Multigenerational responses in the Lymnaea stagnalis freshwater gastropod exposed to diclofenac at environmental concentrations.

Over the last decade, there has been increased concern about the occurrence of diclofenac (DCF) in aquatic ecosystems. Living organisms could be exposed to this "pseudo-persistent" pharmaceutical for more than one generation. In this multigenerational study, we assessed the DCF impact at environmentally relevant concentrations on the life history and behavioral parameters of two offspring generations (F1 and F2) of the Lymnaea stagnalis freshwater gastropod. Snail growth was affected by DCF in the F1 generation, with increased shell sizes of juveniles exposed to 0.1 µg L - 1 concentration and a decreased shell size at 2 and 10 µg L - 1. DCF also lowered food intake, enhanced locomotion activity and reduced the number of eggs/egg mass in the F1 generation. For the F2 generation, shorter time to hatch, faster growth, increased food intake and production of more egg masses/snail were induced by DCF exposure at 10 µg L - 1. Over time, DCF exposure led to maximization of L. stagnalis reproductive function. These results show that multigenerational studies are crucial to reveal adaptive responses to chronic contaminant exposure, which are not observable after short-term exposure.

Metabolism of the aquatic pollutant diclofenac in the Lymnaea stagnalis freshwater gastropod.

The metabolism of organic contaminants in Lymnaea stagnalis freshwater gastropod remains unknown. Yet, pharmaceuticals-like the NSAID diclofenac-are continuously released in the aquatic environment, thereby representing a risk to aquatic organisms. In addition, lower invertebrates may be affected by this pollution since they are likely to bioaccumulate contaminants. The metabolism of pharmaceuticals in L. stagnalis requires further investigation to understand their detoxification mechanisms and characterized the risk posed by contaminant exposure in this species. In this study, a non-targeted strategy using liquid chromatography combined with high-resolution mass spectrometry was applied to highlight metabolites formed in L. stagnalis freshwater snails exposed to 300 µg/L diclofenac for 3 and 7 days. Nineteen metabolites were revealed by this approach, 12 of which were observed for the first time in an aquatic organism exposed to diclofenac. Phase I metabolism involved hydroxylation, with detection of 3'-, 4'-, and 5-hydroxydiclofenac and three dihydroxylated metabolites, as well as cyclization, oxidative decarboxylation, and dehydrogenation, while phase II metabolism consisted of glucose and sulfate conjugation. Among these reactions, the two main DCF detoxification pathways detected in L. stagnalis were hydroxylation (phase I) and glucosidation (phase II).

Long-term exposure to environmental diclofenac concentrations impairs growth and induces molecular changes in Lymnaea stagnalis freshwater snails.

As pharmaceutical substances are highly used in human and veterinary medicine and subsequently released in the environment, they represent emerging contaminants in the aquatic compartment. Diclofenac (DCF) is one of the most commonly detected pharmaceuticals in water and little research has been focused on its long-term effects on freshwater invertebrates. In this study, we assessed the chronic impacts of DCF on the freshwater gastropod Lymnaea stagnalis using life history, behavioral and molecular approaches. These organisms were exposed from the embryo to the adult stage to three environmentally relevant DCF concentrations (0.1, 2 and 10 µg/L). The results indicated that DCF impaired shell growth and feeding behavior at the juvenile stage, yet no impacts on hatching, locomotion and response to light stress were noted. The molecular findings (metabolomics and transcriptomic) suggested that DCF may disturb the immune system, energy metabolism, osmoregulation and redox balance. In addition, prostaglandin synthesis could potentially be inhibited by DCF exposure. The molecular findings revealed signs of reproduction impairment but this trend was not confirmed by the physiological tests. Combined omics tools provided complementary information and enabled us to gain further insight into DCF effects in freshwater organisms.

Effects of the anxiolytic benzodiazepine oxazepam on freshwater gastropod reproduction: a prospective study.

