Pharmacokinetic Characteristics of Florfenicol in Freshwater Crocodiles (Crocodylus siamensis) After Intramuscular Administration

Simple Summary

Pharmacokinetic information on antimicrobial use in reptiles is extremely limited. Often, drug dosages and approaches are extrapolated from other animal species, which can lead to ineffective or unsafe treatments. This study investigated the disposition kinetics of florfenicol after a single intramuscular injection in freshwater crocodiles based on two dosages. Based on the results, the drug was well absorbed, was prolonged in the bloodstream, and reached levels expected to control bacterial infections. These findings have provided essential baseline information that should help to ensure more effective, accurate, and safe antimicrobial use in freshwater crocodiles.

Abstract

Florfenicol (FFC) is widely used to treat bacterial infections in veterinary medicine; however, its pharmacokinetic characteristics in reptiles remain limited. This study investigated the pharmacokinetic profiles of FFC after intramuscular (IM) injection at doses of 20 or 30 mg/kg body weight (b.w.) in freshwater crocodiles (Crocodylus siamensis). A sample of 10 healthy crocodiles was randomly divided into two groups (n = 5 for each group) according to a parallel study design. Blood samples were obtained from pre-dose to 168 h post-administration. Plasma FFC concentrations were quantified using high-performance liquid chromatography with diode array detection (HPLC-DAD) and analyzed by non-compartmental analysis. The mean maximum plasma concentrations of FFC were 4.05 µg/mL and 6.11 µg/mL for the 20 and 30 mg/kg b.w. doses, respectively. The mean elimination half-lives of FFC were long but not significantly different (51 h). The average plasma protein binding was 37.15%. Based on the pharmacokinetics/pharmacodynamics (PK/PD) index, a single dose of FFC via IM elicited plasma concentrations above the MIC₉₀ values reported for several susceptible bacterial pathogens. Consequently, both dose levels provided plasma exposure consistent with previously reported reference MIC values. However, further PK/PD and multiple-dose investigations are needed to refine species-specific dosage regimens.

1. Introduction

The Siamese crocodile (Crocodylus siamensis) is an economically important freshwater crocodile species. These animals are found primarily in Southeast Asia, including in Thailand, Vietnam, Laos, Cambodia, and Indonesia [1]. In Thailand, the freshwater crocodile is the most widely farmed crocodile species and contributes greatly to the value of the country’s exports because of its high-value skin and meat [2]. The crocodile farming industry generates substantial economic revenue, with the export value of crocodile products reported at approximately 3.8 billion baht in 2017 [2] and increasing to up to 7 billion baht in 2024 [3]. Nowadays, the freshwater crocodile qualifies as critically endangered [4], with its population rates declining rapidly in the wild and on specialized crocodile farms. Primarily, this reduction can be attributed to unsustainable hunting pressure, progressive habitat encroachment, and the impact of climate change. In addition, the expansion of intensive farming for commercial purposes and high-density farming practices can cause physiological stress and predispose animals to various diseases, especially bacterial infectious disease, with reported incidence levels up in recent years [5].

