Shedding light on amphipod behaviour: Baseline locomotion behaviour for laboratory ecotoxicology experiments

Behavioural endpoints offer a sensitive, non-invasive alternative to traditional mortality assays. Yet, they remain underutilized in regulatory frameworks due to a lack of standardised methodologies and baseline behavioural data. This study investigates the baseline locomotion of the marine amphipod Marinogammarus marinus to external light triggers to support standardised laboratory behavioural assays. Using an automated observation chamber, here we tracked swimming behaviour in response to light stimuli, quantifying locomotion as distance travelled. Groups of eight amphipods were tested in large crystalizing dishes over four days in an 8-minute trial involving two light exposures. A total of 60 individuals - males, females, and brooding females - were assessed. Light consistently triggered activity, with males showing strong negative phototaxis and brooding females the weakest. A startle response was observed within 3-5 seconds of initial light exposure, though responses diminished with repetition, suggesting potential overstimulation or habituation. Inter-individual and day-to-day variability highlighted the need for repeated measures. Additional trials with 32 males evaluated the effects of experimental conditions. Wall-hugging (thigmotaxis) appeared to be an escape response, not anxiety-related. Notable decreases in dissolved oxygen and pH within 24 hours were attributed to the amphipods' presence rather than activity, while salinity fluctuations due to evaporation did not visibly affect behaviour. Differences between male groups from the two separate experiments suggest behavioural shifts due to seasonal variation. This study establishes baseline behavioural data for M. marinus and demonstrates the value of automated tracking systems in behavioural assays in laboratory settings. It emphasizes the need to account for sex, size, and temporal variation in assay design and supports integrating behavioural endpoints into regulatory protocols.