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Upper Midwest Environmental Sciences Center

Fisheries Restoration

Depletion of Florfenicol Amine from the Fillet Tissue of Rainbow Trout (Oncorhynchus Mykiss) Maintained in a Recirculating Aquaculture System and Treated with Aquaflor®-medicated Feed

Principal Investigator: Jeff Meinertz

Impact of UMESC Science

The results from this study may lead to an expansion of the Aquaflor® label allowing for a higher dose rate that is effective controlling mortality in salmonids infected with various strains of Flavobacterium psychrophilum. Label expansion would enhance salmonid production on all hatcheries throughout the United Sates.

Introduction

Aquaflor®, also marketed as Aquafen® or Florocol®, is a feed premix containing the broad spectrum antibacterial agent florfenicol (FFC; [R-(R*,S*)-2,2-dichloro-N-[1-fluoromethyl-2-hydroxy-2-(4-methylsulfonylphenyl)] ethyl acetamide];) incorporated at a ratio of 50% (w/w).  Aquaflor® is registered for use in Japan (1990), Norway (1993), Chile (1995), Canada (1997), the United Kingdom (1999), Ecuador, United States, Venezuela (2005), Colombia (2006), Brazil, Costa Rica, Vietnam and China (2007) to control various susceptible pathogens in a variety of commercially important freshwater and marine fish.

Florfenicol was first approved for use in freshwater-reared salmonids for the control of mortality due to coldwater disease associated with Flavobacterium psychrophilum in 2007.Subsequent to the approval, there was interest in raising the allowable dose for salmonids [presently 10 mg/kg bodyweight (BW)/d for 10 consecutive days (U.S. FDA 2005; NADA 141-246)] to 15 mg/kg BW/d for 10 consecutive days (R.G. Endris, Intervet, Inc., personal communication) to enhance effectiveness against certain isolates of F. psychrophilum. Florfenicol is effective in controlling mortality in tilapia Oreochromis sp.due to Streptococcus iniae in laboratory and field efficacy trials when fed at a dose rate of 15 mg/kg BW/d for 10 consecutive days (Gaunt et al. 2010; Gaikowski et al. 2008).

Florfenicol administration up to 34.9 mg/kg BW/d for 20 consecutive days in channel catfish Ictalurus punctatus or up to 100 mg/kg BW/d for 10 consecutive days in Atlantic salmon Salmo salar does not result in pathology or alter feed consumption (Gaikowski et al. 2003; Inglis et al. 1991). Since florfenicol is safe for fish at dose rates exceeding 15 mg/kg BW/d and is efficacious controlling salmonid mortality due to coldwater disease associated with F. psychrophilum (R.G. Endris, personal communication), a label expansion for a dose rate for salmonids of 15 mg/kg BW/d is desired. In pursuit of label expansion, a marker residue depletion study with a salmonid dosed with florfenicol at 15 mg/kg BW/d will be needed.

Florfenicol amine (FFA; [R*,S*0]-α-(1-amino-2-fluoroethyl)-4-(methylsulfonyl)-benzenemethanol]) was identified as the primary metabolite of florfenicol in Atlantic salmon muscle tissue (Horsberg et al. 1994). Being the primary metabolite, FFA was subsequently selected as the marker residue of florfenicol. Florfenicol amine is also the marker residue of florfenicol in cattle, swine, sheep, poultry, catfish, salmon and trout (EMEA 2000).

Data from marker residue depletion studies are used to calculate a withdrawal period for a drug, i.e. the time required for the animal to deplete drug residues to a level that is considered safe for human consumption. Regulatory agencies estimate a maximum residue level (MRL) by combining an acceptable daily intake (ADI) concentration developed from toxicology data, with a standard human body mass and a consumption factor. For florfenicol, a 10 µg/kg ADI is multiplied by a standard human body mass of 60 kg then divided by a daily consumption factor (for fish, the consumption factor is 300 g of skin-on fillet) resulting in a MRL for FFA in the fillet tissue of 2 µg/g. The European Agency for the Evaluation of Medical Products (EMEA 2000) and the U.S. Food and Drug Administration (FDA; US FDA 2005) applied an additional safety factor establishing a 1 µg/g MRL for FFA in fillet tissue consumed in Europe and the U.S.

