Development of a Zebrafish Embryo-Based Test System for Thyroid Hormone System Disruption: 3Rs in Ecotoxicological Research.

There is increasing concern regarding pollutants disrupting the vertebrate thyroid hormone (TH) system, which is crucial for development. Thus, identification of TH system-disrupting chemicals (THSDCs) is an important requirement in the Organisation for Economic Co-operation and Development (OECD) testing framework. The current OECD approach uses different model organisms for different endocrine modalities, leading to a high number of animal tests. Alternative models compatible with the 3Rs (replacement, reduction, refinement) principle are required. Zebrafish embryos, not protected by current European Union animal welfare legislation, represent a promising model. Studies show that zebrafish swim bladder inflation and eye development are affected by THSDCs, and the respective adverse outcome pathways (AOPs) have been established. The present study compared effects of four THSDCs with distinct molecular modes of action: Propylthiouracil (PTU), potassium perchlorate, iopanoic acid, and the TH triiodothyronine (T3) were tested with a protocol based on the OECD fish embryo toxicity test (FET). Effects were analyzed according to the AOP concept from molecular over morphological to behavioral levels: Analysis of thyroid- and eye-related gene expression revealed significant effects after PTU and T3 exposure. All substances caused changes in thyroid follicle morphology of a transgenic zebrafish line expressing fluorescence in thyrocytes. Impaired eye development and swimming activity were observed in all treatments, supporting the hypothesis that THSDCs cause adverse population-relevant changes. Findings thus confirm that the FET can be amended by TH system-related endpoints into an integrated protocol comprising molecular, morphological, and behavioral endpoints for environmental risk assessment of potential endocrine disruptors, which is compatible with the 3Rs principle. Environ Toxicol Chem 2024;00:1-18. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Embryonic exposures to chemicals acting on brain aromatase lead to different locomotor effects in zebrafish larvae.

Pathways underlying neurodevelopmental effects of endocrine disruptors (EDs) remain poorly known. Expression of brain aromatase (aroB), responsible for estrogen production in the brain of teleosts, is regulated by estrogenic EDs and could play a role in their behavioral effects. We exposed zebrafish eleutheroembryos (0-120 h post-fertilization) to various concentrations of 16 estrogenic chemicals (incl. bisphenols and contraceptives), and of 2 aroB inhibitors. Behavior was monitored using a photomotor response test procedure. Both aroB inhibitors (clotrimazole and prochloraz) and a total of 6 estrogenic EDs induced significant behavioral alterations, including DM-BPA, BPC and BPS-MPE, three bisphenol substitutes which behavioral effects were, to our knowledge, previously unknown. However, no consensus was reported on the effects among tested substances. It appears that behavioral changes could not be linked to groups of substances defined by their specificity or potency to modulate aroB expression, or by their structure. Altogether, behavioral effects of estrogenic EDs in 120 h post-fertilization larvae appear unrelated to aroB but are nonetheless not to be neglected in the context of environmental safety.

Low concentrations of oxazepam induce feeding and molecular changes in Radix balthica juveniles.

Psychotropics, especially benzodiazepines, are commonly prescribed worldwide. Poorly eliminated at wastewater treatment plants, they belong to a group of emerging contaminants. Due to their interaction with the GABAA receptor, they may affect the function of the nervous system of non-target organisms, such as aquatic organisms. The toxicity of oxazepam, a very frequently detected benzodiazepine in continental freshwater, has been largely studied in aquatic vertebrates over the last decade. However, its effects on freshwater non-vertebrates have received much less attention. We aimed to evaluate the long-term effects of oxazepam on the juvenile stage of a freshwater gastropod widespread in Europe, Radix balthica. Juveniles were exposed for a month to environmentally-relevant concentrations of oxazepam found in rivers (0.8 µg/L) and effluents (10 µg/L). Three main physiological functions were studied: feeding, growth, and locomotion. Additionally, gene expression analysis was performed to provide insights into toxicity mechanisms. There was a strong short-term activation of the feeding rate at low concentration, whereas the high dose resulted in long-term inhibition of food intake. A significant decrease in mortality rate was observed in juveniles exposed to the lowest dose. Shell growth and locomotor activity did not appear to be affected by oxazepam. Transcriptomic analysis revealed global over-expression of genes involved in the nervous regulation of the feeding, digestive, and locomotion systems after oxazepam exposure. The molecular analysis also revealed a possible interference of animal manipulation with the molecular effects induced by oxazepam exposure. Overall, these results improve our understanding of the effects of the psychoactive drug oxazepam on an aquatic mollusc gastropod.

Behavioral responses of three freshwater planaria species to light, visual and olfactory stimuli: Setting the stage for further ecotoxicological studies.

Planarians are freshwater flatworms commonly used as environmental bioindicator due to their sensitivity of response and their ease of culturing in lab. Nevertheless, to date, very few studies describing their behavior have been led. This work aims to fill the literature gap by providing preliminary results through six behavioral challenges (locomotion, exploration, light stress, planarian light/dark test, shoaling and foraging) conducted with three different species Dugesia tigrina, Schmidtea mediterranea and Schmidtea polychroa. The behavioral responses of every species in each of these six assays were recorded and differences between species were highlighted, depending on the assays and conditions. Schmidtea polychroa is less active than the two others and had the highest light aversion. Reactions observed in response to diverse and realistic stimuli helped us to select the most suitable tests and choose the species that seem the most appropriate for future ecotoxicological and neurophysiological tests. Four tests - out of the six tested- seem reliable in order to standardize planarian behavioral tests.