Multilevel assessment of carbamazepine effects: An integrative approach using zebrafish early-life stages.

Carbamazepine (CBZ) is the most commonly used drug in epilepsy treatment, and its metabolites are commonly detected among persistent pharmaceuticals in the aquatic environment. This study aimed to investigate CBZ effects on early-life-stage zebrafish (Danio rerio) (from 2 to 168 hpf) by employing of an integrative approach linking endpoints from molecular to individual level: (i) development; (ii) locomotor activity; (iii) biochemical markers (lactate dehydrogenase, glutathione-S-transferase, acetylcholinesterase and catalase) and (iv) transcriptome analysis using microarray. A 168 h - LC50 of 73.4 mg L-1 and a 72 h - EC50 of 66.8 mg L-1 for hatching were calculated while developmental effects (oedemas and tail deformities) were observed at CBZ concentrations above 37.3 mg L-1. At the biochemical level, AChE activity proved to be the most sensitive parameter, as evidenced by its decrease across all concentrations tested (∼25% maximum reduction, LOEC (lowest observed effect concentration) < 0.6 µg L-1). Locomotor behaviour seemed to be depressed by CBZ although this effect was only evident at the highest concentration tested (50 mg L-1). Molecular analysis revealed a dose-dependent effect of CBZ on gene expression. Although only 25 genes were deregulated in organisms exposed to CBZ when compared to controls, both 0.6 and 2812 µg L-1 treatments impaired gene expression related to development (e.g. crygmxl1, org, klf2a, otos, stx16 and tob2) and the nervous system (e.g. Rtn3, Gdf10, Rtn3), while activated genes were associated with behavioural response (e.g. prlbr and taar). Altogether, our results indicate that environmentally relevant CBZ concentrations might affect biochemical and genetic traits of fish. Thus, the environmental risk of CBZ cannot be neglected, especially in a realistic scenario of constant input of domestic effluents into aquatic systems.

Exposure to tricyclic antidepressant nortriptyline affects early-life stages of zebrafish (Danio rerio).

Psychiatric drugs are among the leading medications prescribed for humans, with their presence in aquatic environments raising concerns relating to potentially harmful effects on non-target organisms. Nortriptyline (NTP) is a selective serotonin-norepinephrine reuptake inhibitor antidepressant, widely used in clinics and found in environmental water matrices. In this study, we evaluated the toxic effects of NTP on zebrafish (Danio rerio) embryos and early larval stages. Developmental and mortality analyses were performed on zebrafish exposed to NTP for 168 h at concentrations ranging from 500 to 46,900 µg/L. Locomotor behaviour and acetylcholinesterase (AChE) activity were evaluated by exposing embryos/larvae to lower NTP concentrations (0.006-500 µg/L). The median lethal NTP concentration after 168 h exposure was 2190 µg/L. Although we did not identify significant developmental changes in the treated groups, lack of equilibrium was already visible in surviving larvae exposed to ≥ 500 µg/L NTP. The behavioural analyses showed that NTP was capable of modifying zebrafish larvae swimming behaviour, even at extremely low (0.006 and 0.088 µg/L) environmentally relevant concentrations. We consistently observed a significant reduction in AChE activity in the animals exposed to 500 µg/L NTP. Our results highlight acute toxic effects of NTP on the early-life stages of zebrafish. Most importantly, exposure to environmentally relevant NTP concentrations may affect zebrafish larvae locomotor behaviour, which in turn could reduce the fitness of the species. More studies involving chronic exposure and sensitive endpoints are warranted to better understand the effect of NTP in a more realistic exposure scenario.

Auramine dyes induce toxic effects to aquatic organisms from different trophic levels: an application of predicted non-effect concentration (PNEC).

