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Florfenicol residue depletion in channel catfish, Ictalurus punctatus (Rafinesque)
Aquaculture 253 (2006) 309 – 316 www.elsevier.com/locate/aqua-online
Florfenicol residue depletion in channel catfish, Ictalurus punctatus (Rafinesque) Christopher Wrzesinskia,*, Louis Croucha, Patricia Gauntb, Dwayne Holifieldc, Nicole Bertrandd, Richard Endrise a
b
Schering-Plough Research Institute, Lafayette, NJ 07848, United States Mississippi State University College of Veterinary Medicine, Stoneville, MS 38776, United States c Delta Western, Indianola, MS 38751, United States d EnviroTest Laboratories, Xenos Division, Nepean, Ontario, Canada, K2E 7L5 e Schering-Plough Animal Health, Union, NJ 07083, United States Received 10 February 2005; received in revised form 2 May 2005; accepted 4 May 2005
Abstract Florfenicol is a broad-spectrum antibiotic currently used in salmon aquaculture and proposed for use in channel catfish. A residue depletion study was performed under commercial aquaculture conditions to determine a withdrawal period. Market weight catfish (~ 2 lb) maintained in a 0.1 acre test pond at a 7000 fish/acre stocking density were dosed with commercially formulated florfenicol in feed for 12 days at an average rate of 9.3 mg/kg b.w./day over the last 10 days, or 93% of the proposed dose. Twenty fish each were collected 1, 2, 4, 7, 14, and 21 days post withdrawal of medicated feed and levels of the marker residue, florfenicol amine (FFA), in muscle tissue were determined via a validated HPLC-UV method. By 4 days average muscle FFA levels were below the proposed 1000 ppb tolerance for FFA and beyond this time point all individual fish were below this level. D 2005 Elsevier B.V. All rights reserved. Keywords: Florfenicol; Antibiotic; Catfish; Residue depletion; Withdrawal; Florfenicol amine
1. Introduction Florfenicol (SCH 25298), [R-(R*,S*)]-2,2-dichloroN-[1(fluoromethyl)-2-hydroxy-2-[4-(methylsulfonyl)phenyl]ethyl]-acetamide, (Fig. 1) is a broad spectrum * Corresponding author. Tel.: +1 973 940 4388; fax: +1 973 940 4159. E-mail address: [email protected] (C. Wrzesinski). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.05.005
antibiotic that is widely used in veterinary medicine. While it is structurally related to the antibiotics chloramphenicol and thiamphenicol used in humans, the potential for microbial resistance impacting human health is limited because both of these are rarely used due to an association with aplastic anemia (Nagabhusahan et al., 1992). As the injectable formulation NuflorR, florfenicol has been approved for use in cattle (US and European Union) and swine (European Union only) for treatment of respiratory disease and/or foot
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O H3C
OH
S O F
HN O
Florfenicol (SCH 25298) CHCl2
O H3C
OH
S O H2N
F
Florfenicol Amine (SCH 40458) Fig. 1. Structure of florfenicol and florfenicol amine.
rot. As the 50% feed premix AquaflorR/AquafenR, florfenicol is approved for use in salmonids (Norway, Chile, Canada, UK) for treatment of furunculosis (Nordmo et al., 1998; Samuelson et al., 1998). Initial studies have demonstrated that Aquaflor administered in feed to channel catfish at a dose rate of 10 mg/kg body weight/day for 10 consecutive days reduces catfish mortality (Gaunt et al., 2004) resulting from enteric septicemia (ESC), the primary disease affecting commercial catfish production in the United States (Hawke et al., 1981; Thune, 1991). Florfenicol has been demonstrated to cause no changes in fish growth and no clinical changes in catfish fed at rates up to 34.9 mg/kg (Gaikowski et al., 2003). Consequently ScheringPlough Animal Health is developing Aquaflor for treatment of ESC in channel catfish. As part of this development effort a final residue depletion study was conducted with florfenicol in catfish. This is a required study for registration of a veterinary drug and is used to determine a withdrawal period (the required length of time after drug administration that an animal must be held from market). In a final residue depletion study the target animal species (catfish in this case) is medicated at the maximum proposed dosing rate and length of administration and
