Spectraa 220fs

The method described by AOAC [13 ] was used for mineral analysis. The sample was ashed at 550°C and the ash boiled with 10 mL of 20% HCl in a beaker and then filtered into a 100 mL standard flask. The minerals (calcium, magnesium, sodium, potassium, iron copper, zinc, and manganese) were determined by an atomic absorption spectrometer (Varian 220FS SpectrAA, Les Ulis, France). All samples were analyzed in triplicate.

Testicular cadmium content was determined by atomic absorption spectrophotometry and by energy-dispersive X-ray spectroscopy (EDS) [6 (link), 25 (link)]. Testis samples were weighed and dried at 70°C until a constant dry weight was achieved. Dried samples were digested (30 min) in Erlenmeyer flasks with 1.5 mL concentrated HNO₃ and 0.5 mL HClO₄ (70%) using a plate heater, where the temperature was gradually increased from 70°C to 90°C. After digestion, the samples were diluted in deionized water (25 mL) and filtered. Cadmium concentration in each sample was determined using an atomic absorption spectrophotometer (Varian 220FS SpectrAA, Palo Alto, California, USA).
Relative Cd dose-dependent testicular accumulation was investigated by EDS using a scanning electron microscope (Leo 1430VP, Carl Zeiss, Jena, Thuringia, Germany) with an attached X-ray detector system (Tracor TN5502, Middleton, Wisconsin, USA) [26 (link)]. Small pieces of the testis from each animal were dehydrated at 60°C and coated with a thin film of evaporated carbon (Quorum Q150 T, East Grinstead, West Sussex, England, UK). The EDS microanalysis was performed at ×1000 magnification, with an accelerating voltage of 20 kV and a working distance of 19 mm [6 (link)]. Cadmium distribution was normalized considering the mean distribution of reference minerals (Na, Ca, K, P, Mg, Fe, and S) [27 (link), 28 (link)].

The concentration of accumulated selenium was determined in wet weight (ww) in the hatch and four-month-old fry euthanised by MS-222 overdose and stunning with the destruction of the brain. For the purpose of mineralisation, 2 g of wet material was collected. The material was mineralised in a MARS-5 microwave oven by CAM (Matthews, USA). In order to check the accuracy of the method, selenium content was determined in a reference material—DOLT-2 from the National Research Council of Canada. Selenium reference content was 6060 ± 409 μg kg⁻¹. The value obtained in the analytical procedure was 5850 ± 432 μg kg⁻¹. Selenium concentrations were determined using hydride generation atomic absorption spectrometry (HG AAS) on a VARIAN SpectrAA 220 FS apparatus (Melbourne, Australia). The methodology of Diaz-Alarcon et al. [71 (link)] was utilised.

The concentrations of Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn in the investigated samples were determined by the fast sequential atomic absorption spectrometer SpectrAA 220 FS (Varian, Australia) equipped with HCL lamps (Varian, Australia) and the Sampling System with Electronic Control Module SIPS-20 (Varian, Australia). The description of the applied conditions is given in Table A1. The calibration was performed using standard analytical solutions (Merck, Germany). Prior to the analysis, the detection method was validated with reference materials: LKSD-1 (sediment), ERM-CE278K (mussel), and BCR-060 (aquatic plant). The recovery rate exceeded 90 % for all determined elements, at low RSD values (below 5 %). The limit of metal detection was 0.01 mg L⁻¹ for water samples and 0.01 mg kg⁻¹ for sediments, bivalve and Phragmites australis samples.

For the extraction of heavy metals (i.e., Cd, Pb, Zn, Ni and Cu), 0.5 ± 0.05 g of soil was processed by means of a digester tube using 3 mL of hydrogen peroxide to remove the organic matter. Soil samples were then mineralized with 9 mL of hydrochloric acid (Baker Instra-Analyzed, Fisher Scientific, Waltham, MA, USA) and 3 mL of nitric acid (68%) for 40 min at 180 °C and then filtered by means of Whatman no. 42 filters (pore size: 2.5 µm). An atomic absorption spectrophotometer (SpectrAA 220 FS, Varian, Santa Clara, CA, USA) was used for the determination of heavy metals. The concentrations of heavy metals in the soil samples were determined by means of calibration curves, and procedural blanks were used to ensure the absence of contamination in the laboratory. Calibration curves were prepared by diluting a multi-Element standard mix of heavy metals in 10% HNO₃ (O2Si Smart Solutions, North Charleston, SC, USA).
The chemical analyses were carried out in triplicate.