# Effects of Diet on Mercury Bioaccumulation in Farmed Gilhead Seabream (_Sparus aurata_)
## 2 Materials and Methods
### Experimental Design
The percentage of inclusion of the main ingredients is not included in the table due to business secrecy; the ingredients are sorted in descending order based on their amount. The composition of the commercial additives used in the diets is reported partially due to patent coverage. Both CW and QW extracts powder (furnished by Silvateam S.p.A, Cuneo, Italy) were previously suspended in soybean oil (1:15) by means of a ball mill, to reduce the size of the powder particles. This technique allows oil dispersions to be stable over time. The composition of the two commercially available additives used in Diet 1 and Diet 2 is reported in Table 1 captions and summarized here:  
* Additive 1, 45% short-chain fatty acids complex (monoglycerides, diglycerides and triglycerides of propionic, butyric, caproic, heptanoic, caprylic, nonanoic, capric and lauric acids); 20% free glycerol; 35% silicon dioxide;
* Additive 2, 20% caprylic acid; 14% capric acid; 20,000 ppm organic iron; 5000 ppm organic zinc; vitamin B complex (B1, B2, B3, B5, B6, B8, B9); _Saccharomyces cerevisiae_ (dry and autolyzed yeast). After 180 days of feeding, half of the specimens for each diet group were exposed for 20 days to 5 ug L\({}^{-1}\) of mercury (II) chloride (HgCl\({}_{2}\)), directly injected in the seawater. The concentration was obtained by spiking 5 mL of 1000 mg/L mercury (II) chloride standard solution (99.5% purity) directly into the 1000 L tanks. The other half were not exposed and used as controls. During this period fish were fed daily with their diets (CD, D1, D2). Each treatment was performed in duplicate, as illustrated in Figure 1.  
The concentrations used in this study are lower than mercury lethal concentration (LC50) of 0.81 mg/L [42], but higher than the range of mercury environmental concentra  
Figure 1: Experimental design; commercial diet (CD), diet 1 with SCFA and extract of _Castanae sativa_ (D1) and diet 2 with _Saccharomyces cerevisiae_, caprylic acid, vitamin B complex and extract of _Schinopsis balansae_ (D2). CTRLs represent fish not exposed to HgCl\({}_{2}\). tions in the Mediterranean Sea of 0.05-2.87 \(\upmu\)g/L [43], thus facilitating the metal bioaccumulation in a shorter period. After mercury administration, seawater recirculating was stopped. An amount of 10% seawater was changed daily to reduce organic pollution and the mercury concentration into each tank was renewed and monitored at each sampling time. The concentration of mercury in seawater was below the detection limit (0.5 \(\pm\) 0.2 \(\upmu\)g L\({}^{-1}\)) in control tanks containing natural seawater, while in exposure tanks the concentration of mercury was 4.9 \(\pm\) 1.2 \(\upmu\)g L\({}^{-1}\) (mean of N. 5 samples). Three fish per tank were sacrificed after 0, 4, 10, and 20 days through an overdose (0.5 g L\({}^{-1}\)) of Tricaine methanesulfonate solution MS222, Tricaine Pharmaq (1000 mg/g). After the exposure period, the other three fishes per tank were sacrificed after 10 days left without mercury input. Fish organs (liver, kidney, gill, gut) and tissues (fragments of muscle) were dissected by stainless steel scissors and stored at \(-80\)\({}^{\circ}\)C until chemical and toxicological analyses. Fish well-being was constantly monitored and no mortalities, external lesions or signs of pain or suffering occurred during the whole treatment. All procedures on fish were carried out in accordance with Italian legislation on animal experimentation (Legislative Decree 26/2014). The experimental protocol was authorized by the Italian Ministry of Health (Ministerial Authorization N. 707/2021-PR).