer material for environment.

Safety Assessment of Graphene-Based Materials: Focus on Human Health and the Environment.

Graphene and its derivatives are heralded as "miracle" materials with manifold applications in different sectors of society from electronics to energy storage to medicine. The increasing exploitation of graphene-based materials (GBMs) necessitates a comprehensive evaluation of the potential impact of these materials on human health and the environment. Here, we discuss synthesis and characterization of GBMs as well as human and environmental hazard assessment of GBMs using in vitro and in vivo model systems with the aim to understand the properties that underlie the biological effects of these materials; not all GBMs are alike, and it is essential that we disentangle the structure-activity relationships for this class of materials.

Transfer and Ecotoxicity of Titanium Dioxide Nanoparticles in Terrestrial and Aquatic Ecosystems: A Microcosm Study.

With the advancement in nanotechnology, particularly the use of TiO2 nanoparticles (NPs), there is a need to study their release into the environment and assess the related risk in an environmentally relevant contamination scenario. In the present study, the transfer and toxicity of TiO2 NPs in microcosms mimicking terrestrial and aquatic ecosystems were evaluated. The contaminated soil was prepared by spiking natural soils, with these then used as the basis for all exposure systems including preparation of soil leachates for amphibian exposure. Results demonstrated significant reductions in bacterial (-45%) and archaeal (-36%) nitrifier abundance; significant translocation of Ti to M. truncatula leaves (+422%); significant reductions in plant height (-17%), number of leaves (-29%), and aboveground biomass (-53%); nonsignificant Ti uptake in snail foot and viscera, and excretion in feces; and genotoxicity to X. laevis larvae (+119% micronuclei). Our study highlights a possible risk of engineered TiO2 NPs in the environment in terms of trophic transfer and toxicity in both terrestrial and aquatic environments.

CeO2 nanoparticle fate in environmental conditions and toxicity on a freshwater predator species: a microcosm study.

We studied the fate and toxicity of two types of CeO2 NPs (bare or citrate-coated) in environmentally relevant conditions, using large indoor microcosms. Long-term exposure was carried out on a three-leveled freshwater trophic chain, comprising microbial communities as primary producers, chironomid larvae as primary consumers, and amphibian larvae as secondary consumers. Whereas coated NPs preferentially sedimented, bare NPs were mainly found in the water column. However, mass balance indicated low recovery (51.5%) for bare NPs, indicating possible NP loss, against 98.8% of recovery for coated NPs. NPs were rather chemically stable, with less than 4% of dissolution. Chironomid larvae ingested large amounts of NPs and were vectors of contamination for amphibian larvae. Although bioaccumulation in amphibian larvae was important (9.47 and 9.74 mg/kg for bare and coated NPs, respectively), no biomagnification occurred through the trophic chain. Finally, significant genotoxicity was observed in amphibian larvae, bare CeO2 NPs being more toxic than citrate-coated NPs. ᅟ.

Environmental impact of engineered carbon nanoparticles: from releases to effects on the aquatic biota.

Nano-ecotoxicology is an emerging science which aims to assess the environmental effect of nanotechnologies. The development of this particular aspect of ecotoxicology was made necessary in order to evaluate the potential impact of recently produced and used materials: nanoparticles (NPs). Among all the types of NPs, carbon nanoparticles (CNPs) especially draw attention giving the increasing number of applications and integration into consumer products. However the potential impacts of CNPs in the environment remain poorly known. This review aims to point out the critical issues and aspects that will govern the toxicity of CNPs in the environment.

Surface Area of Carbon Nanoparticles: A Dose Metric for a More Realistic Ecotoxicological Assessment.

Engineered nanoparticles such as graphenes, nanodiamonds, and carbon nanotubes correspond to different allotropes of carbon and are among the best candidates for applications in fast-growing nanotechnology. It is thus likely that they may get into the environment at each step of their life cycle: production, use, and disposal. The aquatic compartment concentrates pollutants and is expected to be especially impacted. The toxicity of a compound is conventionally evaluated using mass concentration as a quantitative measure of exposure. However, several studies have highlighted that such a metric is not the best descriptor at the nanoscale. Here we compare the inhibition of Xenopus laevis larvae growth after in vivo exposure to different carbon nanoparticles for 12 days using different dose metrics and clearly show that surface area is the most relevant descriptor of toxicity for different types of carbon allotropes.

Toxicity of CeO2 nanoparticles on a freshwater experimental trophic chain: A study in environmentally relevant conditions through the use of mesocosms.

