Phylogenetic diversity and functional characterization of the Manila clam microbiota: a culture-based approach

According to the hologenome theory, the microbiota contributes to the fitness of the holobiont having an important role in its adaptation, survival, development, health, and evolution. Environmental stress also affects the microbiota and its capability to assist the holobiont in coping with stress factors. Here, we analyzed the diversity of cultivable bacteria associated with Manila clam tissues (mantle, gills, hemolymph) in two non-contaminated sites (Portugal and France) and one metal-contaminated site (Portugal). A total of 240 isolates were obtained. Representative isolates (n = 198) of the overall diversity were identified by 16S rDNA sequencing and subjected to functional characterization. Isolates affiliated with Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. Proteobacteria (mostly Pseudoalteromonadaceae and Vibrionaceae) were dominant in non-contaminated sites while Actinobacteria (mostly Microbacteriaceae) dominated in the metal-contaminated site. The main factor affecting the microbiota composition was contamination. No significant differences were observed between clam tissues and geographic regions. Several isolates tested positive for antibacterial activity, biofilm formation, protease, and siderophore production. The results show that the Manila clam harbors a diverse microbiota that may contribute to clam protection and overall fitness, as well as to its adaptation to stressful environments. In addition, the Manila clam microbiota is revealed as a promising source of novel probiotics with potential application in aquaculture.     

Impact of hypoxia on hemolymph contamination by uranium in an aquatic animal, the freshwater clam Corbicula fluminea

Multi-stress situations are a major question and low-oxygenated waters (hypoxia) are a growing problem. Importantly, hypoxia stimulates the ventilatory flow rate in aquatic animals and this increases gill exposure to contaminants. Surprisingly, in the freshwater clam Corbicula fluminea, this is associated with increased bioaccumulation of uranium in gills but not in deep tissues. We searched for an explanation by analyzing hemolymph U-transport in Corbicula exposed to 0.36 μM dissolved uranium at various O₂-levels for 10 days. In hypoxia, one observed an increased U concentration in the arterial hemolymph flowing from gills to tissues but this was not associated with an increased U concentration in the venous hemolymph nor in the other tissues. We conclude that the cardiac flow rate must have decreased to explain this absence of over-accumulation. In addition to its already known deleterious effects, uranium can thus deeply impair cardiac flow rate in exposed aquatic animals during multi-stress exposures.     

Effects of permethrin exposure on antioxidant enzymes and protein status in Mediterranean clams Ruditapes decussatus

The effects of permethrin (PER) on a panel of antoxidant enzymes; superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) and indices of protein oxidation status (carbonylation and free thiols) were determined in digestive gland and gills of the clam Ruditapes decussatus. Animals were exposed to 100 ppb PER for 2 days. These enzyme activities increased significantly in digestive gland (p?<?0.05) after PER treatment and oxidative modification of proteins was detected in both gill and digestive gland extracts using redox proteomics. PER exposure significantly reduced the amount of protein free thiol groups in digestive gland rather than in gill, when compared to controls. Conversely, digestive gland showed significantly higher levels of carbonylated proteins than gill after PER exposure. Some proteins were successfully identified by mass spectrometry of tryptic peptides. Our data suggest that digestive gland of R. decussatus can be used as a model tissue for investigating environmental risk of PER contamination.     

Exposure of caged mussels to metals in a primary-treated municipal wastewater plume

The biological availability of metals in municipal wastewater effluents is strongly influenced by the physical and chemical conditions of both the effluent and the receiving water. Aquatic organisms are exposed to both dissolved and particulate (food ingestion) forms of these metals. In the present study, the distribution of metals in specific tissues was used to distinguish between exposure routes (i.e. dissolved vs. particulate phase) and to examine metal bioavailability in mussels exposed to municipal effluents. Caged Elliptio complanata mussels were deployed at sites located between 1.5 km upstream and 12 km downstream of a major effluent outfall in the St. Lawrence River. Metals in surface water samples were fractionated by filtration techniques to determine their dissolved, truly-dissolved (<10 kDa), total-particulate and acid-reactive-particulate forms. At the end of the exposure period (90 days), pooled mussel soft tissues (digestive gland, gills, gonad, foot and mantle) were analyzed for several metals. The results showed that gills and digestive gland were generally the most important target tissues for metal bioaccumulation, while gill/digestive gland metal ratios suggest that both exposure routes should be considered for mussels exposed to municipal effluents. We also found that Ag and Cd in the dispersion plume nearest the outfall, in contrast to other metals such as Cu and Zn, are more closely associated with colloids and were generally less bioavailable than at the reference site in the St. Lawrence River.     