Florfenicol distribution, metabolism and depletion in a variety of fish following dosing at 10 mg/kg BW/d has been well characterized (Martinsen et al. 1993; Horsberg et al. 1996; Pinault et al. 1997; Samuelsen et al. 2003; Yanong et al. 2005; Wrzesinski et al. 2006; Feng et al. 2008). No studies have been published describing the disposition of florfenicol residues in fillet tissue from fish dosed at higher rates. In addition, currently there are no published data describing the depletion of florfenicol residues from fished dosed in a recirculating aquaculture system.
In this study, we intend to monitor florfenicol residues in fillet tissue from dosed fish by analyzing for FFA. To analyze for FFA, residual florfenicol and other lesser metabolites residing in the tissue along with FFA are converted to FFA through acid hydrolysis (Wrzesinski et al. 2003). Determining the resulting FFA concentrations in the fillet tissue provides a conservative estimate of florfenicol concentrations in the fillet tissue and enables a calculation of a conservative withdrawal period for florfenicol.

To characterize a conservative estimate of florfenicol residue depletion from the fillet tissue of rainbow trout dosed with florfenicol at 15 mg/kg BW/d, we intend to dose rainbow trout at 20 mg/kg BW/d in a recirculating aquaculture system with water that is relatively cold when considering rainbow trout culture. During a post dosing period, fish will be sacrificed at predetermined times, and FFA concentrations determined to estimate the total florfenicol residue burden in fillet tissue through that period. A conservative estimate of florfenicol depletion is likely from this study design for the following 3 reasons.

  1. At any particular sample time, the florfenicol fillet tissue concentrations resulting from a dose of 20 mg/kg BW/d should be greater than the fillet tissue concentrations resulting from a dose rate of 15 mg/kg/ BW/d.
  2. Assuming there is the potential for fish to take up excreted drug residues in a recirculating aquaculture system, florfenicol residues in the fillet tissue of fish in the recirculating system should deplete slower than the residues from fish in a flow through system.
  3. Fish dosed at a relatively cold water temperature should deplete florfenicol residues slower than fish dosed in moderate or relatively warm water temperatures.

In addition to characterizing a conservative estimate of florfenicol residue depletion from rainbow trout, 2 other issues related to dosing fish in recirculating systems will be addressed. The first issue is a concern that in a shared recirculating system, untreated fish may take up through the shared water, florfenicol residues from fish dosed with florfenicol medicated feed. Florfenicol residues may enter the water column from the excretion of florfenicol residues from dosed fish and from the leaching of residues from uneaten feed.To address this issue, the test system for this study will comprise of tanks with fish dosed with florfenicol medicated feed and fish that will not be dosed. All tanks will share the same recirculating water supply. Water will be analyzed for FFA periodically throughout the pre dosing, dosing, and post dosing periods to determine the concentrations of all florfenicol residues in the recirculating water. Untreated fish will be sacrificed during the post dosing period. The fillet tissue of untreated fish will be analyzed for FFA to determine the florfenicol residue concentrations in the fillet tissue.

The second issue is a concern that the depletion rates of florfenicol residues from fish dosed in recirculating and flow through aquaculture systems may differ.The depletion of florfenicol residues from fish dosed in a recirculating aquaculture system may be slowed because fish may be taking up excreted residues from the recirculating water. The depletion of residues from fish in the flow through aquaculture system may only be minimally impacted by excreted residues because the residues are continually being flushed from the aquaculture system. To address this issue, rainbow trout in a flow through aquaculture system will be dosed with florfenicol medicated feed. The water temperature in the flow through system will be consistent with the water temperature in the recirculating aquaculture system. During a post dosing period, fish will be sacrificed at predetermined times, and FFA concentrations determined in the fillet tissue to estimate the total florfenicol residue burden in fillet tissue through that period. The depletion of florfenicol residues from the fillet tissue of these fish will be compared to the depletion of residues from the fillet tissue of fish in the recirculating system.

Objectives

  1. Determine the depletion rate of the florfenicol residues from the fillet tissue of rainbow trout dosed with florfenicol medicated feed in a recirculating aquaculture system.
  2. Determine the florfenicol residue concentrations in the water of the recirculating aquaculture system during and after a dosing rainbow trout with florfenicol medicated feed.
  3. Determine florfenicol residue concentrations in the fillet tissue of non-dosed rainbow trout sharing a recirculating aquaculture system with rainbow trout dosed with florfenicol medicated feed.
  4. Determine the depletion rate of the florfenicol residues from the fillet tissue of rainbow trout dosed with florfenicol medicated feed in a flow through aquaculture system.
  5. Determine if the depletion rate of florfenicol residues from rainbow trout dosed in recirculating and flow through aquaculture systems differ.