Florfenicol (FFC) is an amphenicol antibiotic developed for veterinary use. It is a third-generation derivative from a series of fluorinated analogs of chloramphenicol (CAP; the first generation) and thiamphenicol (TAP; the second generation). FFC inhibits bacterial protein synthesis by reversibly binding to the 50S ribosomal subunit of susceptible bacteria, thereby interfering with peptidyl transferase-mediated reactions between amino acids and tRNA and ultimately preventing peptide bond formation [6]. FFC has broad-spectrum antibacterial activity, acting as a bacteriostatic agent against diverse bacterial groups, including Gram-positive, Gram-negative, and anaerobic bacteria. It is commonly used to manage bacterial infections in cattle, pigs, and fish. It has been approved to treat bacterial respiratory disease, enteric infections, reproductive tract infections, and skin infections [7]. Bacteria are presented as representative examples encompassing the broad antimicrobial spectrum of FFC and include pathogens that have been reported in crocodiles and other reptiles or aquatic species, such as Actinobacillus pleuropneumoniae, Aeromonas spp., Bacillus anthracis, Chlamydia spp., Edwardsiella spp., Escherichia coli, Klebsiella spp., Mannheimia spp., Pasteurella multocida, Proteus spp., Pseudomonas spp., Salmonella spp., Staphylococcus spp., Streptococcus spp., and Vibrio spp. [7,8]. However, pharmacokinetic studies of FFC in reptiles are highly limited, with no published record of its application to freshwater crocodiles (C. siamensis). In another published study (2024), we conducted pharmacokinetic investigations of FFC in two reptilian species, green sea turtles (Chelonia mydas) and hawksbill sea turtles (Eretmochelys imbricata), providing some of the earliest characterizations of this drug in reptiles [9]. Additionally, the pharmacokinetic study of FFC has been reported previously (2003) in loggerhead sea turtles (Caretta caretta) [10]. Therefore, the information obtained from those studies has provided an essential foundation for subsequent research on crocodiles.

Often, antimicrobial treatment in crocodiles is extrapolated from dosages prescribed in other species due to a lack of relevant pharmacokinetic data. Such a shortcoming may compromise treatment efficacy and contribute to antimicrobial resistance [11]. Therefore, to obtain species-specific, accurate, and practically relevant information, the present study investigates the pharmacokinetic profiles of FFC following a single intramuscular (IM) administration at two dose levels of 20 and 30 mg/kg body weight (b.w.) in freshwater crocodiles (C. siamensis).

2. Materials and Methods

2.1. Animals

A sample of 10 healthy and clinically normal freshwater crocodiles (C. siamensis) was used, aged 1.2–1.5 years, with an average body weight (±standard deviation) of 9.05 ± 1.63 kg and body lengths in the range 123–153 cm. All experimental crocodiles were maintained in cement ponds at the Wongveerakit Crocodile Farm in Bo Phloi District, Kanchanaburi Province, Thailand. Pond water was renewed daily to remove excreted drug residues and waste, and the environmental temperature ranged from 25 to 30 °C. The freshwater crocodiles were acclimatized and did not receive any other medications for at least one month prior to study initiation. The study protocol was conducted in strict compliance with Guidelines for the Use of Animals and was approved by the Animal Ethics Research Committee of the Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand (approval code: ACKU68-VET-098). Prior to the study, all experimental crocodiles were confirmed to be clinically normal based on a review of their health history and physical examination findings.

2.2. Drugs and Chemicals

Florfenicol (FFC; Nuflor^TM, 450 mg/mL) for injection was purchased from MSD Animal Health Inc. (Chiyoda-ku, Tokyo, Japan). The FFC reference standard for calibration, with a purity of 99.5% was supplied by Dr. Ehrenstorfer (LGC Labor GmbH, Augsburg, Germany). Chloramphenicol (CAP; purity ≥ 98%) served as the internal standard and was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Purified water was generated using a Milli-Q purification system (Millipore Sigma; Bedford, MA, USA). All other analytical-grade reagents and chemicals were procured from Sigma Chemical Co. (St. Louis, MO, USA).

2.3. Experimental Design

The 10 freshwater crocodiles were weighed and divided randomly into two groups (n = 5 per group) and enrolled in parallel studies. Each group was administered a single IM injection of FFC at either 20 or 30 mg/kg b.w. These dosages were selected based on a recent study in green sea turtles and hawksbill sea turtles [9]. The drug was administered into the biceps muscle using a 22-gauge, 1.5-inch needle. All crocodiles were physically restrained by experienced personnel to minimize stress, and no chemical restraint was used. Blood samples (1.5 mL) were collected from the tail vein at the intercoccygeal region using heparinized syringes prior to dosing and at 5, 15, and 30 min, as well as 1, 2, 4, 8, 10, 24, 48, 72, 96, 120, 144, and 168 h after drug administration. Plasma was separated by centrifugation at 1900× g for 15 min and stored at −20 °C until analysis.

