Screening 800 putative endocrine disrupting chemicals in a representative mammal, bird, and fish using a neurochemical cell-free testing platform.

There is global demand for novel ecotoxicity testing tools that are based on alternative to animal models, have high throughput potential, and may be applicable to a wide diversity of taxa. Here we scaled up a microplate-based cell-free neurochemical testing platform to screen 800 putative endocrine disrupting chemicals from the U.S. Environmental Protection Agency's ToxCast e1k library against the glutamate (NMDA), muscarinic acetylcholine (mACh), and dopamine (D2) receptors. Each assay was tested in cellular membranes isolated from brain tissues from a representative bird (zebra finch = Taeniopygia castanotis), mammal (mink = Neogale vison), and fish (rainbow trout = Oncorhynchus mykiss). The primary objective of this short communication was to make the results database accessible, while also summarising key attributes of assay performance and presenting some initial observations. In total, 7200 species-chemical-assay combinations were tested, of which 453 combinations were classified as a hit (radioligand binding changed by at least 3 standard deviations). There were some differences across species, and most hits were found for the D2 and NMDA receptors. The most active chemical was C.I. Solvent Yellow 14 followed by Diphenhydramine hydrochloride, Gentian Violet, SR271425, and Zamifenacin. Nine chemicals were tested across multiple plates with a mean relative standard deviation of the specific radioligand binding data being 24.6%. The results demonstrate that cell-free assays may serve as screening tools for large chemical libraries especially for ecological species not easily studied using traditional methods.

Early molecular responses of mangrove oysters to nanoplastics using a microfluidic device to mimic environmental exposure.

This study assessed the effects of nanoplastics (NPs) using for the very first time microfluidic devices (chip) mimicking transition waters. Three kinds of NPs were tested: crushed NPs from polystyrene pellets (NP-PS), or from Guadeloupe beaches (NP-G); and latex PS (PSL-COOH). The eluted fractions from the microfluidic device showed a low aggregation of NPs. They remained stable over time in the exposure media, with a stabilization of NPs of small sizes (< 500 nm). These chips were thus used for the toxicological assessment of NPs on swamp oysters, Isognomon alatus. Oysters were exposed for 7 days to the chip elution fraction of either NP-G, NP-PS or PSL-COOH (0.34-333 µg.L-1). Gene transcription analyses showed that the tested NPs triggered responses on genes involved in endocytosis, mitochondrial metabolism disruption, oxidative stress, DNA repair, and detoxification. Highest responses were observed after NP-G exposure at low concentrations (1 µg.L-1), as they are originated from the natural environment and accumulated contaminants, enhancing toxicological effects. As salinity influences aggregation and then the bioavailability of NPs, our results demonstrated the importance of using microfluidic devices for ecotoxicological studies on swamp or estuarine species.

The underestimated toxic effects of nanoplastics coming from marine sources: A demonstration on oysters (Isognomon alatus).

This study aims to assess the potential toxicity of (1) nanoplastics (NPs) issued from the fragmentation of larger plastic particles collected on the Caribbean marine coast (NP-G), and (2) polystyrene NPs (NP-PS), commonly used in the literature, on Caribbean swamp oysters (Isognomon alatus). Oysters were exposed to 7.5 and 15 µg.L-1 of each type of NPs, combined or not with arsenic (As) at 1 mg.L-1 for one week before molecular analyses at gene levels. Overall, the NP-G triggered more significant changes than NP-PS, especially when combined with As. Genes involved in the mitochondrial metabolism were strongly up-regulated in most of the conditions tested (up to 11.6 fold change for the NP-PS exposure at 7.5 µg.L-1 for the 12s). NPs in combination with As or not triggered a response against oxidative stress, with an intense repression of cat and sod1 (0.01 fold-changes for the NP-G condition at 7.5 µg.L-1). Both NP-G and NP-PS combined or not with As led to an up-regulation of apoptotic genes p53 and bax (up to 59.3 fold-changes for bax in the NP-G condition with As). Our study reported very innovative molecular results on oysters exposed to NPs from environmental sources. Our results suggest that the composition, surface charge, size, and the adsorbed contaminants of plastics from the natural environment may have synergic effects with plastic, which are underestimated when using manufactured NPs as NP-PS in ecotoxicological studies.

Bioaccumulation dynamics and gene regulation in a freshwater bivalve after aqueous and dietary exposures to gold nanoparticles and ionic gold.

