## 4 Discussion  
To obtain clues of the mode of action of toxicity of silver and AgNPs, we have performed in-gel assays for three major antioxidant enzymes--CAT, SOD, and GPX--in the tissues of marine bivalves, which often serve as bioindicators of marine pollution. The three methods have been shown to be sensitive and robust, making them suitable for routine use in biological monitoring programs in place of classical enzyme kinetics-based assays. These may have some limitations due to the heterogeneous protein fraction and the need for kinetic detection, leading to possible interference with other enzymes and/or nonlinear kinetics, which complicates the evaluation. The use of an electrophoretic approach allows direct detection immediately after the preparation of a protein fraction. We have substantially revised the native gel electrophoresis method originally developed by [PERSON] [46] and [PERSON] [47] and recently re-proposed by [PERSON] and [PERSON] [44] for the determination of antioxidant protein activity. This method requires a time-consuming procedure with a long duration of pre-electrophoresis and a total time of 6 h using two different running buffers. In our hands, this type of run proved to be insufficient due to sample degradation or sample loss. Therefore, we abandoned the long and ineffective pre-electrophoresis steps and proceeded directly to electrophoresis of proteins in Tris/glycine pH 8.3 in the presence of EDTA for a duration of only 90 min (40 mA, 4 \({}^{\circ}\)C) using precast gels. This procedure proved successful for the sufficient separation of multiple molecular weight markers containing cytochrome c, bovine serum albumin, aprotinin, catalase, and carbonic anhydrase (data not shown). In addition, precast gel technology eliminated the high background that interferes with the quantification of enzymatic activity. This error, reported by [PERSON] and [PERSON] [44] was due to the interference of the polyacrylamide gel catalyst TEMED with the CAT and GPX reaction mixture. This change in protocol allowed for complete elimination of the background, shortening run times while increasing scalability for a large number of samples. Finally, we also increased the hydrogen peroxide concentration from 0.003% to 0.01%, which allowed a better definition of the CAT band in the gel without a loss of linearity and with a significant increase in sensitivity. The design of the protocol for GPX was based on the native method described by [PERSON] et al. [43]. The main improvement was the use of cumene hydroperoxide at a concentration of 0.008%, which allowed us to determine a specific glutathione-dependent peroxidase activity that cannot be detected with hydrogen peroxide as a substrate. We have shown that this is a specific reaction that does not occur in the absence of GSH. Furthermore, three distinct achromatic bands appear in 12% native PAGE, indicating the presence of multiple enzymes with true glutathione-dependent peroxidase activity. Nowadays, nano-silver is one of the most widely used metals in consumer products due to its antibacterial activity and good tolerance [48]. Silver NPs are known to enter the aquatic environment in various ways, mainly through incorporation into commercial products, but also during their use. In addition, AgNPs can be spread through air and deposited in aquatic environments, not to mention the NPs generated by the erosion of polluted soils [24; 49]. The ocean is the ultimate recipient of nanomaterials and is sometimes considered a sink, as its high salinity tends to destabilize, or in the worst case, dissolve nanomaterials [50; 51]. Mussels have been identified as a good indicator of exposure to NPs. Indeed, agglomerate/aggregate particles are taken up by the gills and then partially enter the digestive gland, leading to a series of reactions, lysosomal disorders, changes in the expression of antioxidant- and immune-related genes, and other effects [7]. It is well known that heavy metals can induce oxidative stress in organisms.