Ch3hgcl

For experimental diets, the commercial feed was crushed and divided into four groups. Group 1 (control group) was not mixed with others. Group 2–4 were mixed with 1, 5 and 10 mg methylmercury chloride standard solution (CH₃HgCl; purity 99.6%; Sigma-Aldrich, St. Louis, MO, USA) per kg. Subsequently, the feed were pelleted through a miner; the resulting pellets were dried in an oven at 40 °C and stored in a dark sealed bin. The concentration of MeHg in each feed was confirmed using high performance liquid chromatography–inductively coupled plasma–mass spectrometry (HPLC-ICP-MS).
A total of 87 hybrid groupers (200 ± 24.98 g) were randomly distributed into four groups. Each group included 21 fish maintained in a 270 L tank (90 × 60 × 50 cm) with flow through system, and water exchange was 50% per day. The fish was fed twice daily with the experimental diets at 3% body mass for 28 days. Three fish from each group were sampled on days 0, 1, 2, 7, 14, and 28 to determine immune parameters of head kidney leukocytes and bioaccumulation of MeHg in liver, head kidney, and muscle. The phagocytic activity and O₂⁻ production were measured following the methods described in Section 2.1.2 and Section 2.1.3 using leukocytes isolated from the head kidney. The samples of bioaccumulation were from the same fish which from the nonspecific immune samples.

Mercury resistance levels of each microbial community were determined, as described previously for individual bacteria [26 (link)] for mercuric mercury (HgCl₂) ((Sigma-Aldrich, St. Louis, MO, USA Portugal),) and MeHg (CH₃HgCl) (Sigma-Aldrich, St. Louis, MO, USA), using nominal concentrations ranging from 0.01 to 1003 µg/mL Hg²⁺ and 0.01 to 100 µg/mL CH₃Hg⁺. Determinations of mercury resistance were carried out in duplicate at each concentration tested. After incubation at 37 °C for 24 h in the dark and under aerobic and anaerobic (anaerobic jars with AnaeroGen sachet (Oxoid, Basingstoke, UK)) conditions, bacterial growth was monitored. The mercury resistance was registered as the lowest concentration of test compounds without visible growth. All data points represent the mean ± standard deviation (STD) of 2 independent determinations (each one also performed in duplicate).