The vertebrate thyroid system is important for multiple developmental processes, including eye development. Thus, its environmentally induced disruption may impact important fitnessrelated parameters like visual capacities and behaviour. The present study investigated the relation between molecular effects of thyroid disruption and morphological and physiological changes of eye development in zebrafish (Danio rerio). Two test compounds representing different molecular modes of thyroid disruption were used: propylthiouracil (PTU), which is an enzyme-inhibitor of thyroid hormone synthesis, and tetrabromobisphenol A (TBBPA), which interacts with the thyroid hormone receptors. Both chemicals significantly altered transcript levels of thyroid system-related genes (TR, TR, TPO, TSH, DIO1, DIO2 and DIO3) in a compound-specific way. Despite these different molecular response patterns, both treatments resulted in similar pathological alterations of the eyes such as reduced size, RPE cell diameter and pigmentation, which were concentration-dependent. The morphological changes translated into impaired visual performance of the larvae: the optokinetic response was significantly and concentrationdependently decreased in both treatments, together with a significant increase of light preference of PTU-treated larvae. In addition, swimming activity was impacted. This study provides first evidence that different modes of molecular action of the thyroid disruptors can be associated with uniform apical responses. Furthermore, this study is the first to show that pathological eye development, as it can be induced by exposure to thyroid disruptors, indeed translates into impaired visual capacities of zebrafish early life stages. Abstract:The vertebrate thyroid system is important for multiple developmental processes, including eye development. Thus, its environmentally induced disruption may impact important fitnessrelated parameters like visual capacities and behaviour. The present study investigated the relation between molecular effects of thyroid disruption and morphological and physiological changes of eye development in zebrafish (Danio rerio). Two test compounds representing different molecular modes of thyroid disruption were used: propylthiouracil (PTU), which is an enzyme-inhibitor of thyroid hormone synthesis, and tetrabromobisphenol A (TBBPA), which interacts with the thyroid hormone receptors. Both chemicals significantly altered transcript levels of thyroid system-related genes (TR, TR, TPO, TSH, DIO1, DIO2 and DIO3) in a compound-specific way. Despite these different molecular response patterns, both treatments resulted in similar pathological alterations of the eyes such as reduced size, RPE cell diameter and pigmentation, which were concentration-dependent. The morphological changes translated into impaired visual performance of the larvae: the optokinetic response was significantly and concentration-dependently decreased in both treatments, together with a significant increase of light preference of PT...

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Despite frequent field observations of impaired immune response and increased disease incidence in contaminant-exposed wildlife populations, immunotoxic effects are rarely considered in ecotoxicological risk assessment. The aim of this study was to review the literature on immunotoxic effects of chemicals in fish to quantitatively evaluate (i) which experimental approaches were used to assess immunotoxic effects, (ii) whether immune markers exist to screen for potential immunotoxic activities of chemicals, and (iii) how predictive those parameters are for adverse alterations of fish immunocompetence and disease resistance. A total of 241 publications on fish immunotoxicity were quantitatively analyzed. The main conclusions included: (i) To date, fish immunotoxicology focused mainly on innate immune responses and immunosuppressive effects. (ii) In numerous studies, the experimental conditions are poorly documented, as for instance age or sex of the fish or the rationale for the selected exposure conditions is often missing. (iii) Although a broad variety of parameters were used to assess immunotoxicity, the rationale for the choice of measured parameters was often not given, remaining unclear how they link to the suspected immunotoxic mode of action of the chemicals. (iv) At the current state of knowledge, it is impossible to identify a set of immune parameters that could reliably screen for immunotoxic potentials of chemicals. (v) Similarly, in fish immunotoxicology there is insufficient understanding of how and when chemical-induced modulations of molecular/cellular immune changes relate to adverse alterations of fish immunocompetence, although this would be crucial to include immunotoxicity in ecotoxicological risk assessment.

