Pharmacokinetics and temperature-dependent tissue depletion of florfenicol in pangasius catfish (Pangasianodon hypophthalmus) following oral administration

Introduction

It has been observed repeatedly in aquaculture pharmacology that the metabolic rate of poikilothermic fish and therefore the absorption, distribution, metabolism, and excretion of administered drugs is directly coupled to ambient water temperature, yet most national withdrawal-period regulations specify a single fixed interval irrespective of the thermal regime under which the fish are reared [1,2] . This disconnect between pharmacological reality and regulatory practice is especially consequential for Pangasius catfish production in the Mekong Delta, where water temperatures fluctuate between 24°C in the cool dry season (December-February) and 34°C in the hot wet season (May-August), and where florfenicol is the most widely prescribed antibiotic for the treatment of Edwardsiella ictaluri and Aeromonas hydrophila infections [3,4] . Florfenicol(2,2-dichloro-N-[1-(fluoromethyl)-2-hydroxy-2-[4-(methylsulfonyl)phenyl]ethyl] acetamide) is a synthetic amphenicol approved for veterinary use in multiple fish species but is not subject to a harmonised international MRL for Pangasius. The European Union sets an MRL of 1 000 μg/kg for the sum of florfenicol and its primary metabolite, florfenicol amine, in finfish muscle+skin [5] . Vietnam's National Agro-Forestry-Fisheries Quality Assurance Department (NAFIQAD) currently recommends a withdrawal period of 12 days for florfenicol in Pangasius, based on a single PK study conducted at 28-30°C [6] .

If florfenicol is eliminated more slowly at lower temperatures as general pharmacokinetic principles and analogy with other species predict [7,8] then a 12-day withdrawal period may be adequate for summer production but insufficient for winter production, potentially resulting in MRL violations in exported fillets. Vietnam is the world's largest exporter of Pangasius, shipping approximately 1.8 million tonnes of fillets annually, primarily to the EU, United States, and China, and MRL violations carry severe trade consequences including border rejections, enhanced surveillance, and reputational damage to the national brand [9] . This research therefore determined the full plasma PK profile and tissue depletion kinetics of florfenicol in Pangasius catfish at three water temperatures spanning the production range, with the objective of providing evidence-based, temperature-stratified withdrawal-period recommendations.

Material and Methods

Reagents and Chemicals

Florfenicol analytical standard (≥98% purity) and florfenicol amine standard (≥95%) were purchased from Sigma-Aldrich (Singapore distributor). HPLC-grade acetonitrile and methanol were from Merck (Darmstadt, Germany). Florfenicol for oral administration was obtained as a commercial premix (Aquaflor® 50% Premix, Merck Animal Health, Madison, NJ, USA). Solid-phase extraction (SPE) cartridges (Oasis HLB, 3 cc/60 mg) were from Waters (Milford, MA, USA). Phosphate-buffered saline, trichloroacetic acid, and other reagents were analytical grade.

Climatic and Environmental Conditions

The trial was conducted at the Experimental Aquaculture Station of the Can Tho College of Aquaculture and Fisheries (10.03°N, 105.77°E) between November 2022 and March 2023. Water temperature in the three treatment groups was maintained at 26±0.5, 30±0.5, and 34±0.5°C using thermostatically controlled recirculating systems. Water quality parameters were monitored daily: dissolved oxygen 5.2-6.8 mg/L, pH 7.0-7.4, total ammonia nitrogen <0.5 mg/L. Photoperiod was maintained at 12L:12D throughout.

Material: One hundred and eighty clinically healthy

Pangasius catfish (body weight 450±65 g, total length 32±3 cm) were procured from a disease-free broodstock facility in An Giang Province and acclimated for 21 days. Fish were randomly assigned to three temperature groups (n = 60 per group, held in six 500 L tanks per group, 10 fish per tank). Following acclimation, each fish received a single oral dose of florfenicol at 10 mg/kg body weight, administered via a medicated feed pellet (premix incorporated into a commercial Pangasius grower diet at 200 mg/kg feed, consumption confirmed by visual observation).

