3.1 Method validation
The rates of recovery values ranged from 71.7 ± 12.5% to 84.6 ± 5.6% for both FLU enantiomers in sediment. The LOQs were 8.0µg/L for two enantiomers. Eight concentrations (1, 2, 5,10, 20, 50, 100 and 200 µg/L) of each FLU enantiomers were used to construct the calibration curves (R2 > 0.99). Analytical method validation was also described in our previous study [19].
3.2 Enantioselective adsorption of FLU in Sediment
The changes over time in the concentration of Rac-FLU and FLU enantiomers in three different sediments are shown in Fig. 2 (A-C). The original spiked concentrations of Rac-FLU and each FLU enantiomers were 20 mg/L. However, the concentrations in sediment of all antibiotics, detected at the first sampling event, were much lower than the initial spiked concentrations because of the rapid adsorption to suspended particles and sediment.
In the whole adsorption period of 2# sediment, the adsorption capacity of S-(-)-FLU and R-(+)-FLU are higher than the Rac-FLU (Table 2). And there were significant differences in the adsorption capacity of S-(-)-FLU, R-(+)-FLU and Rac-FLU (P < 0.05), but there were no significant differences between S-(-)-FLU and R-(+)-FLU (P > 0.05).
Table 2
Adsorption capacity(mg/kg)of FLU enantiomers in different sediment samples. Error bars are the standard deviations of the means of adsorption tests on three replicates.
|
1#
|
2#
|
3#
|
Rac-FLU
|
135.0 ± 1.2a1
|
164.0 ± 0.8a2
|
149.0 ± 14.6a3
|
S-(-)-FLU
|
165.6 ± 6.1c1
|
173.2 ± 0.4b2
|
161.4 ± 1.3ab3
|
R-(+)-FLU
|
154.6 ± 1.5b1
|
172.6 ± 2.7b2
|
169.6 ± 0.1b3
|
a, b, c : the same superscripts denoted not significant difference (P > 0.05), different superscripts denoted significant difference (P < 0.05). |
1, 2, 3 Represent the three different sediment samples. |
These results indicated that enantioselectivity existed during the adsorption of FLU enantiomers in 1# sediment (Fig. 2D). In the early stage of the adsorption period, EF values were all below 0.5, so the R-(+)-FLU adsorbed faster than the S-(-)-FLU during this period. After 5 d of the adsorption period, the S-(-)-FLU adsorbed faster than the R-(+)-FLU.
Besides, the EF values fluctuated around 0.5 during the whole adsorption period (Fig. 2E). Therefore, the adsorption behaviors of FLU enantiomers had no enantioselectivity in 2# sediment.
These results of Table 2 and Fig. 2F indicate that enantioselectivity existed during the adsorption of FLU enantiomers in 3# sediment. There was significant difference in the adsorption capacity of Rac-FLU and R-(+)-FLU (P < 0.05). In the early stage of the adsorption period, there was no obvious enantioselectivity of FLU enantiomers. After 5 d of the adsorption period, the R-(+)-FLU adsorbed faster than the S-(-)-FLU. These results indicated that the enrichment of one FLU enantiomer entering the environment [22].
Many studies have shown that the adsorption capacity of antibiotics in sediment may be affected by the pH value of different sediments [23,24]. The higher the pH value, the lower the adsorption capacity of antibiotics in sediments. This is mainly because the adsorption of antibiotics is related to the charged state of sediments, and pH value can substantially contribute to the adsorption behavior by changing the charge state of antibiotics [25–27]. In the view of the obtained results, Table 1 shows that 2# sediment had the lowest pH value, however, the adsorption capacity of FLU in 2# sediment was significantly stronger than the 1# and 3# sediments. Besides, enantioselectivity existed during the adsorption of FLU enantiomers in 1# and 3# sediments, so the stereoselective adsorption differences of FLU enantiomers in sediments is also related to the pH value of sediments.
3.3 Enantioselective degradation of FLU in sediment under sterile condition
The degradation of the FLU enantiomers in three different sediments showed first order kinetic behavior, with the correlation coefficient values (R2) between 0.7235 to 0.9135 (Table 3). The degradation curves of FLU enantiomers were given in Fig. 3A-C, the data show that both R-(+)-FLU and S-(-)-FLU degraded over time and both enantiomers disappeared at similar rates in three different sediments under sterile conditions.
Table 3
The degradation of kinetic equations and half-life period under sterile and natural conditions
Sediment
|
FLU
|
Sterile conditions
|
Natural conditions
|
Kinetic equations
|
R2
|
t1/2(d)
|
Kinetic equations
|
R2
|
t1/2(d)
|
1#
|
S-(-)-FLU
|
Y = -0.0160x − 1.7932
|
0.7235
|
43.31
|
Y = -0.0127x − 1.5467
|
0.8168
|
54.57
|
R-(+)-FLU
|
Y = -0.0177x − 1.6003
|
0.7863
|
39.15
|
Y = -0.0144x − 1.1550
|
0.8017
|
48.13
|
2#
|
S-(-)-FLU
|
Y = -0.0176x − 0.5709
|
0.8059
|
39.38
|
Y = -0.0076x − 0.2486
|
0.5830
|
91.18
|
R-(+)-FLU
|
Y = -0.0202x − 0.6548
|
0.7757
|
34.31
|
Y = -0.0084x − 0.2404
|
0.6223
|
82.50
|
3#
|
S-(-)-FLU
|
Y = -0.0174x − 0.4680
|
0.9063
|
39.83
|
Y = -0.0121x − 0.7678
|
0.8045
|
57.27
|
R-(+)-FLU
|
Y = -0.0182x − 0.3692
|
0.9135
|
38.08
|
Y = -0.0141x − 0.5311
|
0.8875
|
49.15
|
R2 Represent determination coefficient |
As shown in Table 3, the degradation of FLU enantiomers in 2# sediment (t1/2=39.38 days for S-(-)-FLU, 34.31 days for R-(+)-FLU) was slightly faster than those of other sediments. Table 1 shows the lowest pH (7.03), and lowest organic content (10.9687 g/kg) in 2# sediment, therefore, it can be speculated that the pH value and organic content in sediment were the factors affecting the degradation rate of FLU enantiomers in sterile condition. More importantly, the R-(+)-FLU degraded more rapidly than S-(-)-FLU in three sediments.
