Comparison of Different Commercial Nanopolystyrenes: Behavior in Exposure Media, Effects on Immune Function and Early Larval Development in the Model Bivalve Mytilus galloprovincialis

Abstract

In the absence of standard methods for the detection/quantification of nanoplastics (NPs) in environmental samples, commercial nanopolymers are utilized as proxies for toxicity testing and environmental risk assessment. In marine species, a considerable amount of data are now available on the effects of nanopolystyrene (PS-NPs) of different size/surface characteristics. In this work, amino modified PS-NPs (PS-NH₂) (50 and 100 nm), purchased from two different companies, were compared in terms of behavior in exposure media and of biological responses, from molecular to organism level, in the model marine bivalve Mytilus. Different PS-NH₂ showed distinct agglomeration and surface charge in artificial sea water (ASW) and hemolymph serum (HS). Differences in behavior were largely reflected by the effects on immune function in vitro and in vivo and on early larval development. Stronger effects were generally observed with PS-NH₂ of smaller size, showing less agglomeration and higher positive charge in exposure media. Specific molecular interactions with HS components were investigated by the isolation and characterization of the NP-corona proteins. Data obtained in larvae demonstrate interference with the molecular mechanisms of shell biogenesis. Overall, different PS-NH₂ can affect the key physiological functions of mussels at environmental concentrations (10 µg/L). However, detailed information on the commercial NPs utilized is required to compare their biological effects among laboratory experiments.

Keywords: Mytilus; NP-protein corona; charge; hemocytes; immune responses; larvae; nanopolystyrene; size.

Figure 1

Characterization of amino-modified nanopolystyrene (PS-NH₂) of different nominal sizes from (A) Bangs Laboratories, Inc. (Fishers, IN, USA); PS₅₀-B and PS₁₀₀-B; (B) Sigma-Aldrich (Milan, Italy); PS₅₀-S and PS₁₀₀-S. Upper panels: representative SEM images. Scale bar: 100 nm. Lower panels: physicochemical characterization and behavior in different exposure media. PDI = polydispersity index; ζ = zeta potential; MQ = Milli-Q water; ASW = artificial seawater; HS = Mytilus hemolymph serum. ¹ from [28]; ² from [29]; ³ this study.

Figure 2

In vitro effects of PS₅₀-B and PS₁₀₀-B in hemocytes of Mytilus galloprovincialis suspended in either ASW (artificial seawater) or HS (hemolymph serum). Hemocytes were exposed for 30 min to different concentrations of NPs 10, 50, and 100 µg/mL. PS₅₀-B: Lysosomal Membrane Stability (A); Phagocytic activity (B) PS₁₀₀-B: Lysosomal Membrane Stability (C); Phagocytic activity (D). Data are expressed as percent of control. Statistical analyses were performed by non-parametric Kruskal–Wallis followed by the Dunn’s multiple comparisons test (p < 0.05). * All treatments vs. controls; # HS vs. ASW.

Figure 3

In vitro effects of PS₅₀-S and PS₁₀₀-S in hemocytes of Mytilus galloprovincialis suspended in either ASW (artificial seawater) or HS (hemolymph serum). PS₅₀-S: Lysosomal Membrane Stability (A); Phagocytic activity (B) PS₁₀₀-S: Lysosomal Membrane Stability (C); Phagocytic activity (D). Data are expressed as percent of control. Statistical analyses were performed by non-parametric Kruskal–Wallis followed by the Dunn’s multiple comparisons test (p < 0.05). * All treatments vs. controls.

Figure 4

In vivo effects of PS₅₀-B and PS₁₀₀-B on hemolymph immune parameters of Mytilus. Mussels were exposed to both size PS-NH₂ for 24 h at 10 µg/L. Hemocyte lysosomal membrane stability (LMS) (A), phagocytosis (B), ROS production (C), and serum lysozyme activity (D). Data are expressed as percent of control. Statistical analyses were performed by non-parametric Kruskal–Wallis followed by the Dunn’s multiple comparisons test (p < 0.05), * exposed vs. controls.

Figure 5

In vivo effects of PS₅₀-S and PS₁₀₀-S on hemolymph immune parameters of Mytilus. Adult mussels were exposed to both size PS-NH₂ for 24 h at 10 and 50 µg/L. Hemocyte lysosomal membrane stability (LMS) (A), phagocytosis (B), ROS production (C), and serum lysozyme activity (D). Data expressed as percent of control. Statistical analyses were performed by non-parametric Kruskal–Wallis followed by the Dunn’s multiple comparisons test (* p < 0.05).

Figure 6

Effects of PS₅₀-S and PS₁₀₀-S on early larval development of M. galloprovincialis, evaluated in the 48 h embryotoxicity assay. Fertilized eggs were exposed to different concentrations of NPs in ASW (0.001–1000 μg/L). Data, reporting the percentage of normal D-shaped larvae, represent the mean ± SD of four experiments carried out in 96-multiwell plates (six replicate wells for each sample). Statistical analyses were performed by non-parametric Kruskal–Wallis followed by the Dunn’s multiple comparisons test (* p < 0.05).

Figure 7

Effects of PS₅₀-S on shell formation of M. galloprovincialis larvae at 24 (A–D) and 48 hpf (E,F) evaluated by Calcofluor/Calcein staining and confocal microscopy. Fertilized eggs were exposed to 150 μg/L PS₅₀-S in ASW. 24 hpf: light microscopy images of Control (A) and NP-exposed larvae (B); calcofluor staining (blue) of the organic matrix the growing shell of Control (C) and NP-exposed larvae (D). In (E), the effect on PS₅₀-S on the area of the organic matrix is reported (percent values with respect to control). 48 hpf: Calcein staining (green) of calcified shell. Control larvae show extensive calcification with shell accretion rings (F); PS₅₀-S exposure results in irregular calcification and shell malformations (G–I). Scale bar: 20 µm. Statistical analyses were performed by Mann–Whitney U test (* p < 0.05).