## 2 Materials and Methods
### Biochemical Composition Analysis  
The samples were analyzed for their protein (Kjeldahl), ash (gravimetric, 500 \({}^{\circ}\)C, 6 h), carbohydrate (calculated) and total fat (Soxlet) content and their caloric/nutritional value (calculated), while for the fatty acids, a more detailed analysis was performed through gas chromatography to fully identify the fatty acids in the samples. lyophilized microalgae were analyzed for fatty acids according to the method of [PERSON] et al. [21]. For each treatment, three biological repeats were analyzed (\(n=3\)). Briefly, in 15 mg lyophilized microalgae, 0.2 mL chloroform/methanol (2:1 \(v\)/\(v\)) solution (Merck KGaA, Darmstadt, Germany) and 0.3 mL HCl/methanol (5% \(v\)/\(v\)) (Merck KGaA, Darmstadt, Germany) solution were added simultaneously. The mixture was heated for 1 h at 85 \({}^{\circ}\)C in the presence of 250 \(\upmu\)e tridecanoic acid methyl ester (Fluka Analytical, Sigma-Aldrich, St. Louis, MO, USA) as an internal standard. The resulting fatty acid methyl esters (FAMEs) were extracted with 1 mL hexane at room temperature for at least 1 h. The FAMEs were analyzed by GC-FID (Agilent 6890 N, Centerville Road, Wilmington, NC, USA) with an HP 88 column (60 m \(\times\) 0.25 mm i.d. with 0.20 \(\upmu\)m film thickness, Agilent Technologies, Santa Clara, CA, USA). The flame ionization detector temperature was set at 260 \({}^{\circ}\)C, and the chromatographic analysis involved a temperature-programmed run starting at 50 \({}^{\circ}\)C and held for 5 min. The temperature was initially increased at a rate of 5 \({}^{\circ}\)C/min to 150 \({}^{\circ}\)C followed by an increase at a rate of 1.5 \({}^{\circ}\)C/min to 210 \({}^{\circ}\)C, at which point, it was eventually maintained for 5 min. Hydrogen was used as the carrier gas with a linear velocity set at 30 cm/s. Each peak was identified and quantified using a 37-component FAME mix standard (Supelco, Sigma-Aldrich, St. Louis, MO, USA).  
The identification and quantification of fatty acids also allowed for the calculation of important indicators of the nutritional quality of lipids. Specifically, the nutritional quality of the fatty acids was determined by the following values [22; 23]:  
1. AI: atherogenicity index ([PERSON] and [PERSON] 1991) [22]: \[AI=\frac{C12:0+4\times C14:0+C16:0}{\Sigma MUIFAs+\Sigma PUIFAs}\] (1)  
Figure 1: Pearl oyster sampling positions: (EV) Evoikos Gulf; (SAR) Saronic Gulf.  
2. TI: thrombogenicity index ([PERSON] and [PERSON] 1991) [22]: \[TI=\frac{C14:0+C16:0+C18:0}{0.5\times\Sigma MUFAs+0.5\times\Sigma n6 PUFAs+3 \times\Sigma n3 PUFAs+\left(\frac{n3}{m6}\right)}\] (2)
3. HH: hypocholesterolemic/hypercholesterol ratios [23]: \[HH=\frac{C18:1 cis9+C18:2n6+C20:4n6+C18:3n3+C20:5n3+C22:5n3+C22:6n3}{C14:0+C16:0}\] (3) where \(\Sigma MUFAs\) is the quantity of all monounsaturated fatty acids and \(\Sigma PUIFAs\) is the quantity of all polyunsaturated fatty acids. Finally, statistical analysis of the results was carried out with the STATISTICA software (version 10, StatSoft(r)Inc., Palo Alto, CA, USA), specifically using one-way and two-way ANOVA combined with [PERSON]'s test (\(p<0.05\) corresponds to a statistically significant difference) to investigate the correlation of the values of the studied quantities with respect to the change in location, season and year and therefore to evaluate any statistically significant differences between samples.