## 3 Results and Discussion
### Principal Component Analysis (PCA)  
A multivariate analysis approach was applied to all data to help discriminate the main variables responsible for the variance of biological effects detected in mussels (Table 3, Figure 3). The application of PCA to the original 13 variables at the pH bioassay indicates that they can be grouped in two new factors. These new variables explain more than 96% of the total variance of the original data set. In the present study, we selected a group of variables associated with a component loading \(\geq 0.40\), corresponding to an associated explained variance of more than 30%.  
The F1 (61%) includes the lethal response and DNA damage of the mussel. According to [89], the positive LPO 96 h value may demonstrate a relationship between an increase of antioxidant enzymes and LPO levels as defense mechanisms due to possibly being overwhelmed by ROS produced in organism tissues. It was more representative for pH 6.0 with higher positive values (Figure 3).  
The F2 (21%) showed the relationship between NRRT, DNA damage, LPO, mortality and pH reduction. The effects of this factor could be related with acidification by CO2 enrichment because of the significant variance in lysosome membrane stability (NRTT) and the increase in DNA damage, the oxidative stress and the mortality.  
Figure 3: Estimated factor scores in relation to the pH treatments used in the study.  
\begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Variables**} & \multicolumn{2}{c}{**Components**} \\ \cline{2-3}  & **Factor 1** & **Factor 2** \\ \hline NRRT 48 h & & \(-0.92\) \\ NRRT 96 h & \(-0.76\) & \(-0.52\) \\ DNA 48 h & \(0.69\) & \\ DNA 96 h & \(0.91\) & \(0.24\) \\ LPO 48 h & \(-0.76\) & \\ LPO 96 h & \(0.51\) & \(0.72\) \\ Mortality 48 h & & \\ Mortality 96 h & \(0.55\) & \(0.68\) \\ TA & \(0.88\) & \(0.40\) \\ DIC & \(0.89\) & \(0.41\) \\ \(\Omega_{\rm Cal}\) & \(-0.26\) & \(-0.94\) \\ \(\Omega_{\rm Arag}\) & \(-0.26\) & \(-0.94\) \\ \(p\)CO\({}_{2}\) & \(0.91\) & \\ \hline Variance (\%) & \(5.63\) & \(4.51\) \\ Cumulative (\%) & \(0.43\) & \(0.34\) \\ \hline \hline \end{tabular}
\end{table}
Table 3: Sorted rotated factor loadings of 13 variables for the two principal factors.  
The presented results corroborate the toxic effects observed from a decrease in pH value \(\leq\) 6.5, while from pH values \(\leq\) 7.5 a stress showed to be produced through activating enzymatic systems maintained at the lowest pH values. In summary, the stress produced via increasing proton concentration can be noticed in the enzymatic systems of the mussels (NRRT and LPO) in pH values lower than 7.5, being accentuated when the value of the concentration of protons is higher, which produces toxic effects related to pH 6.5 and 6.0.  
A similar integration analysis was carried out for the same pH range, but with other toxicity species and endpoints (amphipods mortality, sea-urchin embryo-larval development and the benthic community) in the Bay of Santos [29]. Similar results were obtained; they determined that pH 6.0 was extremely toxic for marine life.  
The responses of the mussels might be affected by the presence of other toxic compounds in the environment. The Santos Bay is affected by sewage discharges that carry illicit drugs, such as crack cocaine. This, in combination with acidification conditions (also below pH 7.5), provokes alterations in _Perna perna_ reproduction [31].