2.4. Analysis of Florfenicol Concentration in Plasma

The FFC extraction procedure was adapted from the method reported by Sitthiangkool et al. [9]. Frozen freshwater crocodile plasma samples were allowed to thaw at room temperature and vortex-mixed prior to extraction. A 200 µL plasma sample was spiked with 25 µL of CAP internal standard solution (0.5 µg/mL) and extracted twice using 400 µL of ethyl acetate. Samples were mixed for 1 min and centrifuged at 2490× g for 10 min. The ethyl acetate supernatants were pooled and evaporated to dryness under a chemical fume hood. Then, the dried residues were reconstituted with 200 µL of the mobile phase and passed through a 0.22 µm nylon syringe filter. Finally, 25 µL of the processed extract was injected into the high-performance liquid chromatography (HPLC) system for analysis.

Plasma concentrations of FFC were determined using an Agilent 1260 series system (Agilent Technologies; Santa Clara, CA, USA) consisting of a binary pump, an automatic sample injector, a column thermostat, and a diode array detector set to a wavelength of 223 nm. The column was a reverse-phase, ZORBAX Eclipse Plus Rapid Resolution HT (RRHT) C18 column along with a length of 100 mm × 4.6 mm inner diameter, 3.5 µm particle size (Agilent Technologies; Santa Clara, CA, USA) and a ZORBAX RRHT C18 guard column along with a length of 12.5 mm × 4.6 mm inner diameter, 5 µm particle size (Agilent Technologies; Santa Clara, CA, USA). The analytical C18 column was maintained at 35 °C. Isocratic elution was performed using a mobile phase composed of methanol and 10 mM sodium phosphate buffer (NaH₂PO₄-Na₂HPO₄, pH 5) mixed at a 30:70 v/v ratio and delivered at a flow rate of 0.6 mL/min.

2.5. Analytical Method Validation

Method validation was performed according to the EMA/CHMP/ICH/172948/2019 guideline [12]. Calibration standards were prepared by spiking blank plasma with the FFC working standard solution to obtain concentrations ranging from 0.1 to 10 µg/mL (0.1, 0.25, 0.5, 1, 2.5, 5, and 10 µg/mL) with CAP added as the internal standard at a fixed concentration of 0.5 µg/mL. The resulting calibration curve exhibited excellent linearity, with a coefficient of determination (R²) of 0.999. The limits of detection and quantification were 0.075 and 0.1 µg/mL, respectively. Recovery and precision were assessed using quality control samples prepared at three concentration levels (0.1, 1, and 5 µg/mL), with five replicates analyzed per level per day over three different days. Recovery values ranged from 86 to 103%, while the intra-day and inter-day levels of precision were <2.42% and <2.73%, respectively.

2.6. Plasma Protein-Binding Assay

Plasma protein binding was determined based on ultrafiltration using a Vivaspin^® 500 (Sartorius Stedim Lab Ltd.; Sperry Way, Gloucestershire, UK) according to Dow [13] and Toma et al. [14]. Freshwater crocodile blank plasma and 50% methanol were spiked with FFC to achieve nominal concentrations in the range 0.1–10 µg/mL. The prepared samples were subsequently centrifuged at 25,000× g for 30 min. Both the plasma samples and their corresponding ultrafiltrates were analyzed by HPLC as described above. The extent of plasma protein binding was calculated using the following equation:

Protein binding (%) = [(Total concentration − Ultrafiltrate concentration)/Total concentration] × 100

2.7. Pharmacokinetic Analysis

Pharmacokinetic evaluation was conducted using ThothPro^TM T 4.3 software (ThothPro LLC; Gdansk, Poland). Non-compartmental analysis of the plasma concentration–time data was applied to estimate pharmacokinetic parameters, including the maximum concentration (C_max), time at maximum concentration (T_max), the first-order elimination rate constant (λ_z), elimination half-life (t_1/2λz), area under the curve from zero to the last time point (AUC_0–last) and extrapolated to infinity (AUC_0–∞), volume of distribution (V_z/F), clearance (Cl/F), and mean residence time (MRT).

