## 2 Results  
Volatile organic compounds (VOCs) extraction from the headspace (HS) of a vial containing fragments of sponges was performed using four SPME fibers, namely carboxax-polyethylene glycol (PEG), polyacrylate (PA), divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS), and carboxen/polydimethylsiloxane (CAR/PDMS). The obtained SPME-GC-MS chromatographic profiles were always very complex and with a certain variability between species (Figure S1). Among the volatile compounds extracted by the different fibers, species belonging to various chemical classes could be attributed using the NIST library with a probability factor \(\geq 60\%\), including hydrocarbons, alcohols, carbonyl compounds, esters, halogen compounds, ethers, nitrogen and/or sulfur compounds and carboxylic acids. Based on the intensity of the relevant chromatographic peaks, the bipolar fibers DVB/CAR/PDMS and CAR/PDMS provided the most efficient extraction of low molecular weight sulfur compounds, considered the main causes of the nauseating smell of these sponges. In addition, these two fibers allowed us to highlight the differences between the two species, as shown in Table 1, which reports the sulfur compounds identified in _Ircinia variabilis_ (_lv_) and _Sarcotragus spinosus_ (_Ss_) samples. The classes of molecules of the volatile profile of the sponges, characterized using the HS approach, were also detected by DI-SPME-GC-MS. In addition, the DI approach permitted the determination of indole and biogenic amines. Among the fibers tested, the PA coating proved to be more effective for the extraction of the target compounds providing the most intense chromatographic signals and was used for the prosecution of the work. As far as indole determination is concerned, a linear calibration curve was constructed over the investigated concentration range, which was described by the following equation: y = 1.8 \(\times\) 10\({}^{4}\) + 40, with correlation coefficient of 0.9996 and intercept not significantly different from zero at 95% confidence level. The obtained within-day and between-days RSD% values were 4 and 7, respectively, and were not concentration dependent (according to a _t_-test). LOD and LOQ were 0.01 and 0.03 ug/mL, respectively. The estimated indole concentrations in _Ss_ specimens were between 0.05 \(\pm\) 0.01 and 0.48 \(\pm\) 0.13 ug/g (average 0.28 \(\pm\) 0.21 ug/g, \(n\) = 9), whereas it was always at not detectable levels in all _lv_ specimens analyzed. The obtained percentage recovery was 98 \(\pm\) 2% for all samples. As for the determination of the BAs released by the sponges, Figure 1 shows two typical GC-MS chromatograms related to _lv_ and _Ss_ samples subjected to DI-SPME extraction after derivatization with isobutyl chloroformate (IBCF). As evident, the chromatographic profiles for the two species are different and the BAs were identified by comparing both the retention times and the mass spectra of standard solutions analyzed under the same conditions. Table 2 shows the list of IBCF derivates of BAs determined in the analyzed sponge samples, listed in elution order, the main \(m/z\) ions present in the relative mass spectra, and the relative intensities. Calibration curves were constructed in selected ion monitoring \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**RT (min)**} & \multirow{2}{*}{**Compound**} & \multirow{2}{*}{**MF**} & \multirow{2}{*}{**P (\%)**} & \multicolumn{2}{c}{**SPME Fiber Coating**} \\ \cline{5-5}  & & & & **CAR/PDMS** & **DVB/CAR/PDMS** \\ \hline
2.01 \(\pm\) 0.01 & dimethyl sulfide & 915 & 70.1 & \(\textit{lv}/\textit{Ss}\) & \(\textit{lv}/\textit{Ss}\) \\ \hline
4.94 \(\pm\) 0.02 & methyl isothiamate & 875 & 82.5 & _Ss_ & _Ss_ \\ \hline
10.90 \(\pm\) 0.03 & dimethyl trisulfide & 869 & 91.9 & & _Ss_ \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Volatile sulfur compounds extracted from _Ircinia variabilis_ (_lv_) and _Sarcotragus spinosus_ (_Ss_) by the headspace approach using different fibers, and identified by NIST library.