References

EMEA.  2000.  Committee for Veterinary Medicinal Products, Florfenicol (extension to fish), Summary Report 5. EMEA/MRL/760/00-Final. Available at www.emea.europa.eu/pdfs/vet/mrls/076000en.pdf (active as of July 17, 2008).

Feng, J.B., X.P. Jia, and L.D. Li.  2008.  Tissue distribution of florfenicol in tilapia (Oreochromis niloticus x O. aureus) after a single oral administration in freshwater and seawater at 28°C. Aquaculture 276:29-35.
Gaikowski, M.P. S.M. Schleis, B.A. Lasee, and E. Leis.  2008.  Field Effectiveness of Aquaflor® (50% Type A Medicated Article), Florfenicol - SCH25298 administration in feed to control mortality associated with Streptococcus iniae in tilapia (Oreochromis spp.).  Submitted to Schering-Plough Animal Health Corporation.  December 16, 2008. 355 pages.

Gaikowski, M.P., J.C. Wolf, R.G. Endris, and W.H. Gingerich.  2003.  Safety of Aquaflor® (Florfenicol, 50% Type A Medicated Article), Administered in Feed to Channel Catfish, Ictalurus punctatus.  Toxicologic Pathology 31:689-697.

Gaunt, P., R.G. Endris, A. McGinnis, T. Santucci, and J. Cao.  2010.  Determination of dose rate of florfenicol in feed for control of mortality in Nile tilapia, Oreochromis sp., infected with Streptococcus iniae.  Journal of Aquatic Animal Health 22:158-166.

Horsberg, T.E., K.A. Hoff, and R. Nordmo.  1996.  Pharmacokinetics of florfenicol and its metabolite florfenicol amine in Atlantic salmon. Journal of Aquatic Animal Health 8:292–301.

Horsberg, T.E., B. Martinsen, and K.J. Varma.  1994.  The disposition of 14C-florfenicol in Atlantic salmon (Salmo salar).  Aquaculture 122:97–106.

Inglis,V, R.H. Richards, K.J. Varma, I.H. Sutherland, and E.S. Bokken.  1991.  Florfenicol in Atlantic salmon, Salmo salar L., parr: tolerance and assessment of efficacy against furunculosis.  Journal of Fish Diseases 14:343–351.

Martinsen, B., T.E. Horsberg, K.J. Varma, and R. Sams.  1993.  Single dose pharmacokinetic study of florfenicol in Atlantic salmon (Salmo salar) in seawater at 11°C.  Aquaculture 112:1-11.

Pinault, L.P., L.K. Millot, and P.J. Sanders.  1997.  Absolute oral bioavailability and residues of florfenicol in the rainbow trout (Oncorhynchus mykiss).  Journal of Veterinary Pharmacology and Therapeutics 20: (Suppl. 1), 297– 298.

Samuelson, O.B, Ø. Bergh, and A. Ervik.  2003.  Pharmacokinetics of florfenicol in cod Gadus morhua and in vitro antibacterial activity against Vibrio anguillarum.  Diseases of Aquatic Organisms 56:127-133.

U.S. Food and Drug Administration, Center for Veterinary Medicine.  2005.  Original New Animal Drug Application, NADA 141-246 (AQUAFLOR Type A Medicated Article (florfenicol), an Antibiotic.  U.S. Department of Health and Human Services.  Available at www.fda.gov/cvm/drugsuseaqua.htm (active as of April 28, 2008).

Wrzesinski, C., L. Crouch, P. Gaunt, D. Holifield, N. Bertrand, and R. Endris.  2006.  Florfenicol residue depletion in channel catfish, Ictalurus punctatus (Rafinesque).  Aquaculture 253:309–316.

Yanong, R.P., E.W. Curtis, R. Simmons, V.A. Bhattaram, M. Gopalakrishnan, N. Ketabi, N.V. Nagaraja, and H. Derendorf.  2005.  Pharmacokinetic studies of florfenicol in Koi carp and Threespot gourami Trichogaster trichopterus after oral and intramuscular treatment.  Journal of Aquatic Animal Health 17:129-137.

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