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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.

Shape and size of the arenas affect amphipod behaviours: implications for ecotoxicology