## 4 Results and Discussions
### Excitation/Fluorescence Spectrum Matrix

Table 4: Pairs of excitation/emission wavelengths where difference between fluorescence of spectrum is higher, using the standard deviation as selection criteria. Figure 4: Standard deviation of the excitation/emission fluorescence values corresponding to _Nanochloropsis gaditana_, _Isochrysis galbuna_ and _Tetraselmis succia_, calculated with Equation (1). The peaks represent the excitation/emission pairs where fluorescence differs in these three species. Excitation and emission wavelengths are plotted in horizontal axis and standard deviation (as percentage of mean value) in vertical axis. Differences between the species could be detected from Figures 5 and 6. As expected, the higher standard deviations from Table 4 are confirmed in the observed differences of the fluorescence spectra in Figures 5 and 6. We also identified the emission wavelengths where fluorescence is very similar for the studied species, which resulted to be emission wavelengths bellow 660 nm and emission wavelengths above 710 nm. These emission wavelengths can be used as reference. Based on this study, excitation wavelengths of 420 nm, 450 nm and 530 nm and emission wavelengths of 640 nm and 685 nm were chosen as the most suitable to distinguish between these species. In order to confirm if the classification method could be used to discriminate potentially toxic species, the fluorescence spectrum of _Alexandrium tamarense_ reported in [23; 24] was used. Figure 7 shows the _Alexandrium tamarense_ fluorescence  
Figure 5: Fluorescence at 685 nm and 640 nm, when excitation is scanned from 320 nm to 700 nm. The maximum standard deviation (\(n=3\)) of 5% was obtained. Figure 6: Fluorescence spectrum with excitation at: (**a**) 420 nm; (**b**) 450 nm; (**c**) 530 nm. The maximum standard deviation (\(n=3\)) of 5% was obtained. spectrum at 685 nm, when excitation light is scanned from 200 nm to 600 nm. The pairs of excitation/emission are presented in Table 5.  
For each species, nine ratios were derived from the fluorescence values shown in Table 5: six ratios resulting from the four fluorescence values obtained at 685 nm, with the four different excitation wavelengths (320 nm, 420 nm, 450 nm and 530 nm), named FBX/CX, FBX/DX FBX/AX, FCX/DX, FCX/AX and FDX/AX; and three ratios between the emission obtained at 685 nm and 640 nm, for each excitation wavelengths (320 nm, 420 nm, 450 nm and 530 nm), named FBX/BY, FCX/CY and FDX/DY. These ratios are presented in Table 6 for each species. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Fluorescence of _Nannochloropsis gaditana_ **(a.u.)** & \multicolumn{2}{c}{**Emission (nm)**} \\ \hline Excitation (nm) & X: 685 & Y: 640 \\ A: 320 & 1010 & \\ B: 420 & 1332 & 74 \\ C: 450 & 944 & 71 \\ D: 530 & 335 & 109 \\ \hline
**Fluorescence of _Isochrysis gablana_ **(a.u.)** & \multicolumn{2}{c}{**Emission (nm)**} \\ \hline Excitation (nm) & X: 685 & Y: 640 \\ A: 320 & 707 & \\ B: 420 & 768 & 74 \\ C: 450 & 962 & 75 \\ D: 530 & 533 & 110 \\ \hline
**Fluorescence of _Tétraselmis suecia_ **(a.u.)** & \multicolumn{2}{c}{**Emission (nm)**} \\ \hline Excitation (nm) & X: 685 & Y: 640 \\ A: 320 & 805 & \\ B: 420 & 665 & 66 \\ C: 450 & 653 & 62 \\ D: 530 & 251 & 90 \\ \hline
**Fluorescence of _Alexandrium tamarense_ **(a.u.)** & \multicolumn{2}{c}{**Emission (nm)**} \\ \hline Excitation (nm) & X: 685 & Y: 640 \\ A: 320 & 79.84 & \\ B: 420 & 150.4 & 8 \\ C: 450 & 180.8 & 10 \\ D: 530 & 128.8 & 9 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Pairs of excitation/emission wavelengths used to distinguish _Nannochloropsis gaditana_, _Isochrysis gablana_, _Tétraselmis suecia_ and _Alexandrium tamarense_ and respective measured fluorescence value. Figure 7: _Alexandrium tamarense_ fluorescence spectrum with several excitation wavelengths. Reprinted/adapted with permission from Ref. [23]. Copyright © 2005 Elsevier B.V.  
For each ratio, the maximum (M) and minimum (m) values (maximum and minimum at each row) were selected.