The toxicity of CeO2 NPs on an experimental freshwater ecosystem was studied in mesocosm, with a focus being placed on the higher trophic level, i.e. the carnivorous amphibian species Pleurodeles waltl. The system comprised species at three trophic levels: (i) bacteria, fungi and diatoms, (ii) Chironomus riparius larvae as primary consumers and (iii) Pleurodeles larvae as secondary consumers. NP contamination consisted of repeated additions of CeO2 NPs over 4 weeks, to obtain a final concentration of 1 mg/L. NPs were found to settle and accumulate in the sediment. No effects were observed on litter decomposition or associated fungal biomass. Changes in bacterial communities were observed from the third week of NP contamination. Morphological changes in CeO2 NPs were observed at the end of the experiment. No toxicity was recorded in chironomids, despite substantial NP accumulation (265.8 ± 14.1 mg Ce/kg). Mortality (35.3 ± 6.8%) and a mean Ce concentration of 13.5 ± 3.9 mg/kg were reported for Pleurodeles. Parallel experiments were performed on Pleurodeles to determine toxicity pathways: no toxicity was observed by direct or dietary exposures, although Ce concentrations almost reached 100 mg/kg. In view of these results, various toxicity mechanisms are proposed and discussed. The toxicity observed on Pleurodeles in mesocosm may be indirect, due to microorganism's interaction with CeO2 NPs, or NP dissolution could have occurred in mesocosm due to the structural complexity of the biological environment, resulting in toxicity to Pleurodeles. This study strongly supports the importance of ecotoxicological assessment of NPs under environmentally relevant conditions, using complex biological systems.

Toxicity of CeO2 nanoparticles at different trophic levels--effects on diatoms, chironomids and amphibians.

The aim of the present work is to provide wider information on the toxicity of cerium dioxide nanoparticles (CeO2 NPs) in aquatic environments, by studying the toxicity of two types of CeO2 NPs for four species (diatoms Nitzschia palea, the sediment-dwelling invertebrate Chironomus riparius, and the amphibian larvae Xenopus laevis and Pleurodeles waltl.). The two types of CeO2 NPs have different intrinsic properties: some of them are small citrate-coated spheres (2-5 nm), and the others are larger uncoated plates (20-60 nm). Acute toxicity (mortality at 48 or 96 h, depending on the test-organism) was assessed for the four species, from 0.1 to 100 mg L(-1) of NPs. Sub-lethal effects were assessed on chironomids exposed between 0.01 and 1 mg L(-1) of NPs. Mortality, growth inhibition and genotoxic effects were evaluated on amphibian larvae from 0.1 to 10 mg L(-1). Results reveal that no acute toxicity occurs on any species after short exposures, even at the highest concentrations. Mortality (35%) is observed on Xenopus larvae after 12d of exposure at the highest concentration of one type of NPs. No significant effects were observed on chironomids during chronic exposure. Xenopus larvae growth was inhibited from 1 mg L(-1) of both NPs while growth inhibition is observed on Pleurodeles only at the highest concentration of one type of NPs. No genotoxicity was observed on Xenopus but Pleurodeles exhibited dose-dependent genotoxic effects when exposed to one type of NPs. Observed differences in toxicity are discussed focusing on the studied compartment, routes of exposure, species and NPs.

Multi-walled carbon nanotubes, natural organic matter, and the benthic diatom Nitzschia palea: "a sticky story".

Different effects of multi-walled carbon nanotubes (MWCNTs) on the freshwater diatom Nitzschia palea were examined. MWCNTs used in this study (MWCNT) were dispersed either by sonication without (MWCNT sonicated) or with a realistic concentration (10 mg L(-1)) of Natural Organic Matter (MWCNT+NOM). A pocket-size device was designed to distinguish shading effect (using MWCNT suspensions as external filters) from total exposure effect of MWCNT sonicated and MWCNT+NOM on benthic algae. This study demonstrates that cell division was strongly inhibited after a 48 h exposure to MWCNT+NOM compared to MWCNT sonicated. This device did not yield a quantifiable contribution of shading to growth inhibition of MWCNT sonicated and below 10 mg L(-1) of MWCNT+NOM. In all cases, neither lethal effects nor drops in photosynthetic quantum yield were observed. After a 6-d exposure, a complete growth recovery was observed for all conditions except at the highest concentration of MWCNT+NOM. Different microscopic approaches using carbohydrates markers revealed the strong affinity between MWCNT and extracellular polymeric substances (EPS) produced by N. palea. These seem to constitute a defensive mechanism against MWCNT.