Evaluation of absorption and depletion of florfenicol in European seabass Dicentrarchus labrax

Abstract The pharmacokinetic properties and residue elimination of florfenicol (FLO) and its amine were investigated in European seabass Dicentrarchus labrax at 24°C. The trial mainly included analysis of FLO in plasma after a single dose dietary administration of 10 mg/kg and in muscle plus skin following a multiple dosing (10 mg kg−1 day−1 for 7 days) to estimate pharmacokinetics and residue depletion, respectively. The maximum plasma concentration of FLO was measured to be 1.64 μg/ml, 4 hr post administration. The elimination half‐life (t 1/2b) and the area under the concentration‐time curve extrapolated to infinity (AUC0‐∞) were calculated to be 13.0 hr and 34.7 μg h−1 ml−1, respectively. Withdrawal times of FLO and its amine were calculated to be 46.3 degree‐days, indicating a fast removal from the edible tissues of treated European seabass. Overall, FLO can be considered as a potentially efficient antibacterial agent for farmed European seabass provided that additional efforts will be devoted towards its in vitro and clinical efficacy.

(FLO), a synthetic amphenicol, could potentially be an effective solution to the limited range of established antibacterials.
Florfenicol is related to chloramphenicol and exerts broad spectrum antibacterial activity (inhibition of protein synthesis) against gram-negative bacilli, gram-positive cocci and other atypical bacteria (Papich, 2016). It is more active however than either chloramphenicol or thiamphenicol and can even display bactericidal action (Papich, 2016). Moreover FLO is highly lipophilic, and can provide concentrations high enough to treat intracellular pathogens and easily crosses biological barriers. Florfenicol has been entered into Annex I of Council Regulation (EC) No 2377/90 with a maximum residue level (MRL) of 1,000 μg/kg fish muscle plus skin (EMEA, 2002).
The compound is registered for aquatic use in few Mediterranean countries, while it can be prescribed as a drug authorised for other than fish farmed animals by the prescribing cascade mechanism (90/676/EC), where the compound is not labelled. In such cases, a standard withdrawal period is imposed, corresponding to 500 degree-days (dd) in fish (Directive 2004/28/EC).
Florfenicol has been clinically assessed (Gaunt et al., 2003;Soto et al., 2010) and its absorption after oral administration has been investigated in several important marine farmed fish species including Atlantic salmon Salmo salar (Horsberg et al., 1996;Martinsen et al., 1993), turbot Scophthalmus maximus (de Ocenda et al., 2017), orange-spotted grouper Epinephelus coioides (Feng et al., 2018) and hybrid striped bass Morone chrysops × M. saxatilis. The absorption of FLO has been also determined in main freshwater farmed fish species such as Nile tilapia Oreochromis niloticus, hybrid tilapia O. niloticus × O. aureus (Feng & Jia, 2009;Kosoff et al., 2009), rainbow trout, Oncorhynchus mykiss (Pourmolaie et al., 2015), channel catfish Ictalurus punctatus (Gaunt et al., 2012(Gaunt et al., , 2013 and crucian carp Carassius auratus (Zhao et al., 2011). However, pharmacokinetic information of FLO is totally lacking in European seabass regardless of its commercial importance in Mediterranean marine fish farming. Therefore, the aim of the present work was to provide insights into important pharmacokinetic parameters for dosing schedule, such as absorption and depletion, in European seabass following a single and a multiple oral FLO administrations at water temperatures optimum for bacterial outbreaks.

| Experimental fish
Apparently healthy European seabass averaging about 100 ± 12 g were obtained from a local fish farm (Selonda aquaculture S.A) and distributed in two cages (1 m 3 ) located within a 50 m 3 cement tank (85 fish/cage). Water was supplied by open flow and oxygen was provided continuously by bubbling air. The water temperature and salinity were 24 ± 1°C and 38‰, respectively. Fish were allowed to acclimate for 7 days prior to experimentation and fed a drug-free commercial diet at 1.5% body weight (B.W.). To increase acceptance of the therapy, fish were starved for 24 hr prior to administering the medicated feed. Management of experimental animals followed the EU legislation 'on the protection of animals used for scientific

| Medicated feed and drug administration
Fish received a commercial feed (BioMar, Denmark) (Table 1)

| Sampling
Sampling of fish was performed at predetermined time points during and post treatment ( Figure 1)

| Chemicals and reagents
Analytical standards of FLO and its amine were obtained from

| Sample preparation
The extraction and analysis of FLO in plasma samples was carried out according to the procedure of Xie et al. (2011). Briefly, 1 ml of plasma sample was placed in a 10 ml polypropylene centrifuge tube with 500 μl of acetonitrile:water (35:65, v/v). The mixture was vortexed for 30 s, followed by the addition of 5.5 ml of ethyl acetate to deproteinize and extract the FLO. It was then mixed for 2 min and homogenized ultrasonically for 15 min. The homogenised samples were centrifuged (8,000 g for 10 min) and the supernatant was transferred to a 15 ml polypropylene centrifuge tube. The extraction step was repeated twice. The combined extract was then evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was reconstituted by 1 ml of mobile phase solution. Five milliliter of hexane were added into the tube and after mixing it was subjected to centrifugation for 5 min at 2,150 g prior to the removal of the hexane layer. The afore-mentioned de-fatting step was also repeated twice.
The water-based phase was filtered using 0.22 μm nylon filter and the filtrate (200 μl) was then analysed by HPLC.
A modified method of  was used for FLO extraction and analysis in muscle samples. Briefly, muscle plus skin samples were sheared, and subsequently 2 g of ground sample was weighed into a 50 ml centrifuge tube. Ten ml of ethyl acetate were added and the mixture was homogenized with an IKA Ultra-Turrax T25 Disperser (IKA ® -Werke GmbH & Co. KG, Staufen, Germany) for 30 s at 16,000 g. The mixture was agitated for 20 min and then was centrifuged at 3,500 rpm/min for 10 min at 5°C. The supernatant was transferred to a 15 ml polypropylene centrifuge tube and the extraction steps were repeated twice. The combined extract was then evaporated to dryness at 45°C under nitrogen stream. Two milliliter of hexane and 1 ml of mobile phase solution were added in the residue. After gentle agitation for 5 min, the mixture was centrifuged (2,150 g for 5 min). The upper layer (hexane) was discarded and the de-fatting step was repeated. The bottom layer (1 ml) was filtered (0.22 μm nylon filter) and then was subjected to HPLC analysis.