Gold nanoparticles (AuNPs) are being developed and produced for a wide variety of industrial and biomedical applications, which raises the concern about their release and potential effects in the environment. In this study, we aim to assess the effects of PEGylated AuNPs and ionic gold on the freshwater bivalve Corbicula fluminea. As NP bioavailability is conditioned by many factors of variability, we focused on the determination of biodynamic parameters which control AuNP uptake and elimination in bivalves. Three experiments were conducted: (1) a waterborne exposure (0-24 mg/L for AuNPs and 0-12 mg/L for ionic gold), (2) a dietborne exposure (0-48 mg/L for AuNPs and 0-24 mg/L for ionic gold), and (3) an elimination phase (after waterborne exposure to 12 mg/L for AuNPs and 24 mg/L for ionic gold), to calculate rate constants for uptake from water(kuw), from food (kuf), and for the physiological elimination (ke) for AuNPs and AuCl(OH)3-. Jointly, the relative expression of several genes was investigated in the hemolymph cells to relate AuNPs and gold ion exposures to detoxification, oxidative stress, immune, and apoptosis responses in C. fluminea. Results show that kuw and kuf were around 10 and 30 times higher for AuNPs compared to AuCl(OH)3-, respectively. The ke was also faster in clams exposed to AuNPs meaning that they also had greater excretion capacities in comparison to gold ions. Water seems to be the main exposure pathway for C. fluminea according to kuw and kuf values for AuNPs and AuCl(OH)3- (kuw = 0.28 and 0.03, kuf = 0.009 and 0.001, respectively). The gene analyses pointed out important responses against oxidative stress, strong activations of genes of the immunity, and apoptosis after the waterborne exposure to AuNPs and to a lesser extent after exposure to gold ions. Very few responses were observed after the dietary exposure to both forms of gold, probably due to valve closure in response to contamination. While some studies suggest that the toxicity of nanoparticles may come from the release of metal ions, our results showed that the AuNPs we used were very stable (less than 1% of ion release) and generated more effects at the gene level than ionic gold. Therefore these results highlight the strong potential of toxicity of AuNPs compared to ionic gold and raise new concerns about the toxicity inherent to NPs in the environment.

Ecotoxicity of polyethylene nanoplastics from the North Atlantic oceanic gyre on freshwater and marine organisms (microalgae and filter-feeding bivalves).

Each year, 5 to 10 million tons of plastic waste is dumped in the oceans via freshwaters and accumulated in huge oceanic gyres. Under the effect of several abiotic factors, macro plastic wastes (or plastic wastes with macro sizes) are fractionated into microplastics (MP) and finally reach the nanometric size (nanoplastic NP). To reveal potential toxic impacts of these NPs, two microalgae, Scenedemus subspicatus (freshwater green algae), and Thalassiosira weissiflogii (marine diatom) were exposed for up to 48 h at 1, 10, 100, 1000, and 10,000 µg/L to reference polyethylene NPs (PER) or NPs made from polyethylene collected in the North Atlantic gyre (PEN, 7th continent expedition in 2015). Freshwater filter-feeding bivalves, Corbicula fluminea, were exposed to 1000 µg/L of PER and PEN for 48 h to study a possible modification of their filtration or digestion capacity. The results show that PER and PEN do not influence the cell growth of T. weissiflogii, but the PEN exposure causes growth inhibition of S. subspicatus for all exposure concentrations tested. This growth inhibition is enhanced for a higher concentration of PER or PEN (10,000 µg/L) in S. subspicatus. The marine diatom T. weissiflogii appears to be less impacted by plastic pollution than the green algae S. subspicatus for the exposure time. Exposure to NPs does not lead to any alteration of bivalve filtration; however, fecal and pseudo-fecal production increased after PEN exposure, suggesting the implementation of rejection mechanisms for inedible particles.

A cell-free testing platform to screen chemicals of potential neurotoxic concern across twenty vertebrate species.

