Analyst 800

To establish the serum concentrations of Mg, silicon (Si), iron (Fe), copper (Cu), and zinc (Zn), approximately 0.5-ml serum samples were digested using 8-ml concentrated nitric acid in a Microwave Digestion System (Berghof, Eningen, Germany) for 30 min, and diluted at a ratio of 1:10 with distilled water prior to analysis. Lanthanum chloride (Merck, Darmstadt, Germany) was added as an interference suppressant for Mg analyses. The heating program employed was the one described in the oven’s user manual. Sample blanks were similarly prepared using 8-ml nitric acid (65%, w/v). Digested samples and blanks were diluted with ultra-high purity water, and total final volumes, assessed accurately by weight were recorded before analysis. We obtained AAS standard solutions from Merck (Darmstadt, Germany). The mineral levels of the samples were measured using Flame AAS (AAS, Perkin-Elmer, Analyst 800, Norwalk, CT, USA) with flame atomization in an acetylene-air via recognized and fully confirmed procedures, with the Zeeman background correction. The assessments were performed in triplicate. The method was verified with certified reference materials (bovine muscle BCR 184).

The biological oxygen demand (BOD) in sub-surface waters (15–25-cm depth) was quantified as BOD₅ (5-day incubation period; Eaton et al. 1995 ) and dissolved oxygen (DO) was measured following Winkler’s method (APHA 1998 ). The chemical oxygen demand (COD) and the concentration of dissolved organic carbon (DOC) were estimated following potassium dichromate method (APHA 1998 ) and KMnO₄ digestion (Michel 1984 ) respectively. The dissolved oxygen deficit (DOD) was estimated following Sánchez et al. (2007 (link)) method. The water samples were collected from sub-water surface (15–25-cm depth) and the dissolved oxygen deficit was estimated as the difference between dissolved oxygen concentration measured at corresponding locations and the oxygen saturation concentrations of pure water at similar temperature and pressure. For determining the concentration of Cr, Cu, Cd, Zn, Ni, and Pb in water, a tri-acid digestion method (Allen et al. 1986 ) was followed. Samples in tri-acid mixture (high purity HNO₃ (70%), HClO₄ (65%), and H₂SO₄ (70%)) were digested in a microwave digestion system and filtered (Whatman No. 42). The concentrations determined using an AAS (Atomic Absorption Spectrophotometer; Analyst 800, Perkin Elmer).

The iron concentration in the liver was determined using 20 mg of dried liver tissue as previously published8 (link). The iron concentration in the bone was determined using atomic absorption spectroscopy (PerkinElmer Analyst 800) of dried bone tissue (bone marrow-flushed femur and tibia) as previously published69 (link).

Serum Zn levels were measured using an atomic absorption spectrophotometer flame photometer (Analyst 800, Perkin Elmer, Inc., USA). All samples and standards were diluted (rate of 1/4) with 5% glycerol, and serum Zn concentrations (μg/dl) were calculated using a commercial Zn standard (1000 mg/L) curve [21 (link)].