The β-receptor blocker metoprolol alters detoxification processes in the non-target organism Dreissena polymorpha

Due to increasing amounts of pharmaceutically active compounds (PhACs) in the aquatic environment, their largely unknown effects to non-target organisms need to be assessed. This study examined physiological changes in the freshwater mussel Dreissena polymorpha exposed to increasing concentrations (0.534, 5.34, 53.4 and 534 μg L⁻¹) of the β-blocker metoprolol in a flow-through system for seven days. The two lower concentrations represent the environmentally relevant range. Surprisingly, metallothionein mRNA was immediately up-regulated in all treatments. For the two higher concentrations mRNA up-regulation in gills was found for P-glycoprotein after one day, and after four days for pi class glutathione S-transferase, demonstrating elimination and biotransformation processes, respectively. Additionally, catalase and superoxide dismutase were up-regulated in the digestive gland indicating oxidative stress. In all treated mussels a significant up-regulation of heat shock protein mRNA was observed in gills after four days, which suggests protein damage and the requirement for repair processes. Metoprolol was 20-fold bioaccumulated for environmentally relevant concentrations.     

Effects of landfill leachate effluent and bisphenol A on glutathione and glutathione-related enzymes in the gills and digestive glands of the freshwater snail Bellamya purificata

Environmental contaminants with estrogenic activity have recently attracted attention due to their potential detrimental effects on the reproduction of human and wildlife. The aim of this study was to evaluate the use of endogenous glutathione and glutathione-related enzymes as biomarkers of exposure to landfill leachate effluent and bisphenol A (BPA) in the freshwater snail, Bellamya purificata. Following exposure to 1%, 5% and 10% landfill leachate effluent and 1, 10, 50 and 100mugl(-1) BPA for 0, 2, 7 and 15d, activities of glutathione S-transferase (GST), selenium-dependent glutathione peroxidase (SeGPx) and glutathione reductase (GR) and levels of total glutathione were measured in the gills and digestive glands of the snails. GST and total glutathione were the most sensitive parameters in both exposure scenarios. GST activities increased by about 80%, while total glutathione decreased to 70% and 80% in the gills and digestive glands, respectively. In contrast, SeGPx and GR activities remained at the same levels in all the treatment groups compared with those of controls. The results indicated that among glutathione and glutathione-related enzymes, GST activity and total glutathione level, which showed dose-dependent dynamics, could be used as biomarkers of aquatic ecosystems contaminated with landfill leachate.     

Comparative study of the accumulation and detoxification of Cu (essential metal) and Hg (nonessential metal) in the digestive gland and gills of mussels Mytilus galloprovincialis, using analytical and histochemical techniques

The aim of the present study is the comparative examination of accumulation and detoxification of Cu and Hg in digestive gland and gills of mussels Mytilus galloprovincialis, using atomic absorption spectrophotometry and autometallography. Mussels were exposed to 0.08 mg L⁻¹ Cu, 0.08 mg L⁻¹ Hg, as well as to a mixture of 0.08 mg L⁻¹ Hg and 0.08 mg L⁻¹ Cu for 11 d. After the experimental exposure, animals were kept under laboratory conditions for a detoxification period of 7 d. An antagonistic effect of Cu against to Hg accumulation was noted in the digestive gland of mussels after the experimental exposure, as well as after the detoxification period, supporting the protective role of Cu against to Hg toxicity in this tissue. Digestive gland was suggested as a main organ for Hg accumulation and gills as a target position for Cu accumulation. Additionally, lower time was evaluated for Hg detoxification in the digestive gland and gills of mussels, in relation to those addressed for Cu detoxification in the same tissues. The evaluation of black silver deposits (BSD) extent performed in digestive gland and gills was suggested as a less sensitive approach, in relation to atomic absorption spectrophotometry (AAS), to indentify the concentration of heavy metals in tissues of mussels. The toxic effects of Hg, Cu and a mixture of them on lysosomal system of the digestive cells are also discussed.     

