Copper 2 sulfate anhydrous

The commercially available mimosa tannin “Weibull” extract was supplied by the Tanac company (Montenegro, Brazil). The 5-(hydroxymethyl)furfural (5-HMF, ≥ 95%) was acquired from AVA Biochem (Zug, Switzerland). Technical grade acetone (>99%), sodium hydroxide pellets (NaOH) and N,N-Dimethylformamide (DMF, 99.9%) were provided by VWR. Ammonium hydroxide (25% in H₂O), zinc sulfate heptahydrate, Eriochrome^® Black T, ammonium chloride, Murexide, and methyl orange (MO) were obtained from Sigma Aldrich (St. Louis, MO, USA). Copper(II)sulfate (anhydrous) and acetic acid (glacial, 100%) were acquired from Merck (Darmstadt, Germany). Rhodamine B (RB) was provided by Alfa Aesar (Haverhill, MA, USA). Ethylenediaminetetraacetic acid disodium salt solution (0.1 M) was purchased from Fischer Scientific (Hampton, VA, USA). Ethylenediaminetetraacetic dianhydride (EDTAD, 98%) was supplied by ABCR (Karlsruhe, Germany).

A stock standard solution of Cu(II) ions at a concentration of 1000 μg/ml was prepared from copper(II) sulfate anhydrous (≥ 99.0) (Merck), and the working standard solutions were obtained daily by stepwise dilutions of the stock solution with doubly distilled water. The copper concentrations in the digestive gland and gill tissues were estimated using the method by Bagherian et al. [32 (link)]. Digestive gland and gill tissue samples were dried at 60°C, and combustion was performed at 450°C for one day. Then, the resulting samples were dissolved in a hot solution of 1 M HNO₃. The digested samples were adjusted to 50 ml using deionized water in 50 ml volumetric flasks and then analyzed at 324.8 nm using a flame atomic absorption spectrophotometer (Perkin-Elmer, 3100). The absorbance of the sample solution was measured against the blank solution. The difference between the absorbance of the sample and blank solutions at 324.8 nm was used as an analytical signal. A calibration curve was constructed by plotting the analytical signal versus the Cu(II) concentration in a series of working standard solutions. The amounts of copper in the digestive glands and gills are presented as µg/g wet tissue weight.