# The Influence of Silver Nanoparticle Form on the Toxicity in Freshwater Mussels
## 3 Results  
Mussels were exposed to three concentrations (2, 10 and 50 ug/L) of different forms of nAg (spheres, cubes and prisms) and two concentrations of ionic Ag (1 and 10 ug/L). The dimensions of the nAg were in the same range and all were coated with PVP to maximize the influence of nAg geometry (Table 1). The size ranges of the nAg forms were 70 nm diameter for the spheres, 75 nm for the edges of the cubes and 90 nm for the equilateral prisms based on TEM analysis from the supplier. The Zeta potentials of the PVP-coated nAg were in the same range (\(-42\) to \(-37\) mvolt) in MilliQ water. In the exposure media (aquarium water), the Zeta potential was \(-7.5\) mvolts with no evidence of precipitation (i.e., when the Zeta potential nears 0 mvolt). This information led us to prepare the dilutions of the PVP-coated nAg forms at 1 mg/L in MilliQ water prior to the final dissolution in aquarium water. The nominal concentration of Ag was determined after 1 h dissolution and was between 70-90% of the theoretical Ag concentration for each form of Ag, thus confirming the stability of nAg suspensions during the initial exposure to mussels. The hydrodynamic diameter/edge size remained stable after 24 h dissolution in aquarium water. Although they were similar in size and coatings, the surface areas (SA) differed between geometries and were estimated based on a perfect sphere (4\(\pi\)r\({}^{2}\), \(r\) = radius in nm), cube (6\(e^{2}\), \(e\) = edge in nm) and prism (2 \(\times\)_base perimeter_\(\times\)_hi), assuming a height of 10 nm based on TEM analysis. The SA of the nAg was in decreasing order: prims \(<\) sphere \(<\) cube at the same concentration. The calculated SA controlled for concentration revealed that the 10 ug/L spherical nAg (31,000 nm\({}^{2}\)) was similar to the prismatic nAg at 50 ug/L (30,000 nm\({}^{2}\)). Hence, these two groups could be used to verify the hypothesis that SA area is a contributing factor to the geometry of nAg (a significant difference would suggest that other factors than SA were at play). Mussel health status was determined by following the condition factor (mussel weight/shell length) and air survival time following exposures to the various forms of Ag (Figure 1). The data revealed low changes in these responses in exposed mussels, with the exception of a small increase in the condition factor in mussels exposed to 50 ug/L cubic nAg and somewhat decreased air survival time in mussels exposed to 2 ug/L prismatic nAg. Air survival time dropped from 7 days in controls to 6 days in mussels exposed to the low concentration of prismatic nAg. This suggests that the exposure concentration and Ag forms did not have acute lethal impacts on the mussels. Based on added SA, no significant differences were obtained for CF and air survival between nAg of similar SA (10 ug/L spherical and 50 ug/L prismatic nAg), suggesting no other influence (form, reactivity) contributed to the observed effects. Figure 1: Condition factor and air survival time of mussels exposed to nAg geometries. Mussels were collected for condition factor (**A**) and air survival time (**B**). The data represent the mean with the standard error. The star symbol * indicates significant differences from controls. The biochemical properties of the post-mitochondrial fraction (15,000\(\times\) g supernatant) were examined by following changes in viscosity, protein aggregation and neutral lipids levels (Figure 2). Viscosity dropped in mussels exposed to the low concentrations of cubic (2 \(\mu\)g/L) and prismatic (2 and 10 \(\mu\)g/L) nAg relative to control mussels (Figure 2A). However, viscosity returned to control values at higher concentrations for cubic and prismatic nAg. The levels in lipids revealed that prismatic nAg reduced lipids at 10 \(\mu\)g/L and marginally (\(p<0.1\)) increased lipids at 50 \(\mu\)g/L (Figure 2B). Correlation analysis showed a trend between lipids and viscosity (\(r=0.45\)) (Table 2). This suggests that high neutral lipid levels could increase viscosity in the cytoplasm.