The use of behaviour in ecotoxicology is expanding, however the lack of standardisation and validation of these assays currently presents a major drawback in moving forward in the development of behavioural assays. Furthermore, there is a current paucity of control data on test species, particularly invertebrate models. In this study we assessed a range of behaviours associated with spatial distribution and locomotion in relation to arena size and shape in two species of amphipod crustacean (Echinogammarus marinus and Gammarus pulex). Arena shape had significant effects on almost all behavioural parameters analysed. Increasing arena size resulted in an increased mean velocity and activity plus increased proportional use of the central zones. These results indicate that ‘ceiling effects’ may occur in some ecotoxicological studies resulting in potentially ‘false’ negative effects if careful consideration is not paid to experimental design. Differences in behaviours were observed between the two species of amphipod. For example, G. pulex spend approximately five times (∼20%) more of the available time crossing the central zones of the arenas compared to E. marinus (∼4%) which could have implications on assessing anxiolytic behaviours. The results of this study highlight several behaviours with potential for use in behavioural ecotoxicology with crustaceans but also underscore the need for careful consideration when designing these behavioural assays.

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Behavior is a useful endpoint in ecotoxicological research; it links the biochemical effects of contamination with physiology of individuals, which can be applied to higher levels of organization with relevance to ecology. Animals exhibit species-specific and sex specific behaviors. Previous experiments within ecotoxicology using amphipods as models have either not separated by sex or have on the assumption that they may create more variability in the results. Therefore, the objectives of this study were to investigate the effect of time (which controlled light conditions), sex, and the interaction of time and sex on the swimming velocity in males and females of the marine amphipod Echinogammarus marinus. E. marinus exhibited a phototactic response to light, as is consistent with previous findings. It was determined that females swim significantly faster than males and that this response was enhanced during periods when the lights were switched off. This suggests that a greater understanding of the baseline unconditioned behaviors of experimental organisms is needed to avoid impacts of unknown variables on results. The results of this study reveal significant sexual differences in the baseline behavior of E. marinus which has implications for future research in ecotoxicology. It is recommended that in future research specimens be separated by sex prior to experimentation, in order to account for possible behavioral differences such as those observed in the present studies. This study also highlights the need for thorough observation of behavior over shorter time intervals as larger intervals may miss short-term variations.

Animal behaviour is becoming increasingly popular as an endpoint in ecotoxicology due to its increased sensitivity and speed compared to traditional endpoints. However, the widespread use of animal behaviours in environmental risk assessment is currently hindered by a lack of optimisation and standardisation of behavioural assays for model species. In this study, assays to assess swimming speed were developed for a model crustacean species, the brine shrimp Artemia franciscana. Preliminary works were performed to determine optimal arena size for this species, and weather lux used in the experiments had an impact on the animals phototactic response. Swimming speed was significantly lower in the smallest arena, whilst no difference was observed between the two larger arenas, suggesting that the small arena was limiting swimming ability. No significant difference was observed in attraction to light between high and low light intensities. Arena size had a significant impact on phototaxis behaviours. Large arenas resulted in animals spending more time in the light side of the arena compared to medium and small, irrespective of light intensity. The swimming speed assay was then used to expose specimens to a range of psychotropic compounds with varying modes of action. Results indicate that swimming speed provides a valid measure of the impacts of behaviour modulating compounds on A. franciscana. The psychotropic compounds tested varied in their impacts on animal behaviour. Fluoxetine resulted in increased swimming speed as has been found in other crustacean species, whilst oxazepam, venlafaxine and amitriptyline had no significant impacts on the behaviours measured. The results from this study suggest a simple, fast, high throughput assay for A. franciscana and gains insight on the impacts of a range of psychotropic compounds on the swimming behaviours of a model crustacean species used in ecotoxicology studies.

Behavioural responses to contaminants are an important endpoint in ecotoxicology because they link effects at biochemical or cellular levels to impacts on individual fitness. Due to the increasing use of silver in nanomaterials, studies of its effects on the behaviour of aquatic organisms are important to assess the risks of silver nanoparticles (AgNP) released into the environment. The aim of this work was to evaluate the behavioural effects of silver on the marine amphipod Echinogammarus marinus after exposure to AgNO3 via water and AgCl or AgNP via food. Swimming activity of the amphipods was tracked during 6 min alternating dark and light conditions. Animals swam slower and responded less to light at higher concentrations of silver in the water. No differences were found in the behaviour of animals exposed via feeding up to 28 days, hence, longer exposure times may be required for the observation of effects. This is the first work to appraise behaviour effects of silver ions and AgNP on marine amphipods.Although the protocol has been successfully developed for this purpose, specimens appeared to habituate to test conditions during the experiments. Therefore, the need for further understanding of baseline behaviours in these model organisms is discussed.