Freshwater fish are threatened by the cumulative impact of multiple stressors. The purpose of this study was to unravel the molecular and organism level reactions of rainbow trout, Oncorhynchus mykiss, to the combined impact of two such stressors that occur in the natural habitat of salmonids. Fish were infected with either the myxozoan parasite, Tetracapsuloides bryosalmonae, which causes proliferative kidney disease (PKD), or exposed to ethinylestradiol (EE2) an estrogenic endocrine disrupting compound, or to a combination of both (PKD x EE2). PKD is a slow progressive chronic disease here we focused on a later time point (130-day postinfection (d.p.i)) when parasite intensity in the fish kidney has already started to decrease. At 130 d.p.i, RNA-seq technology was applied to the posterior kidney, the main target organ for parasite development. This resulted with 280 (PKD), 14 (EE2) and 444 (PKD x EE2) differentially expressed genes (DEGs) observed in the experimental groups. In fish exposed to the combination of stressors (PKD x EE2), a number of pathways were regulated that were neither observed in the single stressor groups. Parasite infection, alone and in combination with EE2, only resulted in a low intensity immune response that negatively correlated with an upregulation of genes involved in a variety of metabolic and inflammation resolution processes. This could indicate a trade-off whereby the host increases investment in recovery/resolution processes over immune responses at a later stage of disease. When PKD infection took place under simultaneous exposure to EE2 (PKD x EE2), parasite intensity decreased and pathological alterations in the posterior kidney were reduced in comparison to the PKD only condition. These findings suggest that EE2 modulated these response profiles in PKD infected fish, attenuating the disease impact on the fish.

The aim of ecotoxicology is to study toxic effects on constituents of ecosystems, with the protection goal being populations and communities rather than individual organisms. In this ecosystem perspective, the use of in vitro methodologies measuring cellular and subcellular endpoints at a first glance appears to be odd. Nevertheless, more recently in vitro approaches gained momentum in ecotoxicology. In this article, we will discuss important application domains of in vitro methods in ecotoxicology. One area is the use of in vitro assays to replace, reduce, and refine (3R) in vivo tests. Research in this field has focused mainly on the use of in vitro cytotoxicity assays with fish cells as non-animal alternative to the in vivo lethality test with fish and on in vitro biotransformation assays as part of an alternative testing strategy for bioaccumulation testing with fish. Lessons learned from this research include the importance of a critical evaluation of the sensitivity, specificity and exposure conditions of in vitro assays, as well as the availability of appropriate in vitro-in vivo extrapolation models. In addition to this classical 3R application, other application domains of in vitro assays in ecotoxicology include the screening and prioritization of chemical hazards, the categorization of chemicals according to their modes of action and the provision of mechanistic information for the pathway-based prediction of adverse outcomes. The applications discussed in this essay may highlight the potential of in vitro technologies to enhance the environmental hazard assessment of single chemicals and complex mixtures at a reduced need of animal testing.

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The aim of the present study was to investigate the effects of the androgenic endocrine disruptor 17β-trenbolone on the sexual development of zebrafish (Danio rerio) with special emphasis on the question of whether adverse outcomes of developmental exposure are reversible or persistent. An exposure scenario including a recovery phase was chosen to assess the potential reversibility of androgenic effects. Zebrafish were exposed to environmentally relevant concentrations of 17β-trenbolone (1 ng/L-30 ng/L) from fertilization until completion of gonad sexual differentiation (60 d posthatch). Thereafter, exposure was either followed by 40 d of recovery in clean water or continued until 100 d posthatch, the age when zebrafish start being able to reproduce. Fish exposed for 100 d to 10 ng/L or 30 ng/L 17β-trenbolone were masculinized at different biological effect levels, as evidenced from a concentration-dependent shift of the sex ratio toward males as well as a significantly increased maturity of testes. Gonad morphological masculinization occurred in parallel with decreased vitellogenin concentrations in both sexes. Changes of brain aromatase (cyp19b) mRNA expression showed no consistent trend with respect to either exposure duration or concentration. Gonad morphological masculinization as well as the decrease of vitellogenin persisted after depuration over 40 d in clean water. This lack of recovery suggests that androgenic effects on sexual development of zebrafish are irreversible.

Endocrine-disrupting chemicals are known to impact multiple hormonal axes of vertebrates, among which the thyroid system is crucial for multiple developmental and physiological processes. Thus, the present study focused on the semi-quantitative visualization of intrafollicular triiodothyronine (T3) and thyroxin (T4) in zebrafish embryos as a potential test system for the detection of disrupted thyroid hormone synthesis. To this end, an antibody-based fluorescence double-staining protocol for whole-mount zebrafish embryos and larvae was adapted to simultaneously detect intrafollicular T3 and T4. During normal development until 10 days post-fertilization (dpf), the number of thyroid follicles increased along the ventral aorta. Concentrations of T4 and T3, measured by fluorescence intensity, increased until 6 dpf, but decreased thereafter. Exposure of zebrafish embryos to propylthiouracil (PTU), a known inhibitor of TH synthesis, resulted in a significant decrease in the number of follicles that stained for T3, whereas a trend for increase in follicles that stained for T4 was observed. In contrast, fluorescence intensity for both thyroid hormones decreased significantly after exposure to PTU. Overall, the zebrafish embryo appears to be suitable for the simultaneous visualization and detection of changing intrafollicular TH contents during normal development and after PTU treatment.