Psychoactive drugs have emerged as contaminants over the last few decades. These drugs are frequently prescribed and poorly eliminated by wastewater treatment plants, and many are present at non-negligible concentrations in surface waters. Several studies have investigated the non-target organism toxicity of one such drug, oxazepam, a benzodiazepine anxiolytic frequently detected in rivers. However, very little is known about the impact of this drug on reproduction. We investigated the effects of environmentally relevant concentrations of oxazepam on Radix balthica, a freshwater gastropod widespread in Europe. We identified the reproductive organs of Radix balthica. We then exposed this gastropod to oxazepam for two months and assessed several reproductive parameters, from reproductive organ status to behavioral parameters. We found that adults exposed to 10 µg/L oxazepam display an increase in the density of spermatozoa, and that adults exposed to 0.8 µg/L oxazepam displayed a decrease in the number of eggs per egg mass over time. By contrast, oxazepam had no effect on shell length, the size of male reproductive organs or social interactions. Finally, a locomotor activity analysis showed the distance covered over time decreased in all conditions of exposure to oxazepam, potentially reflecting a disturbance of exploratory activity. These results shed light on the effects of oxazepam on the reproduction of a non-target freshwater mollusk.

Low concentrations of oxazepam induce feeding and molecular changes in Radix balthica juveniles.

Psychotropics, especially benzodiazepines, are commonly prescribed worldwide. Poorly eliminated at wastewater treatment plants, they belong to a group of emerging contaminants. Due to their interaction with the GABAA receptor, they may affect the function of the nervous system of non-target organisms, such as aquatic organisms. The toxicity of oxazepam, a very frequently detected benzodiazepine in continental freshwater, has been largely studied in aquatic vertebrates over the last decade. However, its effects on freshwater non-vertebrates have received much less attention. We aimed to evaluate the long-term effects of oxazepam on the juvenile stage of a freshwater gastropod widespread in Europe, Radix balthica. Juveniles were exposed for a month to environmentally-relevant concentrations of oxazepam found in rivers (0.8 µg/L) and effluents (10 µg/L). Three main physiological functions were studied: feeding, growth, and locomotion. Additionally, gene expression analysis was performed to provide insights into toxicity mechanisms. There was a strong short-term activation of the feeding rate at low concentration, whereas the high dose resulted in long-term inhibition of food intake. A significant decrease in mortality rate was observed in juveniles exposed to the lowest dose. Shell growth and locomotor activity did not appear to be affected by oxazepam. Transcriptomic analysis revealed global over-expression of genes involved in the nervous regulation of the feeding, digestive, and locomotion systems after oxazepam exposure. The molecular analysis also revealed a possible interference of animal manipulation with the molecular effects induced by oxazepam exposure. Overall, these results improve our understanding of the effects of the psychoactive drug oxazepam on an aquatic mollusc gastropod.

New protocols for the selection and rearing of Metoncholaimus pristiurus and the first evaluation of oxidative stress biomarkers in meiobenthic nematodes.

Meiobenthic nematodes have been designated as sensitive global models in the development of biomonitoring and ecotoxicology monitoring programs howbeit the sensitivity of these organisms against oxidative stress biomarkers have never been addressed. The present study aimed to decipher this research axis after selecting and culturing a single nematode species from an entire community through original laboratory protocols. The purpose of this investigation was to change the grain size of the sediment into the immediate environment of nematodes by progressively adding a biosubstrate made from Sepia officinalis endoskeletton. At the end of the experiment, Metoncholaimus pristiurus became the unique component of the nematode species when the sediment was enriched with 80% of S. officinalis powder. After the mono-species level had been achieved, the selected species was fed on an another biosubstrate made from bodies of Porcellio scaber under the identical laboratory controlled conditions of light and temperature adopted during the selection process. Accordingly, the bioassay protocol this study layed new foundations for the study of meiobenthic nematodes in the biomarker field. Our results revealed that, in case of M. pritiurus, discernible oxidative stress responses are valid for catalase and gluthatione S-transferase. Indeed, for both enzymes, a clear increase in the activity was recorded, and the response was more reinforced when zinc and permethrin were administrated in combination. The relevance of the protocols proposed in this work parallels their global applicability to reach and maintain the monospecific level in laboratory by using biosubstrates made from animals widely distributed. It is true also that our data provided the first results in terms of biochemical biomarkers for meiobenthic nematodes and showed that the selected taxa, M. pristiurus, could be one of the first marine taxa responding early to the tested stressors, zinc and permethrin, even at very low concentrations.

Evaluation of psychiatric hospital wastewater toxicity: what is its impact on aquatic organisms?

