Materials and methods
2.1 Site description
The CW and the YR sampling sites were in Silong town (36°25.639 N, 104°23.428 E) in the BYR (Fig. 1). Baiyin city is 75 km from Lanzhou, and was one of the first non-ferrous metal smelting and processing areas in China. Large amounts of domestic and industrial wastewater from Baiyin city are discharged into the YR at Silong town, through the East Ditch and the West Ditch, leading to high levels of pollution in the water and soil in the area.
2.2 Wetland design
The CW was 18 m × 15 m, and 1 m deep, and contained 20 cm of homogenous soil planted with cattails Typha angustifolia, a widespread species in BY, in March. The cattails were transplanted ...view middle of the document...
During the experiment, toads were collected from the YR and the CW, at random, at different life stages , which were stage I (Zhao’s stage 26/27: postembryonic development begins), stage II (Zhao’s stage 32/33: five hind toes emerged), stage III (Zhao’s stage 37/38: more than half of the tail reabsorbed), stage IV (metamorphism completed), and stage V (a month after metamorphism completed). The toads were transferred to the laboratory, euthanized by pithing , and their liver coefficients (the liver to total body weight ratio) measured. Blood was collected and the reactive oxygen species (ROS) contents, total antioxidant capacity (T-AOC), and DNA oxidative damage were analyzed. For HM analysis, toad samples from the CW and the YR were randomly chose at different life stages, and were oven dried at 70 °C for 72 h, then ground and passed through a 1.0 mm sieve, before their HM contents were determined.
2.4 Sample analysis
For all the following experiments, toads were caught randomly from CW or the YR, and all the samples were assayed in triplicate.
2.4.1 HM analysis
HM concentrations in water, wetland substrate, and toad samples were analyzed using inductively coupled plasma-atomic emission spectroscopy (IRIS advantage; Thermo Electron, Waltham, MA, USA) at Lanzhou University.
2.4.2 Liver coefficient analysis
After a toad was euthanized, its liver was removed and washed with phosphate-buffered saline (PBS), wiped with a filter paper to remove excess liquid, and weighed. The liver coefficient was determined as the ratio of the liver weight to the total body weight.
2.4.3 ROS content analysis
The ROS contents were determined using 2′,7′-dichlorofluorescein diacetate (DCFH-DA), following the manufacturer’s (Beyotime Institute of Biotechnology, Jiangsu, China)...