Toxic effects of a mixture of five pharmaceutical drugs assessed using Fontinalis antipyretica Hedw.

The potential health risks associated with the pharmaceuticals released into the environment through effluents from sewage treatment plants have become a major cause for concern. Owing to the lack of effective indicators, monitoring the concentration of these pollutants in the aquatic environment is challenging. The aim of this study was to assess the toxicity of a mixture of five pharmaceutical drugs (paracetamol, carbamazepine, diclofenac, irbesartan, and naproxen) using the aquatic moss Fontinalis antipyretica as a bioindicator and bioaccumulator. We examined the effects of the drug mixture on the cellular antioxidant system, chlorophyll content, and morphological traits of F. antipyretica. The plant was exposed for 5 months to three concentrations of the mixture, including the environmental concentration (MX1), and 10- (MX10) and 100-times (MX100) the environmental concentration. The results showed that only carbamazepine and irbesartan were accumulated by the species. The bioconcentration level increased with exposure time, with the maximum uptake at the 4th month of exposure. The increase in bioaccumulation with exposure time was more evident in plants exposed to MX100. Analysis of the activity of antioxidant enzymes showed that superoxide dismutase (SOD, EC 1.15.1.1.) and catalase (EC 1.11.1.6.) were highly sensitive to the drug mixture. The activity of the enzymes was significantly higher in plants exposed to MX100; however, the activity of guaiacol peroxidase (GPX, EC 1.11.1.7.) was not significantly affected. Plants exposed to MX10 and MX100 had significantly lower total chlorophyll content and chlorophyll a/b ratio compared with those of plants in the control group; however, photosynthetic activity was restored after 5 months of exposure. The morphological characteristics of F. antipyretica were less sensitive to the treatment conditions.

Water quality of the Meuse watershed: Assessment using a multi-biomarker approach with caged three-spined stickleback (Gasterosteus aculeatus L.).

The use of a multi-biomarker approach with three-spined sticklebacks (Gasterosteus aculeatus) through an active biomonitoring strategy appears to be a promising tool in water quality assessment. The present work proposes to assess the efficiency of these tools in the discrimination of some sites in a large scale on the Meuse basin in Europe. The study was part of an EU program which aims to assess water quality in the Meuse across the French-Belgian border. Sticklebacks were caged 21 days upstream and downstream from the wastewater treatment plants (WWTPs) of Namur (Belgium), Charleville-Mézières (France), Bouillon (Belgium) and Avesnes-sur-Helpe (France). First, the state of a variety of physiological functions was assessed using a battery of biomarkers that represented innate immunity (leucocyte mortality and distribution, phagocytosis activity, respiratory burst), antioxidant system (GPx, CAT, SOD and total GSH content), oxidative damages to the membrane lipids (TBARS), biotransformation enzymes (EROD, GST), synaptic transmission (AChE) and reproduction system (spiggin and vitellogenin concentration). The impacts of the effluents were first analysed for each biomarker using a mixed model ANOVA followed by post-hoc analyses. Secondly, the global river contamination was assessed using a principal component analysis (PCA) followed by a hierarchical agglomerative clustering (HAC). The results highlighted a small number of effects of WWTP effluents on the physiological parameters in caged sticklebacks. Despite a significant effect of the "localisation" factor (upstream/downstream) in the mixed ANOVA for several biomarkers, post-hoc analyses revealed few differences between upstream and downstream of the WWTPs. Only a significant decrease of innate immune responses was observed downstream from the WWTPs of Avesnes-sur-Helpe and Namur. Other biomarker responses were not impacted by WWTP effluents. However, the multivariate analyses (PCA and HAC) of the biomarker responses helped to clearly discriminate the different study sites from the reference but also amongst themselves. Thus, a reduction of general condition (condition index and HSI) was observed in all groups of caged sticklebacks, associated with a weaker AChE activity in comparison with the reference population. A strong oxidative stress was highlighted in fish caged in the Meuse river at Charleville-Mézières whereas sticklebacks caged in the Meuse river at Namur exhibited weaker innate immune responses than others. Conversely, sticklebacks caged in the Helpe-Majeure river at Avesnes-sur-Helpe exhibited higher immune responses. Furthermore, weak defence capacities were recorded in fish caged in the Semois river at Bouillon. This experiment was the first to propose an active biomonitoring approach using three-spined stickleback to assess such varied environments. Low mortality and encouraging results in site discrimination support the use of this tool to assess the quality of a large number of water bodies.

