# The Influence of Silver Nanoparticle Form on the Toxicity in Freshwater Mussels
## 4 Discussion  
The activity of AChE was readily inhibited by all forms of Ag in the present study. Moreover, inhibition appeared stronger with prismatic and cubic nAg compared to spheric and dissolved Ag, thus allowing the rejection of the null hypothesis that all forms of Ag are equally toxic. Studies comparing the toxicity of Ag forms in aquatic organisms are scarce. In a previous study of PVP-coated nAg of different geometries (spheres, cubes and plates), the activity of pyruvate kinase-lactate dehydrogenase was altered, with corresponding changes in the fractal dimension of the enzyme complexes in exposed mussels [10]. In the present study, decreases in the _fD_ of AChE suggest that the various forms of nAg change the spatial organisation of the enzyme AChE, perhaps by crowding effects. Other enzymes could be affected as well, not only at the neurological level but in other critical physiological systems. Strong inhibition of AChE activity was also observed in the marine clam _Ruditapes philippinarum_ exposed to nAg [14]. Interestingly, while the addition of dissolved organic matter reduced bioavailability and oxidative stress, AChE activity was still affected, suggesting susceptibility of bivalves towards nAg at the neurological level. Disturbance in the cholinergic system in mussels, as determined by AChE activity inhibition, was observed with other nanoparticles, such as gold-coated nTiO\({}_{2}\)[27]. The decrease was accompanied with increased oxidative stress, as determined by catalase, glutathione S-transferase activities and LPO levels in tissues. Behavior was seemingly affected by reduced filtration activity (clearance rate of neutral red particles). In the present study, oxidative stress was not observed given that the nanoparticles were PVP-coated and the surfaces were equally reactive [28]. Although PVP-coated nAg was more bioavailable than citrate-coated nAg, no evidence of oxidative stress was found in the digestive gland and gill tissues. Neurological impacts were also found in the snail _Lymnea stagnalis_ exposed to uncoated 100 nm diameter nAg [29]. A biphasic response in memory function was observed where enhanced memory formation was observed at 10 \(\upmu\)g/L followed by reduced memory at 50 \(\upmu\)g/L. In another study, _Sorbicularia plana_ clams exposed to gold nanoparticles (nAu) also displayed behavioral changes [30]. This study showed increased oxidative stress, including metallothionein induction in clams exposed to relatively high concentrations (100 \(\upmu\)g/L nAu) of different sizes leading to decreased burrowing activity. Interestingly, not all sizes of nAu displayed similar effects, suggesting that size could influence toxicity. For example, metallothioneins were induced with sizes of 5 and 40 nm, while catalase activity was increased by 15 and 40 nm diameter nAu. This suggests that nanoparticles induce size-dependent interaction in tissues. The influence of the size of nAg revealed the importance of protein adsorption at surfaces or corona formation [31]. The curvature of the nanoparticle is stronger in smaller nanoparticles, favoring more stable binding of protein coronae. Stronger binding arises from disruption to the secondary structure of the proteins via cysteine-cystine interactions with Ag at the surface. The stronger curvature found in smaller sizes of nAg (high surface volumes) contributes to a more stable protein corona, thus limiting availability towards cells and toxicity from the release of ionic Ag in cells compared to uncoated nAg. If we extend this reasoning with other geometries, those producing more curvatures (higher surface areas) should produce more stable coronae and be less toxic in mussels. Indeed, inhibition of AChE was less severe with spheric and cubic nAg (high SA, Table 1) than prismatic nAg (lowest SA and curvature). With respect to viscosity, spheric nAg decreased viscosity in a concentration-dependent manner, but viscosity returned to control values at higher concentrations for cubic and prismatic nAg.