## 4 Discussion
### Degradation of DES and E1-3G  
This study investigated the degradation of estrogens in in situ river water. Theoretically, the concentration of estrogen will decrease with time if the degradation occurs; hence, the estrogen concentration ratio (C\({}_{n}\)/C\({}_{0}\)) changed with time, having a negative slope and a \(p\)-value of <0.05 in the linear regression. Figure 3 shows the degradation curves of DES, E1-3G, E1, E2 and E3. The linear regression was statistically tested and the results are listed in Table 4. The linear results show E1-3G had a positive slope value (0.0078) and insignificantly decreased (r = 0.31, \(p\) = 0.56). DES had a negative slope value (\(-\)0.016) and insignificantly decreased (r = 0.60, \(p\) = 0.21). The E1, E2, and E3 had negative slope values and significantly decreased (\(p\) < 0.05) as shown in Table 4. The negative slope value and \(p\) < 0.05 suggests the concentration of E1, E2 and E3 significantly decreased over time. Conjugated estrogen E1-3G had almost no degradation in this study. The initial concentrations of E1-3G ranged from 2.0 to 3.2 ng/L, representing a relatively low concentration. E1-3G can be regarded as residual concentrations and had been through a fast dissociation step after it was released into the river, thus demonstrating slow degradation. This is similar to the [PERSON] and [PERSON] [35] study where additional experiments monitored the E2-3S and E2-3G at every two concentrations of 40 and 4000 ng/g for estrogen degradation and metabolite formation in agricultural soil. At an initial 24 h, more than 95% was removed and reached a steady-state after 36-72 h, with persistence levels remaining after 20 days. Similar results found that the aqueous phase persisted for up to 28 d; E2-3G had additional concentrations of 3.7, 9.1, and 22.5 ug/L [10]. Other possible reasons included the river water lacking a \(\beta\)-glucuronidase enzyme to dissociate Glu-ligand estrogen and the occurrence of reversed reaction between E1-3G and E1 [13].  
Unlike in laboratory experiments, natural intensive feedlot operation receiving environments cannot control experimental factors; they are more complex and continue to be polluted. Compared to the DOC of this study, the estrogen concentration was lower than other carbon sources; at low concentrations, the microorganism may prefer to use the other carbon source [60]. For E2 degradation experiments, previous studies indicated that bacterial strains (_Ochrobarrum_ sp. _Strain FJ1_, E2 degrading bacteria) preferred to use the additional carbon source instead of estradiol [11]. On the other hand, intensive feedlot operations increased the number of veterinary antibiotics used, affecting estrogen persistencewhen released to the aquatic environment. The previous study indicated that veterinary antibiotics used for an animal could affect E2 persistence in swine manure [61]. Therefore, the E1-3G concentrations in the degradation test did not change in 24 h. Additional experiments monitored the E1-3G concentration of 40 and 4000 ng/g for half-lives, 0.81 and 8.56 h\({}^{-1}\), respectively [35]. This study may mean E1-3G was present in the intensive feedlot operation receiving river environment and may have reached a steady-state at low concentration [34; 35].