The behavioural, toxicological, and biochemical effects of caffeine on Lumbriculus variegatus

Caffeine is an emerging contaminant of concern frequently detected in freshwater systems, yet the behavioural, toxicological and biochemical effects of caffeine in aquatic invertebrates remain poorly characterised. Here, we investigate the effects of caffeine exposure on survival, behaviour, locomotion, and energy stores in the freshwater annelid Lumbriculus variegatus . Exposure to ≥5.0 mM caffeine for 10 minutes or ≥3.0 mM for 24 hours reduced stimulated behaviours, with locomotion suppressed at ≥5.0 mM (10 minutes) and ≥1.0 mM (24 hours) ( p <.05, n = 8), which persisted 24 hours after exposure to 10 mM (10 minutes) or 3.5 mM (24 hours). A 24-hour LC 50 of 4.7 mM (95% CI: 4.60–4.70 mM) was observed, with significant lethality after seven days at 4.5 mM ( p <.0001). These findings provide the first characterisation of caffeine’s effects in L. variegatus and inform environmental risk assessment of caffeine in freshwater systems. • Caffeine exposure causes behavioural changes in Lumbriculus variegatus • ≥5.0 mM caffeine for 10 min reduces tactile stimulation response • ≥5.0 mM caffeine for 10 min reduces locomotor activity • Failure to recover behavioural function occurs after 24 hr exposure to 3.5 mM • Behavioural assays are reproducible across different institutes and worm cultures