## 3 Results and Discussion  
### Gamete Fertilization and Embryotoxicity  
The effects of tested MP, CHR-MP and CHR treatments on the mussel gamete fertilization and embryonic development are shown in Figure 1. The percentage of fertilized eggs in control treatments was 88.91%, which is in line with values required for test validation [46]. The results did not highlight differences between the gamete fertilization success attained in control conditions and that measured after exposure to MP, CHR-MP, 0.1 mg/L and 3.78 ug/L CHR (Figure 1A); similarly, the fertilization rate did not change among performed treatments. The impact of plastic debris on gamete fertilization of marine invertebrates may depend on either the size or the degree of functionalization of proffered particles [57; 58]. In a recent study, [PERSON] et al. [59] reported a decreased fertilization success following oyster (_Crassostrea gigas_) gamete exposure to 50-nm aminated PS nanoparticles, while no effect was recorded following exposure to plain (2-um) polystyrene MP. This may denote a relatively high degree of adaptation of mussels' spermatozoa to the physical disturbance induced by micro-sized debris, as well as a negligible effect of the tested level of MP-sorbed and freely dissolved CHR on their motility/viability. However, the current knowledge on MP or POPs toxicity on mussel gamete fertilization is still limited, and even less is known on the possible cumulative effects induced by their interaction. Further investigations are thus essential to clarify these points.  
Figure 1: Mean percentage (\(\pm\) SEM, \(n\) = 5) of fertilized mussel eggs (**A**) and normally developed D-veliger larvae (**B**) measured after exposure to 25 \(\times\) 10\({}^{3}\) units/mL FSW of polystyrene microparticles (MP) and chrysene-loaded microplastics (CHR-MP), 0.1 mg/L and 3.78 ug/L of freely dissolved CHR (without MP). Different letters denote statistically significant differences (\(p\) < 0.05, ANOVA— Bonferroni _post-hoc_ comparison) among treatments.  
Embyrotoxicity data recorded after 48 h of embryo exposure also showed basal levels within the thresholds for test validity (77.50% normally developed D-veligers in the control condition; ASTM [39]) (Figure 1B). The treatment at 0.1 mg/L CHR was the only condition resulting in a significant decrease of normally developed mussel larvae compared to control. The morphology of larvae exposed to this treatment did not differ from that assessed following MP and CHR-MP exposure, but was significantly lower compared to 3.78 ug/L CHR. As highlighted in previous investigations on plastic-associated chemicals [44], the mussel embryonic development generally shows a higher sensitivity to POPs (CHR in this case) compared to gamete fertilization, likely depending on the relatively higher permeability of embryos to hydrophobic substances, as previously speculated [60]. Although further evidence is not reported for CHR, other PAHs such as benzo[a]pyrene, naphthalene, phenanthrene and pyrene are known to elicit embryotoxic effects on marine invertebrates and fish [61; 62; 63]. Embryotoxicity data reported here confirm previous evidence of low to absent toxicity of plain polystyrene MP on the fast-evolving processes governing the mussel embryonic development [64] and indicate the lack of cumulative effects of MP-sorbed CHR on the morphology of mussel (D-shaped) veliger larvae. In line with these observations, the co-exposure of MP and 4-nonylphenol performed by [PERSON] and [PERSON] [65] did not cumulatively increase the effect of polyethylene MP on the embryo-larval development of the sea urchin _Paracentrotus lividus_. Conversely, evidence of increased developmental abnormalities was observed in medaka (_Orylzias latipes_) embryos exposed to benzo[a]pyrene and PFOA-sorbed polyethylene MP compared to virgin particles [66], suggesting that the magnitude of effects induced by POPs -loaded MP may vary depending on specific traits characterizing the ontogeny of marine species.