2.8. Statistical Analysis

The plasma concentration profiles as a function of time for FFC and the pharmacokinetic parameter values were expressed as geometric means, except for the time to reach maximum concentration (T_max) and the elimination half-life (t_1/2λz), which were presented as the median and harmonic mean, respectively. Differences in pharmacokinetic parameters between groups were evaluated using the Mann–Whitney U test. Statistical analyses were performed using GraphPad InStat software (GraphPad Software; Boston, MA, USA). Differences were considered significant at p < 0.05

3. Results

The freshwater crocodiles showed no localized reaction at the injection site, nor were any adverse effects or health alterations related to FFC detected throughout the study period or during the post-study observation period.

The semi-logarithmic plasma concentration–time curves of FFC after a single IM dose of 20 or 30 mg/kg b.w. are plotted in Figure 1. The concentrations of FFC in the plasma samples of the freshwater crocodiles (C. siamensis) were quantifiable from 5 min to 144 and 168 h after administration for the 20 and 30 mg/kg doses, respectively. The pharmacokinetic parameters determined following IM administration of FFC are presented in Table 1. The average maximum concentration (C_max) values at the 20 and 30 mg/kg b.w. doses were 4.05 and 6.11 µg/mL, respectively. There were no significant differences in the pharmacokinetic parameters of two treatment groups of the freshwater crocodiles. FFC had a long elimination half-life of around 51 h. The average value of plasma protein binding for FFC was 37.15%.

Figure 1.

Semi-logarithmic plasma concentration–time curve of florfenicol based on mean values after single-dose intramuscular administration at 20 or 30 mg/kg body weight (b.w.) in Siamese crocodiles (n = 5 per group), where error bars represent SD.

Table 1.

Geometric mean (GM) values evaluated for pharmacokinetic parameters of freshwater crocodiles (C. siamensis) following a single dose of florfenicol administered intramuscularly at two different dosages of 20 and 30 mg/kg body weight (b.w.).

Parameter	Unit	Intramuscular
		20 mg/kg b.w. (n = 5)			30 mg/kg b.w. (n = 5)
		GM	Max	Min	GM	Max	Min
λz	1/h	0.013	0.015	0.011	0.013	0.021	0.010
t1/2λz *	h	51.22	-	-	49.74	-	-
t1/2λz	h	51.50	63.08	46.56	51.36	67.10	32.74
Cmax	µg/mL	4.05	4.26	3.71	6.11	6.56	5.71
Tmax +	h	2.0	2.0	2.0	2.0	2.0	2.0
AUC0–last	µg×h/mL	190.50	217.74	167.11	261.43	279.49	247.19
AUC0–∞	µg×h/mL	225.45	270.75	197.40	296.41	311.80	282.29
AUC%extrap	µg×h/mL	15.32	19.58	12.72	11.11	17.06	6.17
Vz/F	mL/kg	6591.61	6855.24	6140.11	7499.07	9744.01	4756.19
Cl/F	mL/kg/h	88.71	101.32	73.87	101.21	106.27	96.22
MRT	h	76.53	85.94	70.06	79.32	94.74	67.54

Abbreviations: AUC_0–last—area under the curve from zero to the last time point; AUC_0–∞—area under the curve from zero to infinity; AUC_%extrap—area under the curve percentage extrapolated; C_max—maximum concentration; Cl/F—clearance; MRT—mean residence time; t_1/2λz—elimination half-life; λ_z—elimination rate constant; T_max—time at maximum concentration; V_z/F—volume of distribution. * Harmonic mean, ⁺ Median value.