the decline in marker residue concentration over time in the target tissue is determined. The marker residue can be the drug itself or a metabolite and it is chosen because the ratio of its concentration to the concentration of total drug-related residues remains constant over time in the target tissue. The target tissue is the last edible tissue in the target animal in which residues deplete to the permitted safe concentration (US FDACVM Guideline 3, 1994). Thus the target tissue marker residue concentration is used to determine whether total drug-related residues have declined to a safe concentration in all edible tissues of the target animal. As muscle is considered by regulation to be the only edible tissue in catfish, it is the target tissue. Previous studies with florfenicol in cattle, poultry, swine, and salmon have identified florfenicol amine as the marker residue (US FDA NADA 141-063, 1996; EMEA MRL 251/97-Final, 1997; EMEA MRL 589/99Final, 1999; EMEA MRL 591/99-Final, 1999). The sample preparation employed in these studies and in the current study includes an acid hydrolysis step that converts florfenicol and its related metabolites to florfenicol amine (Wrzesinski et al., 2003) (Fig. 1). Therefore florfenicol amine (FFA, SCH 40458) was designated as the marker residue in catfish. Metabolism studies of florfenicol in Atlantic salmon have identified FFA as the major metabolite in muscle tissue although florfenicol is much more predominant in skin (Horsberg et al., 1994; EMEA MRL 251/97Final, 1997). Hormazabel et al. (1993) describe a method that utilized solvent extraction followed by HPLC analysis to determine the concentration of florfenicol and FFA in muscle and liver of rainbow trout. Horsberg et al. (1996) fed Atlantic salmon florfenicol medicated feed at a rate of 10 mg florfenicol/kg b.w./day for 10 days to determine the depletion of FFA in muscle and liver. The analytical method described by Hormazabel et al. (1993) was used for the Horsberg study. Pinault et al. (1997) describe a study in which rainbow trout were fed florfenicol medicated feed at a rate of 10 mg florfenicol/kg b.w./day for 10 days to determine the depletion of FFA in muscle with attached skin. Additionally, in 2 separate studies, Atlantic salmon kept at 58 and at 108 C were dosed with florfenicol medicated feed at 10 mg florfenicol/kg b.w./day for 10 days and the depletion of FFA in muscle and in skin were determined (EMEA MRL 251/97-Final, 1997). The sample preparation in these last 3 studies (Pinault et al., 1997;
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EMEA MRL 251/97-Final, 1997) included an acid hydrolysis step to convert florfenicol and related metabolites to FFA.
2. Materials and method 2.1. Test article and analytical standards AquaflorR 50% Type A Medicated Article, a formulation containing 50% florfenicol, nominal (49.49% assayed), was supplied by Schering-Plough Animal Health and was used in the preparation of medicated feed as described below. Florfenicol and FFA were supplied by the Schering-Plough Research Institute (SPRI) and were used as analytical reference standards during feed analysis and during tissue analysis, respectively. 2.2. Feed preparation and analysis Basal (control) and medicated floating catfish feeds were formulated according to normal aquaculture practices at Delta Western Research Center (Indianola, MS). Fish were fed a commercial diet prior to the start of dosing. The average daily feeding rate of the catfish (the ratio of total weight of feed consumed to the total weight of fish in the pond) as determined for 4 days prior to the start of dosing was 0.21% body weight (based on a 2.02-lb average body weight and 700 total fish in the pond as described below). This required a 4762 ppm concentration of florfenicol in feed to give the desired dose level of 10 mg/kg/day (target dose rate divided by feeding rate multiplied by 100%). However, available Aquaflor was only sufficient to manufacture feed at a 4425 ppm nominal FFC concentration (4.4%). Florfenicol is commercially incorporated into catfish feed at 2–3%. Separate batches of control and medicated feed were mixed in a commercial feed mixer and aliquots were taken from the beginning, middle, and end of each batch. The feed samples were shipped to Xenos Labs (Nepean, Ontario) for drug concentration analysis. After the end of the treatment period, an additional composite sample of medicated feed was obtained according to the AOAC method on the sampling of animal feed (2000) and shipped to Xenos for storage stability analysis.
