Do environmentally relevant concentrations of fluoxetine and citalopram impair stress-related behavior in zebrafish (Danio rerio) embryos?

Selective serotonin reuptake inhibitors (SSRIs) have been shown to interfere with various physiological functions of aquatic organisms, yet the neuroactive potential of low concentrations of SSRIs in the aquatic environment is unclear. The current study investigated the effects of fluoxetine and citalopram on the visual motor response (VMR) of 107 h old zebrafish (Danio rerio) embryos. Results document a reduction in stress-related swimming activity of zebrafish embryos at environmentally relevant concentration levels, with fluoxetine being more effective than citalopram. Further experiments were designed to elucidate (1) if the lower neuroactive potential of citalopram is due to differences in uptake kinetics, (2) if the metabolite of fluoxetine, norfluoxetine, contributes to the neuroactive potential of fluoxetine, (3) and how SSRIs and their metabolites interact in equimolar mixtures. At the stage of 120 h, zebrafish embryos accumulate citalopram at significantly lower rates (up to 127 times) than fluoxetine. Moreover, it was demonstrated that norfluoxetine reduces the embryonic VMR similarly to fluoxetine resulting in additive effects of these substances on stress-related behavior in zebrafish embryos. In contrast, the interaction of fluoxetine, norfluoxetine and citalopram varied with test concentrations of the equimolar mixtures. Findings provide evidence that environmentally relevant concentrations of fluoxetine reduce stress-related behavior of zebrafish embryos, while these effects may be enhanced by the interaction of multiple SSRIs and their metabolites in environmental exposure scenarios.

Norfluoxetine Is the Only Metabolite of Fluoxetine in Zebrafish (Danio rerio) Embryos That Accumulates at Environmentally Relevant Exposure Scenarios.

Fluoxetine has been recognized as one of the most toxic pharmaceuticals in the aquatic environment. Since there is growing evidence that the toxic potential of fluoxetine in surface waters is markedly influenced by its own metabolism in aquatic species, this study investigated the biotransformation of fluoxetine in the zebrafish embryo - an aquatic model organism of intermediate complexity. Zebrafish embryos were exposed to 0.1, 1.0, 10, 50, and 5000 µg/L of fluoxetine from 48 to 120 h post-fertilization (hpf), and the accumulation of fluoxetine and its metabolites was analyzed over time. Additionally, depuration of fluoxetine and its metabolites from 96 to 120 hpf was investigated, and autoinhibitory effects of fluoxetine on phase I biotransformation were analyzed. Exposure to 5000 µg/L fluoxetine resulted in elevated 7-ethoxyresorufin-O-deethylase (EROD) activity of cytochrome P450 enzymes and continuous accumulation of fluoxetine and 11 fluoxetine metabolites. Embryos exposed to 10 and 50 µg/L fluoxetine were able to reduce fluoxetine accumulation from 94 to 120 hpf. During depuration, accumulation of fluoxetine and most metabolites was clearly reduced, and biotransformation shifted in favor of norfluoxetine, the primary fluoxetine metabolite in humans. Findings demonstrated that norfluoxetine is the only metabolite of fluoxetine that accumulates in zebrafish embryos at environmentally relevant exposure scenarios.

Analysis of tail coiling activity of zebrafish (Danio rerio) embryos allows for the differentiation of neurotoxicants with different modes of action.

In (eco)toxicology, there is a critical need for efficient methods to evaluate the neurotoxic potential of environmental chemicals. Recent studies proposed analysis of early coiling activity in zebrafish embryos as a powerful tool for the identification of neurotoxic compounds. In order to demonstrate that the analysis of early tail movements of zebrafish embryos allows for the discrimination of neurotoxicants acting via different mechanisms, the present study investigated the effects of four different neurotoxicants on the embryogenesis (fish embryo toxicity test) and early tail coiling movements of zebrafish embryos. Cadmium predominantly increased the frequency of tail coiling at the late pharyngula stage. Dichlorvos delayed embryonic development and caused convulsive tail movements resulting in prolonged duration of tail coils. Embryos exposed to teratogenic concentrations of fluoxetine and citalopram displayed absence of spontaneous tail movements at 24 h post-fertilization. In contrast, a non-teratogenic test concentration of citalopram decreased coiling frequency at multiple time points. Results demonstrated that the analysis of tail coiling movements of zebrafish embryos has the potential to discriminate neurotoxic compounds with different primary modes of action. In addition, chemical-induced effects on coiling activity were shown to potentially overlap with effects on embryogenesis. Further studies are needed to clarify the interplay of unspecific developmental toxicity of neurotoxic chemicals and effects resulting from specific neurotoxic mechanisms.

Time-course of coiling activity in zebrafish (Danio rerio) embryos exposed to ethanol as an endpoint for developmental neurotoxicity (DNT) - Hidden potential and underestimated challenges.

