Aa 6800

The piglet (feed and feces) samples were dried in a MARCONI model MA-035 (Piracicaba, Brazil) circulating air drying oven, milled in a freezer cryogenic impact mill SPEX model 6750 (Metuchen, EUA) and mineralized in digestion blocks MARCONI model MA-447/6/100 (Piracicaba, Brazil).
An ultrasonic cell disrupter (horn-type ultrasonic probe) UNIQUE model USC-DC (Campinas, Brazil) equipped with a 3 mm titanium probe and operated at frequency of 20 kHz was used in the ultrasound-assisted extraction of the analytes. An ultracentrifuge model Bioagency Bio-Spin-320R (São Paulo, Brazil) was used to separate the ultrasound extracts from the remaining solid phase.
Calcium and magnesium were determined by using an atomic absorption spectrometer SHIMADZU AA-6800 (Tokyo, Japan) equipped with a background absorption correction with a deuterium lamp and self-reverse (SR) system. Phosphorus was measured using UV/Vis spectrophotometer Thermo Spectronic model Genesys 6 (Madison, EUA) equipped with a xenon lamp and an automatic six-cell changer with 1 cm quartz cells.

At the end of each subculture, the total Se content of the oven dried shoots was determined in the acidic solution (mixture of HNO₃ and H₂O₂ (9:1, v/v)) according to the US-EPA method 3052 [45 ] on three samples (2.0 g each one) for each Se concentration. The determination of the Se in digested materials was accomplished by using an atomic absorption spectrophotometer, equipped with a graphite furnish and a deuterium lamp (Shimadzu AA-6800, GF-AAS, “Shimadzu Corp.”, Tokyo, Japan). The background correction was done using a matrix modifier (Pd(NO₃)₂, 0.5 mol M in HNO₃).

Once the experimental site was set up, fresh composite soil samples were collected from the pots using an auger, dried at 40 °C for 24 h, ground and sieved to 2 mm and 250 µm. All soil analyses were conducted following standardized protocols.
Soil particle-size distribution was determined through sedimentation and sieving upon organic matter removal by H₂O₂ following the NFX 31-107 French standard. pH (H₂O) was determined according to ISO 10390 standard by stirring a mixture of soil and deionized water (1:5, v/v).
For determining Cd, Pb, and Zn pseudototal concentrations, soil acid digestion was performed according to Al Souki et al. [3 (link)] with the use of a digestion plate (HotBlock^TM Environmental Express, Charleston, SC, USA). An aqua regia solution (HCl + HNO₃, 3:1, 6 mL) was added and the aliquot was heated at 120 °C for 120 min. The extraction and analysis quality control was provided by introducing two internal reference samples and a certified soil reference (CRM 141, IRMM, Geel, Belgium). Cd, Pb and Zn concentrations in the extracts were detected by atomic absorption spectrophotometry (AA-6800, Shimadzu, Kyoto, Japan).

T.E. concentration in leaves was determined according to Waterlot et al. [38 (link)], in which 300 mg of each sample were acid-digested with nitric acid (HNO₃, 70%) and heated at 95 °C for 75 min. Following the addition of hydrogen peroxide (H₂O₂, 30%), the solution was heated for further 180 min prior to adding osmosed water. Cd, Pb, and Zn concentrations in the solutions were determined by atomic absorption spectrophotometry (AA 6800, Shimadzu, Kyoto, Japan). Quality control for chemical extraction and digestion was performed by including blanks and internal and certified (Polish Virginia tobacco leaves, INCTPVTL–6, Warsaw, Poland) reference materials.

A clay-loam soil was air-dried, passed through a 2 mm sieve and analyzed for its basic physical and chemical properties. Water-holding capacity was 300 mL kg^-1 and the permanent wilting point was 120 mL kg^-1. On a dry weight basis: a pH (H₂O) of 8.3, CaCO₃ concentration of 240 g kg^-1, total organic C of 9.9 g kg^-1, total N concentration of 1.0 g kg^-1, CSC of 20.1 cmol⁽⁺⁾ kg^-1 and total Se concentration of 12.0 mg kg^-1. Soil analyses were performed following official methods (Carter and Gregorich, 2007 ). For the determination of Se concentration, acid digestion of soil [0.25 g was performed with a mixture of HNO₃ and H₂O₂ (8:2, v/v)]. The determination of the Se in digested materials was accomplished by using an atomic absorption spectrophotometer (US Environmental Protection Agency [EPA], 1996 ), equipped with a graphite furnace and a deuterium lamp (Shimadzu AA-6800, GF-AAS, “Shimadzu Corp.”, Tokyo, Japan). The back-ground correction was carried out using a matrix modifier of Pd(NO₃)₂ in 0.5 M in HNO₃ (Sigma-Aldrich, St. Louis, MO, United States).