Antioxidant responses in clam Venerupis philippinarum exposed to environmental pollutant hexabromocyclododecane

The objective of this study was to assess the potential toxic effects of hexabromocyclododecane (HBCD) on tissues of clam Venerupis philippinarum using parameters of antioxidant defenses and oxidative stress. Antioxidant biomarkers including ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), superoxide dismutase (SOD), and glutathione (GSH), as well as DNA damage and lipid peroxidation (LPO) in gills and digestive glands of V. philippinarum, were analyzed after a 1-, 3-, 6-, 10-, and 15-day exposure to seawater containing HBCD at environmentally related concentrations, respectively. The results showed that the activity of most antioxidant enzymes increased, and different trends were detected with exposure time extending. The oxidative stress could be obviously caused in the gills and digestive glands under the experimental conditions. This could provide useful information for toxic risk assessment of environmental pollutant HBCD.     

Ubiquitination and carbonylation of proteins in the clam Ruditapes decussatus, exposed to nonylphenol using redox proteomics

Ubiquitination and carbonylation of proteins were investigated in the gill and digestive gland of Ruditapes decussatus exposed to NP (nonylphenol) (100 μgL(-1)) using redox proteomics. After 21 d of exposure, clams were dissected and cytosolic proteins of both tissues separated by 2DE SDS-PAGE. Protein expression profiles were tissue-dependent and differently affected by NP exposure. Ubiquitination and carbonylation were also tissue-specific. NP exposure induced significantly more ubiquitinated proteins in gills than in digestive glands, compared to controls. Digestive gland showed a significant higher number of carbonylated proteins than gills after NP exposure. Protein ubiquitination and carbonylation are therefore independent processes. Results showed that NP exposure generated ROS in gill and digestive gland of R. decussatus that significantly altered the proteome. Results also highlighted the advantage of using redox proteomics in the assessment of protein ubiquitination and carbonylation, which may be markers of oxidative stress in R. decussatus.     

Effects of different inorganic arsenic species in Cyprinus carpio (Cyprinidae) tissues after short-time exposure: Bioaccumulation, biotransformation and biological responses

Differences in the toxicological and metabolic pathway of inorganic arsenic compounds are largely unknown for aquatic species. In the present study the effects of short-time and acute exposure to As^III and As^V were investigated in gills and liver of the common carp, Cyprinus carpio (Cyprinidae), measuring accumulation and chemical speciation of arsenic, and the activity of glutathione-S-transferase omega (GST Ω), the rate limiting enzyme in biotransformation of inorganic arsenic. Oxidative biomarkers included antioxidant defenses (total glutathione-S-transferases, glutathione reductase, glutathione, and glucose-6-phosphate dehydrogenase), total scavenging capacity toward peroxyl radicals, reactive oxygen species (ROS) measurement and lipid peroxidation products. A marked accumulation of arsenic was observed only in gills of carps exposed to 1000 ppb As^V. Also in gills, antioxidant responses were mostly modulated through a significant induction of glucose-6-phosphate dehydrogenase activity which probably contributed to reduce ROS formation; however this increase was not sufficient to prevent lipid peroxidation. No changes in metal content were measured in liver of exposed carps, characterized by lower activity of GST Ω compared to gills. On the other hand, glutathione metabolism was more sensitive in liver tissue, where a significant inhibition of glutathione reductase was concomitant with increased levels of glutathione and higher total antioxidant capacity toward peroxyl radicals, thus preventing lipid peroxidation and ROS production. The overall results of this study indicated that exposure of C. carpio to As^III and As^V can induce different responses in gills and liver of this aquatic organism.     

