Repeated pollution exposure magnifies the escape behaviour in the avoidance test with enchytraeids

Avoidance testing has become a more popular soil ecotoxicity testing for worms, however, it is unclear the avoidance behaviours for future encounters. Similarly, it is unknown if organisms that initially do not avoid contamination will continue to do so or if they can "learn" to avoid it through phenotypic adaptation. To investigate this, we conducted a series of avoidance experiments using Enchytraeus crypticus in clean versus contaminated soil with silver nanomaterials (Ag NM300K). Initially, we exposed a population of these organisms via a conventional avoidance test to the contaminant and then divided them into two groups: those that avoided the contaminated soil (A group) and those that did not (Non-A group). The A group was exposed (again via an avoidance test) to the contaminated soil three more times, each time continuing only with the organisms that avoided it. In total, they underwent four repeated exposures. The Non-A group was treated similarly, but only the non-avoiding organisms were continued in each subsequent exposure. We found that avoidance behaviour increased with repeated exposures in the A group. However, in the Non-A group, there was no change in avoidance behaviour over the subsequent exposures.