| HPLC analysis
Chromatographic separation of parent compound and its amine was carried out in a HPLC apparatus combining a 600 Pump sys-

| Pharmacokinetic parameters
Calculation of the pharmacokinetic parameters of FLO in European seabass plasma after a single dietary administration of FLO (10 mg/kg) was carried out by the non-compartmental pharmacokinetic model based on the statistical moment theory, according to the method described by Gibaldi & Perrier (1982). The maximum plasma concentration (observed maximal concentration) and the time to reach maximum plasma concentration were measured directly from the mean plasma drug concentration versus time profiles. A semi-logarithmic graph of mean plasma concentration at the elimination phase versus time was used for the elimination half-life (t 1/2β = 0.693/β) calculation. The area under the concentration-time curve (AUC 0-∞ ) was determined using trapezoidal method and was extrapolated to infinity. Calculation of the total body clearance (Cl T /F) was also performed in a model independent way (Ritschel, 1986).

| Withdrawal times (WTs)
Withdrawal times were calculated based on the guidelines of European Medicines Agency (EMA) (2018). The total concentrations of FLO and its amine in muscle plus skin were calculated at each sampling time point post treatment and subjected to a linear regression analysis versus time data from each individual using the statistical program WT1.4 (Hekman, 2004). Withdrawal period was determined at the time when the upper one-sided 95% tolerance limit for the residue was below the MRL with 95% confidence.

| Statistical analysis
Results are presented as means ± SD ( was used for the statistical analysis.   (Table 2). Minimum plasma concentrations of FLO at the 24 hr sampling intervals after multiple oral administrations at 10 mg/kg per day for seven consecutive days are presented in Figure 3. Values ranged from 0.22 to 0.55 μg/ml; however, no statistical difference between time intervals was found.

| Depletion and WTs of FLO
Mean and standard deviations of the parent FLO and its amine residues in muscle plus skin samples are presented in Table 3. The results indicate that the elimination of FLO and its metabolite in edible tissues were rapid as the drug concentrations declined below LOQ 144 hr post treatment. The WTs for of FLO and its metabolite in European seabass muscle plus skin tissues ( Figure 4) were calculated to be 46.32 dd.

| D ISCUSS I ON
this species. The maximum plasma concentration value of FLO in European seabass was found 1.6 μg/ml which is admittedly lower compared to those calculated after dietary delivery in other farmed fish species (1.8-55 μg/ml) held however in different environmental conditions (Table 4). Apparently, as with most pharmacokinetic parameters, maximum plasma concentration values are strongly interspecific as well as experimentally and environmentally dependent (Chang et al., 2019;Huang et al., 2019;Rairat et al., 2019Rairat et al., , 2020 and therefore, should be seriously considered when direct comparisons are attempted among different studies. Water temperature (Chang et al., 2019;Huang et al., 2019;Rairat et al., 2019) and salinity Rairat et al., 2020)   WT, withdrawal times.

TA B L E 4 (Continued)
reflecting similar depletion properties among the two amphenicols in fish circulation.
The WTs of the parent FLO and its major metabolite were also calculated in European seabass herein. Admittedly, the latter is of great interest as a marker residue for FLO even though it has been reported that it virtually lacks antibacterial activity (Park et al., 2006).  Salvo et al., 2013). Generally, the differences in WTs of FLO among several studies (Table 4) are, in addition to variation in experimental set-up, also due to species-specific differences. Moreover environmental parameters such as water temperature and salinity have also proved to have an effect on the drug excretion pathway.
In particular,  reported that the primary route of excretion of FLO and its metabolites following a single oral dose of 10 mg/kg fish in freshwater hybrid tilapia is the bile duct, whereas in seawater tilapia, is mostly the gills. Additionally, Lim et al. (2010) demonstrated higher tissue concentrations of FLO in gills than in the other tested tissue samples in olive flounder Paralichthys olivaceus following multiple oral administrations at 20 mg kg −1 day −1 for three consecutive days. These findings indicate that the elimination of the drug would probably be faster in seawater species compared to the freshwater species. Indeed, the literature has revealed that the WTs of FLO were shorter in marine farmed fish (16-189 hr) compared to those calculated in fresh water (72-360 hr) (Table 4), which is consistent with the results of the present study.
In conclusion, FLO was readily absorbed in circulation and rapidly eliminated from European seabass edible tissues. Minimum inhibitory tests against important bacterial pathogens of European seabass and more importantly, specific clinical trials are needed to acquire a thorough profile of the antibacterial value of FLO. A double FLO dosage, administered twice a day may lead to enhanced circulatory drug levels in European seabass, but this has yet to be experimentally verified.

ACK N OWLED G M ENTS
We acknowledge support of this work by the project "MOdern UNifying Trends in marine biology -MOUNT" (MIS 5002470) which is implemented under the "Action for the Strategic Development on the Research and Technological Sector", funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund). We would like to acknowledge Ms. Margaret Eleftheriou for proofreading the manuscript.