# Risk Assessment of a Coastal Ecosystem from SW Spain
## 2 Materials and Methods
### Statistical Analysis (WOE Approach)  
Figure 3 represents the four LOEs integration used to characterize and distinguish the impacts related to the contamination levels and acidification degrees. The obtained results from the application of each LOE were linked for each component assessed using three different methods (WOE): (a) tabular matrix based on statistical significance (\(p<0.05\)) compared to the reference case (RSP sediment, pH 8.0); (b) pie charts, showing statistical differences (\(p<0.05\) significantly different and positive effect; \(0.01\geq p>0.05\) significantly different but moderate effect; and \(p>0.01\) not significantly different) among the samples sites and pH values compared to the reference case (RSP sediment, pH 8.0); and (c) linking the results using a multivariate analysis using the factor analysis principal component as extraction method [29]. \begin{table}
\begin{tabular}{l l l l l l l} \hline
**Test Organism** & **pH Range** & \
\begin{tabular}{l} **T** \\ (°C)** \\ \end{tabular} & **DO (\%)** & **Duration** & **Endpoint** & **Type** \\ \hline _A. brevicornis_ & 8.0–6.0 & \(19\pm 1\) & \(>\)80 & 10 d & Mortality & Acute \\ _P. lividus_ & 8.0–6.0 & \(18\pm 1\) & \(>\)80 & 48 h & Plateaus stage & Chronic \\ _M. galloprovincialis_ & 8.0–6.0 & \(19\pm 1\) & \(>\)80 & 48 h & D-phase & Chronic \\ _M. galloprovincialis_ & 8.0–6.0 & \(19\pm 1\) & \(>\)80 & 10 d & Mortality & Acute \\ _M. galloprovincialis_ & 8.0–6.0 & \(19\pm 1\) & \(>\)80 & 14 d & Metal concentration & Bioaccumulation \\ _M. galloprovincialis_ & 8.0–6.0 & \(19\pm 1\) & \(>\)80 & 24/96 h, 10 d & LMS & Acute \\ Macrobenthic & 8.0–6.0 & \(19\pm 1\) & \(>\)80 & 21 d & Biological indices &
\begin{tabular}{l} Macro-benthic \\ community \\ Alteration \\ \end{tabular} \\ \hline \end{tabular}  
\end{table} 
Table 1: Summarized description of the biological tests and the ecological integrity assessment tested in this study. Significant differences found between the reference site and the pH treatments were determined by an ANOVA and Dunnet's tests (TOXSTAT software, version 3.5) to develop the pie charts and tabular matrix. The indexes of contamination, toxicity, bioaccumulation, and ecological integrity were calculated as outlined by [11], considering the ratio-to-reference (RTR) values. Results from each LOE and each pH treatment were referred to the reference case and then derived as the minimum value. The RTRs were averaged for contamination in sediments and mobility, acute and chronic toxicity, bioaccumulation, and ecological integrity. The contamination index was obtained regarding the metal concentration in sediments (RTR\({}_{\text{sediment}}\)), and concentration in seawater and elutriates (RTR\({}_{\text{mobility}}\)). The elements As, Cr, Cu, Ni, and Zn were considered to determine the indexes. The acute toxicity index was derived from the average of RTR calculated for amphipod and mussel mortalities and NRRT assays. The values of NRRT were transformed as their inverse to be treated with equal importance. The chronic toxicity index was estimated from the average RTR for larval development inhibition results of mussels and sea urchins. Regarding the index of macrobenthic community structure (ecological integrity), the values were also inverted to be treated with equal importance, and the average of RTR was calculated based on the results of total abundance, diversity, and richness. The bioaccumulation index was derived from the average RTR estimated for the metals bioaccumulated in the tissues of mussels. Furthermore, to develop an integrated and more precise interpretation of the risks associated with CO\({}_{2}\) enrichment in the marine environment, a multivariate analysis (principal-component analysis, PCA-with-varimax rotation) was carried out. Therefore, the correlation between increasing pollution, pH reduction, and biological effects measured in the laboratory was further explained.