Rapid analysis of diclofenac and some of its transformation products in the three-spined stickleback, Gasterosteus aculeatus, by liquid chromatography-tandem mass spectrometry.

Pharmaceuticals are emerging organic contaminants ubiquitously present in the environment due to incessant input into the aquatic compartment mainly resulting from incomplete removal in wastewater treatment plants. One of the major preoccupations concerning pharmaceuticals released into surface waters is their potential for bioaccumulation in biota, possibly leading to deleterious effects on ecosystems especially as they could affect a broad variety of organisms living in or depending on the aquatic environment. Thus, the development of accurate and sensitive methods is necessary to detect these compounds in aquatic ecosystems. Considering this need, this study deals with the analytical development of a methodology to quantify traces of diclofenac together with some of its biotic and abiotic transformation products in whole-body tissue of three-spined stickleback. A simple and reliable extraction method based on a modified QuEChERS extraction is implemented on 200 mg of fish. The detection and quantification of the ten target compounds are performed using liquid chromatography-tandem mass spectrometry. The whole process was successfully validated regarding linearity, recovery, repeatability, and reproducibility. The method limits of detection and quantification do not exceed 1 ng/g. To reproduce environmental conditions, we measured the concentration of DCF and its transformation products in three-spined sticklebacks after a 6-month exposure in mesocosms at several levels of DCF ranging from 0.05 to 4.1 µg/L. The phase I metabolite 4'-hydroxydiclofenac was detected in fish samples exposed at the highest DCF concentration. Graphical abstract Analysis of diclofenac and some of its transformation products in the three-spined stickleback, Gasterosteus aculeatus, by QuEChERS extraction followed by LC-MS/MS.

Effects of chronic exposure to a pharmaceutical mixture on the three-spined stickleback (gasterosteus aculeatus) population dynamics in lotic mesocosms.

Pharmaceutical substances are ubiquitous in the aquatic environment and their concentration levels typically range from ng/L up to several µg/L. Furthermore, as those compounds are designed to be highly biologically active, assessing their impacts on non-target organisms is important. Here, we conducted a mesocosm experiment testing a mixture of five pharmaceuticals (diclofenac, carbamazepine, irbesartan, acetaminophen and naproxen) on fish, three-spined stickleback (Gasterosteus aculeatus). The mixture concentration levels were chosen on the basis of the contamination of the Meuse river in Belgium which had been measured previously during a monitoring campaign undertaken in 2015 and 2016. Three nominal mixture concentration levels were tested: the lowest concentration level mixture was composed by environmentally-relevant concentrations that approximate average realistic values for each pharmaceuticals (Mx1); the two other levels were 10 and 100 times these concentrations. Although no impact on stickleback prey was observed, the mixture significantly impaired the survival of female fish introduced in the mesocosms at the highest treatment level without causing other major differences on fish population structure. Impacts on condition factors of adults and juveniles were also observed at both individual and population levels. Using a modelling approach with an individual-based model coupled to a bioenergetic model (DEB-IBM), we concluded that chronic exposure to environmentally-relevant concentrations of five pharmaceuticals often detected in the rivers did not appear to strongly affect the three-spined stickleback populations. Mechanisms of population regulation may have counteracted the mixture impacts in the mesocosms.

Modelling BPA effects on three-spined stickleback population dynamics in mesocosms to improve the understanding of population effects.

Bisphenol A (BPA), a well-known endocrine-disrupting chemical, is ubiquitously present in the aquatic environment. Its impacts at the population level on three-spined sticklebacks (Gasterosteus aculeatus) have been studied in artificial streams with low-dose BPA concentrations. The causes explaining the observed effects remained unclear. Here, we used an individual-based model coupled to a Dynamic Energy Budget model to (i) assess the potential of modelling to predict impacts at the population level using individual level laboratory ecotoxicological endpoints and (ii) provide insight on the mechanisms of BPA toxicity in these mesocosms. To do that, both direct and indirect effects of BPA on three-spined sticklebacks were incorporated in the model. Indeed, direct BPA effects on fish have been identified based on literature data whereas indirect effects on sticklebacks have been taken into account using sampling data of their prey from the exposed artificial streams. Results of the modelling showed that direct BPA effects on fish (impacts on gonad formation, growth, male reproductive behavior, eggs and larvae survival) mainly explained the three-spined stickleback population structure in the mesocosms, but indirect effects were not negligible. Hence, this study showed the potential of modelling in risk assessment to predict the impacts on fish population viability from behavioral and physiological effects measured on organisms.