There is global demand for new in vitro testing tools for ecological risk assessment. The objective of the present study was to apply a set of cell-free neurochemical assays to screen many chemicals across many species in a relatively high-throughput manner. The platform assessed 7 receptors and enzymes that mediate neurotransmission of γ-aminobutyric acid, dopamine, glutamate, and acetylcholine. Each assay was optimized to work across 20 vertebrate species (5 fish, 5 birds, 7 mammalian wildlife, 3 biomedical species including humans). We tested the screening assay platform against 80 chemicals (23 pharmaceuticals and personal care products, 20 metal[loid]s, 22 polycyclic aromatic hydrocarbons and halogenated organic compounds, 15 pesticides). In total, 10 800 species-chemical-assay combinations were tested, and significant differences were found in 4041 cases. All 7 assays were significantly affected by at least one chemical in each species tested. Among the 80 chemicals tested, nearly all resulted in a significant impact on at least one species and one assay. The 5 most active chemicals were prochloraz, HgCl2 , Sn, benzo[a]pyrene, and vinclozolin. Clustering analyses revealed groupings according to chemicals, species, and chemical-assay combinations. The results show that cell-free assays can screen a large number of samples in a short period of time in a cost-effective manner in a range of animals not easily studied using traditional approaches. Strengths and limitations of this approach are discussed, as well as next steps. Environ Toxicol Chem 2017;36:3081-3090. © 2017 SETAC.

The antidepressant venlafaxine may act as a neurodevelopmental toxicant in cuttlefish (Sepia officinalis).

The Serotonin/Norepinephrine Reuptake Inhibitor (SNRI) antidepressant venlafaxine (VEN, Effexor(®)) has become one of the most common antidepressants detected in North American and European streams. Mammalian research has established that VEN exposure is associated with a range of structural, neurochemical, and functional alterations of the brain in adults and newborns. However, the neurodevelopmental effects of VEN on non-target organisms have never been investigated. The aim of our research was to decrease this gap in knowledge by characterizing the effects of VEN exposure on a cephalopod mollusk, the common cuttlefish Sepia officinalis. This species inhabits VEN-contaminated waters and possesses an unusually sophisticated brain. These characteristics render it a unique invertebrate species for studying the neurodevelopmental effects of VEN. Cuttlefish were exposed to environmentally-relevant concentrations of VEN (Measured concentrations ≈5 and 100ngL(-)(1)) or to filtered natural seawater (control) in a closed-loop system with regular water changes during the first 20days after hatching. We evaluated brain maturation as well as neurochemical changes and behavioral performances during this critical period of development. Our results show that both VEN-exposed groups exhibited a decrease in norepinephrine levels, along with a reduction in the relative number of glutamate NMDA-like receptors binding sites in the group exposed to 5ngL(-1) of VEN after 20days of exposure. Brain regional changes in cellular proliferation were observed in VEN-exposed groups in the vertical lobe (i.e. a key structure involved in cognitive processes) and in the optic lobes (i.e. main visual processing centers) in the absence of significant change in their volume. Along with these neurodevelopmental changes, 20days of exposure to 100ngL(-1) of VEN was associated with a decrease in camouflage ability. Overall, our study suggests that VEN is a neurodevelopmental toxicant in non-target aquatic organisms at environmentally-relevant concentrations.

In vivo and In vitro neurochemical-based assessments of wastewater effluents from the Maumee River area of concern.

Wastewater treatment plant (WWTP) effluents contain potentially neuroactive chemicals though few methods are available to screen for the presence of such agents. Here, two parallel approaches (in vivo and in vitro) were used to assess WWTP exposure-related changes to neurochemistry. First, fathead minnows (FHM, Pimephales promelas) were caged for four days along a WWTP discharge zone into the Maumee River (Ohio, USA). Grab water samples were collected and extracts obtained for the detection of alkylphenols, bisphenol A (BPA) and steroid hormones. Second, the extracts were then used as a source of in vitro exposure to brain tissues from FHM and four additional species relevant to the Great Lakes ecosystem (rainbow trout (RT), river otter (RO), bald eagle (BE) and human (HU)). The ability of the wastewater (in vivo) or extracts (in vitro) to interact with enzymes (monoamine oxidase (MAO) and glutamine synthetase (GS)) and receptors (dopamine (D2) and N-methyl-D-aspartate receptor (NMDA)) involved in dopamine and glutamate-dependent neurotransmission were examined on brain homogenates. In vivo exposure of FHM led to significant decreases of NMDA receptor binding in females (24-42%), and increases of MAO activity in males (2.8- to 3.2-fold). In vitro, alkylphenol-targeted extracts significantly inhibited D2 (66% in FHM) and NMDA (24-54% in HU and RT) receptor binding, and induced MAO activity in RT, RO, and BE brains. Steroid hormone-targeted extracts inhibited GS activity in all species except FHM. BPA-targeted extracts caused a MAO inhibition in FHM, RT and BE brains. Using both in vivo and in vitro approaches, this study shows that WWTP effluents contain agents that can interact with neurochemicals important in reproduction and other neurological functions. Additional work is needed to better resolve in vitro to in vivo extrapolations (IVIVE) as well as cross-species differences.

