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).

Early Biological Modulations Resulting from 1-Week Venlafaxine Exposure of Marine Mussels Mytilus galloprovincialis Determined by a Metabolomic Approach.

There is growing evidence of the presence of pharmaceuticals in natural waters and their accumulation in aquatic organisms. While their mode of action on non-target organisms is still not clearly understood, their effects warrant assessment. The present study assessed the metabolome of the Mediterranean mussel (Mytilus galloprovincialis) exposed to a 10 µg/L nominal concentration of the antidepressant venlafaxine (VLF) at 3 time-points (1, 3, and 7 days). Over the exposure period, we observed up- or down-modulations of 113 metabolites, belonging to several metabolisms, e.g., amino acids (phenylalanine, tyrosine, tryptophan, etc.), purine and pyrimidine metabolisms (adenosine, cyclic AMP, thymidine, etc.), and several other metabolites involved in diverse functions. Serotonin showed the same time-course modulation pattern in both male and female mussels, which was consistent with its mode of action in humans, i.e., after a slight decrease on the first day of exposure, its levels increased at day 7 in exposed mussels. We found that the modulation pattern of impacted metabolites was not constant over time and it was gender-specific, as male and female mussels responded differently to VLF exposure.

Environmental Metabolomics Promises and Achievements in the Field of Aquatic Ecotoxicology: Viewed through the Pharmaceutical Lens.

Scientists often set ambitious targets using environmental metabolomics to address challenging ecotoxicological issues. This promising approach has a high potential to elucidate the mechanisms of action (MeOAs) of contaminants (in hazard assessments) and to develop biomarkers (in environmental biomonitoring). However, metabolomics fingerprints often involve a complex mixture of molecular effects that are hard to link to a specific MeOA (if detected in the analytical conditions used). Given these promises and limitations, here we propose an updated review on the achievements of this approach. Metabolomics-based studies conducted on the effects of pharmaceutical active compounds in aquatic organisms provide a relevant means to review the achievements of this approach, as prior knowledge about the MeOA of these molecules could help overcome some shortcomings. This review highlighted that current metabolomics advances have enabled more accurate MeOA assessment, especially when combined with other omics approaches. The combination of metabolomics with other measured biological endpoints has also turned out to be an efficient way to link molecular effects to (sub)-individual adverse outcomes, thereby paving the way to the construction of adverse outcome pathways (AOPs). Here, we also discuss the importance of determining MeOA as a key strategy in the identification of MeOA-specific biomarkers for biomonitoring. We have put forward some recommendations to take full advantage of environmental metabolomics and thus help fulfil these promises.

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.

An integrated metabolomics and proteogenomics approach reveals molecular alterations following carbamazepine exposure in the male mussel Mytilus galloprovincialis.

Carbamazepine is one of the most abundant pharmaceutical active compounds detected in aquatic systems. Based on laboratory exposures, carbamazepine has been proven to adversely affect aquatic organisms. However, the underlying molecular events remain poorly understood. This study aims to investigate the molecular mechanisms potentially associated with toxicological effects of carbamazepine on the mussel Mytilus galloprovincialis exposed for 3 days at realistic concentrations encountered in coastal environments (80 ng/L and 8 µg/L). An integrated metabolomics and proteogenomics approach, including data fusion strategy, was applied to gain more insight in molecular events and cellular processes triggered by carbamazepine exposure. Consistent metabolic and protein signatures revealed a metabolic rewiring and cellular stress at both concentrations (e.g. intensification of protein synthesis, transport and catabolism processes, disruption of lipid and amino acid metabolisms). These highlighted molecular signatures point to the induction of autophagy, closely related with carbamazepine mechanism of action, as well as a destabilization of the lysosomal membranes and an enzymatic overactivity of the peroxisomes. Induction of programmed cell death was highlighted by the modulation of apoptotic cognate proteins. The proposed integrative omics data analysis was shown to be highly relevant to identify the modulations of the two molecular levels, i.e. metabolites and proteins. Multi-omics approach is able to explain the resulting complex biological system, and document stronger toxicological pieces of evidence on pharmaceutical active compounds at environmental concentrations in sentinel organisms.

In vivo exposure of marine mussels to venlafaxine: bioconcentration and metabolization.