Determination of a new index of sexual maturity (ISM) in zebra mussel using flow cytometry: interest in ecotoxicology.

The global dynamic spread of chemical contamination through the aquatic environment calls for the development of biomarkers of interest. Reproduction is a key element to be considered because it is related to the sustainability of species. Spermatogenesis is a complex process that leads to the formation of mature germ cells, whose steps and impairments need to be finely described in ecotoxicological analyses. The physiological process has been commonly described by histological analyses of gonads in different taxa. In the present paper, we describe the development of a novel technique to characterize spermatogenesis based on the analysis of the DNA content of germ cells by flow cytometry, using a DNA-intercalating agent. This new biomarker, referred to as an index of sexual maturity, proved relevant to describe the seasonal reproductive cycle of the zebra mussel, Dreissena polymorpha (Pallas, 1771), used as a sentinel species in the biomonitoring of continental waters and sensitive to highlight the reprotoxicity of carbamazepine (an anti-epileptic pharmaceutical) tested under ecosystemic conditions (mesocosms).

A long-term copper exposure on freshwater ecosystem using lotic mesocosms: Primary producer community responses.

Copper is commonly used as an algicide and plant herbicide in the aquatic environment. Despite of its frequent application in rivers, ponds and lakes, few studies have been performed at the ecosystem level on primary producers' communities. A long-term lotic mesocosms study was carried on, in 20m long channels, under environmentally realistic concentrations of copper (0, 5, 25 and 75 microgL(-1)) delivered continuously for 18 months. Results showed significant effects at the 25 and 75 microgL(-1) copper treatment levels on the phytoplankton, periphyton and macrophyte community structure. Effects on biomass and abundance were dissimilar depending on the studied community. Phytoplankton showed no abundance variation with treatment, whereas periphyton had higher biomass and macrophytes had lower coverage at 25 and 75 microgL(-1). Taxa richness was significantly affected for all primary producers communities at 25 and 75 microgL(-1). Some taxa seemed to be sensitive to copper, i.e. Cocconeis spp., Epithemia sp., Gomphonema spp., Lemna minor, Nasturtium officinale and filamentous macroalgae. Other taxa appeared to be tolerant or facilitated by indirect effects, i.e. Leptolyngbya sp., Mougeotia sp., Nitzschia palea, Pseudanabaena sp. and Ulothrix sp. and therefore increased in the 25 and 75 microgL(-1) treatments. Important indirect factors influenced the community responses, such as trophic interactions like decrease of grazing pressure, competition for light and nutrients and temperature favouring blue-green algae development. After 18 months of copper exposure, the no observed effect concentration at community level (NOEC(community)) for primary producers was set up at 5 or 4mugL(-1), if we consider the average effective concentration, with a lowest observed effect concentration (LOEC(community)) at 25 or 20 microgL(-1) (AEC).

A long-term copper exposure on freshwater ecosystem using lotic mesocosms: individual and population responses of three-spined sticklebacks (Gasterosteus aculeatus).

Three-spined stickleback (Gasterosteus aculeatus) was used as the highest trophic level predator in an outdoor mesocosm study assessing the effect of environmentally realistic copper concentration (0, 5, 25 and 75 microgL(-1)) over 18 months of continuous exposure. Condition factor, organosomatic indices (HIS, GSI and SSI) as well as copper bioaccumulation in the liver were measured at 15 days, 2, 4, 6, 10, 14 and 18 months after the beginning of the contamination. Population monitoring was realised after 6 and 18 months of contamination, allowing two reproduction periods to be measured. Results showed that condition factor was affected at medium and high copper concentrations and HSI was sporadically affected in all copper exposure, depending on the sex of the fish. GSI did not show any significant differences and SSI was lowered in the medium and high copper levels. Bioaccumulation was significantly different in males and females and fluctuated with season. A negative correlation was observed between copper bioaccumulation in the liver and fish size and a positive correlation with nominal copper concentration in the water was found. There was a negative correlation between condition factor, organosomatic indices and bioaccumulation in the liver. Population monitoring showed a significantly higher fish mean length after 6 months and a higher abundance after 18 months of exposure at the highest copper level. We conclude that indirect effects such as food and habitat availability or lower predation pressure on eggs and juveniles might have led to higher stickleback population abundances at the highest copper level. This highlights the need to study all the trophic levels when monitoring ecosystem health. Considering the population and the individual responses after 18 months of copper exposure, the NOEC for three-spined sticklebacks was 25 microgL(-1) (or 20 microgL(-1) if we consider the average effective concentration), with a LOEC of 75 microgL(-1) (or 57 microgL(-1), AEC).