Cadmium decontamination and reversal potential of teratological forms of the diatom Planothidium frequentissimum (Bacillariophyceae) after experimental contamination.

While the induction of teratology by cadmium (Cd) on diatoms is already known, reversal kinetics are not well documented. This study aims to understand the viability of diatoms exhibiting teratological frustules and their reproduction capacities within a Cd-impacted population to predict their return to normal diatom forms. We worked on a frequently encountered species in French hydrosystems: Planothidium frequentissimum (Lange-Bertalot) Round & L. Bukhtiyarova. First, a 21-d contamination phase highlighted increasing inductionof different teratological types in response to two levels of Cd contamination: 20 and 100 µg · L(-1) . The deformity counting indicated that Cd firstly generated striae and mixed teratologies, then affected the central area and the valves. Second, a 28-d decontamination phase demonstrated the Cd depuration capacity of Planothidium frequentissimum. Cd half-lives appeared relatively low, ~6 d for the 100 µg · L(-1) condition. Moreover, the decontamination phase showed a decrease in teratology abundances, but a still incomplete recovery after 28 d. Deformations of the striae appeared to be the most sustainable phenotype since they were still significantly higher than in reference cultures at the end of the decontamination phase for both Cd cultures.

Recovery potential of periphytic biofilms translocated in artificial streams after industrial contamination (Cd and Zn).

Metal wastes can significantly disturb aquatic communities, particularly photosynthetic organisms, the main primary producers in freshwater running ecosystems. In this study, biofilms and diatoms were used as bioindicators to characterize the kinetics of biofilm recovery. An experimental decontamination study was conducted under laboratory conditions, after biofilm colonisation at a site subject to discharge of industrial metals (Zn and Cd) and in parallel at an upstream site, metal-free, considered as a control. After 24 days of colonisation, biofilms were translocated and maintained in the laboratory for 56 days under clean conditions (control and decontamination) or metal contamination. Various tests were conducted from the community level--measures of metal bioaccumulation, cell densities and taxonomic investigations, to the individual level--measures of teratological forms. After 56 days of decontamination, Zn and Cd concentrations in decontaminated biofilms showed a sharp decline, respectively ranging from 6.7 ± 2 to 4 ± 2.5 mg Zn g⁻¹ DW and from 207.6 ± 24.5 to 45.4 ± 9.9 µg Cd g⁻¹ DW. However, at the end of the experiment bioaccumulations remained significantly higher than concentrations in control biofilms. Despite a diatom evolution in biofilm assemblages, taxonomic inventories did not demonstrate a complete restoration of diatom communities in biofilms under decontamination conditions compared with controls, since metal-resistant species initially present after colonisation at the contaminated site, such as Eolimna minima, persisted in high abundance in decontaminated biofilms. Biofilms kept under metal pressure showed very high bioaccumulation capacities and a sharp decline of species diversity which allowed identification of some resistant species. Regarding these first results on the behaviour of diatom biofilms under experimental decontamination conditions, improvement of the natural hydrosystem's chemical state appears quickly, but an eventual return to good ecological status appears delayed, with the persistence of metal-tolerant species even after 56 days.

Remediation of a watershed contaminated by heavy metals: a 2-year field biomonitoring of periphytic biofilms.

This study focuses on an industrial contamination site subjected to remediation processes since 2007 in the Riou-Mort watershed (southwest France). The purpose was to assess the first impacts of remediation on periphytic biofilms, and was performed during two years of biomonitoring. Periphytic biofilms were collected on glass slides immersed 24 days at different sites along the contamination gradient for 12 colonisation cycles. Metal contaminations (Cd and Zn) were analysed in biofilms and the evolution of diatom communities was assessed, integrating teratology quantifications. Despite remediation work initiated at the industrial site, this study demonstrated the persistence of metal contamination in water, as well as in biofilms. In addition, our data, showed that the remediation process was initially marked by an increase in metal contamination in the river, with increasing diatom community shifts. Metal-contaminated biofilms presented decreasing species diversities and were dominated by metal-resistant species such as Eolimna minima, whom abundances increased in 2010 reaching 57.2±10%. No significant decrease in metal accumulation was observed and total Cd content in biofilms collected downstream the industrial site ranged from 772.7±88 in July 2009 to 636.9±20 µg/gDW in July 2010. Results obtained on artificial substrates were compared with those of natural substrates and showed similar diatom communities and abundances of deformed diatoms but lower diversities. This ensured that glass slide subtrates gave a good representation of periphytic biofilm health. Finally, results were compared to studies performed before the remediation process and this did not reveal a decrease of metal accumulation in biofilms nor shifts in taxonomic composition of the communities, rather the remaining dominance of metal resistant species such as E. minima was confirmed.