Sub-cellular partitioning of Cd, Cu and Zn in tissues of indigenous unionid bivalves living along a metal exposure gradient and links to metal-induced effects

We studied organ and sub-cellular distributions of several trace metals in a freshwater bivalve that has been proposed for use as a metal biomonitor. Specimens of Pyganodon grandis were collected from nine lakes located along a Cd, Cu and Zn concentration gradient (Rouyn-Noranda area, Quebec). Gills and digestive gland were isolated, homogenized and six sub-cellular fractions were separated by differential centrifugation and analysed for their Cd, Cu and Zn content. Metallothionein was quantified independently. Gill tissues contained abundant calcium concretions that accounted for over 60% of the total gill burden of each metal. Cadmium and Zn concentrations in this granule fraction reflected ambient metal concentrations. Metal concentrations in the digestive gland also responded to the metal contamination gradient, but to a lesser extent than the gills, reflecting the lower abundance of granules in the digestive gland. Metals (Cd, Cu) in this organ were present largely in the "heat-stable proteins" fraction, and metal concentrations in this fraction were strongly correlated with those of both metallothionein and, to a lesser extent, the "lysosomes+microsomes" and "mitochondria" fractions. In both organs, Cd concentrations in the "heat-denaturable protein" fraction remained low and constant, suggesting reasonably effective metal detoxification. Some evidence for oxidative stress was noted in the gills but not in the digestive gland. Overall, we conclude that in nature metals in P. grandis are bound differently in the gills and in the digestive gland and that metal detoxification in the former organ may be less effective than in the latter.     

Toxicokinetics/toxicodynamics links bioavailability for assessing arsenic uptake and toxicity in three aquaculture species

The purpose of this study was to link toxicokinetics/toxicodynamics (TK/TD) and bioavailability-based metal uptake kinetics to assess arsenic (As) uptake and bioaccumulation in three common farmed species of tilapia (Oreochromis mossambicus), milkfish (Chanos chanos), and freshwater clam (Corbicula fluminea). We developed a mechanistic framework by linking damage assessment model (DAM) and bioavailability-based Michaelis?CMenten model for describing TK/TD and As uptake mechanisms. The proposed model was verified with published acute toxicity data. The estimated TK/TD parameters were used to simulate the relationship between bioavailable As uptake and susceptibility probability. The As toxicity was also evaluated based on a constructed elimination?Crecovery scheme. Absorption rate constants were estimated to be 0.025, 0.016, and 0.175?mL g^?1 h^?1 and As uptake rate constant estimates were 22.875, 63.125, and 788.318?ng g^?1 h^?1 for tilapia, milkfish, and freshwater clam, respectively. Here we showed that a potential trade-off between capacities of As elimination and damage recovery was found among three farmed species. Moreover, the susceptibility probability can also be estimated by the elimination?Crecovery relations. This study suggested that bioavailability-based uptake kinetics and TK/TD-based DAM could be integrated for assessing metal uptake and toxicity in aquatic organisms. This study is useful to quantitatively assess the complex environmental behavior of metal uptake and implicate to risk assessment of metals in aquaculture systems.     

Metals bioaccumulation and histopathological biomarkers in Carcinus maenas crab from Bizerta lagoon,Tunisia

