## 3 Results and Discussion
### Biological Responses
The results presented in this study for the control sample (81 \(\pm\) 26 min) are reliable and fit with those obtained in previous studies reporting an NRRT ranging from 60 to 90 min in a study on healthy or non-exposed _P. perna_[80] and 60 min in a study using hemolymph of _Perna viridis_[81]. Furthermore, [82] reported NRRTs of 90 min using _Mytilus galloprovincialis_ hemolymph, and 106 min in the [83] study using clam _Ruditapes philipipurarum_ hemolymph. According to the criteria established by [84], in the cytochemical method, animals are considered to be stressed but compensating if NRRT were between 50 and 120 min. However, studies have demonstrated that lysosomal membrane stability differs in tropical zones compared to temperate zones, as higher temperatures in the tropics lead the neutral red dye to be retained in the lysosomes for a shorter period of time [85], agreeing with the results of this work and previous studies on NRRT of _P. perna_ populations from the Brazilian coast [43; 65; 86; 87]. Therefore, the present study may contribute to the development of a health index for tropical mussels. Lysosomal integrity is directly correlated with physiological scope for growth and is also mechanistically linked with the processes of protein turnover [88]. Ref. [89] showed that lysosomal stability in adult oysters is also correlated with larval viability. Therefore, the integrity or stability of this membrane is considered an indicator of the cell \"well-being\", acting as an important cellular and nonspecific biomarker of stress [46] once its activity is directly related to ion transportation [59]. The significant reduction in lysosomal health observed for pH below 7.0 (48 h) may be related to the increase in Ca\({}^{2+}\) concentrations in the hemolymph [75], which can cause lysosomal destabilization [90]. According to [22], decreasing pH promotes the dissolution of shells and, consequently, an increasing concentration of Ca\({}^{2+}\) in the hemolymph, altering the cellular metabolism and reducing immune function. According to [91], extracellular calcium enters in the cells via pinocytosis, and once in the cytosol, Ca\({}^{2+}\) can activate calcium-dependent phospholipase A2, destabilizing the lysosome membrane. The compensation of acid-base imbalance is also of great importance, since it is related to ion transportation through the cell membrane. The CO\({}_{2}\) produced in the cells during routine metabolism is typically hydrated to form bicarbonate and H\({}^{+}\). The H\({}^{+}\) ions are buffered, while the bicarbonate is transported out of the cell in exchange for Cl\({}^{-}\) via ion transport proteins. Refs. [92; 93] indicated that mussels responded to maintain acid  
Figure 1: Mean (and standard error) of LMS assessed through NRRT assay in the hemocytes of _P. perna_ exposed to different pH levels (Dunn’s test: * \(p\) < 0.05; ** \(p\) < 0.01). base balance through carbonic anhydrase activity, so that it was significantly higher in mussels exposed to low pH in comparison with control individuals. As observed in the present study, the increasing proton concentration in the environment, once more, may be associated with a decrease in the lysosome's capacity to retain the dye as a healthy cell. DNA damage results evidenced that \"exposure time\" (F = 0.298; df = 1; \(p\) = 0.58; ES = 0.001) was not significant, presenting a very small influence on DNA-SB. The \"pH values\" variable, on the other hand, demonstrated a large significance (F = 20.766; df = 5; \(p\) < 0.001; ES = 0.47) and the combination of these explanatory variables showed a significantly medium effect (F = 3.727; df = 5; \(p\) = 0.003; ES = 0.11). Lipid peroxidation results showed that \"exposure time\" (F = 34.953; df = 1; \(p\) < 0.001; ES = 0.24), the \"pH values\" (F = 4.940; df = 5; \(p\) < 0.001; ES = 0.15) and the combination of these explanatory variables (F = 5.603; df = 5; \(p\) < 0.001; ES = 0.17) had a large statistical effect on LPO responses.