Detection of developmental neurotoxicity (DNT) has been recognized as a major challenge by regulatory bodies and science. In search of sensitive and specific test methods, spontaneous tail coiling of embryonic zebrafish has been recommended as a promising tool for identification of DNT-inducing chemicals. The present study was designed to develop a protocol for a prolonged test to study neurotoxicity during the entire development of coiling movement in zebrafish embryos. Ambient illumination was found to modulate coiling activity from the very onset of tail movements representing the earliest behavioral response to light possible in zebrafish. In the dark, embryos displayed increased coiling activity in a way known from photokinesis, a stereotypical element of the visual motor response. Elevated coiling activity during dark phases allows for the development of test strategies that integrate later coiling movements under the control of a further developed nervous system. Furthermore, zebrafish embryos were exposed to ethanol, and coiling activity was analyzed according to the new test protocol. Exposure of embryos to non-teratogenic concentrations of ethanol (0.4-1%) resulted in a delay of the onset of coiling activity and heartbeat. Moreover, ethanol produced a dose-dependent increase in coiling frequency at 26 h post-fertilization, indicating the involvement of neurotoxic mechanisms. Analysis of coiling activity during prolonged exposure allowed for (1) attributing effects on coiling activity to different mechanisms and (2) preventing false interpretation of results. Further research is needed to verify the potential of this test protocol to distinguish between different mechanisms of neurotoxicity.

Transformation Products of Fluoxetine Formed by Photodegradation in Water and Biodegradation in Zebrafish Embryos ( Danio rerio).

The present study investigates the transformation of the antidepressant fluoxetine (FLX) by photo- and biodegradation and shows similarities and differences in transformation products (TPs). TPs were identified using LC-high-resolution mass spectrometry with positive and negative electrospray ionization. In a sunlight simulator, photodegradation was carried out using ultrapure water (pH 6, 8, and 10) and surface water (pH 8) to study the effect of direct and indirect photolysis, respectively. The well-known metabolite norfluoxetine (NFLX) proved to be a minor TP in photolysis (≤2% of degraded FLX). In addition, 26 TPs were detected, which were formed by cleavage of the phenolether bond ( O-dealkylation) which primarily formed 3-(methylamino)-1-phenyl-1-propanol (TP 166) and 4-(trifluoromethyl)phenol, by hydroxylation of the benzyl moiety, by CF3 substitution to benzoic aldehyde/acid, and by adduct formation at the amine group ( N-acylation with aldehydes and carboxylic acids). Higher pH favors the neutral species of FLX and the neutral/anionic species of primary TPs and, therefore, photodegradation. In zebrafish embryos, the bioconcentration factor of FLX was found to be 110, and about 1% of FLX taken up by the embryos was transformed to NFLX. Seven metabolites known from photodegradation and formed by hydrolysis, hydroxylation, and N-acylation as well as three new metabolites formed by N-hydroxylation, N-methylation, and attachment of an amine group were identified in zebrafish embryos. The study highlights the importance of considering a broad range of TPs of FLX in fresh water systems and in ecotoxicity tests and to include TP formation in both environmental processes and metabolism in organisms.

Dispersibility and dispersion stability of carbon nanotubes in synthetic aquatic growth media and natural freshwater.

The dispersion behavior of carbon nanotubes (CNTs) is influenced by both their physicochemical properties and by the aqueous media properties (e.g. ionic strength, presence of divalent cations and natural organic matter) in which they are dispersed. In the current study, the dispersibility and dispersion stability of four multi-walled CNTs (MWCNT) and a single walled CNT (SWCNT) with different physicochemical properties were investigated in three freshwater growth media (with and without natural organic matter; NOM) used in algae and daphnia ecotoxicity studies. CNT dispersion behavior was also investigated in a natural freshwater for comparison. SWCNTs and non-functionalized MWCNTs showed similar dispersibility irrespective of the media type (SWCNTs = 0.5-0.9 mg/L; MWCNTs = 1.5-2.8 mg/L). Functionalized MWCNTs exhibited higher dispersion concentrations, but were more dependent upon the ionic strength and divalent cation concentration of each media (MWCNT-COOH = 3.0-6.6 mg/L). In contrast, CNT surface oxygen content had no influence on CNT dispersibility in the natural water (all MWCNTs = 0.9-1.4 mg/L). Functionalized MWCNTs were affected more by the differences in media properties than non-functionalized MWCNTs. The dispersed CNT concentration decreased over time for all CNT types and in all media due to sedimentation, but was influenced by both CNT and media properties. The study shows how a complex interplay between CNT and media properties can influence the environmental fate of CNTs. Furthermore, the study demonstrates how different CNT types and/or ecotoxicological media in aquatic tests influences the dispersion behavior of the CNTs, and thus their exposure and toxicity to aquatic organisms.

Evaluation of methods to determine adsorption of polycyclic aromatic hydrocarbons to dispersed carbon nanotubes.