Pharmaceuticals are present in natural waters, thus contributing to the general exposure of aquatic organisms, but few data are available on the accumulation of these substances in marine organisms. The present study evaluated the in vivo bioconcentration of an antidepressant-venlafaxine (VLF)-in marine mussels (Mytilus galloprovincialis) during 7 days of exposure at nominal 10 µg/L concentration, followed by a 7-day depuration period. The bioconcentration factor (BCF) was 265 mL/g dry weight (dw). VLF accumulation reached an average tissue concentration of 2146 ± 156 ng/g dw within 7 days, showing a first-order kinetics process. N-desmethylvenlafaxine (N-VLF) and O-desmethylvenlafaxine (O-VLF) metabolites were quantified in mussel tissues, whereas N,N-didesmethylvenlafaxine (NN-VLF) was only recorded as being detected. These three metabolites were also quantified in water, indicating an active metabolism and VLF excretion in Mediterranean mussels. Complementary experiments conducted at nominal concentrations of 1, 10, and 100 µg/L for 7 days confirmed the VLF bioconcentration and metabolism and allowed us to quantify a supplementary metabolite, i.e., N,O-didesmethylvenlafaxine (NO-VLF), in mussel tissues. These results encourage further research on a more complete characterization of metabolism and on any disturbances linked to bioconcentration of VLF on bivalves.

Occurrence, distribution, and ecological risk assessment of emerging and legacy contaminants in the Kadicha river in Lebanon.

The Kadicha river basin in Northern Lebanon is an illustrative example of multiple pressures encountered in the Mediterranean region: it is a small coastal river affected by rapid urbanization, population growth (drastically impacted by the influx of Syrian refugees), and a chronic default of wastewater treatment. In this context, multiple classes of contaminants may attain the river accumulating in sediment. However, very little information is available in the literature on the contamination status in such stressed Mediterranean contexts. This study proposed a first contamination evaluation of a small Mediterranean river submitted to multiple pressures. Two sediment sampling campaigns along sites impacted by increasing urban gradient within the Kadicha river basin were performed to determine the occurrence and the environmental risks of both emerging and legacy contaminants. The results revealed the detection of the 41 studied compounds. The highest concentrations were attained by PAHs and polycyclic musks (up to 311.79, 94.22, and 81.13 ng/g of dry weight for PAH, cashmeran, and galaxolide, respectively). The discontinuous urbanized upstream area and the estuary were the most contaminated areas of the river. An environmental risk assessment showed a hazard quotient (HQ) higher than 1 for both legacy and emerging compounds (EHMC and 4-MBC), indicating a potential risk to benthic species. Monitoring campaigns and implementation of wastewater treatment plants should be encouraged as the anthropogenic pressure on small Mediterranean rivers will increase over the years.

Multifactorial Analysis of Environmental Metabolomic Data in Ecotoxicology: Wild Marine Mussel Exposed to WWTP Effluent as a Case Study.

Environmental metabolomics is a powerful approach to investigate the response of organisms to contaminant exposure at a molecular scale. However, metabolomic responses to realistic environmental conditions can be hindered by factors intrinsic to the environment and the organism. Hence, a well-designed experimental exposure associated with adequate statistical analysis could be helpful to better characterize and relate the observed variability to its different origins. In the current study, we applied a multifactorial experiment combined to Analysis of variance Multiblock Orthogonal Partial Least Squares (AMOPLS), to assess the metabolic response of wild marine mussels, Mytilus galloprovincialis, exposed to a wastewater treatment plant effluent, considering gender as an experimental factor. First, the total observed variability was decomposed to highlight the contribution of each effect related to the experimental factors. Both the exposure and the interaction gender × exposure had a statistically significant impact on the observed metabolic alteration. Then, these metabolic patterns were further characterized by analyzing the individual variable contributions to each effect. A main change in glycerophospholipid levels was highlighted in both males and females as a common response, possibly caused by oxidative stress, which could lead to reproductive disorders, whereas metabolic alterations in some polar lipids and kynurenine pathway were rather gender-specific. This may indicate a disturbance in the energy metabolism and immune system only in males. Finally, AMOPLS is a useful tool facilitating the interpretation of complex metabolomic data and is expected to have a broad application in the field of ecotoxicology.

Metabolomics approach reveals disruption of metabolic pathways in the marine bivalve Mytilus galloprovincialis exposed to a WWTP effluent extract.

Conventional wastewater treatment plants (WWTPs) discharge a highly diverse range of organic contaminants in aquatic environments, including marine waters. The health of marine ecosystems could be threatened by contaminants release. Environmental metabolomics can be helpful to assess the effects of multi-contamination on marine organisms without any a priori information since it is able to provide meaningful information on the biochemical response of organisms to a stress. The aim of the present study was to evaluate the potential of metabolomics to highlight key metabolites disrupted by a WWTP effluent extract exposure and then elucidate the biological effects of such exposure on Mediterranean mussels (Mytilus galloprovincialis). Exposed male mussels showed numerous metabolites altered in response to WWTP effluent exposure. The highlighted metabolites belong mainly to amino acids metabolism (e.g. tyrosine, phenylalanine, leucine, proline, etc.), neurohormones (dopamine and a serotonin metabolite), purine and pyrimidine metabolism (e.g. adenosine, adenine, guanine, uracil etc.), citric acid cycle intermediates (e.g. malate, fumarate), and a component involved in oxidative stress defense (oxidized glutathione). Modulation of these metabolites could reflect the alteration of several biological processes such as energy metabolism, DNA and RNA synthesis, immune system, osmoregulation, byssus formation and reproduction, which may lead to a negative impact of organism fitness. Our study provided further insight into the effects of WWTP effluents on marine organisms.

