Enantioselective Behavior of Flumequine Enantiomers and Metabolites' Identification in Sediment

The enantioselective adsorption, degradation, and transformation of flumequine (FLU) enantiomers in sediment were investigated to elucidate the enantioselective environmental behaviors. The results of adsorption test showed that stereoselective differences of FLU enantiomers in sediment samples and the adsorbing capacity of S-(−)-FLU and R-(+)-FLU are higher than the racemate, and the pH values of the sediment determined the adsorption capacity. Enantioselective degradation behaviors were found under nonsterilized conditions and followed pseudo-first-order kinetic. The R-(+)-FLU was preferentially degraded, and there was significant enantioselectivity of the degradation of FLU. It can be concluded that the microorganism was the main reason for the stereoselective degradation in sediments. The physicochemical property of sediments, such as pH value and organic matter content, can affect the degradation rate of FLU. In addition, the process of transformation of FLU enantiomers in water-sediment system had enantioselective behavior, and R-(+)-FLU was preferential transformed. Meanwhile, the main metabolites of FLU in the sediment were decarboxylate and dihydroxylation products. This study contributes the evidence of comprehensively assessing the fate and risk of chiral FLU antibiotic and enantioselective behavior in the environment.


Introduction
In the past several decades, the residues of the veterinary antibiotics used to treat bacterial infections in humans and animals have been extensively detected in various aquatic and sediment environments [1,2]. Flumequine (FLU), a broad-spectrum antibiotic agent of the fuoroquinolone family, has been commonly used in poultry and aquatic animals especially against Gram-negative bacteria [3]. Te main mechanism of FLU is based on inhibiting the nucleic acid synthesis of bacterial action to terminating the normal DNA replication and synthesis [4]. Particularly, FLU is directly applied as a feed additive in aquaculture, which might be retained in the surrounding waters or sediments, owing to their poor bioavailability in aquatic animals. Te low bioavailability may result in high concentrations of FLU residues in the aquatic and sediment environments [5].
Approximately 50% of quinolone drugs are chiral compounds and have at least one chiral center in the chemical structure, and most are dispensed and manufactured in the racemic form [6][7][8]. For many enantiomeric drugs, although the enantiomers have similar physical and chemical properties, they not only produce diferent pharmacological and toxicological efects but also can be subject enantiospecifc metabolism and pharmacokinetic in living systems [9,10].
FLU has one chiral center and the two enantiomers (Figure 1). Its absolute confguration was confrmed with S-(−)-FLU and R-(+)-FLU [11]. Studies have shown that the antibacterial activity of FLU enantiomers is signifcantly diferent. Wang et al. found that photolysis is the main degradation of FLU in seawater, and the existence of microorganisms led to the diference in degradation of FLU enantiomers [12]. Li studied the stereoselective behaviors of FLU residues in red sea bream after intragastric administration [11]. Tey found that the half-lives of S-(−)-FLU and R-(+)-FLU were 12.4 h and 11.2 h, respectively. Plasma concentration of S-(−)-FLU is always higher than that of R-(+)-FLU.
Te degradation behaviors of Rac-FLU in water and sediment are afected by environmental factors such as light [5] and microbial activities [13,14]. However, the investigations of FLU at the enantiomeric level are limited, especially for some complex matrices such as sediments and the water-sediment system.
In addition, FLU is commonly employed in aquaculture as the racemic form and its enantiomers are frequently ignored, so the risk assessment of FLU in the traditional racemic level is inaccurate [15]. Terefore, it is an important consideration to elucidate the fate, enantioselective behaviors, and ecotoxicological efects of FLU enantiomers in water and the sediment [16,17].
Te aim of this study was, therefore, to identify the environmental behaviors of enrichment, degradation, and transformation and mechanism at the enantiomer level. Simultaneously, its metabolite will be identifed in order to understand the fate and efects of FLU enantiomers pollution on the environment, especially in the sediment.

Materials and Methods
Te materials and methods are described in the following sections.

Preparation of Standard Stock Solutions.
Standard solutions of Rac-FLU and S-(−) and R-(+)-FLU enantiomers were prepared in pure ACN to achieve a fnal concentration of 200.0 mg/L. All solutions were protected against light and stored in the dark at 4°C.