Metals concentrations and histolopathological lesions of gills and digestive gland were investigated in Carcinus maenas crabs sampled from Bizerta Lagoon and Kuriat Island (Tunisia) as control site. The concentrations of trace metals varied between tissues, sites and sampling time. The highest levels of the analysed metals in gills and digestive gland were noted in Menzel Bourguiba and Cimentery sites at both sampling times (February and July). The higher metals loads were associated with severe and various tissues alterations in contaminated crabs. We particularly noted in the gills a haemocytic infiltration, distension and enlargement of the lamellae, lifting of lamellar epithelium, necrotic lesions and fusion of lamellae in the most polluted sites (Menzel Bourguiba and Cimentery). Moreover, others pathological alterations were observed in digestive gland of crabs collected from polluted sites and with a severity site dependent. We observed necrotic tubules containing tissue debris in the lumen with more intensity in crabs collected from Cimentery site in both sampling times. The thickened basal laminae and the walling off of the tubules by haemocytes around the thickened basal laminae were more abundant at Menzel Bourguiba than at others sites. The coagulation in the thickened basal laminae was observed only at Cimentery in February. Tissues histopathological lesions were sensitive to discriminate crabs of different sites and demonstrated its usefulness in this biomonitoring study. We recommend the association of histopatholocial lesions to biochemical biomarkers in future biomonitoring studies.     

Single and combined effects of aluminum (Al<Subscript>2</Subscript>O<Subscript>3</Subscript>) and zinc (ZnO) oxide nanoparticles in a freshwater fish, <Emphasis Type="Italic">Carassius auratus</Emphasis>

The increasing use of nanoparticles (NPs) worldwide has raised some concerns about their impact on the environment. The aim of the study was to assess the toxicity of metal oxide nanoparticles, singly or combined, in a freshwater fish (Carassius auratus). The fish were exposed for 7, 14, and 21 days to different concentrations of NPs (10 μg Al₂O₃.L^?1, 10 μg ZnO.L^?1, 10 μg Al₂O₃.L^?1 plus 10 μg ZnO.L^?1, 100 μg Al₂O₃.L^?1, 100 μg ZnO.L^?1, and 100 μg Al₂O₃.L^?1 plus 100 μg ZnO.L^?1). At the end of each exposure period, antioxidant enzyme activity (catalase, glutathione-S-transferase, and superoxide dismutase), lipid peroxidation, and histopathology were assessed in the gills and livers of C. auratus. The results show an increase in catalase (CAT) and superoxide dismutase (SOD) activity in the gills and livers of fish, especially after 14 days of exposure to single and combined NPs, followed by a reduction at 21 days. An increase in glutathione S-transferase (GST) was observed in gills after 7 days for all tested NP concentrations (single and combined); while in livers, a significant increase was determined after 14 days of exposure to 100 μg.L^?1 of both single ZnO and Al₂O₃ NPs. Lipid peroxidation (LPO) significantly increased in gills after 7 days of exposure to 100 μg.L^?1 Al₂O₃ NPs (single or combined). In livers, LPO increased significantly after 7 days of exposure to all tested concentrations of both single ZnO and Al₂O₃ (except for 10 μg Al₂O₃.L^?1), and after 14 days of exposure to ZnO (10 and 100 μg.L^?1) and Al₂O₃ (100 μg.L^?1)_. The results from histological observations suggest that exposure to metal oxide NPs affected both livers and gills, presenting alterations such as gill hyperplasia and liver degeneration. However, the most pronounced effects were found in gills. In general, this study shows that the tested NPs, single or combined, are capable of causing sub-lethal effects on C. auratus, but when combined, NPs seem to be slightly more toxic than when added alone.     

Integrated spatial health assessment of yellow perch (<Emphasis Type="Italic">Perca flavescens</Emphasis>) populations from the St. Lawrence River,Quebec, Canada) part A: physiological parameters and pathogen assessment