Diclofenac in the marine environment: A review of its occurrence and effects.

Interest in the presence and effects of diclofenac (DCF) and other pharmaceutical products (PPs) in the aquatic environment has been growing over the last 20 years. DCF has been included in the First Watch List of the EU Water Framework Directive in order to gather monitoring data in surface waters. Despite PP input in water bodies, few studies have been conducted to determine the extent of DCF occurrence and effects on marine ecosystems, which is usually the final recipient of surface waters. The present article reviews available published data on DCF occurrence in marine water, sediment and organisms, and its effects on marine organisms. The findings highlight the scarcity of available data on the occurrence and effects of DCF in marine ecosystems, and the need for further data acquisition to assess the risks associated with the presence of this compound in the environment.

Polar pesticide contamination of an urban and peri-urban tropical watershed affected by agricultural activities (Yaoundé, Center Region, Cameroon).

Urban agriculture is crucial to local populations, but the risk of it contaminating water has rarely been documented. The aim of this study was to assess pesticide contamination of surface waters from the Méfou watershed (Yaoundé, Cameroon) by 32 selected herbicides, fungicides, and insecticides (mainly polar) according to their local application, using both grab sampling and polar organic compounds integrative samplers (POCIS). Three sampling campaigns were conducted in the March/April and October/November 2015 and June/July 2016 rainy seasons in urban and peri-urban areas. The majority of the targeted compounds were detected. The quantification frequencies of eight pesticides were more than 20% with both POCIS and grab sampling, and that of diuron and atrazine reached 100%. Spatial differences in contamination were evidenced with higher contamination in urban than peri-urban rivers. In particular, diuron was identified as an urban contaminant of concern because its concentrations frequently exceeded the European water quality guideline of 0.200 µg/L in freshwater and may thus represent an ecological risk due to a risk quotient > 1 for algae observed in 94% of grab samples. This study raises concerns about the impacts of urban agriculture on the quality of water resources and to a larger extent on the health of the inhabitants of cities in developing countries. Graphical abstract ᅟ.

Diurnal variations in personal care products in seawater and mussels at three Mediterranean coastal sites.

The presence of personal care products (PCPs) in the marine environment is of major concern. PCPs, UV filters, and musks can enter the marine environment indirectly through wastewater or directly via recreational activities. We conducted this study to document patterns in the occurrence of seven PCPs at three coastal sites impacted by recreational activities during 1 day. The study focused on diurnal variations in these seven PCPs in seawater and indigenous mussels. In seawater, UV filters showed diurnal variations that mirrored variations in recreational activities at the sites. Ethylhexyl methoxycinnamate (EHMC) and octocrylene (OC) water concentrations increased from under the limit of quantification in the morning to 106 and 369 ng/L, respectively, when recreational activities were the highest. In mussels, diurnal variations in OC were observed, with the lowest concentrations recorded in the morning and then increasing throughout the day. As Mytilus spp. are widely used as sentinels in coastal pollution monitoring programs (mussel watch), our findings on diurnal variations could enhance sampling recommendations for recreational sites impacted by PCPs.

Exposure of marine mussels to diclofenac: modulation of prostaglandin biosynthesis.

Human pharmaceuticals, such as nonsteroidal anti-inflammatory drugs (NSAIDs), are an emerging threat to marine organisms. NSAIDs act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid into prostaglandins. One experiment was carried out whereby marine mussels were exposed for 72 h to 1 and 100 µg/L diclofenac (DCF). A specific and sensitive method using liquid chromatography high-resolution tandem mass spectrometry was developed to quantify DCF in mussel tissues. The developed method could also clearly identify and quantify COX products, i.e., prostaglandin levels, and be used to assess their modulation following DCF exposure. Prostaglandin-D2 (PGD2) was always found below the detection limit (20 µg/kg dry weight (dw)). Basal prostaglandin-E2 (PGE2) concentrations ranged from below the detection limit to 202 µg/kg dw. Exposure of 100 µg/L resulted in a significant reduction in PGE2 levels, whereas a downward trend was observed at 1 µg/L exposure. No difference was observed for prostaglandin-F2α (PGF2α) levels between controls and exposed organisms.

Metabolomics assessment of the effects of diclofenac exposure on Mytilus galloprovincialis: Potential effects on osmoregulation and reproduction.

