Aanalyst 600

Before plant exposition, the bioavailable fraction of Cd and As was quantify in control and contaminated soils following the protocol of Lindsay and Norwell [13] . Briefly, 5 g of soil were extracted with 10 ml of 5 mM DTPA (Sigma-Aldrich), 0.1 M trietanolamine (Sigma-Aldrich) and 0.01 M CaCl₂ (Sigma-Aldrich), for 2 h at 20°C under stirring. Samples were then filtered and metal concentrations were determined by graphite furnace atomic absorption spectroscopy (GFAAS; AAnalyst600, Perkin-Elmer). Nine soil samples for each treatment were processed.
Cd and As were also quantified in plant organs applying the USEPA 3051a protocol. The harvested plants were carefully washed with tap water and then with distilled water to remove soil debries before analysis. All the samples were dried at 100 °C overnight. For each sample 10 mL of HNO3 and 2 mL of HClO3 were added to 0.2 g of dry plant matter. The samples were digested by using the ETHOS HPR 100/10 microwave lab station (FKV, Bergamo, Italy) reaching the 180°C temperature. After their complete mineralization, they were opportunely diluted and analysed by graphite furnace atomic absorption spectroscopy (GFAAS; AAnalyst600, Perkin-Elmer). Standards (from ENEA Research Centre, Roma, Italy) and blanks were run with all sample series for quality control.

Cadmium content in tissues was analyzed by graphite furnace atomic absorption spectrophotometry (GFAAS) (PerkinElmer AAnalyst 600), according to the technique described by Christian, 1969 [33 (link)] and using the conditions described by Eller and Haartz (1977) [34 (link)]. The brain regions were dissected out in parietal cortex (Cx), striatum (St), hippocampus (Hp), and cerebellum (Ce) [35 (link)]. Tissue samples were digested in 1 mL of concentrated HNO₃. Calibration curves were constructed using an aqueous Cd reference standard (GFAA mixed standard, PerkinElmer). As biological-matrix external standard, we used nitric acid-digested bovine liver (NIST 1577b) standard reference material, and this certified standard was analyzed in every analytical session; values obtained from that analysis were 95–105% from those reported by manufacturer. Cd content was expressed as μg of Cd/g wet tissue. All glassware was cleaned by soaking in 3% nitric acid and rinsing several times in deionized water. The limit of quantification for Cd analysis was 0.01 μg/g wet tissue.