The primary source of pharmaceuticals to the aquatic environment is the discharge of wastewater effluents. Pharmaceuticals are a large and diverse group of compounds. Among them, psychotropic substances are particularly interesting to study due to their specific known mode of action. The present study was performed to investigate the effects of wastewater effluents from a psychiatric hospital wastewater treatment plant (WWTP) on several aquatic organisms. All the analyzed pharmaceuticals (10 compounds) were detected in WWTP effluents as well as in the receiving river. Although the environmental concentrations were generally at trace levels (ng L-1 to µg L-1), induce toxic effects were observed. This study showed the effects of the WWTP effluents on the oogenesis and/or embryogenesis of amphipod crustacean Gammarus fossarum, Japanese fish medaka Oryzias latipes, mollusk Radix peregra, and planarian Schmidtea polychroa. A decrease of the number of oocytes and produced embryos was observed for G. fossarum and S. polychroa. Similarly, the hatching rate of R. peregra was affected by effluents. In the receiving river, the macroinvertebrate community was affected by the wastewater effluents discharge.

Correction to: Assessment of Lemna minor (duckweed) and Corbicula fluminea (freshwater clam) as potential indicators of contaminated aquatic ecosystems: responses to presence of psychoactive drug mixtures.

The correct presentation of the Author names are shown in this paper.

Assessment of Lemna minor (duckweed) and Corbicula fluminea (freshwater clam) as potential indicators of contaminated aquatic ecosystems: responses to presence of psychoactive drug mixtures.

The pharmaceutical products are emerging pollutants continuously released into the environment, because they cannot be effectively removed by the wastewater treatment plants. In recent years, questions have been raised concerning the environmental risks related to these pollutants. The goal of this research was to evaluate the responses in Lemna minor after 7 days and in Corbicula fluminea after differing durations (1, 3, 7, and 19 days) of exposure to the psychoactive drug mixture (valproic acid, citalopram, carbamazepine, cyamemazine, hydroxyzine, oxazepam, norfluoxetine, lorazepam, fluoxetine, and sertraline) in different concentrations (0, 0 + ethanol, drug concentration (DC) 1 = river water concentration, DC2 = effluent concentration, and DC3 = 10× effluent concentration). In this aim, growth parameters of L. minor, gluthathione S-transferase (GSTs), catalase (CAT), ethoxyresorufin-O-deethylase (EROD) and/or gene expressions (pi-gst, cat, cytochrome P450 4 (cyp4), multidrug resistant 1 (mdr1), and superoxide dismutase (sod)) were measured. GST activities increased significantly in L. minor exposed to DC3, but no changes were found in CAT activity. In C. fluminea, EROD activity was induced significantly in both gill and digestive gland tissues after 3 days' exposure to DC3, while a GST increase was observed only in digestive gland tissues, suggesting that these pharmaceuticals induced an oxidative effect. Gene expression analysis revealed transient transcriptomic responses of cyp4, sod, and mdr1 under drug concentrations 2 or 3 and no change of expression for the other genes (cat and pi-gst) or condition (environmental drug concentration) tested. Finally, the data reported in this study represent important ecotoxicological information, confirming that this enzyme family (cyp4, sod, and mdr1) may be considered as a sensible and early indicator of exposure to drugs and emphasizing the involvement of selected genes in detoxification pathways.

De novo transcriptome sequencing and analysis of freshwater snail (Radix balthica) to discover genes and pathways affected by exposure to oxazepam.

Pharmaceuticals are increasingly found in aquatic ecosystems due to the non-efficiency of waste water treatment plants. Therefore, aquatic organisms are frequently exposed to a broad diversity of pharmaceuticals. Freshwater snail Radix balthica has been chosen as model to study the effects of oxazepam (psychotropic drug) on developmental stages ranging from trochophore to hatching. In order to provide a global insight of these effects, a transcriptome deep sequencing has been performed on exposed embryos. Eighteen libraries were sequenced, six libraries for three conditions: control, exposed to the lowest oxazepam concentration with a phenotypic effect (delayed hatching) (TA) and exposed to oxazepam concentration found in freshwater (TB). A total of 39,759,772 filtered raw reads were assembled into 56,435 contigs having a mean length of 1579.68 bp and mean depth of 378.96 reads. 44.91% of the contigs have at least one annotation. The differential expression analysis between the control condition and the two exposure conditions revealed 146 contigs differentially expressed of which 144 for TA and two for TB. 34.0% were annotated with biological function. There were four mainly impacted processes: two cellular signalling systems (Notch and JNK) and two biosynthesis pathways (Polyamine and Catecholamine pathways). This work reports a large-scale analysis of differentially transcribed genes of R. balthica exposed to oxazepam during egg development until hatching. In addition, these results enriched the de novo database of potential ecotoxicological models.