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A 250 g aliquot of each feed sample was homogenized in a Braun coffee grinder to a particle size of 1–2 mm and a 10-g subsample was extracted with 100 mL acetonitrile/water (1:1). The subsample was sonicated at 50 8C for 5 min, placed on a wrist-action shaker at the highest setting for 10 min and then sonicated and placed on the shaker one more time. After allowing the extract to settle, approximately 50 mL of the supernatant was decanted and centrifuged. A 5 mL aliquot of the centrifuged extract was transferred onto an Envi-Carb cartridge (0.5 g, 6 mL, Supelco) and the eluate was collected in a 10-mL volumetric flask. The cartridge was rinsed with additional extraction solvent and the volume was adjusted to 10 mL. For the medicated feed, the sample extract was diluted five fold prior to HPLC analysis. 2.3. In-life phase See Table 1 for a calendar of in-life events. Unsexed food size fish (fish weighing z 1 lb) were raised in a Mississippi Agricultural Forestry and Extension Service (MAFES) pond at the Mississippi State University Delta Research Experimental Center (DREC). Three samples of 100 food size fish were collected from a seine and weighed to determine mean weight (2.02 F 0.10 lb/fish). These fish were then released back into the pond and were not used for this study. Fish for the study were then collected from the same seine and weighed to yield a weight equivalent to ~ 700 fish (1428 lb total weight, 707 actual calculated number of fish). These ~ 700 fish were placed in an oxygenated well in a live hauler and transported to a 0.1 acre pond at Delta Western. The Table 1 Calendar of in-life activities Activity
Date
Start of acclimation Control (Group VII) fish collected Preparation of medicated feed First day of dosing Last day of dosing Group I fish (1 day post withdrawal) Group II fish (2 days post withdrawal) Group III fish (4 days post withdrawal) Group IV fish (7 days post withdrawal) Group V fish (14 days post withdrawal) Group VI fish (21 days post withdrawal)
8/30/00 9/21/00 10/13/00 10/13/00 10/24/00 10/25/00 10/26/00 10/28/00 10/31/00 11/7/00 11/14/00
collected collected collected collected collected collected
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stocking density was 7000 fish/acre and therefore similar to commercial stocking practices. Enteric septicemia is most prevalent when water temperatures are 20–28 8C (Waltman and Shotts, 1986). Additionally the rate of residue depletion in fish decreases with decreasing water temperature (Bjo¨rklund and Bylund, 1990; Bjo¨rklund et al., 1992; Sohlberg et al., 1994; Namdari et al., 1996). Therefore this study was conducted in the fall, when water temperatures at the site were at the lower end of or below the 20–28 8C range and the rate of residue depletion would be the lowest. Pond water temperature and pond water oxygen content were monitored at least daily. Supplemental aeration was provided when dissolved oxygen levels could not be determined (due to inclement weather) or were approaching b 4 ppm. Catfish were fed once a day to approximate satiation and the amount of feed consumed was recorded daily. Prior to dosing with medicated feed the fish were allowed to acclimate for 44 days during which they were fed a commercial diet. During acclimation 10 control fish were collected and muscle sampled to check for possible interference with the analytical method and to serve as control matrix for analytical method performance checks. After acclimation the fish were fed medicated feed for 12 days (extended from the originally intended 10 days due to poor feeding during the initial 2 days of medication) and then were fed the prepared control feed (equivalent to a commercial diet) during withdrawal. Twenty fish were arbitrarily collected at 1, 2, 4, 7, 14, and 21 days post-withdrawal of florfenicol-medicated feed and were transported live in ice water to DREC. At DREC collected fish were euthanized via pithing (severing of the brain and spinal cord), sexed via internal examination, and 2 skinless muscle fillets were taken from each. One of the fillets from each fish was then shipped to SPRI for homogenization and residue analysis.
a Nucleosil C18 guard column, 30 3 mm i.d. The flow rate was 0.8 mL/min, the detection wavelength was 225 nm, the injection volume was 20 AL, and no column temperature control was used. The mobile phase was 2:1/0.01 M sodium acetate/acetonitrile (pH = 4.4) with a total run time of 15 min. HPLC analysis of tissue samples was performed using Perkin-Elmer Series 200 Pump and Autosampler, and UV detector set at 220 nm. Perkin-Elmer Turbochrom software (v. 6.1.1.0.0) was used to quantitate residue peaks. The method used was previously reported. 2.5. Residue analysis Skinless catfish fillets were homogenized to a fine powder using a Robot Coupe processor and dry ice. The homogenized samples were transferred to storage bottles and stored at 40 8C. After placement in the freezer the bottles were capped loosely to allow the subliming dry ice to escape. After ~ 24 h the caps were completely tightened. The residue analysis method used has been reported elsewhere (Wrzesinski et al., 2003). Briefly, a 2 g (F 0.2 g) aliquot of catfish muscle homogenate was hydrolyzed with 6 N HCl in a shaking water bath set at 100 8C, for ~ 3 h. The hydrolysate was then extracted with ethyl acetate followed by basification. The extracted basified hydrolysate was applied to a Varian Chem ElutR CE1020 diatomaceous earth sorbent column and allowed to adsorb. Residues were eluted from the column with ethyl acetate, the eluate was taken to dryness, and the residues were resuspended in mobile phase for HPLC analysis. Florfenicol and its related metabolites are converted to FFA during the method’s acid hydrolysis step (US FDA NADA 141-063, 1996; EMEA MRL 251/97-Final, 1997; EMEA MRL 589/ 99-Final, 1999; EMEA MRL 591/99-Final, 1999) and it is FFA that is quantitated.