The dyes Auramine and Auramine O are used in several industrial products, despite the scarce information regarding their ecotoxicity. The aim of the present study was to assess the acute and chronic toxicity of both dyes to aquatic organisms from different trophic levels (Raphidocelis subcapitata, Daphnia similis, Hydra attenuata, and Danio rerio) and calculate their predicted non-effect concentrations (PNEC). Auramine and Auramine O induced toxicity to all selected test organisms with L(E)C50 values ranging from 300 to 4800 ug/L. Both dyes induced inhibition in the growth rate of exposed algae, negatively affecting the reproduction of D. similis and induced deformities in H. attenuata (clubbed tentacles and shortened tentacles) and D. rerio (edemas, tail malformation and delay in yolk sac absorption). PNEC values of 0.92 µg/L and 4.0 µg/L were obtained for Auramine and Auramine O, respectively, based on results of the most sensitive test system (algae). Test results were analyzed using the Criteria of Reporting and Evaluating Ecotoxicity Data (CRED), confirming their reliability and relevance. Thus, PNEC values can be used in future risk assessments of those substances in freshwater systems.

Degradation evaluation and toxicity profile of bilobol, a promising eco-friendly larvicide.

Aedes aegypti is the main arbovirus vector transmitting chikungunya, Zika and dengue. The current vector control strategies are limited due to multiple insecticide resistance, deleterious impacts on the environment, and toxicity to non-target organisms. Bilobol, an alkylresorcinol isolated from the plant species Schinus terebinthifolia, demonstrated larvicidal activity against Aedes aegypti (LC50 7.67 mg/L in less than 24 h). To ensure that bilobol presents a viable alternative as an eco-friendly larvicide, this study aimed to explore the degradation process and acute toxicity of this alkylresorcinol in zebrafish, a non-target organism. A quantification method with validated parameters was developed and used to evaluate bilobol degradation in water over time. The Fish Embryo Toxicity (FET) test was applied to evaluate the acute toxicity of bilobol together with its degradation derivates. Results demonstrated that bilobol gradually degrades over time and almost completely disappears after 96 h, turning into small aliphatic chains which are less toxic than bilobol in its fundamental form. Therefore, it was possible to conclude that bilobol does not present significant toxicity to zebrafish embryos nor does it show signs of persistence in the environment. Additionally, bilobol can be found in high quantities not only in S. terebinthifolia, but also in cashew nut industry waste. Thus, bilobol constitutes an alternative environmentally friendly insecticide because it is not persistent, has indications of low toxicity to non-target organisms and presents a way to exploit massive quantities of material discarded by the food industry.

CNTs coated charcoal as a hybrid composite material: Adsorption of fluoxetine probed by zebrafish embryos and its potential for environmental remediation.

Although traditional water treatment systems can remove various substances from wastewater, these conventional systems fail to remove many chemical molecules that pose potential ecological and health risks. Carbon nanotubes (CNTs) appear attractive to adsorption of many substances, but CNTs adsorbed with toxic substances becomes a nanocomposite still more toxic. Here, we employ zebrafish embryos as biosensor to examine how a hybrid micro/nanostructured carbonaceous material (HMNC) derived from a combination of activated carbon (AC) with hydrophilic carbon nanotubes (CNTs) can remediate wastewater contaminated with the pharmaceutical fluoxetine hydrochloride (FLX). AC and HMNC are practically non-toxic to zebrafish embryos (LC50 > 1000 mg.L-1). HMNC addition to culture medium containing FLX significantly reduces sublethal effects and lethality. Interaction between FLX and HMNC involves chemical adsorption such that embryo co-exposure to HMNC adsorbed with FLX in the range of concentrations evaluated herein does not elicit any behavioral changes in zebrafish.

Exposure to dilute concentrations of bupropion affects zebrafish early life stages.