4. Discussion

Knowledge of pharmacokinetics is essential for responsible drug use and the establishment of appropriate dosage regimens. To date, no known pharmacokinetic studies of FFC have been reported for freshwater crocodiles. The results from the present study have provided valuable information regarding the absorption, distribution, metabolism, and excretion of FFC, which should contribute to a better understanding of drug behavior, safe treatment strategies, and improved effective antimicrobial use in freshwater crocodiles.

Following IM administration, both the AUC_0–last and AUC_0–∞ values increased in a dose-dependent manner. The AUC_0–last value at 30 mg/kg b.w. (261.43 µg×h/mL) was higher than that at 20 mg/kg b.w. (190.50 µg×h/mL); similarly, the AUC_0–∞ value at 30 mg/kg b.w. (296.41 µg×h/mL) exceeded that observed at 20 mg/kg b.w. (225.45 µg×h/mL). In another study, we investigated the pharmacokinetics of FFC at the same dosages and route in green sea and hawksbill sea turtles [9], where a similar trend was observed, indicating that FFC might have a dose-dependent increase in both freshwater crocodiles and sea turtles. However, the present study reported AUC_0–last and AUC_0–∞ values higher than in green sea turtles (138.32 µg×h/mL and 142.59 µg×h/mL for 20 mg/kg b.w. dose, respectively, and 231.20 µg×h/mL and 235.83 µg×h/mL for 30 mg/kg b.w. dose, respectively) and hawksbill sea turtles (119.69 µg×h/mL and 123.19 µg×h/mL for 20 mg/kg b.w. dose, respectively and 183.41 µg×h/mL and 187.53 µg×h/mL for 30 mg/kg b.w. dose, respectively) [9]. Based on these results, there was higher systemic exposure in freshwater crocodiles than in sea turtles. These differences may reflect species-specific physiological, metabolic variations, and plasma protein binding [15,16]. In addition, reptiles have a renal portal system that has been hypothesized to influence drug disposition by directing caudal venous blood through the kidneys prior to systemic circulation [17]. However, a recent pharmacokinetic study of ceftazidime in freshwater crocodiles by Sorn et al. (2025) [18] reported no evidence of altered pharmacokinetics between forelimb and hindlimb administration, despite blood sampling from the tail vein, suggesting that the renal portal system has a limited influence on drug disposition. Therefore, the higher AUC_0–last and AUC_0–∞ values observed in the present study are more likely attributable to species-specific physiological and metabolic characteristics rather than renal portal effects alone. Moreover, the extended sampling duration up to 168 h allowed adequate characterization of the terminal elimination phase, with the percentage of AUC extrapolation accounting at 15.32% and 11.11% for the 20 and 30 mg/kg b.w. doses, respectively. These values are below the commonly accepted threshold of 20% for AUC extrapolation [19], supporting the reliability of the AUC_0–∞ estimates in the present study.

The observed C_max values of FFC in the freshwater crocodiles at 20 and 30 mg/kg b.w. doses were 4.05 µg/mL and 6.11 µg/mL, respectively. These values were in agreement with the findings reported via FFC administered following an IM route in green sea turtles (3.26 µg/mL at 20 mg/kg b.w. and 5.99 µg/mL at 30 mg/kg b.w.) and hawksbill sea turtles (2.52 µg/mL at 20 mg/kg b.w. and 6.01 µg/mL at 30 mg/kg b.w.) [9], beef bulls (2.83 µg/mL at 20 mg/kg b.w.) [20], heifers (3.2 µg/mL at 20 mg/kg b.w.) [21], white-spotted bamboo sharks (4.5 µg/mL at 30 mg/kg b.w.) [22], and alpacas (6.0 µg/mL at 20 mg/kg b.w.) [23]. Nevertheless, the C_max values observed in freshwater crocodiles were lower than those reported in other species, such as pigs (8.15 µg/mL at 30 mg/kg b.w.) [24] and New Zealand White rabbits (8.65 µg/mL at 25 mg/kg b.w.) [25]. These differences might have been due to temperature-dependent pharmacokinetics. The freshwater crocodile is an ectothermic animal whose drug absorption, distribution, and tissue perfusion fluctuate with environmental conditions. Consequently, the C_max values of FFC may vary considerably more than in an endothermic species [16]. The C_max values observed in the freshwater crocodiles in the present study were considerably higher than those obtained in loggerhead sea turtles (0.65 µg/mL at 30 mg/kg b.w. IM), another ectothermic species [10]. These differences may have resulted from the different species, experimental design, sample size, and sampling time points. In the present study, the T_max values were not significantly different between the experimental groups in the freshwater crocodiles (2 h) and were in line with those reported in green sea and hawksbill sea turtles (1 h) [9] and loggerhead sea turtles (1 h) [10]. The pharmacokinetic parameters of FFC in freshwater crocodiles are compared with those in other ectothermic and endothermic animal species in Table 2.