2.4. Instrumentation 3. Results HPLC analysis of medicated feed was performed using a Varian 2010 HPLC pump, a Varian 2050 UV detector, a manually operated Valco C6W injector valve with 20 AL sample loop, and a Varian 4270 Integrator. Separation was accomplished on a Nucleo˚ , 5 Am), 250 4.6 mm i.d. with sil C18 column (100 A
3.1. Feed analysis results The average florfenicol level found in the 3 medicated feed samples taken prior to the start of dosing (beginning, middle, and end) was 3872F 328 ppm (87.5 F
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7.42% of the 4425 ppm nominal concentration). Drug levels in the non-medicated feed were below the 13 ppm limit of detection. Drug levels in feed storage stability samples taken after the completion of dosing averaged 3906 F 41.3 ppm, in close agreement with the original results and demonstrating the stability of florfenicol in the feed over the course of dosing. Procedural recoveries from fortified control catfish feed analyzed concurrently with the medicated feed were 98.5–106% indicating that the assay was working correctly. 3.2. In-life results In the initial 2 days of treatment, fish consumed less florfenicol-medicated feed than was anticipated and therefore treatment was extended from 10 to 12 days. Over the entire 12-day treatment period, the fish consumed an average of 3.03 lb of feed/day equivalent to a 0.21% feeding rate (based upon the 700 total fish and an average 2.02 lb body weight determined prior to the start of acclimation) and an 8.1 mg florfenicol/kg b.w./ day dosing rate. Over the last 10 days of medication, the fish consumed an average of 3.39 lb of feed/day equivalent to a 0.24% feeding rate and a 9.3 mg florfe-
313
nicol/kg b.w./day dosing rate (93% of target). The total florfenicol consumed over the entire 12-day treatment period was 97.2 mg/kg which is 97.2% of the 100 mg/ kg therapeutic dose (10 mg/kg/day over 10 days). Water temperature averaged 20.7 8C and 17.9 8C during the treatment and withdrawal periods, respectively, and remained below 28 8C for the entire course of treatment and withdrawal. At the end of the in-life phase the test pond was drained and the remaining fish were counted and weighed. The total number of fish accounted for in the pond (fish remaining plus fish removed) was 709, in very close agreement to the original determination of 707 fish. The average weight of the remaining fish was 2.10 lb, approximately 4% greater than the initial 2.02 lb average weight. This growth rate could be expected of catfish maintained at the cooler temperatures observed over the course of this study. 3.3. Tissue analysis results All control tissue samples were analyzed one time prior to analysis of tissues from treated fish. There were no apparent residues greater than or equal to the method
Table 2 Florfenicol amine residue level ranges, average residue levels, and quartile values in catfish fillet by withdrawal day and by sex Withdrawal day/sex
n
Residue level range (ppb)
Average
Standard deviation
%CV
Median (ppb)
Q1 (ppb)
Q3 (ppb)
Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day
20 15 5 20 12 8 20 12 8 20 10 10 20 11 9 20 15 5
bLODa–27,650 bLODa–27,650 523–10,832 bLOQb–11,151 bLOQb–11,151 329–1900 250–2051 250–2051 254–715 bLODa–424 93–424 bLODa–335 bLODa–317 bLODa–317 bLOQb–173 bLOQb–285 bLOQb–230 112–285
5378 5785 4238 2303 3362 847 876 1196 533 232 228 238 168 165 147 169 163 183
7014 7872 4218 2959 3560 482 537 722 187 109 121 97 80 73 38 50 41 72
130.42 136.08 99.53 128.48 105.89 56.91 61.30 60.37 35.08 46.98 53.07 40.76 47.62 44.24 25.85 29.59 25.15 39.34
2684 2669 4526 831 2043 766 702 1104 592 185 211 181 142 146 137 151 149 167
620 757 541 543 533 550 516 623 355 102 111 67 76 75 38 128 130 119
5620 9110 7791 2784 3989 957 1344 1857 702 300 320 216 173 195 173 200 199 256
1/All 1/Male 1/Female 2/All 2/Male 2/Female 4/All 4 /Male 4/Female 7/All 7/Male 7/Female 14/All 14/Male 14/Female 21/All 21/Male 21/Female
Values b LOQ and b LOD were not used in calculating average. a Less than the 44 ppb limit of detection. b Less than the 75 ppb limit of quantitation.
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A FFA
mAU
150.00
10.97 min
100.00
50.00
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
4
6
8
10
12
14
16
18
20
22
24
26
28
14
16
18
20
22
24
26
28
B
mAU
200.00
150.00
100.00
50.00 0
2
C
mAU
200.00
FFA 11.01 min
150.00
100.00
50.00 0
2
4
6
8
10
12
Time (minutes) Fig. 2. Representative chromatograms of a 2000 ng/mL florfenicol amine standard (A), extract from control catfish muscle sample (B), and extract from a Withdrawal Day 2 catfish muscle sample (C).
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315
8000 catfisha troutb salmonc
7000 6000 5000 4000 3000 2000 1000 0 0
5
10
15
20
Withdrawal Day Fig. 3. Residue depletion curve for florfenicol amine in catfish, trout, and salmon muscle following administration in feed. All 3 species dosed at a nominal 10 mg/kg b.w./day rate in feed for 10 days. (a) 20 fish per time point. Catfish maintained in outside pond. Water temperature during treatment and withdrawal averaged 20.7 8C and 17.9 8C, respectively. (b) 10 fish per time point. Trout maintained in tanks at 10 8C (Pinault et al., 1997). (c) 6 fish per time point. Salmon maintained in tanks at 10 8C. For all time points after 7 days, average residue levels were below the 300 ppb limit of quantitation (EMEA MRL 251/97-Final, 1997).