Sediment Sample Collection and Preparation.
Sediment samples were obtained from three diferent pools (0-20 cm surface layer) in Jinghai District, Tianjin City, China, using the bottom sampler to collect sediment samples at the bottom of pools. All sediment samples were randomly collected in triplicate from an area of approximately 1 m 2 in the center of each sediment site. All samples were refrigerated in storage at 4°C and returned to the laboratory. Te sediment samples were air dried frst, and the particle size refers to the original particle size. Te physicochemical properties of sampled sediments are listed in Table 1. Tese samples did not contain the target analytes. After the natural drying process, the sediment samples were homogenized into powder and were passed through the mesh sieve and stored in the refrigerator at −20°C until analysis. Te sample collection and preparation progress are similar to our previously published report [18].

Adsorption Experiment.
Te background solution was prepared with 0.005 mol/L CaCl 2 (maintaining ion concentration) and 100 mg/L NaN 3 (inhibiting microbial activity). Rac-FLU and S-(−) and R-(+)-FLU enantiomers were added to the background solution, respectively. Finally, the concentration of FLU solution used in the experiment was 20 mg/L.
A total of 20 mL FLU solution and 1.0 g sediment sample (dry weight) were added to 50 mL centrifuge tubes with a screw cap. All the centrifuge tubes were shaken at 25°C in a temperature-controlled shaking incubator at a shaking speed of 230 rpm. Te shaking incubator was covered with a black cloth, and all procedures were conducted in the dark to avoid photodegradation. Sampling after shaking starts at 30 9 d, and 10 d. Ten, these samples were centrifuged at 8000 rpm for 10 min. Te supernatant was then fltered through a 0.22 μm syringe flter before Q-TOF/MS analysis. All experiments were conducted in triplicates. Blank samples contain the same concentration of FLU and a total background solution volume of 20 mL (without sediment). Te procedure was consistent with the above.
Te amount of FLU adsorbed to the sediment was calculated as where Cs (mg/kg) is the uptake amount of the FLU at equilibrium, Ci (mg/L) and Ce (mg/L) are the initial and equilibrium concentrations of FLU in solution, M(g) is the mass of the sorbent, and V(L) is the volume of the solution.
2.6. Degradation Experiment. Te degradation experiment of FLU enantiomers were examined under both sterilized and nonsterilized condition in three diferent sediments. Te sterilized experiment represents abiotic degradation only, 250 g sediment (dry weight) was weighed into 500 mL conical fask bottles, and the sediment was sterilized at 120°C for 15 min and then poured into 125 mL sterile water prior to the addition of the FLU enantiomers. Te sediment-to-solution ratios adopted were 2 : 1 (2 g of sediment to 1 mL of solution). Te initial concentration of Rac-FLU and S-(−) and R-(+)-FLU enantiomers (20 mg/kg) was used by adding into each conical fask, respectively. Both sterilized and nonsterilized conical fasks were sealed with cotton wool. After that, they were put into a thermotank at 35°C and prevented from light exposure (the sterilized group were placed in a sterile thermotank). For both sterilized and nonsterilized degradation experiments, samples were collected on 0, 3, 7, 14, 28, 42, 56, 70, 84, 98, 128, 158, and 188 d after treatment and stored at −80°C until analysis. All experiments were conducted in triplicates. For the nonsterilized experiment, the sediment and water used in experiment were not sterilized. Other experimental procedures are the same as the sterilized experiment.
Data from the degradation experiments were ftted to the frst-order equations: where C t is the concentrations of antibiotics (mg/kg) for time t (days), C 0 is the initial antibiotics concentration (mg/ kg), and k is the degradation coefcient. Half-lives (t 1/2 , d) were calculated by the equation: t 1/2 � ln 2/k.

Transformation Experiment.
Sediments can serve as a source in processes involving the migration and transformation of antibiotics [19]. Two rectangle water tanks were used to perform the migration and transformation experiment, and the tank is made of glass in order to reduce the sorption of antibiotics. Te tanks were housed in a large laboratory, with the temperature of the room kept at 20 ± 2°C. About 2 kg of sediment were laid evenly at the bottom of the tank, and 3 kg of water were then slowly added to the tank, about 50 mm above the surface of sediment.
Ten, 20 mg/kg of Rac-FLU antibiotics were dissolved and spiked into the water. 5 g of sediment samples were accurately weighed and collected in diferent sampling periods to observe the migration of the FLU antibiotics from water to sediment, and these results could be useful for assessing the migration and fate of commonly used antibiotics in watersediment systems.