Table 2.

Comparison of florfenicol pharmacokinetic parameters between freshwater crocodile (C. siamensis) and other animal species.

Species	Dose (mg/kg)	Cmax (µg/mL)	AUC0–last (µg×h/mL)	AUC0–∞ (µg×h/mL)	t1/2λz (h)	Vz/F (L/kg)	Cl/F (L/kg/h)	Reference
Green sea turtles	20 (IM) 30 (IM)	3.26 5.99	138.32 231.20	142.59 235.85	19.67 25.85	3.61 5.53	0.13 0.14	[9]
Hawksbill sea turtles	20 (IM) 30 (IM)	2.52 6.01	119.69 183.41	123.19 187.53	23.59 23.90	5.68 5.74	0.16 0.16	[9]
Loggerhead sea turtles	30 (IM)	0.79	NA	NA	3.2	NA	NA	[10]
White-spotted bamboo sharks	30 (IM)	4.50	NA	NA	-	NA	NA	[22]
Asian seabass	15 (PO)	NA	NA	NA	8.07	1.19	0.11	[26]
Nile tilapia	10 (PO)	NA	NA	NA	1.49	0.80	NA	[27]
Alpacas	20 (IM)	6.00	-	-	16.7	NA	NA	[23]
Beef bulls	20 (IM)	2.83	-	-	-	NA	NA	[20]
Heifers	20 (IM)	3.20	-	-	6.8	-	0.20	[21]
New Zealand White rabbits	25 (IM)	8.65	NA	NA	-	NA	NA	[25]
Pigs	30 (IM)	8.15	-	-	18.19	0.19	0.19	[24]

Abbreviations: AUC_0–last—area under the curve from zero to the last time point; AUC_0–∞—area under the curve from zero to infinity; C_max—maximum concentration; Cl/F—clearance; t_1/2λz—elimination half-life; V_z/F—volume of distribution; IM (intramuscular administration); PO (oral administration). - indicates parameters reported in the referenced studies but not included in the present comparison. NA indicates parameters not reported or not available in the referenced studies.

Prolonged t_1/2λz values of FFC were observed in freshwater crocodiles, measuring 51.50 h and 51.36 h for the 20 and 30 mg/kg b.w. doses, with no significant differences between the experimental groups. These values were considerably greater than those reported in other reptiles, including green sea turtles (19.67–25.85 h) and hawksbill sea turtles (23.59–23.90 h) [9], as well as loggerhead sea turtles (3.2 h) [10]. Furthermore, they were substantially higher than those reported in mammal and aquatic species, such as heifers (6.8 h) [21], alpacas (16.7 h) [23], pigs (18.19 h) [24], Asian seabass (8.07 h) [26], and Nile tilapia (1.49 h) [27]. These findings indicated that there were slower elimination rates (λ_z) and longer t_1/2λz values of FFC in reptiles than in mammals and fish. These differences may be attributed to the ectothermic physiology in the former, where lower metabolic rates, reduced enzymatic activity, temperature-dependent biochemical processes, reduced hepatic and renal blood flow, and variations in cardiovascular shunting mechanisms influenced the extended half-life and slow drug clearance in the freshwater crocodiles [28,29,30]. Furthermore, the longer t_1/2λz values of FFC in the freshwater crocodiles were consistent with the large AUC_0–last and AUC_0–∞ values observed in the present study, suggesting that prolonged systemic exposure may contribute to enhanced drug accumulation and potentially longer therapeutic duration [31]. Although the prolonged terminal phase observed after IM administration may raise the theoretical possibility of flip-flop kinetics [32], the present data do not provide clear evidence of absorption-limited elimination. This observation may therefore be more plausibly attributed to ectothermic physiological factors rather than definitive flip-flop kinetics.

