## 3 Results and Discussion
### Biological Responses  
The percentage of mussel mortality for each treatment assay is shown in Table 2. In general, the mortality increases as pH values decrease. For pH values up to 7.5, no mortality was registered, while for pH values below 7.0, mortality was recorded at a minimum of 20% after 96 h of exposure. These results concur with study [77], in which the clam _Srochicularia plana_ showed a mortality of 20% at the pH value of 7.1 at 96 h of exposure. Lysosomes are central to many key biological processes, including reproduction, digestion and immune response; thus, any deleterious changes to their function can have serious consequences for those processes. The general health status of the mussels was therefore considered in terms of changes to the lysosomal system of the blood cells [75]. The NRT assay is based on the principle that only lysosomes in healthy cells take up and retain the vital dye neutral red. Lysosomal membrane damage caused by the impact of xenobiotics can decrease the NRT times through inducing the leaking of lysosomal components. Lysosomal membrane damage caused by the impact of xenobiotics [78] or by CO\({}_{2}\)-induced seawater acidification [75] can decrease the NRRT through inducing the leaking of lysosomal components [79]. The results of the biomarker of exposure NRRT assay for _Perna perna_ involving acidified seawater are shown in Figure 1. The \"exposure times\" presented very small estimates (F = 0.197; df = 1; \(p\) = 0.65; ES = 0.001), while the isolated \"pH values\" (F = 10.328; df = 5; \(p\) < 0.001; ES = 0.37) and the combination of both explanatory variables (F = 3.518; df = 5; \(p\) = 0.007; ES = 0.15) also evidenced a large effect on NRRT responses. The range in NRRT \begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline \multirow{2}{*}{
\begin{tabular}{c} **pH** \\ **Treatment** \\ \end{tabular} } & **TA** & **TCO\({}_{2}\)** & **TIC/DIC** & **HCO\({}_{3}\)** & **CO\({}_{3}\)** & **CO\({}_{2}\)** & **CO\({}_{2}\)** & **\(p\)CO\({}_{2}\)** & **\(\Omega_{\text{Cal}}\)** & **\(\Omega_{\text{Arag}}\)** \\  & **(\(\mu\)mol/kg)** & **(\(\mu\)mol/kg)** & **(\(\mu\)mol/kg)** & **(\(\mu\)mol/kg)** & **(\(\mu\)mol/kg)** & **(\(\mu\)mol/kg)** & **(\(\mu\)atom)** & & \\ \hline
8.3 & 1644 & 1404 & 1404 & 1249 & 147 & 8 & 262 & 3.53 & 2.31 \\
8.0 & 1662 & 1511 & 1511 & 1397 & 99 & 14 & 472 & 2.38 & 1.56 \\
7.5 & 1731 & 1718 & 1718 & 1629 & 39 & 50 & 1672 & 0.93 & 0.61 \\
7.0 & 1682 & 1828 & 1828 & 1650 & 12 & 165 & 5451 & 0.29 & 0.19 \\
6.5 & 2483 & 3249 & 3249 & 2469 & 6 & 774 & 25,996 & 0.14 & 0.09 \\
6.0 & 3172 & 5996 & 5996 & 3167 & 3 & 2827 & 97,130 & 0.06 & 0.04 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Carbonate system speciation in assays exposed to the different pH treatments for both bioassays of fertilization and embryo-larval development. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**pH Value**} & \multicolumn{2}{c}{**Mortality (\%)**} \\ \cline{2-3}  & **48 h** & **96 h** \\ \hline
8.3 & 0 & 0 \\
8.0 & 0 & 0 \\
7.5 & 0 & 0 \\
7.0 & 0 & 20 \\
6.5 & 10 & 20 \\
6.0 & 0 & 30 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: _Perna perna_ mortality percentage registered after 48-and 96 h exposure for the different pH levels (8.0, 7.5, 7.0, 6.5, and 6.0) and control (8.3 with no CO\({}_{2}\) added). values observed for lysosomes from control mussels (\(n\) = 10; no CO\({}_{2}\) added) at time zero, 48 h and 96 h of assay were 81 \(\pm\) 26 min, 93 \(\pm\) 23 min and 77 \(\pm\) 24 min, respectively. In the first 48 h, the RT significantly (\(p<0.05\)) decreased at pH 7.0 and 6.5. After 96 h, the organisms exposed to different pH values showed a significant decrease (\(p<0.01\)) in the dye retention time in lysosomes only at pH 6.0. There was no significant difference between RT of the dye in the control group for the highest pH values (8.0 and 7.5).