The presence of pharmaceutically active compounds in aquatic environments has become a major concern over the past 20years. Elucidation of their mode of action and effects in non-target organisms is thus now a major ecotoxicological challenge. Diclofenac (DCF) is among the pharmaceutical compounds of interest based on its inclusion in the European Union Water Framework Directive Watch List. In this study, our goal was to investigate the potential of a metabolomic approach to acquire information without any a priori hypothesis about diclofenac effects on marine mussels. For this purpose, mussel's profiles were generated by liquid chromatography combined with high resolution mass spectrometry. Two main metabolic pathways were found to be impacted by diclofenac exposure. The tyrosine metabolism was mostly down-modulated and the tryptophan metabolism was mostly up-modulated following exposure. To our knowledge, such DCF effects on mussels have never been described despite being of concern for these organisms: catecholamines and serotonin may be involved in osmoregulation, and in gamete release in mollusks. Our results suggest potential impairment of mussel osmoregulation and reproduction following a DCF exposure.

Metabolic profiling identification of metabolites formed in Mediterranean mussels (Mytilus galloprovincialis) after diclofenac exposure.

Despite the growing concern on the presence of pharmaceutically active compounds in the environment, few studies have been conducted on their metabolism in marine organisms. In this study, a non-targeted strategy based on the generation of chemical profiles generated by liquid chromatography combined with high resolution mass spectrometry was used to highlight metabolite production by the Mediterranean mussel (Mytilus galloprovincialis) after diclofenac exposure. This method allowed revealing the production of 13 metabolites in mussel tissues. Three of them were phase I metabolites, including 4'-hydroxy-diclofenac and 5-hydroxy-diclofenac. The remaining 10 were phase II metabolites, including sulfate and amino acids conjugates. Among all of the metabolites highlighted, 5 were reported for the first time in an aquatic organism exposed to diclofenac.

Occurrence of PPCPs in the marine environment: a review.

Little research has been conducted on the occurrence of pharmaceuticals and personal care products (PPCPs) in the marine environment despite being increasingly impacted by these contaminants. This article reviews data on the occurrence of PPCPs in seawater, sediment, and organisms in the marine environment. Data pertaining to 196 pharmaceuticals and 37 personal care products reported from more than 50 marine sites are analyzed while taking sampling strategies and analytical methods into account. Particular attention is focused on the most frequently detected substances at highest concentrations. A snapshot of the most impacted marine sites is provided by comparing the highest concentrations reported for quantified substances. The present review reveals that: (i) PPCPs are widespread in seawater, particularly at sites impacted by anthropogenic activities, and (ii) the most frequently investigated and detected molecules in seawater and sediments are antibiotics, such as erythromycin. Moreover, this review points out other PPCPs of concern, such as ultraviolet filters, and underlines the scarcity of data on those substances despite recent evidence on their occurrence in marine organisms. The exposure of marine organisms in regard to these insufficient data is discussed.

Reducing PEC uncertainty in coastal zones: a case study on carbamazepine, oxcarbazepine and their metabolites.

Concentrations of the antiepileptic drugs carbamazepine (Cbz), oxcarbazepine (OxCz) and their main metabolites were predicted in a wastewater treatment plant (WTP) and in the vicinity of its submarine outfall located in a Mediterranean coastal zone. Refined predicted environmental concentrations (PECs) were calculated in effluents based on consumption data and human excretion rates. PECs were estimated in the sea using the hydrodynamic MARS 3D model integrating meteorological data, oceanic conditions (wind, tide, atmospheric pressure), freshwater and sewage inputs. Measured environmental concentrations (MECs) were compared to PECs to assess the estimation relevance. In the coastal zone, PEC and MEC were in the same magnitude range. Modeling of Cbz diffusion and advection just above the submarine outfall showed the influence of the thermocline during summer, with low diffusion of Cbz from the bottom to the surface. This work allowed understanding the dispersion of target compounds and deserved further development for a better acknowledgement of vulnerability at local scales.

Affinity purification using recombinant PXR as a tool to characterize environmental ligands.

Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 µM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 µM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis.

Chiral signature of venlafaxine as a marker of biological attenuation processes.

The chiral signature of the antidepressant venlafaxine was used in this study to gain insight into biological attenuation processes and to differentiate abiotic and biotic transformation processes in water. Laboratory scale experiments revealed that sorption and phototransformation processes were not enantioselective while venlafaxine was enantioselectively biotransformed into O-desmethylvenlafaxine. The enantiomeric fraction (EF) variations of venlafaxine appeared to be proportional to its microbial fractional conversion. Enantioselective biotransformation of venlafaxine was also investigated in a eutrophic French river. Venlafaxine was found to be racemic at the output of the main wastewater treatment plant discharging into the river, independently of the sampling date during the year. An analysis of EF variations might provide evidence of biodegradation along a 30 km river stretch.