Cortisol Quantification for Assessing Stress-Induced Changes in Zebrafish Larvae.

The stress response, mainly mediated by cortisol, plays a critical role in the regulation of physiological and behavioral homeostasis through a variety of mechanisms. Different aquatic animal models have been widely employed to understand the pathobiology of stress and stress-related brain disorders. The early life stress can affect the hypothalamic-pituitary-interrenal (HPI) axis and induce cellular and molecular impairments that impact the brain functioning later in life. However, these alterations have been poorly explored mainly due to the lack of suitable models. In this chapter, the vortex flow stimulation, an acute stress that causes a forced swimming and activates the HPI axis, is described and its correlations with behavioral outputs reported. To this end, the early life stages of zebrafish are used as animal models for modeling stress disorders experimentally. The behavioral despair model can be employed as an initial screening tool for assessing neural circuit activation and motor alterations. Taken together, the implementation of this strategy in this animal model allows the analysis of stress responses in a simple manner and its correlation with neural circuitries and motor alterations.

Behavioural impairment and oxidative stress by acute exposure of zebrafish to a commercial formulation of tebuconazole.

Tebuconazole is a systemic follicular fungicide known to cause diverse problems in non-target organisms namely associated to the pure active ingredient. As such, the objective of this work was to evaluate developmental changes induced by a tebuconazole commercial formulation to a non-target animal model. Zebrafish embryos at ± 2 h post-fertilization were exposed to tebuconazole wettable powder concentrations (0.05, 0.5 and 5 mg L-1) for 96 h with developmental toxicity assessed throughout the exposure period and biochemical parameters evaluated at the end of the exposure. Behavioural assessment (spatial exploration and response to stimuli) was conducted 24 h after the end of the exposure. While no developmental and physiological alterations were observed, exposure to tebuconazole resulted in an increased generation of reactive oxidative species at the 0.05 and 0.5 mg L-1 concentrations and a decreased GPx activity at the 0.5 mg L-1 concentration suggesting a potential protection mechanism. There was also a change in the avoidance-escape behaviour supporting an anxiolytic effect suggesting possible alterations in the central nervous system development demanding further studies.

Embryonic zebrafish response to a commercial formulation of azoxystrobin at environmental concentrations.

Azoxystrobin is a broad-spectrum strobilurin fungicide for use on a wide range of crops available to end-users as formulated products. Due to its extensive application, it has been detected in aquatic ecosystems, raising concerns about its environmental impact, which is still poorly explored. The objective of this work was to study the effects of a commercial formulation of azoxystrobin in the zebrafish embryo model. Sublethal and lethal effects were monitored during the exposure period from 2 h post fertilisation (hpf) to 96 hpf after exposure to azoxystrobin concentrations (1, 10 and 100 µg L-1). The responses of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)) as well as detoxifying enzymes (glutathione-s-transferase (GST) and carboxylesterase (CarE)) were evaluated at 96 hpf. Similarly, glutathione levels (reduced (GSH) and oxidised (GSSG) glutathione), neurotransmission (acetylcholinesterase (AChE)) and anaerobic respiration (lactate dehydrogenase (LDH)) -related enzymes were assayed. At 120 hpf, larvae from each group were used for behaviour analysis. Results from this study showed concentration-dependent teratogenic effects, particularly by increasing the number of malformations (yolk and eye), with a higher prevalence at the highest concentration. However, it was found that the lowest concentration induced a high generation of reactive oxygen species (ROS) and increased activity of SOD, GST, and CarE. In addition, GR and GSSG levels were decreased by the lowest concentration, suggesting an adaptive response to oxidative stress, which is also supported by the increased AChE activity and absence of behavioural changes. These findings advance the knowledge of the azoxystrobin developmental and environmental impacts, which may impose ecotoxicological risks to non-target species.