A long-term copper exposure in a freshwater ecosystem using lotic mesocosms: Invertebrate community responses.

A lotic mesocosm study was carried out in 20-m-long channels, under continuous, environmentally realistic concentrations of copper (Cu) in low, medium, and high exposures (nominally 0, 5, 25, and 75 µg L-1 ; average effective concentrations <0.5, 4, 20, and 57 µg L-1 respectively) for 18 mo. Total abundance, taxa richness, and community structure of zooplankton, macroinvertebrates, and emerging insects were severely affected at Cu treatment levels of 25 and 75 µg L-1 . Some taxa were sensitive to Cu, including gastropods such as Lymnaea spp. and Physa sp., crustaceans such as Chydorus sphaericus, Gammarus pulex, and Asellus aquaticus, rotifers such as Mytilina sp. and Trichocerca sp., leeches such as Erpobdella sp., and the emergence of dipteran insects such as Chironomini. Other taxa appeared to be tolerant or favored by indirect effects, as in Chironimidae larvae, the emergence of Orthocladiinae, and the zooplankter Vorticella sp., which increased in the 25 and 75 µg L-1 treatments. After approximately 8 mo of Cu exposure, the macroinvertebrate community in the high treatment was decimated to the point that few organisms could be detected, with moderate effects in the medium treatment, and very slight effects in the low-Cu treatment. Subsequently, most taxa in the high-Cu exposure began a gradual and partial recovery. By the end of the study at 18 mo, macroinvertebrate taxa richness was similar to control richness, although overall abundances remained lower than controls. After 18 mo of copper exposure, a no-observed-effect concentration at the community level for consumers was set at 5 µg L-1 (4 µg L-1 as average effective concentration), and a lowest-observed-effect concentration at 25 µg L-1 (20 µg L-1 as average effective concentration). Environ Toxicol Chem 2017;36:2698-2714. © 2017 SETAC.

Analysis of community-level mesocosm data based on ecologically meaningful dissimilarity measures and data transformation.

The principal response curve (PRC) method is a constrained ordination method developed specifically for the analysis of community data collected in mesocosm experiments, which provides easily understood summaries and graphical representations of community response to stress. It is a redundancy analysis method and is usually performed on log-transformed abundance data. The choice of a measure of dissimilarity between samples and the choice of the data transformation significantly affect the results of multivariate analysis. Dissimilarity measures that are more ecologically meaningful than the Euclidean distance can be incorporated into the PRC using distance-based redundancy analysis. The present study investigates the ordinations produced by a small selection of dissimilarity measures: the Euclidean distance using log-transformed and Hellinger-transformed data and the Bray-Curtis dissimilarity using raw and log-transformed data. It compares 2 data sets from experiments on the effect of the anti-inflammatory drug diclofenac and the insecticide chlorpyrifos on macroinvertebrate communities. The choice of dissimilarity measure can determine the outcome of a risk assessment. For the diclofenac data set, the PRCs were different depending on the dissimilarity measure: the community no-effect concentration was lowest for the Bray-Curtis on log-transformed data and Hellinger dissimilarity measures. For chlorpyrifos, however, the PRCs were similar for all dissimilarity measures. Environ Toxicol Chem 2017;36:1667-1679. © 2016 SETAC.

Determination of carbamazepine and 12 degradation products in various compartments of an outdoor aquatic mesocosm by reliable analytical methods based on liquid chromatography-tandem mass spectrometry.

