Novaa300

Total Hg and Al were determined in Danio rerio’s head and body by atomic absorption spectrometry (AAS) with atomization in a graphite oven (GF-AAS) and cold steam (CV-AAS) using Analytik Jena spectrometers (NovAA 300 models with hydride generator module HS60 and ContrAA 700). The analyzed samples underwent pre-treatment for total digestion in a microwave oven (Anton Paar brand, Graz, Austria). The samples were digested by acid using the oxidant mixture HNO₃ + H₂O₂ according to the EPA3050B reference method described by the EPA [47 ]. After total digestion, the samples were diluted to 25 mL in ultrapure water (Milli-Q) and analyzed according to the methodologies validated for each element. For the determination of total Hg, mol/L HCl and NaBH₄ (0.25% w/v) solutions were used as acidic and reducing media, respectively. Hollow cathode lamps were operated at 4 mA. The wavelength was set to 253.7 nm for Hg and 309.3 nm for Al, and the spectral band passes at 0.5 nm for both metals. The final concentration of each element was calculated in µg/g according to the wet mass used in each digested sample (~300 mg).

Soil pH was determined by a combination of glass electrodes using a 1: 2.5 (w: v) ratio of soil to distilled water (Mi et al., 2018 (link)). Soil total C (TOC) and N (TON) were measured by LECO CNS Combustion Analyzer (LECO, CNS 2000, LECO Corporation, Michigan, United States) following manufacturer protocol. Available phosphorus (AP) in soil was determined by ammonium fluoride extraction (Bray and Kurtz, 1945 (link)). Soil available K⁺, Ca²⁺, and Mg²⁺ were extracted using 1 M KCl (1:10), and determined by using atomic absorption spectrophotometry (NovAA300, Analytik Jena, Germany).

Soil organic matter was determined using the K₂Cr₂O₇ titration method. Soil available P was extracted with 0.5 M NaHCO₃ (pH 8.5) and then determined using the Olsen method [37 ], and soil available K was extracted with 1 M ammonium acetate (pH 7.0), and measured by atomic absorption spectrometry (NovAA300, Analytik Jena AG). The total N content and the atom% ¹⁵N of soils and plants were determined using a stable isotope ratio mass spectrometer (Isoprime100 and Vario Pyrd/Cube, Elementar, Germany). Soil mineral N content (N_min: NH₄⁺-N and NO₃^–-N) was extracted with 1M KCl and determined using a flow injection analyzer (FLA star 5000 Analyzer, Foss, Denmark). Soil microbial biomass N content (MBN) was determined by chloroform fumigation-extraction protocol, extracted with 0.5M K₂SO₄, and analyzed using a multi N/C analyzer (Multi N/C 3100/HT1300, Analytik Jena AG, Germany). The atom% ¹⁵N of N_min and MBN was determined using a stable isotope ratio mass spectrometer (Isoprime100 and Vario Pyrd/Cube, Elementar, Germany).

Acid digestion was used to determine the amount of metals, including lead, copper and iron (Pb, Cu, and Fe), contained in the VCO according the method reported by Ang and Lee (2005). About 0.5 g of VCO was added into 9 ml of freshly‐prepared mixture of HNO₃ (63%) and HCl (37%) at 1:3 ratio in a digestion flask. The mixture was boiled gently over a water bath at a temperature of 80–90°C for 4–5 hr until the sample had completely dissolved. Once digestion has completed, the mixture was then cooled down to room temperature and filtered through filter paper (Whatman No. 42; 2.5‐μm particle retention). The extract was then evaporated to remove excessive acid, and the volume was topped up to 50 ml with distilled water. The metals in the VCO were quantitatively measured using Nov AA 300 (Analytik Jena, Germany) atomic absorption spectrometer (AAS). The measurement condition was optimized for the determination of the metals with the limit of detection of 1 μg/l. The standard calibration of the metals was measured from 0 to 10 ppm.

For measurement to Na⁺ and K⁺. the 3-d-old seedlings grown under normal conditions were transferred to the MS medium containing 0 or 150 mM NaCl and then grown for 2 d. The shoots and roots from these 5-d-old plant were separately harvested, dried for 48 h at 80 °C and then ground to powder. The same mass tissue powder was digested in concentrated (69%, v/v) HNO₃ for 24 h at room temperature for elemental extraction. Na⁺ and K⁺ concentrations was determined by atomic absorption spectrophotometry (novAA300, analytikjena).

The composition of LB (Luria-Bertani) liquid medium was 5.0 g/L beef extract, 10.0 g/L peptone, 5.0 g/L NaCl, pH 7.0. Taking 79 mL LB liquid medium into each beaker flask, all of the media were autoclaved at 121 °C for 20 min, 1 mL of the logarithmic phase bacteria was added in the liquid medium, the initial optical densities (OD₆₀₀) were all kept at 0.03, and 10 mL of 20 mM CdCl₂·2.5H₂O solution and 20.0 g/L of urea solution filtered through 0.22 µm filter were added, respectively. Then, the samples were incubated (170 rpm) at 37 °C for 120 h. Samples were taken at regular time intervals and the pH was measured. The concentrations of Cd²⁺ in the samples were measured using an atomic absorption spectrometer (novAA300, Analytik Jena AG, Jena, Germany).