Validating Enteroid-Derived Monolayers from Murine Gut Organoids for Toxicological Testing of Inorganic Particles: Proof-of-Concept with Food-Grade Titanium Dioxide.

Human exposure to foodborne inorganic nanoparticles (NPs) is a growing concern. However, identifying potential hazards linked to NP ingestion often requires long-term exposure in animals. Owing these constraints, intestinal organoids are a promising alternative to in vivo experiments; as such, an in vitro approach should enable a rapid and reliable assessment of the effects of ingested chemicals on the gut. However, this remains to be validated for inorganic substances. In our study, a transcriptomic analysis and immunofluorescence staining were performed to compare the effects of food-grade TiO2 (fg-TiO2) on enteroid-derived monolayers (EDMs) from murine intestinal organoids to the known impacts of TiO2 on intestinal epithelium. After their ability to respond to a pro-inflammatory cytokine cocktail was validated, EDMs were exposed to 0, 0.1, 1, or 10 µg fg-TiO2/mL for 24 h. A dose-related increase of the muc2, vilin 1, and chromogranin A gene markers of cell differentiation was observed. In addition, fg-TiO2 induced apoptosis and dose-dependent genotoxicity, while a decreased expression of genes encoding for antimicrobial peptides, and of genes related to tight junction function, was observed. These results validated the use of EDMs as a reliable model for the toxicity testing of foodborne NPs likely to affect the intestinal barrier.

Dietary Amino Acid Source Elicits Sex-Specific Metabolic Response to Diet-Induced NAFLD in Mice.

SCOPE: Non-alcoholic fatty liver disease (NAFLD) is a sexually dimorphic disease influenced by dietary factors. Here, the metabolic and hepatic effects of dietary amino acid (AA) source is assessed in Western diet (WD)-induced NAFLD in male and female mice. METHODS AND RESULTS: The AA source is either casein or a free AA mixture mimicking the composition of casein. As expected, males fed a casein-based WD display glucose intolerance, fasting hyperglycemia, and insulin-resistance and develop NAFLD associated with changes in hepatic gene expression and microbiota dysbiosis. In contrast, males fed the AA-based WD show no steatosis, a similar gene expression profile as males fed a control diet, and a distinct microbiota composition compared to males fed a casein-based WD. Females are protected against WD-induced liver damage, hepatic gene expression, and gut microbiota changes regardless of the AA source. CONCLUSIONS: Free dietary AA intake prevents the unhealthy metabolic outcomes of a WD preferentially in male mice.

A 90-day oral exposure to food-grade gold at relevant human doses impacts the gut microbiota and the local immune system in a sex-dependent manner in mice.

BACKGROUND: Edible gold (Au) is commonly used as a food additive (E175 in EU) for confectionery and cake decorations, coatings and in beverages. Food-grade gold is most often composed of thin Au sheets or flakes exhibiting micro- and nanometric dimensions in their thickness. Concerns about the impact of mineral particles used as food additives on human health are increasing with respect to the particular physico-chemical properties of nanosized particles, which enable them to cross biological barriers and interact with various body cell compartments. In this study, male and female mice were exposed daily to E175 or an Au nanomaterial (Ref-Au) incorporated into food at relevant human dose for 90 days in order to determine the potential toxicity of edible gold. RESULTS: E175 or Ref-Au exposure in mice did not induce any histomorphological damage of the liver, spleen or intestine, nor any genotoxic effects in the colon and liver despite an apparent higher intestinal absorption level of Au particles in mice exposed to Ref-Au compared to the E175 food additive. No changes in the intestinal microbiota were reported after treatment with Ref-Au, regardless of sex. In contrast, after E175 exposure, an increase in the Firmicutes/Bacteroidetes ratio and in the abundance of Proteobacteria were observed in females, while a decrease in the production of short-chain fatty acids occurred in both sexes. Moreover, increased production of IL-6, TNFα and IL-1ß was observed in the colon of female mice at the end of the 90-day exposure to E175, whereas, decreased IL-6, IL-1ß, IL-17 and TGFß levels were found in the male colon. CONCLUSIONS: These results revealed that a 90-day exposure to E175 added to the diet alters the gut microbiota and intestinal immune response in a sex-dependent manner in mice. Within the dose range of human exposure to E175, these alterations remained low in both sexes and mostly appeared to be nontoxic. However, at the higher dose, the observed gut dysbiosis and the intestinal low-grade inflammation in female mice could favour the occurrence of metabolic disorders supporting the establishment of toxic reference values for the safe use of gold as food additive.

The food additive titanium dioxide hinders intestinal production of TGF-ß and IL-10 in mice, and long-term exposure in adults or from perinatal life blocks oral tolerance to ovalbumin.

