## 4 Discussion  
In the present work, selected biomarker data from five ecotoxicological contributions on emerging contaminants (three pharmaceuticals, one detergent and one herbicide), were subjected to integrated response analyses using three methods, the integrated biomarker response (IBR) [5], the principal components analysis (PCA)-based index and the bioeffect assessment index (BAI) [19], to devise which could be more suitable in delivering an accurate measurement on the physiological stress response of diatom cell cultures under pollutant exposure. Greater emphasis on evaluating the suitability of the already existing indexes prior to developing new ones is essential [16]. The set of biomarkers should be selected with flexibility considering cell function, biological level and pollutant affinity, in order to calculate relevant integrated responses of organisms to different contamination compounds, given  
Figure 5: Spearman correlations heatmaps between the calculated index values and the applied exogenous concentrations for each applied compound. (*) denote significant differences at \(p<0.05\) for all tested compounds. Indexes include the integrated biomarker response (IBR) index; the principal component analysis (PCA)-based index; and the bioeffect assessment index (BAI), whilst (C) refers to the gradient of exposure concentration. that certain marker responses could emphasize certain functions or systems, while other levels of sensitivity could be at stake. Additionally, attention to the proper interpretation of biomarker responses integrating the index construction as parameters, and later, interpreting the derived index scores, is paramount [35]. In sum, a representative index is able to expose toxically induced response trends in natural systems which, therefore, are relevant to policy resolutions. Previous contributions concerning aquatic environmental contamination, based their analysis on biomarker responses, proving that the adverse effects resulting from a wide variety of pollutants can be evaluated based on an array of specific and general exposure biomarkers, such as oxidative stress biomarkers, exhibiting sensitivity and accuracy in developing valid results [6, 43], which are essential for the correct computation of the indexes [35]. Here, the efficiency of the applied set of biomarkers was determined by CAP (canonical analysis of principal coordinates) multivariate statistical approach. The antioxidant enzymes CAT, SOD and APX, and the non-enzymatic TBARS oxidative stress biomarkers, involved in the stress response physiological processes of a cosmopolitan marine diatom species, _Phaeodactylum tricornutum_, revealed an overall suitability for characterizing the different exogenous contaminants and the applied concentration gradients. This set of biomarkers produced statistically significant dose-response relations for four out of the five pollutants, fitting the experimental purpose, as determined by CAP efficiency results. The IBR index is one of the most widely used and proficient indexes for evaluating good ecological status and contamination levels of sites and in fact, available literature on biological effects following chemical exposure integrate the IBR index as an important tool for contamination assessment [5, 6, 7, 35, 44, 45, 46, 47]. IBR appears as a useful tool to mark pollution in field assessments, as it reflects contaminant levels at sites, despite the variability of biomarkers used for its calculation. Field and biomonitoring studies, concerning environmental pollution, rely on IBR for result support [48, 49, 50, 51, 52]. Fewer laboratory studies testify to IBR's applicability within the scope of treatment comparisons [47, 53]. One of the premises of the IBR index is the \"user judgment step\", as one should decide the weight of each biomarker for the index construction a priori, as it may positively or negatively influence the index, depending on an induction or an inhibition in terms of physiological response to contamination.