Antenna regeneration as an ecotoxicological endpoint in a marine amphipod: a proof of concept using dimethyl sulfoxide and diflubenzuron.

Regeneration is a widely spread process across the animal kingdom, including many species of marine crustaceans. It is strongly linked to hormonal cycles and, therefore, a great endpoint candidate for toxicology studies. We selected the amphipod Parhyale hawaiensis as test organism, already used in ecotoxicological studies and able to regenerate its body appendages. We are proposing a protocol to use the antenna regeneration as a toxicity endpoint. First, we evaluated differences in time of completion of regeneration in males and females after the amputation of one antenna of 6 months old animals. Then we compared the influence of different testing volumes in the regeneration process (100 and 5 mL). We used as testing substances, dimethyl sulfoxide (DMSO) and diflubenzuron, a chitin synthesis inhibitor. The most suitable protocol consisted of volumes of 5 mL in 12-well microplates, with 1 organism per well, 12 organisms per concentration (1:1 females/males) and test time duration of around 5 weeks. DMSO accelerated regeneration time with a NOEC of 0.06%. Diflubenzuron inhibited the time necessary to its completion with a NOEC of 0.32 µg L-1. We conclude that the Parhyale hawaiensis antenna regeneration protocol proposed here is a potential tool in ecotoxicology, but more studies are required for its validation not only to verify its utility for testing chemicals but also environmental samples.