LOD of 44 ppb (data not shown). FFA residue level ranges are presented in Table 2 for all treated fish and also by sex. See Fig. 2 for example analytical standard and control and treated tissue sample chromatograms. Residue levels of the FFA marker residue in catfish muscle averaged 5378 ppb 1 day after dosing. They declined rapidly during the first week of withdrawal to an average of 2303 ppb, 876 ppb, and 232 ppb at 2, 4, and 7 days, respectively, after dosing. Muscle residue levels plateaued at 2 weeks after dosing, at an average of 168 ppb. A residue depletion curve for this study (average FFA residue level vs. withdrawal day) is presented in Fig. 3. 3.4. QC sample analysis As a part of each specimen assay set quality control (QC) samples were analyzed. Duplicate aliquots of control tissue were fortified at 250 ppb and 2000 ppb and processed along with the treated fish tissue samples and an unfortified control sample. At the 250 ppb fortification level, recoveries ranged from 88.6% to 127.6% (102.1 F11.0% average recovery). At the 2000 ppb fortification level, recoveries ranged from
79.2% to 104.8% (94.1 F 6.1% average recovery). For every set analyzed, recoveries from at least 3 out of the 4 fortified QC samples were z 80% and V 110%. For every sample set analyzed, residue levels for the unfortified control samples were all b LOQ (75 ppb) (data not shown).
4. Discussion Individual muscle residue levels varied considerably within each interval during the initial week of withdrawal. On day 1, levels ranged from b LOD (44 ppb) to 27,650 ppb, an approximate 600-fold difference. This high variability is to be expected in a study conducted under field conditions as not all fish consume the same amount of feed and not all fish feed every day. However, average residue levels declined rapidly so that by 4 days the mean concentration of FFA was below the 1000 ppb proposed tolerance level and by 7 days all individual fish were below it (Table 2). Pinault et al. (1997) reported rapidly declining mean residue levels in rainbow trout maintained in
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freshwater tanks at 10 8C and treated with florfenicolmedicated feed by essentially the same dosing regimen. Average residue levels were 3.37 F 1.17 ppm, 1.08 F 0.54 ppm, 0.21 F 0.11 ppm, and 0.15 F 0.05 ppm at 1, 3, 8, and 15 days post withdrawal (10 fish per time point). In Atlantic salmon held in seawater tanks maintained at 10 8C and treated with florfenicolmedicated feed by essentially the same dosing regimen, muscle residue levels of FFA were 7.59 F 1.39 ppm, 7.90 F 2.23 ppm, 6.71 F 0.69 ppm, 2.24 F 0.78 ppm, and 1.31 F1.25 ppm at 3 h, 12 h, and 1, 3, and 7 days after withdrawal, respectively (EMEA MRL 251/ 97-Final, 1997) (values for 3 and 12 h, 3 days, and for standard deviations are personal communication, A. Bova). At 15 days post withdrawal and beyond, average residue levels were below the 300 ppb limit of quantitation (6 fish per time point). No information on the sex of the trout or salmon was provided. A comparison of the depletion curves for catfish, trout and salmon demonstrate they are closely correlated for the 3 species (Fig. 3). By 7 days after treatment the average FFA muscle residue levels were below 1000 ppb for both catfish and trout and only slightly above this level for salmon. By 14 days the residue levels were significantly below 1000 ppb for all 3 species of fish. As with catfish, residue levels in salmon and in trout varied considerably during the first week of withdrawal.
References Bjo¨rklund, H., Bylund, G., 1990. Temperature-related absorption and excretion of oxytetracycline in rainbow trout (Salmo gairdneri R.). Aquaculture 84, 363 – 372. Bjo¨rklund, H.V., Eriksson, A., Bylund, G., 1992. Temperaturerelated absorption and excretion of oxolinic acid in rainbow trout. Aquaculture 102, 17 – 27. EMEA MRL 251/97-FINAL (1997) http://www.emea.eu.int/pdfs/ vet/mrls/025197en.pdf. EMEA MRL 589/99-FINAL (1999) http://www.emea.eu.int/pdfs/ vet/mrls/058999en.pdf. EMEA MRL 591/99-FINAL (1999) http://www.emea.eu.int/pdfs/ vet/mrls/059199en.pdf. FDA NADA 141-063 FOI Summary (1996) http://www.fda.gov/ cvm/efoi/section2/141063053196.html.
