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).

Enantioselectivity in the Ecotoxicity of Amphetamine Using Daphnia magna as the Aquatic Model Organism: Morphophysiological, Behavioral, Reproductive and Biochemical Parameters.

Amphetamine (AMP) is a chiral psychoactive substance that exhibits enantioselectivity in its pharmacological properties. It has been detected in wastewaters and surface waters and can occur as enantiomeric mixtures, but little is known about its environmental risk and potential enantioselective toxicity to aquatic organisms. Our study aimed to target enantioselectivity in AMP toxicity to the freshwater invertebrate Daphnia magna. Daphnids were subchronically exposed to the racemate (rac-AMP: 0.1, 1.0, and 10 µg/L) and pure enantiomers, (R)-AMP and (S)-AMP (0.1, and 1.0 µg/L, respectively), for 8 days. Morphophysiological, swimming behavior, reproductive and biochemical variables were evaluated during critical life stages (juveniles vs. adults). Some responses were context-dependent and often enantioselective, varying between racemate and enantiomers and across the life stage of the organisms. Overall, rac-AMP stimulated D. magna growth, decreased heart rate and area, affected behavior, and stimulated reproduction. The effect of enantiomers was totally or partially concordant with rac-AMP, except for swimming behavior and reproduction. Enantioselectivity was observed for body size, number of eggs/daphnia, and heart rate (steeper decrease caused by (R)-AMP on day 3). Changes in biochemical parameters were also observed: AMP caused a significant decrease in catalase activity as racemate or pure enantiomers, whereas a decrease in acetylcholinesterase activity was found only for rac-AMP. Evidence for oxidative stress was contradictory, although both enantiomers caused a significant decrease in reactive oxygen species (unlike rac-AMP). Overall, these results show that AMP can interfere in an enantioselective way with aquatic organisms at low concentrations (e.g., 0.1 µg/L), demonstrating the relevance of this kind of study to an accurate environmental risk assessment regarding medium- to long-term exposure to this psychoactive drug. Environ Toxicol Chem 2023;42:1743-1754. © 2023 SETAC.

Integrated Approach for Synthetic Cathinone Drug Prioritization and Risk Assessment: In Silico Approach and Sub-Chronic Studies in Daphnia magna and Tetrahymena thermophila.

Synthetic cathinones (SC) are drugs of abuse that have been reported in wastewaters and rivers raising concern about potential hazards to non-target organisms. In this work, 44 SC were selected for in silico studies, and a group of five emerging SC was prioritized for further in vivo ecotoxicity studies: buphedrone (BPD), 3,4-dimethylmethcathinone (3,4-DMMC), butylone (BTL), 3-methylmethcathinone (3-MMC), and 3,4-methylenedioxypyrovalerone (MDPV). In vivo short-term exposures were performed with the protozoan Tetrahymena thermophila (28 h growth inhibition assay) and the microcrustacean Daphnia magna by checking different indicators of toxicity across life stage (8 days sublethal assay at 10.00 µg L-1). The in silico approaches predicted a higher toxic potential of MDPV and lower toxicity of BTL to the model organisms (green algae, protozoan, daphnia, and fish), regarding the selected SC for the in vivo experiments. The in vivo assays showed protozoan growth inhibition with MDPV > BPD > 3,4-DMMC, whereas no effects were observed for BTL and stimulation of growth was observed for 3-MMC. For daphnia, the responses were dependent on the substance and life stage. Briefly, all five SC interfered with the morphophysiological parameters of juveniles and/or adults. Changes in swimming behavior were observed for BPD and 3,4-DMMC, and reproductive parameters were affected by MDPV. Oxidative stress and changes in enzymatic activities were noted except for 3-MMC. Overall, the in silico data agreed with the in vivo protozoan experiments except for 3-MMC, whereas daphnia in vivo experiments showed that at sublethal concentrations, all selected SC interfered with different endpoints. This study shows the importance to assess SC ecotoxicity as it can distress aquatic species and interfere with food web ecology and ecosystem balance.

Toxicity of the 3,4-Methylenedioxymethamphetamine and Its Enantiomers to Daphnia magna after Isolation by Semipreparative Chromatography.

MDMA (3,4-methylenedioxymethamphetamine) is a chiral psychoactive recreational drug sold in illicit markets as racemate. Studies on the impact of MDMA on aquatic organisms are scarce. While enantioselectivity in toxicity in animals and humans has been reported, none is reported on aquatic organisms. This study aimed to investigate the ecotoxicological effects of MDMA and its enantiomers in Daphnia magna. For that, enantiomers (enantiomeric purity > 97%) were separated by liquid chromatography using a homemade semipreparative chiral column. Daphnids were exposed to three concentrations of (R,S)-MDMA (0.1, 1.0 and 10.0 µg L-1) and two concentrations of (R)- and (S)-enantiomers (0.1 and 1.0 µg L-1) over the course of 8 days. Morphophysiological responses were dependent on the substance form and daphnia development stage, and they were overall not affected by the (R)-enantiomer. Changes in swimming behaviour were observed for both the racemate and its enantiomers, but enantioselective effects were not observed. Reproductive or biochemical changes were not observed for enantiomers whereas a significant decrease in acetylcholinesterase and catalase activity was noted at the highest concentration of (R,S)-MDMA (10 µg L-1). Overall, this study showed that sub-chronic exposure to MDMA racemate and its enantiomers can interfere with morphophysiological and swimming behaviour of D. magna. In general, the (R)-enantiomer demonstrated less toxicity than the (S)-enantiomer.

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.

Ketamine and Norketamine: Enantioresolution and Enantioselective Aquatic Ecotoxicity Studies.

Ketamine is a chiral drug used for various clinical purposes but often misused. It is metabolized to norketamine, an active chiral metabolite. Both substances have been detected in environmental matrices, but studies about their enantioselective toxic effects are scarce. In the present study, the enantiomers of ketamine and norketamine were separated by a semipreparative enantioselective liquid chromatography method, and their toxicity was investigated in different aquatic organisms. The enantioseparation was achieved using a homemade semipreparative chiral column. Optimized conditions allowed the recovery of compounds with enantiomeric purity higher than 99%, except for (R)-ketamine (97%). The absolute configuration of the enantiomers was achieved by experimental electronic circular dichroism (ECD). The ecotoxicity assays were performed with the microcrustacean Daphnia magna and the protozoan Tetrahymena thermophila using Toxkit MicroBioTests. Different concentrations were tested (0.1-10 000 µg/L) to include environmental levels (~0.5-~100 µg/L), for racemates (R,S) and the isolated enantiomers (R or S) of ketamine and norketamine. No toxicity was observed in either organism at environmental levels. However, at greater concentrations, (R,S)-ketamine presented higher mortality for D. magna compared with its metabolite (R,S)-norketamine (85 and 20%, respectively), and the (S)-ketamine enantiomer showed higher toxicity than the (R)-ketamine enantiomer. In addition, (S)-ketamine also presented higher growth inhibition than (R)-ketamine for T. thermophila at the highest concentrations (5000 and 10 000 µg/L). Contrary to D. magna, growth inhibition was observed for both enantiomers of norketamine and in the same magnitude order of the (S)-ketamine enantiomer. The results showed that the 2 organisms had different susceptibilities to norketamine and that the toxicity of ketamine at high concentrations is enantioselective for both organisms. Environ Toxicol Chem 2022;41:569-579. © 2020 SETAC.