Confounding factors affect hemocyte responses of mussels Mytilus galloprovincialis upon foodborne exposure to polystyrene nanoplastics of three sizes

Information regarding the impact of nanoplastics (NPs, ≤ 1 μm) on marine organisms is insufficient. This work aimed to investigate the differential toxicity of three sizes of polystyrene (PS) NPs (50, 200 and 1000 nm) on the hemocytes and condition index (CI) of sentinel marine mussels Mytilus galloprovincialis. Mussels were dietarily exposed for 7 days through microalgae to PS at a low dose (LD: 10³ NPs/mL) and a high dose (HD: 10⁸ NPs/mL of 50 and 200 nm NPs, and 10⁶ NPs/mL of 1000 nm NPs). Each experiment (E) was performed twice, as follows: E1 and E4 for 50 nm PS NPs, E2 and E5 for 200 nm PS NPs and E3 and E6 for 1000 nm PS NPs. Despite the six experiments were run in less than a month, there were significant differences between the independent experiments of the different sizes of PS NPs. No differences in CI were found within each experiment, but differences were observed between independent experiments, possibly due to the spawning events observed during the acclimation of mussels of E3-E4. Similarly, a higher ROS production occurred in hemocytes of control mussels of E3-E4 compared to E1-E2 and E5-E6. But no differences in ROS were recorded in NPs treatments compared to controls within experiments. Cell viability of control hemocytes decreased in E3-E4 and E5-E6 in comparison to E1-E2. This could mask the size-dependent response to the NPs, as in E1 hemocytes showed lower viability and phagocytic activity than those from control mussels, but not in E4. Similarly, hemocytes of mussels exposed to HD in E2 showed lower viability than controls, but the contrary was observed in E5. In conclusion, a trend for size-dependent effects on mussel hemocytes was observed. However, hemocyte responses varied along time, possibly due to seasonal variations in mussels physiology connected with the gametogenic cycle.