Comparison of periphytic biofilm and filter-feeding bivalve metal bioaccumulation (Cd and Zn) to monitor hydrosystem restoration after industrial remediation: a year of biomonitoring.

Despite a significant decrease in the metallic waste emissions from an industrial site and a remediation process initiated in 2007, the Riou-Mort watershed (southwest France) still exhibits high Cd and Zn concentrations. Metal wastes have long been proven to significantly disturb aquatic communities. In this study, bioaccumulation capacities and responses to the chemical improvement of the hydrosystem were assessed for a year along the contamination gradient through the comparison of two biological models: Corbicula fluminea and periphytic biofilms, both considered as good bioindicators. Bioaccumulation results confirmed the persistence of water contamination in Corbicula fluminea and biofilms with, respectively, maximum Cd concentrations reaching 80.6 and 861.2 µg gDW(-1), and Zn concentrations 2.0 and 21.3 mg gDW(-1). Biofilms exhibited bioaccumulation in close correlation with water contamination, while Corbicula fluminea presented Cd bioaccumulation clearly regulated by water temperature and metal concentrations, affecting the ventilatory activity, as revealed by condition indices measurements. Also, a linear regression using Cd bioaccumulation and temperature () showed that below approximately 6 °C Corbicula fluminea did not appear to accumulate metals. Bioconcentration factors (BCFs) were higher in biofilms in comparison with Corbicula fluminea and showed the great accumulation capacity of suspended particulate matter in biofilms. However, bioaccumulation capacities are known to be influenced by many factors other than metal concentrations, such as temperature, water oxygenation or plankton and nutrient concentrations. Thus, this study demonstrates the power of a combined assessment using both Corbicula fluminea and biofilms as bioindicators to give a more integrated view of water quality assessment. Finally, when comparing our results with previous studies, the start of hydrosystem restoration could be shown by decreasing bioaccumulation in organisms.

Early genotoxic effects in gill cells and haemocytes of Dreissena polymorpha exposed to cadmium, B[a]P and a combination of B[a]P and Cd.

The aim of this study was to assess the genotoxic potential of environmentally relevant concentrations of Cd on the zebra mussel, an important freshwater sentinel organism, and to determine the stability of DNA damage in gill cells and haemocytes. The oxidative DNA damage and the co-genotoxicity of Cd in combination with B[a]P were investigated. We measured DNA damage in haemocytes and gill cells of zebra mussels exposed for 11 days to a constant concentration of Cd (10µg/L), B[a]P (10µg/L) or the two combined chemicals (10µg/L+1µg/L). Enzymatic dissociation of gills with dispase gave the lower percentage DNA in tail, compared with collagenase/dispase or collagenase. Bioaccumulation of cadmium in the soft tissues of mussels exposed to CdCl(2) or CdCl(2)+B[a]P increased in a time-dependent manner indicating that both exposures were effective. Cd (10µg/L) is genotoxic only during the first 3 days of exposure in gill cells, while in haemocytes the genotoxicity of Cd was observed later. B[a]P (10µg/L) induced an early increase of DNA damage in gill cells (after 10h and 1 day), while in both gill cells and haemocytes, B[a]P caused a marked increase of DNA damage after 3 days of exposure. The Cd+B[a]P mixture decreased the DNA-damaging effect of Cd and B[a]P in both cell types. Cd induced an increase of DNA damage in Fpg-treated slides, indicating that Cd contributed to oxidative DNA damage. Cadmium induced a cytogenetic effect in gill cells, assessed by the number of micronuclei, throughout the duration of the exposure, while B[a]P did not induce any cytogenetic effect. B[a]P, Cd and Cd+B[a]P induced a transient increase in the number of bi-nucleated cells. Our data clearly show that gills are more sensitive to Cd and B[a]P, which makes them more suitable for future bio-monitoring studies.