A number of methods have been reported for determining hydrophobic organic compound adsorption to dispersed carbon nanotubes (CNTs), but their accuracy and reliability remain uncertain. We have evaluated three methods to investigate the adsorption of phenanthrene (a model polycyclic aromatic hydrocarbon, PAH) to CNTs with different physicochemical properties: dialysis tube (DT) protected negligible depletion solid phase microextraction (DT-nd-SPME), ultracentrifugation, and filtration using various types of filters. Dispersed CNTs adhered to the unprotected polydimethylsiloxane (PDMS)-coated fibers used in nd-SPME. Protection of the fibers from CNT adherence was investigated with hydrophilic DT, but high PAH sorption to the DT was observed. The efficiency of ultracentrifugation and filtration to separate CNTs from the water phase depended on CNT physicochemical properties. While non-functionalized CNTs were efficiently separated from the water phase using ultracentrifugation, incomplete separation of carboxyl functionalized CNTs was observed. Filtration efficiency varied with different filter types (composition and pore size), and non-functionalized CNTs were more easily separated from the water phase than functionalized CNTs. Sorption of phenanthrene was high (< 70%) for three of the filters tested, making them unsuitable for the assessment of phenanthrene adsorption to CNTs. Filtration using a hydrophilic polytetrafluoroethylene (PTFE) filter membrane (0.1 µm) was found to be a simple and precise technique for the determination of phenanthrene adsorption to a range of CNTs, efficiently separating all types of CNTs and exhibiting a good and highly reproducible recovery of phenanthrene (82%) over the concentration range tested (70-735 µg/L).

An optimized method to assess ototoxic effects in the lateral line of zebrafish (Danio rerio) embryos.

In order to clarify the suitability of the lateral line of zebrafish (Danio rerio) embryos as a model for the screening of ototoxic (neurotoxic) effects, existing neuromast assays were adapted, improved and validated with a series of chemicals known or unknown for their ototoxic potential (caffeine copper sulfate, dichlorvos, 2.4-dinitrotoluene, neomycin, 4-nonylphenol, perfluorooctanesulfonic acid). Present methods were improved by (1) the introduction of a 4-step scoring system, (2) the selection of neuromasts from both the anterior and posterior lateral line systems, (3) a combined DASPEI/DAPI staining applied after both a continuous and pulse exposure scenario, and (4) an additional screening for nuclear fragmentation. Acute toxicities of the model substances were determined by means of the fish embryo test as specified in OECD TG 236, and EC10 concentrations were used as the highest test concentration in the neuromast assay. The enhanced neuromast assay identified known ototoxic substances such as neomycin and copper sulfate as ototoxic at sensitivities similar to those of established methods, with pulse exposure leading to stronger effects than continuous exposure. Except for caffeine, all substances tested (dichlorvos, 2.4-dinitrotoluene, 4-nonylphenol, perfluorooctanesulfonic acid) produced significant toxic effects in neuromasts at EC10 concentrations. Depending on the test substances and their location along the lateral line, specific neuromasts differed in sensitivity. Generally, neuromasts proved more sensitive in the pulse exposure scenario. Whereas for neomycin and copper sulfate neuromasts located along the anterior lateral line were more sensitive, posterior lateral line neuromasts proved more sensitive for the other test substances. Nuclear fragmentation could not only be associated with all test substances, but, albeit at lower frequencies, also with negative controls, and could, therefore, not be assigned specifically to chemical damage. The study thus documented that for a comprehensive evaluation of lateral line damage both neuromasts from the anterior and the posterior lateral line have to be considered. Given the apparently rapid regeneration of hair cells, pulse exposure seems more appropriate for the identification of lateral line neurotoxicity than continuous exposure.

Phenanthrene Bioavailability and Toxicity to Daphnia magna in the Presence of Carbon Nanotubes with Different Physicochemical Properties.

Studies investigating the effect of carbon nanotubes (CNTs) on the bioavailability and toxicity of hydrophobic organic compounds in aquatic environments have generated contradictory results, and the influence of different CNT properties remains unknown. Here, the adsorption of the polycyclic aromatic hydrocarbon phenanthrene (70-735 µg/L) to five types of CNTs exhibiting different physical and chemical properties was studied. The CNTs were dispersed in the presence of natural organic matter (nominally 20 mg/L) in order to increase the environmental relevance of the study. Furthermore, the bioavailability and toxicity of phenanthrene to Daphnia magna in the absence and presence of dispersed CNTs was investigated. Both CNT dispersion and adsorption of phenanthrene appeared to be influenced by CNT physical properties (diameter and specific surface area). However, dispersion and phenanthrene adsorption was not influenced by CNT surface chemical properties (surface oxygen content), under the conditions tested. Based on nominal phenanthrene concentrations, a reduction in toxicity to D. magna was observed during coexposure to phenanthrene and two types of CNTs, while for the others, no influence on phenanthrene toxicity was observed. Based on freely dissolved concentrations, however, an increased toxicity was observed in the presence of all CNTs, indicating bioavailability of CNT-adsorbed phenanthrene to D. magna.