Gaikowski, M.P., Wolf, J.C., Endris, R.G., Gingerich, W.H., 2003. Safety of Aquaflor (Florfenicol 50% Type A Medicated Article), Administered in Feed to Channel Catfish, Ictalurus punctatus. Toxicol. Pathol. 31, 689 – 697. Gaunt, P.S., Khoo, L., Howard, R., McGinnis, A.L., Santucci, T.D., Katz, T., Endris, R.G., 2004. Determination of dose rate and duration of administration of florfenicol in feed for control of mortality in channel catfish, Ictalurus punctatus (Rafinesque), infected with Edwardsiella ictaluri, etiological agent of enteric septicemia. J. World Aquac. Soc. 35, 279 – 289. Hawke, J.P., McWhorter, A.C., Steigerwalt, A.G., Brenner, D.J., 1981. Edwardsiella ictaluri sp. Nov., the causative agent of enteric septicemia in catfish. Int. J. Syst. Bacteriol. 31, 396 – 400. Hormazabel, V., Steffenak, I., Yndestad, M., 1993. Simultaneous determination of residues of Florfenicol and the metabolite Florfenicol Amine in fish tissues by high performance liquid chromatography. J. Chromatogr. 616, 161 – 165. Horsberg, T.E., Martinsen, B., Varma, K.J., 1994. The disposition of 14 C-Florfenicol in atlantic salmon (Salmo salar). Aquaculture 122, 97 – 106. Horsberg, T.E., Hoff, K.A., Nordmo, R., 1996. Pharmacokinetics of Florfenicol and its Metabolite Florfenicol Amine in Atlantic Salmon. J. Aquat. Anim. Health 8, 292 – 301. Nagabhusahan, T., Miller, G.H., Varma, K.J., 1992. Chloramphenicol and analogues. In: Howe-Grant, Mary (Ed.), Encyclopedia of Chemical Technology, Vol. 2. John Wiley & Sons, Hoboken, NJ, pp. 961 – 978. Namdari, R., Abedini, S., Law, F.C.P., 1996. Tissue distribution and elimination of oxytetracycline in seawater chinook and coho salmon following medicated feed treatment. Aquaculture 144, 27 – 38. Nordmo, R., Holth Riseth, J.M., Varma, K.J., Sutherland, I.H., Brokken, E.S., 1998. Evaluation of florfenicol in Atlantic salmon, Salmo salar L.: efficacy against furunculosis due to Aeromonas salmonicida and cold water vibriosis due to Vibro salmonicida. J. Fish Dis. 21, 289 – 297. Pinault, L.P., Millot, L.K., Sanders, P.J., 1997. Absolute oral bioavailability and residues of Florfenicol in the rainbow trout (Oncorhynchus mykiss). J. Vet. Pharmacol. Ther. 20 (Suppl. 1), 297 – 298. Samuelson, O.B., Hjeltnes, B., Glette, J., 1998. Efficacy of orally administered florfenicol in the treatment of furunculosis in Atlantic salmon. J. Aquat. Anim. Health 10, 56 – 61. Sohlberg, S., Aulie, A., Soli, N.E., 1994. Temperature-dependent absorption and elimination of Flumequine in rainbow trout in fresh water. Aquaculture 119, 1 – 10. Thune, R.L., 1991. Major infectious and parasitic diseases of channel catfish. Vet. Hum. Toxicol. 33 (Supplement 1), 14 – 18. Waltman, W.D., Shotts, E.B., 1986. Antimicrobial susceptibility of Edwardsiella ictaluri. J. Wildl. Dis. 22, 173 – 177. Wrzesinski, C.L., Crouch, L.S., Endris, R., 2003. Determination of Florfenicol Amine in channel catfish muscle by liquid chromatography. JAOC 86, 515 – 520.