Psychotropic drugs in mixture alter swimming behaviour of Japanese medaka (Oryzias latipes) larvae above environmental concentrations.

Psychiatric pharmaceuticals, such as anxiolytics, sedatives, hypnotics and antidepressors, are among the most prescribed active substances in the world. The occurrence of these compounds in the environment, as well as the adverse effects they can have on non-target organisms, justifies the growing concern about these emerging environmental pollutants. This study aims to analyse the effects of six psychotropic drugs, valproate, cyamemazine, citalopram, sertraline, fluoxetine and oxazepam, on the survival and locomotion of Japanese medaka Oryzias latipes larvae. Newly hatched Japanese medaka were exposed to individual compounds for 72 h, at concentrations ranging from 10 µg L(-1) to 10 mg L(-1). Lethal concentrations 50 % (LC50) were estimated at 840, 841 and 9,136 µg L(-1) for fluoxetine, sertraline and citalopram, respectively, while other compounds did not induce any significant increase in mortality. Analysis of the swimming behaviour of larvae, including total distance moved, mobility and location, provided an estimated lowest observed effect concentration (LOEC) of 10 µg L(-1) for citalopram and oxazepam, 12.2 µg L(-1) for cyamemazine, 100 µg L(-1) for fluoxetine, 1,000 µg L(-1) for sertraline and >10,000 µg L(-1) for valproate. Realistic environmental mixture of the six psychotropic compounds induced disruption of larval locomotor behaviour at concentrations about 10- to 100-fold greater than environmental concentrations.

Responses of cytochrome P450, GST, and MXR in the mollusk Corbicula fluminea to the exposure to hospital wastewater effluents.

Pharmaceutical products are a major group of chemical compounds that are continuously released into the environment. The primary pathway of pharmaceuticals to the aquatic environment is the discharge of wastewater effluents. The Psychiatric hospital of Montpon (Dordogne, France) operates with its wastewater treatment plant. We first evaluated the presence and concentrations of 27 pharmaceuticals compounds in these effluents. All of the 27 compounds were detected in these wastewater effluents at concentrations ranging between 37,500 ng L(-1) (paracetamol) and 150 ng L(-1) (citalopram). The aim of the study was then to evaluate the exposure effects of the effluents on cytochrome P450, GST, and MXR responses in Corbicula fluminea gills and digestive glands. Experiments on clams exposed during 1, 3, 7 14, and 21 days revealed a strong and continuous overexpression of mdr1 (multidrug resistant 1) gene expression in gills and transitory variations in pi-gst expression and GST activity. EROD activity increased also transitory after 1 day in the digestive gland of exposed clams. These results indicated that in the effluent, some molecules have undergone metabolism of phase 1 and/or phase 2.

Environmental and health impacts of fine and ultrafine metallic particles: assessment of threat scores.

This study proposes global threat scores to prioritize the harmfulness of anthropogenic fine and ultrafine metallic particles (FMP) emitted into the atmosphere at the global scale. (Eco)toxicity of physicochemically characterized FMP oxides for metals currently observed in the atmosphere (CdO, CuO, PbO, PbSO(4), Sb(2)O(3), and ZnO) was assessed by performing complementary in vitro tests: ecotoxicity, human bioaccessibility, cytotoxicity, and oxidative potential. Using an innovative methodology based on the combination of (eco)toxicity and physicochemical results, the following hazard classification of the particles is proposed: CdCl2~CdO>CuO>PbO>ZnO>PbSO(4)>Sb(2)O(3). Both cadmium compounds exhibited the highest threat score due to their high cytotoxicity and bioaccessible dose, whatever their solubility and speciation, suggesting that cadmium toxicity is due to its chemical form rather than its physical form. In contrast, the Sb(2)O(3) threat score was the lowest due to particles with low specific area and solubility, with no effects except a slight oxidative stress. As FMP physicochemical properties reveal differences in specific area, crystallization systems, dissolution process, and speciation, various mechanisms may influence their biological impact. Finally, this newly developed and global approach could be widely used in various contexts of pollution by complex metal particles and may improve risk management.