Methods

Blood samples (0.5 mL, caudal vein) were collected from three fish per temperature group at 0, 1, 2, 4, 6, 8, 12, 24, 36, 48, 72, and 96 hours post-dose for plasma PK analysis. Fish were sampled only once (destructive sampling). Plasma was separated by centrifugation (3 000 g, 10 min, 4°C). Tissue samples (muscle, liver, kidney, skin+fat) were collected from three fish per group at 1, 3, 5, 7, 10, 14, and 21 days postdose. Florfenicol and florfenicol amine were quantified by HPLC-UV (Shimadzu LC-20AT, 227 nm) after SPE cleanup. The method was validated according to VICH GL49 guidelines [10] , with limits of quantification of 10 μg/kg (tissue) and 0.05 μg/mL (plasma), recoveries of 82-94%, and intra-day CVs <8%. Non-compartmental PK analysis was performed using PKSolver [11] . Withdrawal periods were calculated as the time at which the upper 99th percentile of the tissue concentration distribution (with 95% confidence) fell below the EU MRL of 1 000 μg/kg, using the WT1.4 software of the European Medicines Agency [12] . Statistical comparisons of PK parameters across temperatures used oneway ANOVA with Tukey's post-hoc test in R v4.

Results

Florfenicol was rapidly absorbed after oral administration, with peak plasma concentrations occurring at 2 hours postdose at all three temperatures. Both absorption and elimination were accelerated at higher water temperatures, consistent with the expected effect of temperature on metabolic rate in poikilotherms.

Discussion:

The temperature dependence of florfenicol pharmacokinetics in Pangasius observed in this research is consistent with the fundamental physiological principle that metabolic rate in poikilotherms approximately doubles for each 10°C increase in temperature (Q10 ≈ 2) [1,13] . The Q10 for florfenicol clearance calculated from these data (1.31 per degree, or approximately 1.9 over a 10°C range) is within the expected range and comparable to values reported for oxytetracycline in channel catfish (Q10 = 1.7) and enrofloxacin in tilapia (Q10 = 2.1) [7,14] . Calculated withdrawal periods (days) for muscle, skin +fat, and liver at three water temperatures. Liver consistently required the longest withdrawal period, and all tissues showed shorter withdrawal at higher temperatures.

Fig 3:

Proportional distribution of florfenicol residues across tissues at 24 hours post-dose (all temperatures combined). Liver contained the highest proportion of total residues (31.2%), followed by muscle (28.4%), kidney (18.7%), skin+fat (14.3%), and plasma (7.4%). The clinical implication is straightforward: a withdrawal period that is safe at 30°C is not safe at 26°C. The current NAFIQAD recommendation of 12 days is adequate at 30°C, where muscle residues fell below the MRL by day 10 (providing a 2-day safety margin), but at 26°C, muscle residues did not reach sub-MRL levels until day 12, leaving no safety margin. Liver residues, which exceeded the MRL until day 18 at 26°C, represent a greater concern: although Pangasius fillets are the primary export product, viscera may enter the domestic food chain, and EU inspectors may test composite tissue samples [5,15] . The higher Cmax at higher temperatures (12.4 vs. 16.8 μg/mL at 26 vs. 34°C) is attributable to faster gastrointestinal absorption, which in fish is driven by increased intestinal motility and mucosal blood flow at elevated temperatures [16] .

The lower AUC at higher temperatures (284 vs. 218 μg•h/mL) reflects the faster clearance, meaning that although peak exposure is higher at 34°C, total drug exposure and therefore residue burden is lower. This paradox of higher Cmax but lower AUC at higher temperatures is characteristic of drugs whose elimination is more temperature-sensitive than their absorption [2,17] . From a food-safety regulatory perspective, we propose temperature-stratified withdrawal recommendations: 12 days for water temperatures ≥30°C, 15 days for 26-29°C, and 18 days for <26°C. This tiered approach has precedent in Norwegian salmon aquaculture, where withdrawal periods for oxolinic acid and flumequine are adjusted by temperature band [18] .

Conclusion

Water temperature exerts a profound and predictable influence on florfenicol pharmacokinetics in Pangasius catfish, with plasma elimination half-life decreasing by 43% and muscle withdrawal time by 33% across the 26-34°C production temperature range.

The current Vietnamese regulatory withdrawal period of 12 days is adequate for summer production (≥30°C) but provides no safety margin at 26°C and is insufficient for liver residue compliance at any temperature below 30°C. A temperaturestratified withdrawal system analogous to those used in salmonid aquaculture in northern Europe would better protect consumers and trade partners. These data also have implications for antimicrobial stewardship: the lower total drug exposure (AUC) at higher temperatures means that the duration of therapeutically effective plasma concentrations is shorter in summer, potentially increasing the risk of subtherapeutic exposure and selection for resistant bacteria. Dose optimisation studies that simultaneously address both the food-safety (residue) and antimicrobial-resistance dimensions of florfenicol use in Pangasius aquaculture are urgently needed.

Acknowledgement

Acknowledgements Institutional SupportThe authors thank the Can Tho College of Aquaculture and Fisheries for providing access to the Experimental Aquaculture Station and HPLC instrumentation.

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