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Using advanced techniques for the development of behavioural assays in ecotoxicology: assessing the impacts of psychotropic compounds on crustacean behaviour

Student thesis: Doctoral Thesis

  • Shanelle Amber Kohler
Behaviour in ecotoxicology is expanding as it provides a link between the biochemical and physiological effects of environmental contaminants. This has been facilitated by advancements in computational automaton and the increased prevalence of behavioural modulating compounds in the environment. Despite increased interest, the inclusion of behavioural endpoints in environmental risk assessment is hindered by a lack of standardisation of behavioural assays, a paucity of information on the baseline behaviours of non-target organisms, and the speed of data collection.

The main aims of this project were to develop standardised behavioural assays for ecotoxicology and to assess the effects of psychotropic compounds on the behaviours of model crustaceans. Assays were developed for behaviours associated with stress or anxiety, translating methods from pharmacology studies on vertebrates. This was followed by exposure to environmentally relevant concentrations of psychotropic compounds to a range of species, varying in size to allow for the high-throughput assessment of behavioural impacts.

Amphipods are crustaceans that are ubiquitous to all aquatic systems and play key ecological roles across multiple levels of organisation. A local marine and freshwater species (Echinogammarus marinus and Gammarus pulex respectively) were used for the development of behavioural assays. To date, the antidepressant fluoxetine and the anxiolytic oxazepam are the most studied psychotropic compounds in the literature and so were selected as reference drugs for assay validation. Following assay development, methods were translated to the brine shrimp Artemia franciscana to allow for the simultaneous, high-throughput screening of multiple behavioural modifying compounds on swimming behaviours.

Results suggest that multiple factors can impact the results of behavioural assays including the shape and size of arenas, and intensity of stimuli. Differences were also observed in the baseline unconditioned behaviours of closely related species. These results highlight the importance of carefully designed, highly standardised assays, as well as an understanding of the baseline behaviours of test species, for reliable and repeatable results. Exposure to psychotropic compounds had significant impacts on crustacean behaviours, however, results were not consistent both between and within experiments. The inconsistencies in organism response to psychotropic compounds is thought to be the result of multiple factors including subtle differences in experimental design, inter- and intra- species variation in baseline behaviours, and the use of environmentally relevant concentrations which are close to doses previously reported to have no effects on wildlife in standard toxicity tests.

All of these factors can create ‘noise’ between data sets, and highlights some of the current issues which must be addressed for the continued development of behaviour as an assay for regulatory toxicity testing. Behaviours represent a useful endpoint in ecotoxicology and an important addition to adverse outcomes pathway for the assessment of environmental risk. This thesis goes some way to addressing the current limitations of behavioural assays and provides guidance for future work. Multiple behaviours can be measured successfully in crustaceans, with some endpoints proving to be more suitable than others. Psychotropic compounds may represent a risk to aquatic invertebrates but the nature and extent of which is not yet clear.
Original languageEnglish
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Award dateSep 2019

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