3.3. Mussel (Mytilus galloprovincialis) Larvae Anti-Settlement Activity

Mussel (Mytilus galloprovincialis) plantigrades were collected in juvenile aggregates during low neap tides at Memória beach, Matosinhos, Portugal (41°13′59″ N; 8°43′28″ W). In the laboratory, mussel plantigrade larvae (0.5–2 mm) were isolated in a binocular magnifier (Olympus SZX2-ILLT, Tokyo, Japan) to a petri dish with filtered seawater, and those with functional foot and competent exploring behavior were selected to the bioassays. Compounds were screened at 50 µM in 24-well microplates with 4-well replicates per condition and 5 larvae per well, for 15 h, in the darkness at 18 ± 1 °C, according to the previously reported [35,36]. All compounds that caused more than 60% of settlement inhibition (≤40% of settlement) in the screening bioassay were considered active and selected for the determination of the semi-maximum response concentration that inhibited 50% of the larval settlement (EC₅₀), at compounds concentrations of 3.125, 6.25, 12.5, 25, 50, 100, and 200 μM.