The V_z/F values in the present study were 6.59 L/kg for 20 mg/kg b.w. and 7.49 L/kg for 30 mg/kg b.w, which were higher than those reported in green sea turtles (3.61–5.53 L/kg) and hawksbill sea turtles (5.68–5.74 L/kg) [9], as well as in Asian seabass (1.19 L/kg) [26], Nile tilapia (0.80 L/kg) [27], and mammals such as pigs (0.19 L/kg) [24]. The Cl/F values of FFC in the freshwater crocodiles were relatively low at 0.08 L/kg/h for 20 mg/kg b.w. and 0.10 L/kg/h for 30 mg/kg b.w. These values were lower than those reported in other ectothermic species, including green sea turtles (0.13–0.14 L/kg/h), hawksbill sea turtles (0.16 L/kg/h) [9], and Asian seabass (0.11 L/kg/h) [26] as well as in mammals, such as heifers (0.20 L/kg/h) [21] and pigs (0.19 L/kg/h) [24]. Therefore, the greater V_z/F values combined with the slower Cl/F values observed in the present study suggested that FFC had distributed extensively into tissues while being eliminated more slowly. These findings corresponded with the prolonged t_1/2λz values of FFC observed in the freshwater crocodiles.

As freshwater crocodiles are food-producing species in several countries, including Thailand, where meat, skin, and blood are commercially utilized [2], the absence of tissue residue and depletion data represents an important limitation of the present study. In addition to clinical and regulatory considerations, FFC has also been reported to persist in aquatic environments and undergo transformation into its metabolites, which may remain in water and sediments and contribute to environmental exposure, as demonstrated in recent studies on aquatic species. Bardhan et al. (2024) reported that FFC and its residual products can persist in aquatic systems, suggesting potential toxicological and environmental relevance [33]. The relatively long elimination half-life and high systemic exposure of FFC observed in freshwater crocodiles suggest the potential for prolonged persistence of the drug in edible tissues. However, these pharmacokinetic data should not be directly extrapolated to establish withdrawal periods or to support clinical or regulatory decisions related to food safety. From a regulatory perspective, the lack of species-specific residue data complicates compliance with food safety and international export standards, as maximum residue limits (MRLs) established for other food-producing species may not be applicable to crocodilians. Therefore, further tissue residue and depletion studies are necessary to determine appropriate withdrawal intervals, to ensure safe use recommendations and consumer protection.

Nevertheless, this study has several limitations that should be acknowledged. The relatively small sample size, although common and ethically appropriate for crocodilian pharmacokinetic studies, may limit the full characterization of inter-individual variability. This is particularly relevant in ectothermic species, in which physiological factors such as body temperature, metabolic rate, and tissue perfusion can markedly influence drug disposition [16,34]. Therefore, the present pharmacokinetic findings should be interpreted with caution, and further studies with larger sample sizes are warranted to better characterize variability in this species. In addition, the ambient temperature during the study, which ranged from 25 to 30 °C, may have contributed to variability in ectothermic pharmacokinetic characteristics [35]. These findings suggest that the pharmacokinetic parameters reported in this study are therefore applicable primarily under similar thermal conditions.

