## 3 Results and Discussion
### Toxic Effects of Sediment Porewater on Zebrafish Embryos  
In this research, the effects of 35 sediment porewater samples on the development of zebrafish in different regions of Taiwan were evaluated after four days of exposure. The results of mortality, hatchability, and deformity rate on zebrafish embryos were recorded daily to evaluate the toxic effects of the sediment porewater as shown in Figures 3-5. Embryos of nearly all fertilized eggs hatched on the third day of the general culture and the hatchability reached more than 98% in cases. In some instances, the control group could also show abnormal development due to physical differences between the organisms. However, the deformity ratio of the control group was low at about 1%. The samples tested were based on the results of mortality and nine sediment porewater samples, HJ7, DB 1-5, and SY 1-3 were classified as being acutely toxic where all zebrafish embryos died after one day of immersion. The toxicity of four samples from YS4, WR1, WR 3, and WR 6 showed the second highest toxicity, which could also be classified as the highest  
Figure 2: Survival rate of _Hyallella zateca_ exposed to 35 sediment porewater samples in eight river basins. Error bars represent standard deviations (n = 2). toxicity due to the fact that all zebrafish embryos were dead after a two-day exposure. When zebrafish embryos were exposed to porewater samples from HJ3, DG1, LK1, LK2, HJ2, LK3, YS1-3, WR2, WR 4, and WR 5, either all of them died or the survival rate was lower than 20% on the third day. We classified such porewater samples as moderately toxic. The other 10 porewater samples (HJ1, DG3, HJ5, DG4, YS5, HJ6, AGD1, AGD2, HJ4, and DG2) were classified as less toxic. The survival rate here was still roughly higher than 15% on the fourth day of exposure. The embryonic development of zebrafish lasted for about 2 days and the main organs of zebrafish were developing thereafter until larval fishes hatched from their eggs. The development of zebrafish was seriously affected by pollutants contained in the porewater samples, causing incomplete development of organs in the fish embryos as shown in Figures 3-5. In terms of deformity, a high deformity ratio was discovered in the moderate toxicity samples HJ3 and YS3. At site HJ3, 78% of the samples showed deformation while the survival rate was still below 69% on the second day. Recent research found that there were up to 85% deformities in the surviving individuals of the porewater sample from YS3. The remaining less toxic samples showed that mortality and deformity rates increased at longer treatment periods. Moreover, the hatching rate was low and showed delays until hatching. Although less toxic samples were classified with low toxicity, they still showed a significant toxic effect compared to the control group. Figure 4: Toxic effects on survival, hatching rate, and deformity rate of zebrafish embryos of river sediment porewater from different sampling sites in Kaohsiung city. Error bars represent standard deviations (n = 3). Figure 3: Toxic effects on survival, hatching rate, and deformity rate of zebrafish embryos of river sediment porewater from different sampling sites in Tainan city. Error bars represent standard deviations (n = 3). ### Toxic Effects of Sediment Porewater on Zebrafish Embryos  
In general, zebrafish hatch after 2-3 days post-fertilization (2 dpf), when most of the organs are developed. In our study, the morphology of zebrafish was observed on the 2 nd and 3 rd day of sediment porewater immersion with results shown in Figures S1 and S2. The zebrafish in the control group show normal development on the 2 nd day. Their eyes, trunk, and yolk sacs indicate a clear formation of melanin; however, most samples from sediment porewater of different rivers show developmental stagnation (such as sample HJ3-1, HJ3-2, HJ2, HJ3, and HJ5-2) and effects of delay. Deformity of spinal cord and tail curvature could also be observed (sample HJ5-1, DG4-1, DG4-2, YS1-1, WR4-3, and others).