## 4 Discussion
In particular, ionic silver is one of the most toxic metals for aquatic organisms, it is persistent and accumulates in aquatic systems, water, sediments, soil, and organisms, and causes ecological damage in the environment [48]. In this study, we determined the activities of antioxidant enzymes and the relative abundances of HSP70 in response to 28-day exposure to silver nitrate and silver nanoparticles in two of the most responsive tissues of marine mussels. The concentration range of silver used in the current work (0.2-20 \(\mu\)gL\({}^{-1}\)) is from a previous work in which weevaluated the ecotoxicological range of silver nitrate and silver nanoparticles for acute and chronic effects. All concentrations are completely sublethal, but in the case of silver nitrate, the highest two concentrations (20 and 2 \(\mathrm{\SIUnitSymbolMicro\text{gL}^{-1}}\)) are within the range of an effective concentration for the inhibition of byssus synthesis (below EC50) [39]. Our data (this work) show that silver nitrate is capable of eliciting biochemical effects even at lower doses (e.g., 0.2 \(\mathrm{\SIUnitSymbolMicro\text{gL}^{-1}}\)), as noted for CAT and SOD activities in both gills and digestive glands. The two patterns were very similar (Figures 5 and 6), showing a dose-dependent effect or a threshold-like effect in the gills or digestive glands. GPX also responded to silver nitrate, but only in the gills and at the highest exposure concentration. On the other hand, silver nitrate nanoparticles had no significant effect on any of the biochemical variables tested. It should be mentioned that previous data from our research group have shown that silver nitrate and silver nanoparticles target different organs in terms of bioaccumulation. In fact, the nitrate form is preferentially taken up in the gills, probably due to the stability of complex ionic silver forms in seawater [52]. On the other hand, particulate silver, i.e., nanoparticles, are also found in the digestive gland, probably following the feeding pathway after having undergone an increase in size in seawater due to the loss of zeta-potential. Our best explanation for this difference between the two metal forms--in terms of biochemical effects--is based on the higher reactivity and bioavailability of silver nitrate compared to silver nanoparticles, which, as mentioned, tend to be more unstable in seawater [39]. Indeed, silver nanoparticles tend to form aggregates when the surface potential suddenly changes due to pH and ionic strength [48]. In addition, it should be considered that a long exposure time (28 days) may have allowed a compensatory response of the antioxidant enzyme system, which could have occurred earlier with nanoparticle exposure essentially because of the overall lower dose, due to the aggregation and precipitation of nanoparticles [39]. Previously published data on the effects of 3-15-day exposure of silver NP in marine mollus showed that CAT, SOD, and GPX activity in the gills increased linearly with time [27], but in another study using similar concentrations of a silver NP for 94 h, no effects were reported for CAT and very little change for GST, another common antioxidant system [9]. Regarding ionic silver, [PERSON] et al. [27] reported a significant induction effect of CAT activity in the digestive gland on the fifteenth day, a decrease in GPX in the gills, and no effects for SOD. [PERSON] et al. [53] also reported increased CAT activity in larvae of _M. galloprovincialis_ exposed to ionic silver. In the soft tissues of other marine bivalves, such as the endothelin _S. plana_, aqueous nano- or ionic silver affected CAT activity at day fourteen, but not SOD and several other biochemical markers (GST, MT, LDH) [54]. When we compare these data with our results in detail, we cannot ignore the fact that the exposure time is shorter in almost all these papers and therefore a complete comparison is not possible. In fact, the observed differences should reflect the different stages of a dynamic process of adaptation to stress conditions.