# Effects of Diet on Mercury Bioaccumulation in Farmed Gilhead Seabream (_Sparus aurata_)
## 4 Discussion
### Effects of the Yeast S. cerevisiae, Tannin from Plant Extract and SCFA on Hg Bioaccumulation  
In this study, fish fed with diet D2 exhibited lower mean mercury concentrations in all tissues (except muscle) compared to those fed with D1 and the control diet (CD). Statistical significant differences were observed in the gut and liver.  
These findings represent the first demonstration that the combined addition of the yeast _S. cerevisiae_ and extract of _S. balansae_ in the fish diets can effectively reduce inorganic mercury bioaccumulation in different tissues through mercury absorption and then elimination by feces.  
The efficiency of the yeast _S. cerevisiae_ to remove trace metals (especially mercury) from aqueous solution is well documented [52]. It is noteworthy that inorganic mercury has strong affinity to thiol groups and is one of the main mechanisms by which mercury accumulates and exerts its toxic activity in the eukaryotic cells [53]. The negative surfacecharge of yeast cells produced by phosphodiester bridges and the abundance of thiol, carboxyl, hydroxyl, amino and phosphate groups are the principal factors responsible for its biosorption capability for trace metal cations [54, 55]. The bioremediation ability of yeast depends on various factors such as initial yeast biomass, initial toxic metal concentration, pH, ambient temperature, presence of other heavy metal ions and contact time [56]. A previous study found that the yeast-metal complex was reversible under simulated gastrointestinal conditions, suggesting that biosorption by yeast may not represent a safe or reliable strategy for removing toxic metal from food [57]. This lower efficacy can possibly be attributed to the formation of complexes of mercury with amino acids, polypeptides, or proteins solubilized during the digestive process. For example, a simulated digestion experiment using mercury standards and cysteine demonstrated that this amino acid significantly reduced the ability of yeast to retain mercury during the digestive process [58]. In spite of these considerations, the reduction in mercury bioaccumulation found here in fish tissues allows us to speculate that the combined action of the yeast and the tannins in the extract of _S. balansae_ could overcome this limitation.  
It is noteworthy that tannins' polyphenic hydroxyl groups create stable compounds with metal ions [59] and especially with mercury [60]. A factor affecting this adsorption ability could be high pH solutions (greater than 5) that can cause metal ions precipitation and a consequent oxidation of the phenyl tannin hydroxyl groups [61]. In our experimental conditions, the complex between mercury and tannins' hydroxyl groups were probably formed in the stomach of fishes during digestion of the enriched food. The pH of the fish stomach ranges typically from 3.4 to 4.5 [62], thus not affecting the absorption ability of tannins.  
Surprisingly, as shown in Figure 2, the diet enriched with short-chain fatty acids and extract of _C. sativa_ seems to have no effect on mercury bioaccumulation in fish tissues despite the extract of chestnut wood being rich in tannins. SCFA were added in D1 because they improve the immune function and protect fish against oxidative stress by increasing the antioxidant ability and scavenging of free radicals [63, 64, 65]. However, their affinity for mercury [66] could have led to the formation of organometallic compounds adsorbed in the fish organs, thus contrasting the efficiency of tannins contained in the extract of _C. sativa_ on mercury removal. Future experiments are needed to investigate the potential interference of SCFAs with the mercury-removal capability of tannins.