The average plasma protein binding (PPB) of FFC in the freshwater crocodiles was 37.15%, which was higher than reported in green sea turtles (20.65%) and hawksbill sea turtles (18.59%) [9]. Based on these data, FFC was bound more strongly to plasma proteins in the freshwater crocodiles than in sea turtle species. Consequently, the higher PPB observed in the freshwater crocodiles could play a role in the pharmacokinetic profiles of FFC, including the longer t_1/2λz values and greater area-under-the-curve values [36]. Furthermore, higher PPB may reduce the unbound fraction of the drug available for distribution into tissues [37], suggesting that plasma concentrations alone may not fully represent drug exposure at infection sites. This consideration is particularly relevant when interpreting subsequent PK/PD analyses based on plasma concentrations.

Currently, there are no known minimum inhibitory concentrations (MICs) of FFC for bacterial isolates from C. siamensis. Therefore, the MIC values discussed below are derived from studies conducted in other animal species and are used here only as reference data. Other studies have reported MIC values of FFC in the range 0.5–4 µg/mL for many susceptible microorganisms, including Aeromonas spp., Chlamydia spp., Escherichia coli, Pasteurella multocida, Salmonella spp., and Staphylococcus spp. [38]. FFC is classified as a time-dependent antibiotic; the PK/PD index time above MIC (T > MIC ratio) is a major parameter used to determine drug efficacy [39]. For Chlamydia spp., an MIC value of 2 µg/mL has been proposed as the MIC₉₀ breakpoint, representing the concentration required to inhibit 90% of isolates [40]. Therefore, based on the results of the present study, the effective plasma concentration of FFC in the freshwater crocodiles remained above the MIC₉₀ for approximately 24 h and 48 h at doses of 20 and 30 mg/kg b.w. after a single IM administration, respectively. However, this PK/PD interpretation should be considered preliminary, as pathogen-specific MIC data from freshwater crocodiles are currently unavailable.

5. Conclusions

Florfenicol was detected in the plasma of freshwater crocodiles (C. siamensis) following IM administration at two dose levels of 20 and 30 mg/kg b.w., characterized by a prolonged elimination half-life, high systemic exposure, and slow clearance. A single IM administration of FFC remained plasma concentrations above the reference MIC₉₀ values reported for clinically important pathogens for approximately 24 h at 20 mg/kg b.w. and 48 h at 30 mg/kg b.w. However, due to the lack of crocodile-specific MIC data for FFC and the influence of ectothermic physiology, the clinical applicability of these results should be interpreted with caution. Further multiple-dose studies, including studies focused on drug accumulation, metabolic disposition, and PK/PD integration, are warranted to confirm and refine species-specific dosage regimens of FFC in freshwater crocodiles.

Author Contributions

P.S., conceptualization, investigation, formal analysis, methodology, data curation, and writing; S.P., conceptualization, investigation, formal analysis, methodology, data curation, validation, review, editing, funding acquisition, and project administration; A.P., conceptualization, investigation, data curation, validation, review, editing, and supervision; N.K., N.P. and T.W., investigation and methodology; P.M. data curation, validation, review, editing, and supervision. M.G., B.L.-W. and M.P.-L., validation, review, editing and supervision. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The study protocol was conducted in accordance with the Guidelines for Animal Experiments and approved by the Animal Ethics Research Committee of the Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand (Approval code: ACKU68-VET-098).

Informed Consent Statement

Informed consent was obtained from the farm owner.

Data Availability Statement

The authors confirm that all data supporting the findings of this research are included within the article.

Conflicts of Interest

Tara Wongwaipairoj is employed by the Wongveerakit Crocodile Farm. The funders had no role in the design of the study, the collection, analyses, or interpretation of data, the writing of the manuscript, or the decision to publish the results.

Funding Statement

This research was funded by the Graduate School, Kasetsart University and the Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.