Sample Extraction and Purifcation.
Te FLU enantiomers in sediment and water were determined according to the procedures described in our previous study [18]. Briefy, dry sediment samples (2.00 ± 0.01 g) were weighed into a 50 mL centrifuge tube, and then these sediment samples were extracted three times with 30 mL ACN and EDTA-Mcllvaine bufer solution (40 : 60, v/v). Te extract solution for each sample was evaporated and diluted to 30 mL with Milli-Q water. Te extracts were then passed through Cleanert PEP (polar enhanced polymer) cartridges for purifcation. Te analytes were eluted from each cartridge with 6 mL methanol and dried under a gentle nitrogen stream. Ten the resultant residue was fnally redissolved in 1 mL methanol and fltered through a 0.22 μm flter for HPLC-Q-TOF/MS analysis and quantifcation.

Enantiomer Q-TOF-MS Determination.
Te chromatographic analysis of the FLU enantiomers was performed on an accurate mass tandem quadrupole-time-offight (Q-TOF) mass spectrometer with a chiral Lux Cellulose-2 column. Te mobile phase consisted of 0.2% acetic acid in water as solvent A and acetonitrile as solvent B. Te gradient elution program was as follows: 0-20 min, A : B (45 : 55, V/V); 20-24 min, A : B (5 : 95, V/V); and 24-25 min, A : B (45 : 55, V/V). Te injected volume was set at 1 μL, and the total run time was 30 min at a fow of 1 mL/ min [18].
Te enantiomeric fraction (EF) was used to measure the enantioselectivity of FLU in the sediment during these experiments. Te EF was described by the following equation: Te EF value ranges from 0 to 1, and EF � 0.5 represents the racemate.

Results and Discussion
Te results and discussion of the study are explained in the following sections.  [18]. Besides, the EF values fuctuated around 0.5 during the whole adsorption period (Figure 2(e)). Terefore, the adsorption behaviors of FLU enantiomers had no enantioselectivity in 2# sediment.

Enantioselective
Tese results of Table 2 and Figure 2(f) indicate that enantioselectivity existed during the adsorption of FLU enantiomers in 3# sediment. Tere was signifcant diference 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. Tese results indicated that the enrichment of one FLU enantiomer entering the environment [20].
Many studies have shown that the adsorption capacity of antibiotics in the sediment may be afected by the pH value of diferent sediments [21,22]. Te higher the pH value, the lower the adsorption capacity of antibiotics in sediments. Tis 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 [23][24][25]. 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 signifcantly 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 diferences of FLU enantiomers in sediments is also related to the pH value of sediments.

Enantioselective Degradation of FLU in the Sediment under Sterile
Condition. Te degradation of the FLU enantiomers in three diferent sediments showed frst-order kinetic behavior, with the correlation coefcient values (R 2 ) between 0.7235 and 0.9135 (Table 3)

. Te degradation curves of FLU enantiomers were given in Figures 3(a)-3(c), and the data show that both R-(+)-FLU and S-(−)-FLU degraded over time and both enantiomers disappeared at similar rates in three diferent sediments under sterile conditions.
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). Tere were signifcant diferences in the adsorption capacity of S-(−)-FLU, R-(+)-FLU and Rac-FLU (P < 0.05), but there were no signifcant differences between S-(−)-FLU and R-(+)-FLU (P > 0.05).
As shown in Table 3, the degradation of FLU enantiomers in 2# sediment (t 1/2 � 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 the sediment were the factors afecting 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 kinds of test sediments, the EF values (Figures 3(d)-3(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 the sediment under sterilized condition due to no microbial activity. Tus, microbial decomposition can play an important role in stereoselective metabolism of FLU degradation in the three sediments.