A multi-disciplinary approach was used to evaluate the health of yellow perch (Perca flavescens) in the St. Lawrence River (Quebec, Canada), which is experiencing a severe population decline in the downstream portion of the river. Physiological parameters, liver alterations, trace metal concentrations, parasite prevalence and abundance, stable isotope composition, and the presence/absence of the viral hemorragic septicemia virus (VHSV) were evaluated in perch collected at six sites along the river: Lake St. François, Lake St. Louis (north and south), Beauregard Island, and Lake St. Pierre (north and south). Trace metal concentrations in surface water were higher in Lake St. Louis and downstream of a major urban wastewater treatment plant discharge, indicating that this effluent was a significant source of Cu, As, Ag, Zn, and Cd. Levels of Pb in surface water exceeded thresholds for the protection of aquatic life in Lake St. Louis and were negatively correlated with body condition index in this lake. In Lake St. Pierre, Cu, Ag, and Cd bioaccumulated significantly in perch liver and lower body condition index and greater liver damage were observed compared to upstream sites. Parasite analyses indicated a higher abundance of metacercariae of the trematodes Apophallus brevis and Diplostomum spp. in Lake St. Louis, and VHSV was not detected in the liver of yellow perch for all studied sites. Overall, results suggested that the global health of yellow perch from Lake St. Pierre is lower compared to upstream studied sites, which could contribute to the documented population collapse at this site.     

Impact of common cytostatic drugs on pollen fertility in higher plants

Cytostatic drugs are among the most toxic chemicals which are produced. Many of them cause damage of the genetic material which may affect the fertility of higher organisms. To study the impact of the widely used anticancer drugs [cisplatin (CisPt), etoposide (Et), and 5-fluorouracil (5-FU)] on the reproduction of higher plants, pollen abortion experiments were conducted with species which belong to major plant families, namely with Tradescantia paludosa (Commelinaceae), Arabidopsis thaliana (Brassicaceae), Chelidonium majus (Papaveraceae), and Alisma plantago-aquatica (Alismataceae). All compounds increased the frequencies of abortive grains. The lowest effective doses were in general in a narrow range (i.e., 1 and 10 mg/kg of dry soil). The effects of the individual drugs were similar in T. paludosa, A. plantago-aquatica, and Ch. majus, while A. thaliana was consistently less sensitive. The highest abortion rate was obtained in most experiments with CisPt, followed by 5-FU and Et. Comparisons of the doses which caused effects in the present experiments in the different species with the predicted environment concentrations and with the levels of the cytostatics which were detected in hospital wastewaters show that the realistic environmental concentrations of the drugs are 4–6 orders of magnitude lower. Therefore, it is unlikely that these drugs affect the fertility of higher plants in aquatic and terrestrial ecosystems.     

Effect of tea saponin on phytoremediation of Cd and pyrene in contaminated soils by <Emphasis Type="Italic">Lolium multiflorum</Emphasis>

Tea saponin (TS), a kind of green biosurfactant produced by plants, was added into the Cd–pyrene co-contaminated soils to evaluate its influence on phytoremediation of Cd and pyrene by Lolium multiflorum. The results showed that the accumulation of pyrene in L. multiflorum was significantly promoted by the TS. Compared with no TS treatments (PL and ML), the aboveground concentrations of pyrene in TS treatments (PLT and MLT) increased by 135 and 30%, respectively, and the underground concentrations of pyrene in TS treatments (PLT and MLT) increased by 40 and 25%. The concentrations of Cd in the aboveground and underground parts in single contaminated treatments were all significantly more than those in co-contaminated treatments, while the situation of pyrene was quite the reverse. Besides, the addition of TS enhanced activities of dehydrogenase and polyphenol oxidase in soils and increased the biomass of L. multiflorum. The micromorphology of L. multiflorum was not affected by TS. The study suggests that the use of L. multiflorum with TS is an alternative technology for remediation of Cd–pyrene co-contaminated soils.     

Evaluation of the giant reed (<Emphasis Type="Italic">Arundo donax</Emphasis>) in horizontal subsurface flow wetlands for the treatment of recirculating aquaculture system effluent

Introduction

Two emergent macrophytes, Arundo donax and Phragmites australis, were established in experimental subsurface flow, gravel-based constructed wetlands (CWs) receiving untreated recirculating aquaculture system wastewater.

Materials and methods

The hydraulic loading rate was 3.75 cm day^?1. Many of the monitored water quality parameters (biological oxygen demand [BOD], total suspended solids [TSS], total phosphorus [TP], total nitrogen [TN], total ammoniacal nitrogen [TAN], nitrate nitrogen [NO₃], and Escherichia coli) were removed efficiently by the CWs, to the extent that the CW effluent was suitable for use on human food crops grown for raw produce consumption under Victorian state regulations and also suitable for reuse within aquaculture systems.