Comparison of mineral-based amendments for ex-situ stabilization of trace elements (As, Cd, Cu, Mo, Ni, Zn) in marine dredged sediments: a pilot-scale experiment.

Trace element pollution of marine dredged sediments is an emerging problem all over the world. Comparing to other wastes, trace elements stabilization is more difficult both due to the wide range of contaminants present in the marine sediments and their inherent physicochemical properties. In this study, a pilot-scale experiment was performed to stabilize As, Cd, Cu, Mo, Ni, and Zn in a multi-contaminated sediment sample using hematite, zero-valent iron and zeolite. Results showed that iron-based amendments were able to reduce the leaching and the bioavailability of trace elements in the sediment sample, while zeolite was unsuitable. Chemical stabilization through iron-based amendments seems to be a promising approach as a low-cost alternative to traditional stabilization methods involving chemical reagents.

Batch and column studies of the stabilization of toxic heavy metals in dredged marine sediments by hematite after bioremediation.

The management of dredged sediments is an important issue in coastal regions where the marine sediments are highly polluted by metals and organic pollutants. In this paper, mineral-based amendments (hematite, zero-valent iron and zeolite) were used to stabilize metallic pollutants (As, Cd, Cu, Mo, Ni, Pb, and Zn) in a contaminated marine sediment sample. Mineral-based amendments were tested at three application rates (5 %, 10 %, and 15 %) in batch experiments in order to select the best amendment to perform column experiments. Batch tests have shown that hematite was the most efficient amendment to stabilize inorganic pollutants (As, Cd, Cu, Mo, Ni, Pb, and Zn) in the studied sediment. Based on batch tests, hematite was used at one application rate equal to 5 % to conduct column experiments. Column tests confirmed that hematite was able to decrease metal concentrations in leachates from stabilized sediment. The stabilization rates were particularly high for Cd (67 %), Mo (80 %), and Pb (90 %). The Microtox solid phase test showed that hematite could decrease significantly the toxicity of stabilized sediment. Based on batch and column experiments, it emerged that hematite could be a suitable adsorbent to stabilize metals in dredged marine sediment.

Use of ecotoxicity test and ecoscores to improve the management of polluted soils: case of a secondary lead smelter plant.

With the rise of sustainable development, rehabilitation of brownfield sites located in urban areas has become a major concern. Management of contaminated soils in relation with environmental and sanitary risk concerns is therefore a strong aim needing the development of both useful tools for risk assessment and sustainable remediation techniques. For soils polluted by metals and metalloids (MTE), the criteria for landfilling are currently not based on ecotoxicological tests but on total MTE concentrations and leaching tests. In this study, the ecotoxicity of leachates from MTE polluted soils sampled from an industrial site recycling lead-acid batteries were evaluated by using both modified Escherichia coli strains with luminescence modulated by metals and normalized Daphnia magna and Alivibrio fischeri bioassays. The results were clearly related to the type of microorganisms (crustacean, different strains of bacteria) whose sensitivity varied. Ecotoxicity was also different according to sample location on the site, total concentrations and physico-chemical properties of each soil. For comparison, standard leaching tests were also performed. Potentially phytoavailable fraction of MTE in soils and physico-chemical measures were finally performed in order to highlight the mechanisms. The results demonstrated that the use of a panel of microorganisms is suitable for hazard classification of polluted soils. In addition, calculated eco-scores permit to rank the polluted soils according to their potentially of dangerousness. Influence of soil and MTE characteristics on MTE mobility and ecotoxicity was also highlighted.

Arsenic in marine sediments from French Mediterranean ports: geochemical partitioning, bioavailability and ecotoxicology.

