# Could Acidified Environments Intensity Illicit Drug Effects on the Reproduction of Marine Mussets?
## 3 Results and Discussion
### Toxicity Assays
#### 3.2.2 Embryo-Larval Toxicity Test  
The results of this toxicity assay using _Perna perna_ in reconstituted seawater (N) with different CC concentrations are presented in Figure 4. Significant differences were observed in CC concentrations of 12.5, 25 and 50 mg L\({}^{-1}\) when compared with N (no CC concentration added). The authors of [31] found significant differences in _P. perna_ embryo-larval development when exposed to 1.25 mg L\({}^{-1}\) CC. The results of embryo-larval successes applying the two different methods of acidification (CO\({}_{2}\) and HCl) are shown in Figure 5. It was demonstrated that the CO\({}_{2}\) acidification method promoted a significant decrease (with respect to control treatment) (\(p<0.05\)) for CC concentrations up to 12.5 mg L\({}^{-1}\). Results obtained for HCl acidification methodology showed significant differences for CC concentrations up to 6.25 mg L\({}^{-1}\) at pH 7.7 compared to control treatment. In the figure were pH values of 8.3 (N-control), and 8.0 and 7.7, excluding pH 7.5 (and less), since this pH value was highly toxic and presented no normal larval development for both acidification strategies. The IC\({}_{50}\) was calculated from the results of embryo-larval assay and presented in Table 3. The different methods of acidification presented different values of IC\({}_{50}\) associated with CC, evidencing that HCl is more toxic than CO\({}_{2}\) when associated with the same concentrations of CC. Figure 4: _Perna perna_ embryo–larval development results after exposure to the different CC concentrations in reconstituted seawater (pH 8.3) without acidification and considered as negative control. Asterisks indicate significant differences (\(p<0.05\)). Regarding pH effects, Table 4 shows the EpH\({}_{50}\) and E[H\({}^{+}\)]50 derived from the different treatments; that is, acidification causes effects in more than 50% of the embryos after 44 h exposure. The HCl acidification method presented a greater effect on the organisms (including control groups) when compared to the CO\({}_{2}\) method. In addition, CC showed more severe toxic effects when associated with acidification by HCl (Tables 3 and 4). This increase in toxicity may be related to the chemical reaction of the HCl acid, which released protons H\({}^{+}\) and ions of Cl\({}^{-}\), against the bicarbonate (HCO\({}_{3}\)\({}^{-}\)) released from CO\({}_{2}\). According to [52], two reasons might be promoting the morphological abnormalities of the larvae: (a) damage to the embryonic ectodermic cells rendering them unable to produce sufficient amorphous calcium carbonate, avoiding the proper development of the shell, or (b) shell dilution due to corrosion caused by acidified seawater. However, these factors are the result of an incomplete calcification process, which would not be enough to cover the entire mantle of the larva [55]. This feature would also promote a swimming skill decrease in the larvae, i.e., a fitness decrease [55,56]. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**CC (mg L\({}^{-1}\))**} & \multicolumn{2}{c}{**CO\({}_{2}\)**} & \multicolumn{2}{c}{**HCl**} \\ \cline{2-5}  & **EpH\({}_{50}\)** & **E[H\({}^{+}\)]50 (mol kg\({}^{-1}\))** & **EpH\({}_{50}\)** & **E[H\({}^{+}\)]50 (mol kg\({}^{-1}\))** \\ \hline Control & 7.34 & 4.56 \(\times\) 10\({}^{-8}\) & 7.53 & 2.95 \(\times\) 10\({}^{-8}\) \\
6.25 & 7.55 & 2.81 \(\times\) 10\({}^{-8}\) & 7.65 & 2.21 \(\times\) 10\({}^{-8}\) \\
12.5 & 7.61 & 2.41 \(\times\) 10\({}^{-8}\) & 8.18 & 0.66 \(\times\) 10\({}^{-8}\) \\
25 & 7.58 & 2.61 \(\times\) 10\({}^{-8}\) & - & - \\
50 & 8.18 & 0.66 \(\times\) 10\({}^{-8}\) & - & - \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Values of EpH\({}_{50}\) and E[H\({}^{+}\)]50 for the experimental treatments. Figure 5: _P. perma_ larval development success results after exposure to CC and acidification at two different pH values (8.0 on the (**left**) and 7.7 on the (**right**)).