Florfenicol residue depletion in channel catfish, Ictalurus punctatus (Rafinesque) Christopher Wrzesinskia,*, Louis Croucha, Patricia Gauntb, Dwayne Holifieldc, Nicole Bertrandd, Richard Endrise a
b
Schering-Plough Research Institute, Lafayette, NJ 07848, United States Mississippi State University College of Veterinary Medicine, Stoneville, MS 38776, United States c Delta Western, Indianola, MS 38751, United States d EnviroTest Laboratories, Xenos Division, Nepean, Ontario, Canada, K2E 7L5 e Schering-Plough Animal Health, Union, NJ 07083, United States Received 10 February 2005; received in revised form 2 May 2005; accepted 4 May 2005
Abstract Florfenicol is a broad-spectrum antibiotic currently used in salmon aquaculture and proposed for use in channel catfish. A residue depletion study was performed under commercial aquaculture conditions to determine a withdrawal period. Market weight catfish (~ 2 lb) maintained in a 0.1 acre test pond at a 7000 fish/acre stocking density were dosed with commercially formulated florfenicol in feed for 12 days at an average rate of 9.3 mg/kg b.w./day over the last 10 days, or 93% of the proposed dose. Twenty fish each were collected 1, 2, 4, 7, 14, and 21 days post withdrawal of medicated feed and levels of the marker residue, florfenicol amine (FFA), in muscle tissue were determined via a validated HPLC-UV method. By 4 days average muscle FFA levels were below the proposed 1000 ppb tolerance for FFA and beyond this time point all individual fish were below this level. D 2005 Elsevier B.V. All rights reserved. Keywords: Florfenicol; Antibiotic; Catfish; Residue depletion; Withdrawal; Florfenicol amine
1. Introduction Florfenicol (SCH 25298), [R-(R*,S*)]-2,2-dichloroN-[1(fluoromethyl)-2-hydroxy-2-[4-(methylsulfonyl)phenyl]ethyl]-acetamide, (Fig. 1) is a broad spectrum * Corresponding author. Tel.: +1 973 940 4388; fax: +1 973 940 4159. E-mail address: [email protected] (C. Wrzesinski). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.05.005
antibiotic that is widely used in veterinary medicine. While it is structurally related to the antibiotics chloramphenicol and thiamphenicol used in humans, the potential for microbial resistance impacting human health is limited because both of these are rarely used due to an association with aplastic anemia (Nagabhusahan et al., 1992). As the injectable formulation NuflorR, florfenicol has been approved for use in cattle (US and European Union) and swine (European Union only) for treatment of respiratory disease and/or foot
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O H3C
OH
S O F
HN O
Florfenicol (SCH 25298) CHCl2
O H3C
OH
S O H2N
F
Florfenicol Amine (SCH 40458) Fig. 1. Structure of florfenicol and florfenicol amine.
rot. As the 50% feed premix AquaflorR/AquafenR, florfenicol is approved for use in salmonids (Norway, Chile, Canada, UK) for treatment of furunculosis (Nordmo et al., 1998; Samuelson et al., 1998). Initial studies have demonstrated that Aquaflor administered in feed to channel catfish at a dose rate of 10 mg/kg body weight/day for 10 consecutive days reduces catfish mortality (Gaunt et al., 2004) resulting from enteric septicemia (ESC), the primary disease affecting commercial catfish production in the United States (Hawke et al., 1981; Thune, 1991). Florfenicol has been demonstrated to cause no changes in fish growth and no clinical changes in catfish fed at rates up to 34.9 mg/kg (Gaikowski et al., 2003). Consequently ScheringPlough Animal Health is developing Aquaflor for treatment of ESC in channel catfish. As part of this development effort a final residue depletion study was conducted with florfenicol in catfish. This is a required study for registration of a veterinary drug and is used to determine a withdrawal period (the required length of time after drug administration that an animal must be held from market). In a final residue depletion study the target animal species (catfish in this case) is medicated at the maximum proposed dosing rate and length of administration and
the decline in marker residue concentration over time in the target tissue is determined. The marker residue can be the drug itself or a metabolite and it is chosen because the ratio of its concentration to the concentration of total drug-related residues remains constant over time in the target tissue. The target tissue is the last edible tissue in the target animal in which residues deplete to the permitted safe concentration (US FDACVM Guideline 3, 1994). Thus the target tissue marker residue concentration is used to determine whether total drug-related residues have declined to a safe concentration in all edible tissues of the target animal. As muscle is considered by regulation to be the only edible tissue in catfish, it is the target tissue. Previous studies with florfenicol in cattle, poultry, swine, and salmon have identified florfenicol amine as the marker residue (US FDA NADA 141-063, 1996; EMEA MRL 251/97-Final, 1997; EMEA MRL 589/99Final, 1999; EMEA MRL 591/99-Final, 1999). The sample preparation employed in these studies and in the current study includes an acid hydrolysis step that converts florfenicol and its related metabolites to florfenicol amine (Wrzesinski et al., 2003) (Fig. 1). Therefore florfenicol amine (FFA, SCH 40458) was designated as the marker residue in catfish. Metabolism studies of florfenicol in Atlantic salmon have identified FFA as the major metabolite in muscle tissue although florfenicol is much more predominant in skin (Horsberg et al., 1994; EMEA MRL 251/97Final, 1997). Hormazabel et al. (1993) describe a method that utilized solvent extraction followed by HPLC analysis to determine the concentration of florfenicol and FFA in muscle and liver of rainbow trout. Horsberg et al. (1996) fed Atlantic salmon florfenicol medicated feed at a rate of 10 mg florfenicol/kg b.w./day for 10 days to determine the depletion of FFA in muscle and liver. The analytical method described by Hormazabel et al. (1993) was used for the Horsberg study. Pinault et al. (1997) describe a study in which rainbow trout were fed florfenicol medicated feed at a rate of 10 mg florfenicol/kg b.w./day for 10 days to determine the depletion of FFA in muscle with attached skin. Additionally, in 2 separate studies, Atlantic salmon kept at 58 and at 108 C were dosed with florfenicol medicated feed at 10 mg florfenicol/kg b.w./day for 10 days and the depletion of FFA in muscle and in skin were determined (EMEA MRL 251/97-Final, 1997). The sample preparation in these last 3 studies (Pinault et al., 1997;
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EMEA MRL 251/97-Final, 1997) included an acid hydrolysis step to convert florfenicol and related metabolites to FFA.