This work investigates arsenic mobility, bioavailability and toxicity in marine port sediments using chemical sequential extraction and laboratory toxicity tests. Sediment samples were collected from two different Mediterranean ports, one highly polluted with arsenic and other inorganic and organic pollutants (Estaque port (EST)), and the other one, less polluted, with a low arsenic content (Saint Mandrier port (SM)). Arsenic distribution in the solid phase was studied using a sequential extraction procedure specifically developed for appraising arsenic mobility in sediments. Toxicity assessment was performed on sediment elutriates, solid phases and aqueous arsenic species as single substance using the embryo-toxicity test on oyster larvae (Crassostrea gigas) and the Microtox test with Vibrio fischeri. Toxicity results showed that all sediment samples presented acute and sub-chronic toxic effects on oyster larvae and bacteria, respectively. The Microtox solid phase test allow to discriminate As-contaminated samples from the less contaminated ones, suggesting that toxicity of whole sediment samples is related to arsenic content. Toxicity of dissolved arsenic species as single substance showed that Vibrio fischeri and oyster larvae are most sensitive to As(V) than As(III). The distribution coefficient (Kd) of arsenic in sediment samples was estimated using results obtained in chemical sequential extractions. The Kd value is greater in SM (450 Lkg(-1)) than in EST (55 Lkg(-1)), indicating that arsenic availability is higher for the most toxic sediment sample (Estaque port). This study demonstrates that arsenic speciation play an important role on arsenic mobility and its bioavailability in marine port sediments.

Ex situ remediation of contaminated sediments using mineral additives: assessment of pollutant bioavailability with the Microtox solid phase test.

The aim of this work is to assess the potential ecotoxicological effects of contaminated sediments treated with mineral additives. The Microtox solid phase test was used to evaluate the effect of mineral additives on the toxicity of sediment suspensions. Four Mediterranean port sediments were studied after dredging and bioremediation: Sample A from navy harbor, sample B from commercial port and samples C and D from pleasure ports. Sediment samples were stabilized with three mineral additives: hematite, zero-valent iron and zeolite. Results show that all studied mineral additives can act as stabilizer agent in highly contaminated sediments (A and C) by decreasing dissolved metal concentrations and sediment toxicity level. On the contrary, for the less contaminated samples (B and D) hematite and zeolite can provoke toxic effect towards Vibrio fischeri since additive particles can favor bacteria retention and decrease bioluminescence emission.

The toxicity of composted sediments from Mediterranean ports evaluated by several bioassays.

This work investigates the ecotoxicological evaluation of contaminated dredged sediments from French Mediterranean navy harbour (A), commercial port (B) and two composite specimens (C) and (D) coming from the mixture of A and B with other port sediments. The toxicity of elutriates from these sediments is estimated using embryo-toxicity test, Microtox® solid phase test, LuminoTox, phytotoxicity tests and genotoxicity test. Bioassay responses are not clearly correlated with chemical contamination in the whole sediment and vary as a function of tested organisms. The highest contaminated samples (A and C) are almost always more toxic than the less contaminated samples (B and D). Among composite sediments, the mixture effect with other sediments is not efficient to decrease toxicity in sample C, suggesting that other parameters influence toxicity level such as particle size or organic matter content. These parameters should be taken into consideration in order to improve the efficiency of the mixture process and produce composite sediments with low toxicity.

Does zinc produce reactive oxygen species in Ruditapes decussatus?

Zinc is an essential trace element with many enzymatic functions that include antioxidant properties. To investigate whether an excess of Zn in the cells produces cytotoxicity or tissue damage or an imbalance in the antioxidant systems, marine clams (Ruditapes decussatus) were exposed to two sublethal Zn concentrations (100 and 1000 microg L(-1)) for 28 days. The effects of Zn on the activities of protective antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase, both total and selenium-dependent), lipid peroxidation, and metallothionein induction were followed in the gills and digestive gland of these clams. The results indicate that the effect of Zn exposure in this clam species depends not only on the tissue but also on the Zn concentration present. In the gills, catalase activity was enhanced by Zn exposure, whereas total glutathione peroxidase activity was inhibited. Lipid peroxidation occurred only in the clams exposed to the highest Zn concentration. In the digestive gland, the impact of Zn exposure on metabolic activity was less evident than in the gills. The most evident effect in both tissues was the enhancement of catalase activity by Zn exposure. Catalase and total glutathione peroxidase activities as well as lipid peroxidation are promising biomarkers to assess the effects of Zn in the gills of R. decussatus.