Enantioselective Degradation of FLU in the Sediment under Natural Condition. Figures 4(a)-4(c)
show the degradation curves of both FLU enantiomers under natural conditions in the three diferent sediments, and it can be seen that both enantiomers disappeared over time. However, in 2# sediment, FLU enantiomers were degraded to about 10 mg/L, and then, the concentration of enantiomers increased signifcantly after 56 days of degradation. After that, the concentration of both enantiomers dropped to 3 mg/L. As it is well known, the environmental sediments are very complex and they have diferent compositions and present high variability [26]. So, the microorganism action and diferences in the composition of sediments could play a role in this change [22]. Terefore, except for 2# sediment, the degradation of both FLU enantiomers in 1# and 3# sediment under natural conditions followed frst-order kinetics with R 2 ranging from 0.8017 to 0.8875 (Table 3), and the frstorder rate constants were derived from ln(C 0 /C) versus t plots by regression analysis for each experiment.
Te enantiomers have the similar half-life in 1# and 3# sediments; however, the observed diferences of the half-life in 2# sediment (t 1/2 � 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 was observed.   Te EF values (Figures 4(d)-4(f )) showed that enantioselectivity existed during the degradation process of FLU enantiomers in diferent sediments. Tere 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. Te data suggest the slow degradation of S-(−)-FLU. Te enantioselective degradation rate of FLU enantiomers is diferent between three diferent 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 diferences in the degradation behavior, and the pH value probably plays an important role in enantioselectivity of FLU enantiomers across diferent sediments [21,22,27].
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. Te 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.

Enantioselective Transformation of FLU in the Water-
Sediment System. Te change over time in the concentration of FLU in the sediment of the water-sediment system is shown in Figure 5(a). Te original spiked concentrations of the FLU in the overlying water were 20 mg/kg. Te concentration of the sediment of FLU, detected at the earlier sampling event (7 days), was much lower than the initial spiked concentrations. However, because of the rapid sorption to suspended particles and sediment, the concentration of FLU in the sediment rapidly increased.
Concentration profles in the overlying water and sediment suggested that the difusive transfer of FLU into the sediment was a quick process, with the FLU enantiomers generally detected in the 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, and this may be attributed to microbial degradation. Tese results also suggest that the sediment can potentially act as a signifcant secondary source of antibiotics that can be released into water [28,29].
Te EF values (around 0.5) in Figure 5(b) show that the transformation behavior of FLU enantiomers had no enantioselectivity in the water-sediment system before 150 days. However, the stereoselective transformation behavior occurred after 150 days because of an increase in the EF values' level. Te results indicated that the transformation of FLU enantiomers in the water-sediment system had enantioselective behavior, and R-(+)-FLU transformed faster than S-(−)-FLU.
3.6. Main Metabolites of FLU Identifcation. Identifcation of molecular ions representing possible metabolites is an indispensable step in the overall identifcation procedure of drug metabolites using LC/MS/MS approaches [30]. 13 Figure 7(b) shows that the metabolite degradation maybe due to the microorganism action. Tese were demonstrated that the main metabolites of FLU in the sediment were decarboxylate and dehydroxylation.

Conclusion
In the present study, a chiral residue analysis method was successfully used to the study of enantioselective adsorption, degradation, and transformation behaviors of FLU enantiomers in diferent sediments. Te results indicated that the FLU enantiomers generated stereoselective behavior in the adsorption of sediment, and the adsorption capacity of R-(+)-FLU and S-(−)-FLU were much higher than the Rac-FLU in three diferent sediments; meanwhile, there was signifcant diference in the adsorption capacity between Rac-FLU and R-(+)-FLU or S-(−)-FLU. Te pH value of the sediment had an infuence on the adsorption capacity and enantioselective adsorption of FLU.
Trough the degradation test, the degradation of FLU in the sterilized sediment would not be enantioselective. Te degradation of FLU enantiomers complied with frst-order kinetics and showed stereoselective under nonsterilized condition, which demonstrated that the R-(+)-FLU degraded faster than S-(−)-FLU. Besides, the degradation rates of both FLU enantiomers were diferent under sterile and natural conditions. Tese results indicated that stereoselective degradation and enantioselective diferences of FLU enantiomers may depend on the pH and organic content when diferent microorganisms are involved in the sediment [31]. In addition, stereoselective behavior also occurred in the transformation of FLU in the water-sediment system, and R-(+)-FLU transformed faster from water to sediment. Furthermore, the main metabolites of FLU in the sediment were decarboxylate and dehydroxylation products.
Tese results might be helpful to evaluate the environmental behaviors of chiral FLU, providing the basic data for the evaluation of environmental and ecological risk assessment and the rational suggestions for optically pure antibiotic development and application.

Data Availability
Te data used to support the fndings of the study can be obtained from the corresponding author upon request.

Disclosure
Tis study has previously been published in a preprint [32].

Conflicts of Interest
Te authors declare that they have no conficts of interest.