2. Materials and method 2.1. Test article and analytical standards AquaflorR 50% Type A Medicated Article, a formulation containing 50% florfenicol, nominal (49.49% assayed), was supplied by Schering-Plough Animal Health and was used in the preparation of medicated feed as described below. Florfenicol and FFA were supplied by the Schering-Plough Research Institute (SPRI) and were used as analytical reference standards during feed analysis and during tissue analysis, respectively. 2.2. Feed preparation and analysis Basal (control) and medicated floating catfish feeds were formulated according to normal aquaculture practices at Delta Western Research Center (Indianola, MS). Fish were fed a commercial diet prior to the start of dosing. The average daily feeding rate of the catfish (the ratio of total weight of feed consumed to the total weight of fish in the pond) as determined for 4 days prior to the start of dosing was 0.21% body weight (based on a 2.02-lb average body weight and 700 total fish in the pond as described below). This required a 4762 ppm concentration of florfenicol in feed to give the desired dose level of 10 mg/kg/day (target dose rate divided by feeding rate multiplied by 100%). However, available Aquaflor was only sufficient to manufacture feed at a 4425 ppm nominal FFC concentration (4.4%). Florfenicol is commercially incorporated into catfish feed at 2–3%. Separate batches of control and medicated feed were mixed in a commercial feed mixer and aliquots were taken from the beginning, middle, and end of each batch. The feed samples were shipped to Xenos Labs (Nepean, Ontario) for drug concentration analysis. After the end of the treatment period, an additional composite sample of medicated feed was obtained according to the AOAC method on the sampling of animal feed (2000) and shipped to Xenos for storage stability analysis.
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A 250 g aliquot of each feed sample was homogenized in a Braun coffee grinder to a particle size of 1–2 mm and a 10-g subsample was extracted with 100 mL acetonitrile/water (1:1). The subsample was sonicated at 50 8C for 5 min, placed on a wrist-action shaker at the highest setting for 10 min and then sonicated and placed on the shaker one more time. After allowing the extract to settle, approximately 50 mL of the supernatant was decanted and centrifuged. A 5 mL aliquot of the centrifuged extract was transferred onto an Envi-Carb cartridge (0.5 g, 6 mL, Supelco) and the eluate was collected in a 10-mL volumetric flask. The cartridge was rinsed with additional extraction solvent and the volume was adjusted to 10 mL. For the medicated feed, the sample extract was diluted five fold prior to HPLC analysis. 2.3. In-life phase See Table 1 for a calendar of in-life events. Unsexed food size fish (fish weighing z 1 lb) were raised in a Mississippi Agricultural Forestry and Extension Service (MAFES) pond at the Mississippi State University Delta Research Experimental Center (DREC). Three samples of 100 food size fish were collected from a seine and weighed to determine mean weight (2.02 F 0.10 lb/fish). These fish were then released back into the pond and were not used for this study. Fish for the study were then collected from the same seine and weighed to yield a weight equivalent to ~ 700 fish (1428 lb total weight, 707 actual calculated number of fish). These ~ 700 fish were placed in an oxygenated well in a live hauler and transported to a 0.1 acre pond at Delta Western. The Table 1 Calendar of in-life activities Activity
Date
Start of acclimation Control (Group VII) fish collected Preparation of medicated feed First day of dosing Last day of dosing Group I fish (1 day post withdrawal) Group II fish (2 days post withdrawal) Group III fish (4 days post withdrawal) Group IV fish (7 days post withdrawal) Group V fish (14 days post withdrawal) Group VI fish (21 days post withdrawal)
8/30/00 9/21/00 10/13/00 10/13/00 10/24/00 10/25/00 10/26/00 10/28/00 10/31/00 11/7/00 11/14/00
collected collected collected collected collected collected
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stocking density was 7000 fish/acre and therefore similar to commercial stocking practices. Enteric septicemia is most prevalent when water temperatures are 20–28 8C (Waltman and Shotts, 1986). Additionally the rate of residue depletion in fish decreases with decreasing water temperature (Bjo¨rklund and Bylund, 1990; Bjo¨rklund et al., 1992; Sohlberg et al., 1994; Namdari et al., 1996). Therefore this study was conducted in the fall, when water temperatures at the site were at the lower end of or below the 20–28 8C range and the rate of residue depletion would be the lowest. Pond water temperature and pond water oxygen content were monitored at least daily. