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Title
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Interactive effects of metal ions and other environmental stressors on zebrafish (**Danio rerio**) : evidence from toxicological and behavioural approaches
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Author
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Abstract
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| Freshwater ecosystems are under threat from the effects of multiple stressors. However, classic aquatic toxicology studies on metal ions often utilize tests in which organisms are subjected to chemicals under otherwise constant and favourable experimental conditions. Therefore, the objective of the first part of this doctoral thesis was to evaluate the effect of metal ion toxicity and its interaction with environmental stressors (temperature and water hardness) on the zebrafish (Danio rerio) as a prominent vertebrate model organism. To this end, adult zebrafish were exposed to copper (Cu(II)) and cadmium (Cd(II)) in single or binary exposures in different water hardness treatments or thermal regimes and a range of endpoints including mortality, body condition factor, metal accumulation and whole body major cation content were measured. Results showed that the environmental stressors (water hardness and temperature) influence the toxicity of metal ions to zebrafish. Specifically, the toxicity of metal ions was higher at elevated water softnesses. In terms of temperature, the effect was dependent on the applied scenario: acute pre-incubation at high temperatures increased mortality during subsequent metal ion exposure at optimum temperature, while acute pre-incubation at cold temperatures mitigated the effect of subsequent metal ion exposure at optimum temperature. During simultaneous metal ion exposure and ambient temperature alterations the lowest mortality was observed at the optimum temperature. In general, irrespective of the environmental conditions, zebrafish were found to be more sensitive to Cu(II) than to Cd(II) on the basis of single metal ion exposures. Furthermore, mixture exposures revealed a robust synergistic toxic effect of Cu(II) and Cd(II) on zebrafish at the mortality level. In terms of metal body burden, although the exposure conditions (concentration, water hardness and temperature) affected the whole body metal accumulation in zebrafish, it was not a predictor of mortality and other effects in our study, that is, the whole body burden of dead fish was generally comparable with that of the surviving fish. Finally, we have observed a significant decrease in whole body Na+ level of dead fish in comparison to surviving fish, irrespective of the exposure conditions; such an effect was not observed for the other major cations (K+, Mg2+, Ca2+). Accordingly, the ability to maintain Na+ homeostasis was identified as a crucial factor for survival under the applied multi-stress conditions. In the second part of this thesis, we have evaluated some of the major behavioural traits of zebrafish and the effect of Cu(II) exposure on these responses by utilizing two behavioural assays; namely the novel tank diving test and T-maze assay. The novel tank diving test is based on an instinctive tendency of zebrafish to seek safety upon introduction to a novel environment by diving and dwelling at the bottom of the new tank. To examine the effect of Cu(II) exposure on behavioural responses of zebrafish in the novel tank test and to evaluate the degree of habituation of zebrafish to this process, we have conducted repeated tests throughout the Cu(II) exposure and the behaviour of zebrafish was recorded and analysed by behavioural video tracking software. Results demonstrated anxiety-like behaviour of zebrafish upon introduction to a novel environment. Moreover, a robust habituation response of zebrafish was observed in repeated tests, that is, the exploratory behaviour of the fish increased by repeating the assay over the experimental phase. Finally, the effect of Cu(II) exposure on these behavioural phenotypes was found to be minimal. Furthermore, in the present study, we have determined the effect of a prior Cu(II) exposure on associative learning capabilities of zebrafish by using a T-maze assay based on Pavlovian conditioning; a form of learning in which a neutral or arbitrary stimulus (conditioned stimulus, e.g., green colour) becomes associated with a stimulus of some significance to the animal (unconditioned stimulus, e.g. food), so that the conditioned stimulus becomes synonymous with the unconditioned one, thereby evoking the same innate, reflexive behavioural responses. Results indicated the associative learning capability of zebrafish, specifically, zebrafish in the control group achieved a significant number of correct choices (leading to food award) throughout the conditioning sessions in the T-maze. Moreover, Cu(II) exposure at the applied conditions was found to negatively affect the learning capacities of zebrafish. Finally, we have also assessed the effect of Cu(II) exposure on chemical communication patterns, precisely alarm substance response of zebrafish. Alarm substances are released following mechanical damage to the epidermal cells and when sensed by other conspecifics (and in some cases also heterospecifics) in the environment, they trigger an innate behavioural response (alarm response) mainly characterized by decreased swimming and feeding activities. The behavioural response of zebrafish exposed to alarm substance in the novel tank diving test demonstrated increased level of anxiety, more specifically, alarm substance administration significantly decreased the total distance travelled, the time spent in upper zones of the tank, and the number of shifts between the zones, and significantly increased latency to enter the upper zone and freezing duration. Moreover, Cu(II) contamination was found to disrupt the instinctual behavioural response of zebrafish to alarm substance treatment, that is, following Cu(II) exposure zebrafish lost its sensitivity towards alarm substance treatment and a stress response was not evoked, presumably as a consequence of Cu(II) induced olfaction impairment. Overall, the present thesis provides an insight into the context of multi-stressors and the effect of metal ion toxicity on the behaviour of zebrafish. Specifically, we have documented the determinative impact of environmental stressors on metal ion toxicity. This finding highlights the importance of considering the effect of environmental conditions in designing standard toxicology tests to derive water quality guidelines that are protective for environmentally realistic conditions. At the simplest level, this could involve conducting toxicity tests under various suboptimal environmental conditions. Ultimately, the goal is to develop more sophisticated mechanistic understanding of the processes involved to enable reliable prediction of multi-stressor effects. On the other hand, our findings from behavioural approaches, in agreement with a substantial body of literature, suggested that the behavioural toxicity tests, if properly designed, can be utilized in conjunction with classical toxicology endpoints (e.g. mortality, cellular and biochemical changes and growth rate) to add evidence from higher level of organisation. Indeed, it is not possible to establish the connection between the subtle cellular and biochemical changes within an organism and the ecological changes unless studies at the level of the individual are included. In this way, behaviour serves as a useful tool for linking the responses at lower levels of organisation (cellular and biochemical changes) with organismal responses. Furthermore, alterations in behavioural patterns represent an integrated, whole-organism response which may be an indicator of reduced fitness and survival, resulting in adverse effects at organismal level. From another point of view, our findings showed that toxicants can be considered as useful tools for evaluating the behavioural mechanisms and limitations of fishes in adapting to stressors (e.g. high levels of metal ions) in their natural environment. |
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Language
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English
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Publication
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Antwerpen
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Universiteit Antwerpen, Faculteit Wetenschappen, Departement Biologie
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2020
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Volume/pages
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271 p.
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Note
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Blust, Ronny [Supervisor]
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Town, Raewyn M. [Supervisor]
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Full text (open access)
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