Food hypersensitivities are increasing in industrialized countries, and foodborne nanoparticles (NPs) are suspected as co-factors in their aetiology. Food-grade titanium dioxide (fg-TiO2), a food colouring agent, is composed of NPs with immunomodulatory properties. We investigated whether fg-TiO2 may compromise the establishment of oral tolerance (OT) to food proteins using a model of OT induction to ovalbumin (OVA) in mice, and whether a perinatal exposure could trigger this effect. In pregnant mice fed a TiO2-enriched diet, ICP-MS and TEM-EDX analyses showed passage of TiO2 NPs into the foetus. When their weaned offspring were fed the same diet, a breakdown in OT to OVA was observed at adulthood, characterized by a high anti-OVA IgG production compared to controls. However, adult mice directly exposed to fg-TiO2 did not induce OT to OVA either, ruling out a developmental origin for these effects. When these mice were orally challenged with OVA, intestinal inflammation demonstrated hypersensitivity to OVA. In OVA-naïve mice, fg-TiO2 exposure impaired intestinal TGF-ß and IL-10 production, of key role in OT induction and maintenance. These findings showed that long-term exposure to TiO2 as food additive alters anti-inflammatory cytokine profile, and leads to OT failure regardless of the timing of TiO2 exposure throughout life.

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.

Dysregulation along the gut microbiota-immune system axis after oral exposure to titanium dioxide nanoparticles: A possible environmental factor promoting obesity-related metabolic disorders.

Food additives are one major hallmark of ultra-processed food in the Western-diet, a food habit often associated with metabolic disorders. Among these additives, the whitener and opacifying agent titanium dioxide (TiO2) raises public health issues due to the ability of TiO2 nanoparticles (NPs) to cross biological barriers and accumulate in different systemic organs like spleen, liver and pancreas. However before their systemic passage, the biocidal properties of TiO2 NPs may alter the composition and activity of the gut microbiota, which play a crucial role for the development and maintenance of immune functions. Once absorbed, TiO2 NPs may further interact with immune intestinal cells involved in gut microbiota regulation. Since obesity-related metabolic diseases such as diabetes are associated with alterations in the microbiota-immune system axis, this raises questions about the possible involvement of long-term exposure to food-grade TiO2 in the development or worsening of these diseases. The current purpose is to review the dysregulations along the gut microbiota-immune system axis after oral TiO2 exposure compared to those reported in obese or diabetic patients, and to highlight potential mechanisms by which foodborne TiO2 NPs may increase the susceptibility to develop obesity-related metabolic disorders.

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.

Federating young researchers in microbial ecotoxicology: EcotoxicoMicYR 2021, the first international webinar organized for and by young microbial ecotoxicology researchers.

The EcotoxicoMicYR group was initially composed of 4 Ph.D. students and 4 post-doctoral researchers. In brief, the EcotoxicoMicYR webinar took place three Monday afternoons in a row from November 22 to December 6, 2021. These three half-day webinars reached a success beyond our expectations with 25 countries and 41 presentations. Keynote lectures were delivered by Dr Fabio Roldan (Pontificia Universidad Javeriana, Colombia), Dr Belinda Ferrari (The University of New South Wales, Australia), and Dr Ahmed Tlili (Eawag, Switzerland). Their presentations provided an insight on latest research developments in the microbial ecotoxicology field and highlighted their specific contribution to this discipline. Twenty-two oral presentations and 16 pre-recorded presentations were diffused.

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.

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.

Field biomonitoring using the zebra mussel Dreissena polymorpha and the quagga mussel Dreissena bugensis following immunotoxic reponses. Is there a need to separate the two species?

The zebra mussel, Dreissena polymorpha constitutes an extensively used sentinel species for biomonitoring in European and North American freshwater systems. However, this invasive species is gradually replaced in freshwater ecosystem by Dreissena bugensis, a closely related dreissenid species that shares common morphological characteristics but possess some physiological differences. However, few are known about differences on more integrated physiological processes that are generally used as biomarkers in biological monitoring studies. Declining of zebra mussel populations raises the question of the sustainability of using one or both species indifferently to maintain the quality of environmental pollution monitoring data. In our study, we performed a field comparative study measuring immune-related markers and bioaccumulation of PCBs, PAHs and PBDEs in sympatrically occurring mussel populations from three sites of the St. Lawrence River. For tested organisms, species were identified using RFLP analysis. Measurement of bioaccumulated organic compounds indicated a higher accumulation of PCBs and PBDEs in D. bugensis soft tissues compared to D. polymorpha while no differences were noticed for PAHs. Results of hemocytic parameters highlighted that differences of hemocyte distributions were associated to modulations of phagocytic activities. Moreover, marked differences occurred in measurement of hemocytic oxidative activity, indicating divergences between the two species for ROS regulation strategies. This physiological characteristic may deeply influence species responses facing environmental or pollution related stress and induce bias if the two species are not differentiated in further biomarker or bioaccumulation measurement-based studies.

Differential sensitivity to cadmium of immunomarkers measured in hemocyte subpopulations of zebra mussel Dreissena polymorpha.