Results and discussion

The BOD, TSS, TP, TN, TAN, and E. coli removal in the A. donax and P. australis beds was 94%, 67%, 96%, 97%, 99.6%, and effectively 100% and 95%, 87%, 95%, 98%, 99.7%, and effectively 100%, respectively, with no significant difference (p?>?0.007) in performance between the A. donax and P. australis CWs. In this study, as expected, the aboveground yield of A. donax top growth (stems + leaves) (15.0?±?3.4 kg wet weight) was considerably more than the P. australis beds (7.4?±?2.8 kg wet weight). The standing crop produced in this short (14-week) trial equates to an estimated 125 and 77 t ?ha^?1 year^?1 biomass (dry weight) for A. donax and P. australis, respectively (assuming that plant growth is similar across a 250-day (September–April) growing season and a single-cut, annual harvest).

Conclusion

The similarity of the performance of the A. donax- and P. australis-planted beds indicates that either may be used in horizontal subsurface flow wetlands treating aquaculture wastewater, although the planting of A. donax provides additional opportunities for secondary income streams through utilization of the energy-rich biomass produced.

     

Proposal to optimize ecotoxicological evaluation of wastewater treated by conventional biological and ozonation processes

A mixture of urban and hospital effluents (50 % v/v) was evaluated for ecotoxicity with an advanced bioassay battery. Mixed effluents were tested before any treatment, after biological treatment alone, and after biological treatment followed by a tertiary ozonation (15 mg O₃/L). Laying a high value on the continuance of organisms’ fitness, essential to preserve a healthy receiving ecosystem, the main objective of this study was to combine normalized bioassays with newly developed in vivo and in vitro tests in order to assess alteration of embryo development, growth and reproduction, as well as genotoxic effects in aquatic organisms exposed to complex wastewater effluents. Comparison of the bioassays sensitivity was considered. Contrary to the lack of toxicity observed with normalized ecotoxicity tests, endpoints measured on zebrafish embryos such as developmental abnormalities and genotoxicity demonstrated a residual toxicity in wastewater both after a biological treatment followed or not by a tertiary O₃ treatment. However, the ozonation step allowed to alleviate the residual endocrine disrupting potential measure in the biologically treated effluent. This study shows that normalized bioassays are not sensitive enough for the ecotoxicological evaluation of wastewaters and that there is a great need for the development of suitable sensitive bioassays in order to characterize properly the possible residual toxicity of treated effluents.     

Cytotoxicity assessment of four pharmaceutical compounds on the zebra mussel (Dreissena polymorpha) haemocytes, gill and digestive gland primary cell cultures

Pharmaceutical compounds are considered the new environmental pollutants but at present few studies have evaluated their ecotoxicity on aquatic invertebrates. This study was aimed to investigate the in vitro cytotoxicity of four common drugs, namely atenolol (ATL), carbamazepine (CBZ), diclofenac (DCF) and gemfibrozil (GEM), on three different cell typologies from the zebra mussel (Dreissena polymorpha): haemocytes, gill and digestive gland cells. Results obtained by the Trypan blue exclusion test revealed that exposure to increasing concentrations (0.001; 0.01; 0.1; 1 and 10 mg L⁻¹) of CBZ, DCF and GEM were able to significantly decrease the viability of each cell type, while the MTT (3(4,5-dimethyl-2thiazholyl)-2,5-diphenyl-2H-tetrazolium bromide) reduction assay highlighted only a slight reduction of mitochondrial activity of gill and digestive gland cells. Overall, DCF was the most cytotoxic drug for zebra mussel cells, followed by GEM, CBZ, while ATL has not a noteworthy toxic potential. Our preliminary results lay the groundwork for further in vitro evaluations, which will allow a better definition of the potential toxicity of these drugs.