The aims of this work are to develop suitable analytical methods to determine the widely used anticonvulsant carbamazepine and 12 of its degradation/transformation products in water, sediment, fish (Gasterosteus aculeatus) and mollusc (Dreissena polymorpha). Protocols based on solid phase extraction for water, pressurized-liquid extraction for sediments and QuEChERS (quick easy cheap efficient rugged and safe) extraction for both organisms followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) are developed, validated and finally applied to samples collected during a 6-month experiment in outdoor mesocosms. Very low detection limits are reached, allowing environmentally realistic doses (namely, 0.05, 0.5 and 5 µg/L nominal concentrations) to be employed. The results indicate several metabolites and/or transformation products in each compartment investigated, with concentrations sometimes being greater than that of the parent carbamazepine. Biotic degradation of carbamazepine is demonstrated in water, leading to 10,11-dihydrocarbamazepine and 10,11-epoxycarbamazepine. In sediment, the degradation results in the formation of acridine, and 2- and 3-hydroxycarbamazepine. Finally, in both organisms, a moderate bioaccumulation is observed together with a metabolization leading to 10,11-epoxycarbamazepine in fish and 2-hydroxycarbamazepine in mollusc. Acridone is also present in fish. This study provides new and interesting data, helping to elucidate how chronic exposure to carbamazepine at relevant concentrations may affect impact freshwater ecosystems.

Development of a multi-residue analysis of diclofenac and some transformation products in bivalves using QuEChERS extraction and liquid chromatography-tandem mass spectrometry. Application to samples from mesocosm studies.

Pharmaceuticals are ubiquitously present in the aquatic environment, mainly due to insufficient removal in wastewater treatment plants. Although these compounds are often found at trace levels in waters, long-term exposure can have negative impacts on biotic communities due to their inherent biological activity. The non-steroidal anti-inflammatory drug diclofenac (DCF) is one of the most frequently detected human pharmaceuticals in water and has recently been included in the "watch" list of the European Union. However little data are available on the detection of this substance and its transformation products in aquatic organisms. In this context, an analytical methodology has been developed to quantify traces of DCF along with its biotic and abiotic transformation products in a wild species of bivalve, the zebra mussel Dreissena polymorpha. A modified QuEChERS extraction was implemented on a small quantity of soft bivalve tissue (100mg). This was followed by liquid chromatography coupled to tandem-mass spectrometry (LC-MS/MS) with electrospray ionization in positive mode (ESI+). Whole analytical method was validated on spiked real samples, with regard to linearity (from 1 to 50 or 100ng/g depending on the target compounds, R(2)>0.99), intra-day precision (relative standard deviation (RSD)<18%), inter-day precision (RSD <25%), (recoveries 78-117%), and limits of detection and of quantification (both inferior or equal to 1ng/g). The optimized method was successfully applied to organisms collected from mesocosm experiments. Bioconcentration factors comprised between 4 and 13 were observed for DCF in the zebra mussels. To the best of our knowledge, the product 2-indolone was for the first time detected in bivalves, with levels up to 6ng/g.

Effects of bisphenol A on different trophic levels in a lotic experimental ecosystem.

Bisphenol A (BPA) is commonly used by manufacturers and can be found in many aquatic ecosystems. Data relative to BPA ecotoxicity are only available for studies in laboratory conditions on macro-invertebrates and fish. There is thus a lack of information for other trophic levels such as macrophytes. Moreover, the impacts of BPA within an ecosystem context, i.e. with populations from different trophic levels studied at long term in environmental conditions, have never been assessed. We carried out a long-term lotic mesocosm study in 20 m long channels under three exposure concentrations of BPA (nominal concentrations of 0, 1, 10 and 100 µg/L) delivered continuously for 165 days. Three trophic levels were followed: macrophytes, macro-invertebrates (with a focus on Radix balthica) and fish (Gasterosteus aculeatus). Significant effects were shown at 100 µg/L BPA on the three trophic levels. BPA had a direct impact on macrophyte community structure, direct and indirect impacts on macro-invertebrates and on fish population structure. Gonad morphology of fish was affected at 1 and 10 µg/L of BPA, respectively for female and male sticklebacks. In addition to these ecotoxicity data, our results suggest that fish are good integrators of the responses of other communities (including macro-invertebrates and macrophytes) in mesocosm systems.