Increasing discharge of industrial wastes into the environment results in pollution transfer towards hydrosystems. These activities release heavy metals such as cadmium, known as persistent pollutant that is accumulated by molluscs and exercise immunotoxicological effects. Among molluscs, the zebra mussel, Dreissena polymorpha constitutes a suitable support for freshwater ecotoxicological studies. In molluscs, homeostasis maintain is ensured in part by hemocytes that are composed of several cell populations involved in multiple physiological processes such as cell-mediated immune response or metal metabolism. Thus, hemocytes constitute a target of concern to study adverse effects of heavy metals. The objectives of this work were to determine whether immune-related endpoints assessed were of different sensitivity to cadmium and whether hemocyte functionalities were differentially affected depending on hemocyte subpopulation considered. Hemocytes were exposed ex vivo to concentrations of cadmium ranging from 10-6 M to 10-3 M for 21h prior flow cytometric analysis of cellular markers. Measured parameters (viability, phagocytosis, oxidative activity, lysosomal content) decreased in a dose-dependent manner with sensitivity differences depending on endpoint and cell type considered. Our results indicated that phagocytosis related endpoints were the most sensitive studied mechanisms to cadmium compared to other markers with EC50 of 3.71±0.53×10-4M for phagocytic activity and 2.79±0.19×10-4M considering mean number of beads per phagocytic cell. Lysosomal content of granulocytes was less affected compared to other cell types, indicating lower sensitivity to cadmium. This suggests that granulocyte population is greatly involved in metal metabolism. Mitochondrial activity was reduced only in blast-like hemocytes that are considered to be cell precursors. Impairment of these cell functionalities may potentially compromise functions ensured by differentiated cells. We concluded that analysis of hemocyte activities should be performed at sub-population scale for more accurate results in ecotoxicological studies.

Functional features of hemocyte subpopulations of the invasive mollusk species Dreissena polymorpha.

Dreissena polymorpha is a mussel species that invaded many lotic and lentic inland waters in Western Europe and North America. Its positive or negative interactions with biotic and abiotic components of ecosystems are numerous, making this bivalve the subject of numerous studies in ecology, ecophysiology and ecotoxicology. In these contexts, the functional characterization of the zebra mussel hemocytes is of particular interest, as hemocytes are central cells involved in vital functions (immunity, growth, reproduction) of molluscan physiology. Dreissena polymorpha circulating hemocytes populations were characterized by a combination of structural and functional analysis. Assessments were performed during two contrasted physiological periods for mussels (gametogenesis and spawning). Three hemocyte types were identified as hyalinocytes and blast-like cells for agranular hemocytes and one granulocyte population. Flow cytometry analysis of hemocytes functionalities indicated that blast-like cells had low oxidative and mitochondrial activities and low lysosomal content. Hyalinocytes and granulocytes are fully equipped to perform innate immune response. Hyalinocytes exhibit higher oxidative activity than granulocytes. Such observation is not common since numerous studies show that granulocytes are usually cells that have the highest cellular activities. This result demonstrates the significant functional variability of hemocyte subpopulations. Moreover, our findings reveal that spawning period of Dreissena polymorpha was associated with an increase of hyalinocyte percentage in relation to low levels of biological activities in hemocytes. This reduction in hemocyte activity would reflect the important physiological changes associated with the spawning period of this invasive species known for its high reproductive potential.

Sub-chronic exposure to fluoxetine in juvenile oysters (Crassostrea gigas): uptake and biological effects.

The bioconcentration potential of fluoxetine (FLX) and its biological effects were investigated in juvenile Pacific oyster exposed for 28 days to environmentally relevant concentrations of FLX (1 ng L(-1), 100 ng L(-1) and up to 10 µg L(-1)). FLX bioaccumulated in oyster flesh resulting in 28-day bioconcentration factors greater than 2,000 and 10,000 by referring to wet and dry weights, respectively. Nevertheless, FLX did not induce oyster mortality, delayed gametogenesis, or lead to adverse histopathological alterations. At the two highest concentrations, despite non-optimal trophic conditions, FLX stimulated shell growth but only in a transient manner, suggesting a role of serotonin in the regulation of feeding and metabolism in bivalves. Those high concentrations seemed to drive bell-shaped responses of catalase and glutathione S-transferase activities throughout the exposure period, which may indicate the activation of antioxidant enzyme synthesis and then an enhanced catabolic rate or direct inhibition of those enzymes. However, no clear oxidative stress was detected because no strong differences in thiobarbituric acid-reactive substance (TBARS) content (i.e. lipid peroxidation) were observed between oyster groups, suggesting that cellular defence mechanisms were effective. These results demonstrate the importance of considering additional biomarkers of oxidative stress to obtain a comprehensive overview of the FLX-induced changes in marine bivalves exposed under realistic conditions. Considering the battery of biomarkers used, FLX appears to induce little or no effects on oyster physiology even at a concentration of 10 µg L(-1). These results do not confirm the lowest observed effect concentration (LOEC) values reported by some authors in other mollusc species.