Psychiatric pharmaceuticals are one of the most prescribed active substances globally. Bupropion (BPP) is an antidepressant that acts via inhibition of norepinephrine and dopamine reuptake. It has been found in various water matrices, and thus its effects on aquatic organisms must be studied. The present study aimed to evaluate the acute toxic effects of BPP on zebrafish (Danio rerio) early life stages. For developmental analysis, organisms were exposed for 168 h to concentrations ranging from 0 to 82000 µg/L. Two other experiments were performed by exposing embryos to a wide range of concentrations (from 0 to 50000 µg/L) in order to evaluate BPP effects on embryonic behavior, using the Zebrabox and testing at the biochemical level (acetylcholinesterase, glutathione-S-transferase, lactate dehydrogenase and catalase). Developmental analysis indicated that BPP had low acute toxicity with a calculated 168 h-LC50 of 50346 µg/L. Concentrations equal to or above 44800 µg/L elicited several effects such as hatching delay, edemas and tail deformities. However, concentrations from 7300 µg/L upwards elicited equilibrium alteration. Behavioral analysis showed that BPP affected zebrafish locomotor behavior by decreasing activity at 0.6 µg/L, increasing activity at 8.8 and 158 µg/L, and decreasing activity at 50000 µg/L. Biochemical analysis showed an increase of AChE activity at 158 and 2812 µg/L, an increase in GST at the highest concentrations, CAT alteration and increase of LDH at 0.6, 2812 and 50000 µg/L. We can conclude that BPP affects zebrafish early life stages at environmental concentrations.

Exposure to low concentration of fluoxetine affects development, behaviour and acetylcholinesterase activity of zebrafish embryos.

Fluoxetine (FLX) is a selective serotonin reuptake inhibitor (SSRI) antidepressant widely used in clinics and very often found in environmental samples of urban aquatic ecosystems in concentrations ranging from ng/L to µg/L. Fish populations might be especially susceptible to FLX due to the presence of conserved cellular receptors of serotonin. Neurotoxic effects on fish biota of polluted water bodies may be expected, but there are no sufficient studies in the current literature to elucidate this hypothesis. Batteries of embryo larval assays with zebrafish were performed to evaluate the potential effects of FLX exposure, including environmentally relevant concentrations. Evaluated parameters included survival, development, behaviour and neuronal biochemical markers. Regarding acute toxicity, a 168 h-LC50 value of 1.18 mg/L was obtained. Moreover, hatching delay and loss of equilibrium were observed, but at a concentration level much higher than FLX measured environmental concentrations (>100 µg/L). On the other hand, effects on locomotor and acetylcholinesterase activity (≥0.88 and 6 µg/L, respectively) were found at levels close to the maximum reported FLX concentration in surface waters. Altogether, these results suggest that FLX is neurotoxic to early life stages of zebrafish, in a short period of time causing changes in important ecological attributes which can probably be linked from molecular to population level.

Chronic effects of carbamazepine on zebrafish: Behavioral, reproductive and biochemical endpoints.

Carbamazepine (Cbz), one of the most prescribed pharmaceuticals in the world is often detected in surface waters and sediments. However, few studies addressed its chronic effects in fish. In the present study, Danio rerio adults were exposed for 63 days to Cbz (0 - control, 10 µg L-1 - concentration found in effluents, and 10,000 µg L-1 - 5% of LC50 at 72 h). Assessed endpoints were: feeding behavior, growth rate, number of eggs produced and their viability, histological alterations in female gonads, and biochemical biomarkers associated with antioxidant defenses (catalase - CAT, and glutathione S-transferase - GST activities), neurotransmission (acetylcholinesterase activity - AChE) and metabolism (lactate dehydrogenase - LDH). Cbz exposure increased the total time for food intake but did not affect D. rerio growth. Although the total number of eggs was not affected by Cbz exposure, the eggs viability was significantly impaired. Exposure to Cbz caused alterations in the female gonads follicular stages. In terms of biochemical endpoints, CAT activity in liver and gills, was sensitive to the pharmaceutical exposure presenting a decreased activity. AChE activity was induced in the head (both concentrations) and muscle (10,000 µg L-1). GST activity was increased in gills (both concentrations) but inhibited in the intestine. Concerning LDH, enzymatic activity was increased in the liver and decreased in muscle and gills. Several of the above-mentioned effects can be directly linked with effects at population level (e.g. feeding behavior) and occurred at environmental concentrations (the lowest concentration tested), thus serious concerns regarding risks posed by Cbz residues to fish populations arise with this study.