Life history and behavior effects of synthetic and natural dyes on Daphnia magna.

Azo dyes are the largest class of dyes extensively used by industries despite their mutagenic potential for humans. As such, natural dyes have been reemerging as an important alternative to human safety. However, limited studies have focused on the effect of dyes on the environment, thus their ecotoxicological investigation is imperative. Here, we aimed to evaluate toxic effects induced by the synthetic azo dye Basic Red 51 (BR51) in comparison with natural dye erythrostominone (Ery) in the microcrustacean Daphnia magna, a standard organism used to assess the risk of chemicals to aquatic organisms. The colorless product formed after the photodegradation of Ery (DEry) was also evaluated, addressing an easy and low cost alternative for industrial effluent treatments. The results showed that both dyes are acutely toxic to D. magna. BR51 and Ery reduced the intrinsic rate of D. magna population increase, which generated fewer neonates per brood. BR51 also increased daphnids respiration rates. In contrast, DEry did not alter any of the analyzed parameters. No locomotor changes were observed when daphnids were exposed to sub-lethal concentrations of Ery or BR51. These results indicate that both dyes can induce deleterious consequences for daphnids including population level effects, but the natural dye Ery presents 100-fold lower toxicity in comparison with the azo dye BR51. Also, that photodegradation of Ery is an efficient method to reduce and prevent previously observed toxic effects, suggesting an inexpensive, fast and easy alternative for treatment of effluents containing this natural dye.

Cannabidiol did not induce teratogenicity or neurotoxicity in exposed zebrafish embryos.

Cannabidiol (CBD) is a non-psychotomimetic compound of the Cannabis sativa that has been used for the treatment of severe epilepsy as well as other diseases of nervous system. However, toxicity studies of CBD have great relevance to guarantee the patients safety. In this context, morphological analyses of zebrafish can contribute to evaluate the teratogenic potential, as well as evaluation of acetylcholinesterase activity and motor activity of zebrafish are valuable tools to verify the neurotoxicity potential. In the present work, we use this methodology to test the toxicity of CBD to zebrafish embryos. No malformation was observed in morphological analysis of embryos exposed to all tested concentrations of CBD. Although, twenty per cent of embryos exposed to maximal dose of CBD (300 µg/L) hatched after 96hpf, while embryos in control solution had already hatched in this period. Embryos exposed to CBD did not show differences in acetylcholinesterase activity, but embryos exposed to CBD 20-300 µg/L were 1.4 up to 1.7-fold more active when compared to the control. Despite that, at 48 hpf, motor activity returned to control values. Our results suggest that the effects observed after CBD exposure are intimately related to CB1 receptor that is present in zebrafish since early stages of development. The present work showed early light effects induced by CBD exposure in concentrations that did not alter biochemical activity.

Toxicity of dyes to zebrafish at the biochemical level: Cellular energy allocation and neurotoxicity.

Dyes are widely distributed worldwide, and can be found in wastewaters resulting from industrial or urban effluents. Dyes are of particular concern as contaminants of the aquatic environment, since their toxicity remain poorly understood. Thus, the current study was designed to assess the effects induced by the synthetic azo dye Basic Red 51 (BR51) and by the natural naphthoquinone dye erythrostominone (ERY) on zebrafish early life stages (Danio rerio) at different biological organization levels, i.e., studying how changes in biochemical parameters of important physiological functions (neurotransmission and cellular energy allocation) may be associated with behavior alterations (swimming activity). This approach was also used to assess the effects of ERY after its photodegradation resulting in a colorless product(s) (DERY). Results showed that after 96 h exposure to BR51 and Ery, zebrafish embryos consumed less energy (LOEC = 7.5 mg/L), despite the unaltered levels of available energy (carbohydrates, lipids and proteins). Hence, cellular energy allocation (CEA) was significantly increased. On the other hand, only ERY decreased the acetylcholinesterase activity (LOEC = 15 mg/L). Despite that, zebrafish larvae exposed to both dyes until 144 h were less active. In contrast, DERY did not affect any parameter measured. These results indicate an association between a decrease consumption of energy and decrease swimming activity resulting from an environmental stress condition, independently of the neurotoxicity of the dyes. Degradation of ERY by light prevented all toxic effects previously observed, suggesting a cheap, fast and easy alternative treatment of effluents containing this natural dye. All tools assessed in our current study were sensitive as early-warning endpoints of dyes toxicity on zebrafish early life stages, and suggest that the CEA assay might be useful to predict effects on locomotor activity when cholinergic damage is absent.

Biochemical approaches to assess oxidative stress induced by exposure to natural and synthetic dyes in early life stages in zebrafish.

Zebrafish early life stages were found to be sensitive to several synthetic dyes widely used in industries. However, as environmental concentrations of such contaminants are often at sublethal levels, more sensitive methods are required to determine early-warning adverse consequences. The aim of this study was to utilize a multibiomarker approach to examine underlying oxidative stress mechanisms triggered by sublethal concentrations of synthetic azo dye Basic Red 51 (BR51), the natural dye erythrostominone (ERY), and its light-degraded product using zebrafish embryos. Biochemical biomarkers included parameters of detoxification and markers of antioxidant system, as well as oxidative damage. Results showed pro-oxidant mechanisms attributed to BR51 and ERY as evidenced by increased glutathione S-transferase (GST) activity, a phase II detoxification enzyme related to reactive oxygen species detoxification. BR51 also elevated total glutathione (GSH+GSSG) levels and catalase activity. However, both dyes induced oxidative damage as evidenced by elevated lipid peroxidation content. In contrast, when the natural dye was photodegraded, no marked effects were observed for all biomarkers assessed. Data indicate that such dyes are pro-oxidants at sublethal concentrations, predominantly involving GSH and/or related enzymes pathway.

Toxicological and behavioral responses as a tool to assess the effects of natural and synthetic dyes on zebrafish early life.

Organic dyes extracted from natural sources have been widely used to develop safety and eco-friendly dyes as an alternative to synthetic ones, since the latter are usually precursors of mutagenic compounds. Thereby, toxicity tests to non-target organisms are critical step to develop harmless dyes to environment and in this context, zebrafish early life stages are becoming an important alternative model. We aimed to assess the toxic effects of the synthetic dye Basic Red 51 (BR51, used in cosmetic industry), the natural dye erythrostominone (ERY, a potential commercial dye extracted from fungi) and its photodegradation product (DERY), using zebrafish early life assays. Developmental malformations on embryos and behavioral impairment on larvae were explored. Our results showed that embryos exposed to BR51 and ERY exhibited a large yolk sac (LOEC = 7.5 mg L-1), possibly due to a deformity or delayed resorption. ERY also induced pericardial and yolk sac edemas at high concentrations (LOEC = 15 and 30 mg L-1, respectively). Moreover, larvae swan less distance and time when exposed to ERY (LOEC = 7.5 mg L-1) and BR51 (LOEC = 1.875 mg L-1). The lowest larvae locomotion have been associated with impairment of the yolk sac, important tissue of the energy source. Interestingly, DERY did not affect neither development nor behavior of zebrafish, showing that ERY photodegradation is sufficient to prevent its toxic effects. In conclusion, both natural and synthetic dyes impaired development and behavior of zebrafish early life, therefore, a simple treatment of the natural dye can prevent the aquatic life impact.