Alkaline Comet Assay to Assess Genotoxicity in Zebrafish Larvae.

The zebrafish (Danio rerio) is a model organism widely used in several research fields due to its characteristics and numerous advantages, such as optical embryo transparency, fully sequenced genome, orthologous genes to humans, small size, high reproductive rate, easy gene editing and relatively low costs. Thus, a number of protocols have been developed that allow the use of this vertebrate model for toxic effect evaluation at various biological levels, including genotoxicity, using the comet assay technique.The comet assay or single-cell gel electrophoresis is a popular and sensitive method to study DNA damage in cells, which is described in this chapter. Briefly, cells suspended in agarose on a microscope slide are lysed, denatured, electrophoresed, neutralized, and stained to study the migration of DNA strand breaks. As a result, cells with increased DNA damage present a high fluorescence intensity and an increase of comet tail length. For the visual score, comets are classified according to the head integrity, tail intensity, and tail length into five classes, namely, class 0 until class 4 (comets with high damage and with almost all the DNA in the tail). These data are used to calculate the Genetic Damage Index (GDI) expressed as Arbitrary Units (AU).

Effects of acute metaphedrone exposure on the development, behaviour, and DNA integrity of zebrafish (Danio rerio).

The presence of new psychoactive substances (NPS), like metaphedrone (3-MMC), in aquatic environments raises concern about the potential negative effects on ichthyofauna. Therefore, the aim of this study was to evaluate the potential effects of 3-MMC on zebrafish embryonic development, behaviour, and DNA integrity. For that, embryos were exposed during 96 h post-fertilization to 3-MMC (0.1, 1, 10, and 100 µg/L). Overall, an increase in the eye area of zebrafish larvae was observed for the concentrations of 1 µg/L (increase of 24%) and 100 µg/L (increase of 25%) in comparison with the control group. Genetic damage was noted at the highest concentration (100 µg/L) with an increase of DNA damage (increase of 48%) and hyperactivity and disorganised swimming pattern characterised by an increase in speed (increase of 49%), total distance moved (increase of 53%), and absolute turn angle (increase of 48%) of zebrafish larvae. These findings pointed that, at environmental low levels, 3-MMC harmful effects are not expected to occur during critical development life stages of fish.

Enantioselective Ecotoxicity of Venlafaxine in Aquatic Organisms: Daphnia and Zebrafish.

Venlafaxine is a chiral antidepressant detected in aquatic compartments. It was recently included in the 3rd Watch List from the European Union. The present study aimed to investigate venlafaxine toxicity effects, targeting possible enantioselective effects, using two aquatic organisms, daphnia (Daphnia magna) and zebrafish (Danio rerio). Specimens were exposed to both racemate, (R,S)-venlafaxine (VEN), and to pure enantiomers. Acute assays with daphnia showed that up to 50 000 µg/L of the (R,S)-VEN induced no toxicity. Organisms were also exposed to sublethal concentrations (25-400 µg/L) of (R,S)-, (R)- and (S)-VEN, for 21 days. No significant effects on mortality, age at first reproduction, and size of the first clutch were observed. However, a decrease in fecundity was observed for both enantiomers at the highest concentration. Regarding zebrafish, the effects of venlafaxine on mortality, embryo development, behavior, biochemistry, and melanin pigmentation were investigated after 96 h of exposure to the range of 0.3-3000 µg/L. (R)-VEN significantly increased the percentage of malformations in comparison with (S)-VEN. Behavior was also enantiomer dependent, with a decrease in the total distance moved and an increase in avoidance behavior observed in organisms exposed to (R)-VEN. Despite the biochemical variations, no changes in redox homeostasis were observed. (R)-VEN also led to an increase in zebrafish pigmentation. The different susceptibility to venlafaxine and enantioselective effects were observed in zebrafish. Our results suggest that at environmental levels (R,S)-VEN and pure enantiomers are not expected to induce harmful effects in both organisms, but (R)-VEN increased malformations in zebrafish larvae, even at reported environmental levels. These results highlight the importance of including enantioselective studies for an accurate risk assessment of chiral pollutants. Environ Toxicol Chem 2022;41:1851-1864. © 2022 SETAC.