Abstract
Copper-based antifouling paints supplemented with organic booster biocides are a major source of chemical contamination in coastal marine ecosystems. However, the combined effects of copper(I) and emerging booster biocides on non-target marine organisms remain insufficiently understood. Here, we assessed the toxicity of copper(I) chloride (CuCl) and the booster biocide dichlofluanid, both individually and as released from antifouling coatings, on fertilization success and early embryo-larval development of the sea urchin Paracentrotus lividus , a widely used bioindicator species inhabiting ports and coastal areas affected by maritime traffic. Copper(I) significantly impaired fertilization in a concentration-dependent manner (EC 50 = 0.72 ± 0.10 μM), with complete inhibition at 10 μM. The restoration of fertilization rates in the presence of Cu(I)-specific chelator bathocuproine disulfonate (BCS) confirmed that toxicity was driven by bioavailable Cu(I). Dichlofluanid also reduced fertilization success, with significant effects from 0.01 μM (EC 50 = 0.062 ± 0.007 μM). The two contaminants exhibited distinct effects on embryonic development. Copper(I) caused developmental arrest at the blastula stage, whereas dichlofluanid primarily delayed larval progression without completely inhibiting embryogenesis, and, at 0.1 µM, caused skeletal malformations in plutei. Measured copper concentrations declined over time in seawater, emphasizing the importance of metal speciation and bioavailability in toxicity assessments. Exposure to antifouling paint-coated surfaces demonstrated that all tested Cu 2 O-based formulations released sufficient copper to inhibit normal development. Release dynamics were strongly influenced by paint matrix composition: rosin-based self-polishing coatings produced faster and higher copper release than acrylic-based formulations, which may represent a more environmentally sustainable alternative. Notably, commercial multi-biocide paints — Cu 2 O combined with dichlofluanid and additional compounds such as diuron or zineb — induced the most severe effects, with embryonic development blocked from the earliest cleavage stages, indicating potential synergistic interactions among co-formulants. Overall, these findings demonstrate that copper-based antifouling systems, particularly when combined with booster biocides, pose a significant risk to early life stages of non-target marine invertebrates. The study highlights the need to incorporate mixture toxicity and realistic exposure scenarios into ecological risk assessment frameworks for coastal marine environments.