# Effects of Diet on Mercury Bioaccumulation in Farmed Gilhead Seabream (_Sparus aurata_)
## 2 Materials and Methods  
### Experimental Design  
A total of 144 specimens of gilthead seabream (weight 139 \(\pm\) 98 g) obtained from the Acqua Azzurra fish farm (Pachino, Italy; lat. 36.70976 N, long. 15.11641 E) were transferred, by means of oxygenated transport tanks (500 L), at the Experimental Aquaculture Facility of the IRBIM-CNR (Messina). After two weeks' acclimation period, fish in good health status (i.e., without external signs or behavioral anomalies presumptive of potential illness or poor welfare) were randomly separated into twelve 1.4 m\({}^{3}\) twin fiberglass tanks connected to a recirculating system equipped with a sand mechanical filter and UV lamp. The process of random grouping was conducted ensuring that each specimen has an equal chance of being selected for a group (simple random sampling). Chemical-physical parameters were monitored daily (T = 16.3 \({}^{\circ}\)C \(\pm\) 1.5; pH = 7.7 \(\pm\) 0.2; PSU salinity = 39.5 \(\pm\) 0.3; oxygen = 6.2 \(\pm\) 0.7 mg L\({}^{-1}\)). Photoperiod followed local seasonal changes (November-June; latitude: 38\({}^{\circ}\)11\({}^{\prime}\)38.18\({}^{\prime\prime}\) N). At the end of this period, fish were randomly divided into three groups, each composed of 48 individuals, and fed with three different diets (from 0.8 to 1.7% body weight day\({}^{-1}\), according to the temperature and rationing table for this species). Diets, based on the nutritional requirements of gilthead sea bream (43.0% crude protein; 21.0% crude fats; and 19.76 mJ kg\({}^{-1}\) digestible energy) were produced by Agricola Italiana Alimentare S.p.A. (AIA). The composition of the diets is reported in Table 1, where the main ingredients, equal within the diets, are declared in descending order, based on their amount, due to business secrecy. One diet was used as control (CD), while the other two diets, each one supplemented with a different commercially available additive (Additive 1 and Additive 2), were further supplemented by \"spray-drying\" with two plant-derived tannin-rich extracts, as follows:  
* Diet 1 (D1), with chestnut wood (_Castanea sativa_) extract (CW, 1 g kg\({}^{-1}\));
* Diet 2 (D2), with quebracho Colorado wood (_Schinopsis balunsae_) extract (QW, 1 g kg\({}^{-1}\)). \begin{table}
\begin{tabular}{l l l} \hline  & Fish meal & \\  & Dehulled soybean meal & \\  & Corn gluten & \\ Main ingredients & Fish oil & Sorted in descending order based on their amount \\  & Sunflower seeds meal & \\  & Wheat flour & \\  & Soy oil & \\ \hline  & Vitamin A (U.I.) & 12,000 \\ Vitamins & Vitamin D3 (U.I.) & 2000 \\ (kg\({}^{-1}\)) & Vitamin C (mg) & 160.00 \\  & Vitamin E (mg) & 160.00 \\ \hline  & Zn (mg) & 60.00 \\  & Mn (mg) & 45.00 \\ Minerals & Fe\({}_{2}\) (mg) & 20.00 \\ (kg\({}^{-1}\)) & Cu\({}_{2}\) (mg) & 9.00 \\  & I (mg) & 2.00 \\  & Se (\(\mu\)g) & 160.00 \\ \hline  & & CTRL & Diet 1 & Diet 2 \\ Additives & \({}^{1}\) Additive 1 (g) & - & 10 & - \\ (kg\({}^{-1}\)) & \({}^{2}\) Additive 2 (g) & - & - & 5 \\  & \({}^{3}\) CW (g) & - & 1 & - \\  & \({}^{4}\) QW (g) & - & - & 1 \\ \hline  & Crude protein \% & 43.00 & \\  & Oils and crude fats \% & 21.00 & \\ Proximate & Crude cellulose \% & 2.50 & \\ composition & Crude ash \% & 5.60 & \\ (kg\({}^{-1}\)) & Total carbohydrates \% & 18.90 & \\  & Phosphorus \% & 0.90 & \\  & Digestible energy (mJ/kg) & 19.76 \\ \hline \end{tabular}  
* An amount of 4%; short-chain fatty acids complex (monoglycerides, diglycerides and triglycerides of propionic, butyric, caprico, heptanoic, caprylic, nonanoic, capric and lauric acids); 20% free glycerol; 35% silicon dioxide.
* An amount of 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). \({}^{3}\) Chestnut wood (CW) (_Castane sativu_) extract. \({}^{4}\) Quebracho Colorado wood (QW) (_Schinopsis balunsae_) extract.  
\end{table} 
Table 1: Ingredients and proximal composition of the diets tested in this study.