Comparative analysis of metal toxicity responses in aquatic invertebrate and vertebrate model organisms

Abstract

The presence of metals in the environment has often been associated with various adverse effects on living systems, including humans. Given their ubiquitous nature, metals are omnipresent and often occur as mixtures and have therefore always been of concern. Despite the considerable attention given to this issue, there remains a lack of sufficient knowledge of the mechanisms underlying toxicological outcomes specific to each metal and/or each mixture. In addition, knowledge of the toxicity of metal mixtures in taxonomically distinct species is inadequately explored. Addressing these key questions is important for gaining a better understanding of the magnitude and mechanisms of toxicity in biological systems. In this context, our study aims to explore how exposures in a single and binary metal pollution scenario differentially affect aquatic vertebrate and invertebrate organisms across various biological levels. The study focuses on copper (Cu) and cadmium (Cd) as metal toxicants in three animal models: the zebrafish (Danio rerio), the water flea (Daphnia magna) and the planarian flatworm (Schmidtea mediterranea). In the first chapter of this thesis, experiments were performed on the fertilised embryos and adults of zebrafish (Danio rerio). Both, embryos, and adults were exposed to sublethal levels of Cu and Cd as single and binary mixtures. Developmental, morphological, and functional endpoints were assessed in embryos at 96 hpf (hours post fertilisation). The results of this part of the work shows an increased sensitivity of both embryos and adults that were simultaneously exposed to both metals. Based on the transcriptional pattern of genes, the underlying mechanisms activated during the combined exposure were related to DNA damage and oxidative stress. In adults, the degree of toxicity and the underlying mode of action depended on the specific organ. Alterations in the activity of antioxidant genes were detected in all organs, but DNA damage was only detected in the gills under the given experimental conditions. Furthermore, the results indicate the inadequacy of relying solely on the extent of metal accumulation as a predictor of toxicity. In the second chapter of this thesis, we conducted a comparative analysis of the individual and joint toxicity of Cu and Cd in the water flea (Daphnia magna). Our investigation focused on exploring the role of exposure pathways during the early and adult life stages. Animals were exposed to single and mixture of Cu and Cd through three distinct pathways: aqueous (dissolved in water), dietary (spiked with food) and their combination (water + food), over a —7 period of 7-days. The main objective of evaluating these exposure pathways, was to determine the relative importance of each in contributing to overall toxicity. Toxicity endpoints, including survival, growth, and reproduction were evaluated. The results of this part of the work show that the mixed exposure to Cu and Cd increased toxicity for both age groups of Daphnia magna compared to single exposures. However, neonates showed more sensitivity to stress, when exposed to similar metal concentrations compared to adults. In addition, the severity of stress varied between different exposure pathways and was higher when metal treatments occurred in a combined exposure scenario. Oxidative stress was found to be a common toxicity biomarker of Cu and Cd co-exposure in both neonates and adults exposed via combined pathways. The aim of the third chapter of this thesis was to determine the potential developmental and physiological perturbations in regenerating and intact (adult) planarian flatworms (Schmidtea mediterranea). Regenerating planarians were used as a proxy for developmental toxicity. Underlying cellular and molecular events were assessed following single and mixed exposures to Cu and Cd over 7 and/or 14 days. The mixed exposure resulted in pronounced lethal and non-lethal morphological changes, neuroregenerative impairments, altered behaviour, and a decrease in survival. The mixed exposure additionally impaired the recovery in regenerating animals, indicating the severity of toxicity. Oxidative stress appeared as an underlying mechanism for toxicity in all exposure conditions. The transcriptional pattern of DNA repair genes in adult animals indicated adverse effects on DNA. Overall, our results demonstrate that the toxic effects of the individual metals increase significantly in mixed exposure scenarios, and that developing organisms are more susceptible than adults. Overall, our study contributes to a better understanding of the single and mixture effects of metal toxicity across different biological levels and emphasises the importance of an in-depth approach in assessing the risks associated with metal pollution. The findings presented in this work show a stronger action of Cu and Cd as a mixture in different aquatic organisms under controlled conditions. In particular, our study reveals important developmental, behavioural, and molecular changes with some effects more manifested in mixtures compared to single metal exposures. Furthermore, our study highlights that relying solely on metal accumulation levels as a reliable predictor of toxicity is insufficient; however, it can still serve as a valuable biomarker of exposure. The absence of a clear relationship —8 between metal concentrations in tissues and observed effects emphasises the influence of internal compartmentalisation and the intricate molecular defence mechanisms involved in damage control and repair processes.