Behavioral responses of juvenile largemouth bass after acute Cd poisoning
The appearance characteristics of juvenile largemouth bass under Cd exposure are shown in Fig. 1. After Cd exposure, largemouth bass at two low concentrations of 85mg/L and 90mg/L were able to maintain vitality and swim normally without obvious damage to their appearance in the early stage of exposure. After 24 h Cd exposure group the juvenile largemouth bass was relatively anxious and started to die. A layer of pinkish-white mucus was formed on the skin at three high exposures of 105mg/L, 100mg/L and 95mg/L. After a certain time of Cd exposure, the swimming direction of the fish becomes confused and cannot swim smoothly, they will rush to the water surface at a faster speed and stay or stick to the glass tank wall. With increasing exposure time, the fish reach the limit of their tolerance to Cd as they swim sluggishly or stay quietly in the tank and almost stop swimming. Largemouth bass juveniles die of Cd poisoning with a large mouth spread. The dead largemouth bass has a lot of mucus inside the throat and outside the body, the whole head is slightly red, and there is red blood around the inside of the muzzle, the outside of the eye and the gill cover. moreover, part of the tail will show a curved state. After dissection, the gills of the fish were found to be edematous, the inside of the gills contained a large amount of transparent mucus, the liver had deep red color, and after a certain time of death, a yellowish fluid would flow from the abdomen. At the later stage of the experiment, the white foam was floating on the water surface, a layer of the scale was left on the fish tank wall, the color of the water was slightly yellow and emitted a fishy smell.
LC50 of Cd in juvenile largemouth bass
The average mortality in each group of juvenile largemouth bass under acute Cd exposure is shown in Table 2. Information related to the LC50 of juvenile largemouth bass is shown in Table 3. The LC50 results for 48h, 72h and 96h under cadmium exposure were 88.90mg/L, 86.74mg/L and 86.65mg/L respectively which were in the upper and lower LC50 range and the safe concentration was 8.67mg/L.
Table 2
The average mortality of juvenile largemouth bass exposed to Cd for 24h, 48h, 72h and 96h
Exposure time (h)
|
85mg/L(tail)
|
90mg/L(tail)
|
95mg/L(tail)
|
100mg/L(tail)
|
105mg/L(tail)
|
24
|
0
|
1
|
2
|
4
|
9
|
48
|
0
|
3
|
6
|
8
|
9
|
72
|
2
|
5
|
7
|
8
|
9
|
96
|
2
|
6
|
8
|
9
|
9
|
Table 3
Linear regression calculation of LC50 results
Exposure time
(h)
|
Regression equation
|
LC50
(mg/L)
|
LC50 lower limit (mg/L)
|
LC50 upper limit (mg/L)
|
48
|
y=22.282+10.556x
|
88.90
|
58.23
|
119.58
|
72
|
y=11.468+11.993x
|
86.74
|
64.30
|
109.18
|
96
|
y=11.018+11.054x
|
86.65
|
64.42
|
108.87
|
Pathological effects of Cd on the liver of juvenile largemouth bass
The liver pathology sections of juvenile largemouth bass after cadmium exposure are shown in Fig. 2. Compared with a and b in Figure 2, the hepatocytes in a are well-defined, but the hepatocytes in b are vacuolated (indicated by the arrow in b) and the nucleus is squeezed to the edge of the cell, each vacuole is well-defined, which can be observed even under 10x microscope (indicated by the arrow in c). The central vein shows vacuole-like degeneration and disorganized arrangement of hepatic corpuscles (indicated by the arrow in d). The liver tissue in e has a more obvious damage condition, the cells are widely spaced, the central vein is dilated, some cells are detached (indicated by the arrow in e), and the hepatocytes are edematous (indicated by the arrow in f).
Pathological effects of cadmium on the spleen of juvenile largemouth bass
The spleen pathology sections of juvenile largemouth bass after Cd exposure are shown in Fig. 3. As seen in Figure 3, the boundaries of splenic white marrow and splenic red marrow can be distinguished from each other, and splenic cord cells are arranged in an orderly manner at the periphery of the splenic sinus. The lymphocytes in b are increased and the splenocyte spacing is increased (indicated by the arrow in b). The splenic cord cells in c are disorganized and difficult to distinguish (indicated by arrow 5), and macrophages are increased (indicated by arrow 4).
Effect of Cd exposure on DNA damage in the liver of largemouth bass
DNA damage to the liver of largemouth bass by Cd exposure is shown in Figure 4. After processing with the Comet Analysis software CASP, it can be visualized that the liver cells of the control group are a smooth red orb (shown in a). The cell trailing at 85 mg/L is short and dense (shown in b), the cells at 90 mg/L start to spread and become longer (shown in c), and the cells at 95 mg/L have long trailing edges and spread in an inverted triangular shape (shown in d). The tails of the liver cells at 100 mg/L and 105 mg/L were trailing increasingly longer and more severely fragmented. At the same time the cells in the head of the comet are breaking up and the edges are starting to become mutilated (shown in e, f). The tail length, tail moment, tail DNA and olive tail moment in Table 4 were obtained using SPSS23 analysis. When the exposure concentration reached 90 mg/L, tail length, tail moment, tail DNA and olive tail moment of juvenile largemouth bass were significantly higher than those of the control group (P < 0.05).
Table 4
Effect of Cd exposure on DNA damage of largemouth bass
Items
(mg/L)
|
Tail length
(um)
|
Tail moment
(um)
|
Tail DNA
(%)
|
Olive tail moment(um)
|
0
85
|
3.00±0.0
4.33±1.53
|
0.01±0.01
0.59±0.35
|
0.36±0.26
6.24±1.23*
|
0.07±0.05
1.53±0.10*
|
90
|
12.33±3.51*
|
5.33±3.24*
|
15.24±5.91*
|
3.63±0.60*
|
95
|
21.00±4.00*
|
13.76±2.04*
|
23.79±2.64*
|
8.83±2.36*
|
100
|
31.67±4.51*
|
18.99±2.41*
|
41.12±3.45*
|
15.52±1.96*
|
105
|
47.67±6.66*
|
27.23±4.90*
|
57.16±10.55*
|
20.75±2.65*
|
*Significant difference (P < 0.05).