In the three kind of test sediments, the EF values (Fig. 3D-F) were nearly 0.5 during the whole period. It can make a conclusion that R-(+)-FLU and S-(-)-FLU degradation were not enantioselective in sediment under sterilized condition due to no microbial activity. Thus, microbial decomposition can play an important role in stereoselective metabolism of FLU degradation in three sediments.
3.4 Enantioselective degradation of FLU in sediment under natural condition
Figure 4A-C show the degradation curves of both FLU enantiomers under natural conditions in three different sediments, it can be seen that both enantiomers disappeared over time. However, in 2# sediment FLU enantiomers were degraded to about 10mg/L and then the concentration of enantiomers increased significantly after 56 days of degradation. After that the concentration of both enantiomers dropped to 3 mg/L. As is well known, the environmental sediments are very complex, they have different compositions and present high variability [28]. So, the microorganism action and differences in the composition of sediments could play a role in this change [24]. Therefore, except for 2# sediment, the degradation of both FLU enantiomers in 1# and 3# sediment under natural conditions followed first-order kinetics with R2 ranging from 0.8017 to 0.8875 (Table 3) and the first-order rate constants were derived from ln (C0/C) versus t plots by regression analysis for each experiment.
The enantiomers have the similar half-life in 1# and 3# sediments, however, the observed differences of the half-life in 2# sediment (t1/2=91.18 days for S-(-)-FLU, 82.50 days for R-(+)-FLU) may be determined by the complex organic matrix and pH value. Compared with the half-life of FLU enantiomers in sterile condition, a slower dissipation of FLU enantiomers in sediments under natural condition were observed.
The EF values (Fig. 4D-F) showed that enantioselectivity existed during the degradation process of FLU enantiomers in different sediment. There was an increasing trend of EF value with time in 1# sediment, that indicate the S-(-)-FLU degraded more rapidly. However, the EFs were under 0.5 (after 28 days in 2# sediment) in 2# and 3# sediment and decreased with time. The data suggest that the slow degradation of S-(-)-FLU. The enantioselective degradation rate of FLU enantiomers is different between three different sediments, probably because the chemical or physical activities of high organic matter in 1# sediment.
It is clear that microbial activities played a major role in enantioselective degradation of FLU. Moreover, the organic content of sediments is important to explain the differences in the degradation behavior, and pH value probably play an important role in enantioselectivity of FLU enantiomers across different sediments [24,29,30].
In addition, the structure of chiral compounds is not stable, so more research had been done to clarify whether there are underlying processes of enantiomeric inversion and transformation in the environment. The S-(-)-FLU (or R-(+)-FLU) was respectively added into the sediment, and the results showed that no R-(+)-FLU (or S-(-)-FLU) was detected at any time during the whole degradation process under natural or sterilized conditions.
3.5 Enantioselective transformation of FLU in water-sediment system
The change over time in the concentration of FLU in the sediment of the water-sediment system are shown in Fig. 5A. The original spiked concentrations of the FLU in the overlying water were 20 mg/kg. The concentrations in sediment of FLU, detected at the earlier sampling event (7 days), were much lower than the initial spiked concentrations. However, because of the rapid sorption to suspended particles and sediment, the concentration of FLU in sediment rapidly increased. Concentration profiles in the overlying water and sediment suggested that the diffusive transfer of FLU into sediment was a quick process, with the FLU enantiomers generally detected in sediment at a maximum concentration about 14 mg/kg at a very short sampling interval. After that, the degradation was observed during the experiment period, this may be attributed to microbial degradation. These results also suggest that sediment can potentially act as a significant secondary source of antibiotics that can be released into water [31,32].
The EF values (around 0.5) in Fig. 5B show that, the transformation behavior of FLU enantiomers had no enantioselectivity in water-sediment system before 150 days. However, the stereoselective transformation behavior occurred after 150 days because of an increase of EF values level. The results indicated that the transformation of FLU enantiomers in water-sediment system had enantioselective behavior, and R-(+)-FLU transformed faster than S-(-)-FLU.
3.6 Main metabolites of FLU identification
Identification of molecular ions representing possible metabolites is an indispensable step in the overall identification procedure of drug metabolites using LC/MS/MS approaches [33]. 13C labeled FLU in sediment samples were analyzed. We obtained fragmentation patterns, showing intense ion at m/z 265 (13C-FLU), m/z 207, m/z 247 (Fig. 6A-C).
Figure 7A describes the concentration of m/z 207 (265-COOH) metabolite increased during the experiment period. While the content of m/z 247 (265-OH) metabolite rapidly increased and then gradually declined (Fig. 7B), the metabolite degradation maybe due to the microorganism action. These were demonstrated that the main metabolites of FLU in sediment were decarboxylate and dehydroxylation.