Fig 1.
SEM images and UV absorption spectrum of the maltose-stabilized Ag NPs.
SEM images of Ag20-Mal (A), Ag40-Mal (B) and Ag100-Mal (C) NPs and their respective UV absorption spectrum (D) in distilled water.
Fig 2.
DLS patterns of size distribution of the maltose-stabilized Ag NPs.
Ag20-Mal (1), Ag40-Mal (2) and Ag100-Mal (3) NPs in distilled water. d.nm = diameter in nm.
Fig 3.
TEM images of the commercial Ag NPs.
Ag20 NPs (A and B), Ag80 NPs (C and D) and bulk Ag (E and F).
Table 1.
Temporal release of Ag ions as % of total starting mass of nanoparticle material in artificial seawater containing a 0.1 mM maltose-stabilized Ag NPs suspension.
Fig 4.
Effects of ionic and bulk Ag on mussel cells viability.
Cell viability (NR and MTT assays) in mussel hemocytes and gill cells exposed to ionic Ag (A and B) and bulk Ag (C and D) for 24 h. Data are given as absorbance values (means ± confidence intervals). Stars indicate significant differences (p<0.05) in treated cells with respect to controls according to the bootstrap analysis followed by Bonferroni’s correction. Black stars correspond to the NR assay and grey stars to the MTT assay. n = 6 replicates per treatment.
Fig 5.
Effects of maltose-stabilized Ag NPs on hemocytes viability.
Cell viability based on NR and MTT assays in mussel hemocytes exposed to Ag20-Mal (A and B), Ag40-Mal (C and D) and Ag100-Mal (E and F) NPs and to pure maltose (at the same concentrations present in corresponding maltose-stabilized Ag NPs suspensions) for 24 h. Data are given as absorbance values (means ± confidence intervals). Stars indicate significant differences (p<0.05) in treated cells with respect to controls according to the bootstrap analysis followed by Bonferroni’s correction. Black stars correspond to the maltose-stabilized Ag NPs and grey stars to the maltose results. n = 6 replicates per treatment.
Fig 6.
Effects of maltose-stabilized Ag NPs on gill cells viability.
Cell viability based on NR and MTT assays in mussel gill cells exposed to Ag20-Mal (A and B), Ag40-Mal (C and D) and Ag100-Mal (E and F) NPs and to pure maltose (at the same concentrations present in corresponding maltose-stabilized Ag NPs suspensions) for 24 h. Data are given as absorbance values (means ± confidence intervals). Stars indicate significant differences (p<0.05) in treated cells with respect to controls according to the bootstrap analysis followed by Bonferroni’s correction. Black stars correspond to the maltose-stabilized Ag NPs and grey stars to the maltose results. n = 6 replicates per treatment.
Fig 7.
Effects of commercial Ag NPs on mussel cells viability.
Cell viability (NR and MTT assays) in mussel hemocytes and gill cells exposed to Ag20 (A and B) and Ag80 (C and D) NPs for 24 h. Data are given as absorbance values (means ± confidence intervals). Stars indicate significant differences (p<0.05) in treated cells with respect to controls according to the bootstrap analysis followed by Bonferroni’s correction. Black stars correspond to the NR assay and grey stars to the MTT assay. n = 6 replicates per treatment.
Fig 8.
Effects of ionic Ag, bulk Ag and Ag NPs on ROS production in mussel cells.
ROS production in mussel hemocytes and gill cells exposed to ionic Ag (A and B), bulk Ag (C and D) and Ag20-Mal NPs (E and F) for 1, 3, 6, and 24 h. Data are given as fluorescence values (means ± confidence intervals). Asterisks indicate significant differences (p<0.05) between times of exposure and stars indicate significant differences (p<0.05) in treated cells with respect to controls according to the bootstrap analysis followed by Bonferroni’s correction. n = 6 replicates per treatment.
Fig 9.
Effects of ionic Ag, bulk Ag and Ag NPs on CAT activity in mussel cells.
CAT activity in mussel hemocytes and gill cells exposed to ionic Ag (A), bulk Ag (B) and Ag20-Mal NPs (C) for 24 h. Data are given as absorbance values (means ± confidence intervals). Stars indicate significant differences (p<0.05) in treated cells with respect to controls according to the bootstrap analysis followed by Bonferroni’s correction. n = 4 replicates per treatment.
Fig 10.
DNA damage in mussel cells exposed to ionic Ag, bulk Ag and Ag NPs.
Results of the Comet assay in mussel hemocytes and gill cells exposed to ionic Ag (A), bulk Ag (B) and Ag20-Mal NPs (C) for 24 h. Data are given as arbitrary values for tail DNA (means ± confidence intervals). Cells exposed to 50 μM H2O2 were used as positive control. Stars indicate significant differences (p<0.05) in treated cells with respect to controls according to the bootstrap analysis followed by Bonferroni’s correction. n = 50 cells analyzed per treatment.
Fig 11.
Effects of ionic Ag, bulk Ag and Ag NPs on AcP activity in mussel cells.
Lysosomal AcP activity in mussel hemocytes and gill cells exposed to ionic Ag (A), bulk Ag (B) and Ag20-Mal NPs (C) for 24 h. Data are given as absorbance values (means ± confidence intervals). Stars indicate significant differences (p<0.05) in treated cells with respect to controls according to the bootstrap analysis followed by Bonferroni’s correction. n = 6 replicates per treatment.
Fig 12.
Effects of ionic Ag, bulk Ag and Ag NPs on MXR activity in mussel cells.
MXR transport activity in mussel hemocytes and gill cells exposed to ionic Ag (A), bulk Ag (B) and Ag20-Mal NPs (C) for 24 h. Data are given as fluorescence values (means ± confidence intervals). Stars indicate significant differences (p<0.05) in treated cells with respect to controls according to the bootstrap analysis followed by Bonferroni’s correction. n = 6 replicates per treatment.
Fig 13.
Effects of ionic Ag, bulk Ag and Ag NPs on Na-K-ATPase activity in mussel cells.
Na-K-ATPase activity in mussel gill cells exposed to ionic Ag (A), bulk Ag (B) and Ag20-Mal NPs (C) for 24 h. Data are given as absorbance values (means ± confidence intervals). Stars indicate significant differences (p<0.05) in treated cells with respect to controls according to the bootstrap analysis followed by Bonferroni’s correction. n = 6 replicates per treatment.
Fig 14.
Effects of ionic Ag, bulk Ag and Ag NPs on the actin cytoskeleton of hemocytes.
Actin cytoskeleton (labelled with TRITC-conjugated phalloidin) of untreated mussel hemocytes (A and B) and hemocytes treated with ionic Ag (C-E), bulk Ag (F and G) and Ag20-Mal NPs (H-J). Hyal, hyalinocyte; Gran, granulocyte; L, lamellipodia; M, microspikes. Scale bars = 20 μm.
Fig 15.
Effects of ionic Ag, bulk Ag and Ag NPs on phagocytic activity in hemocytes.
Phagocytic activity in mussel hemocytes exposed to ionic Ag (A), bulk Ag (B) and Ag20-Mal NPs (C) for 24 h. Data are given as absorbance values (means ± confidence intervals). Stars indicate significant differences (p<0.05) in treated cells with respect to controls according to the bootstrap analysis followed by Bonferroni’s correction. n = 6 replicates per treatment.
Table 2.
Summary table of the and LC50 values (in mg Ag/L) obtained in hemocytes and gill cells exposed to ionic Ag, bulk Ag, Ag NPs (Ag20-Mal, Ag40-Mal, Ag100-Mal, Ag20, Ag80) and pure maltose based on NR and MTT assays.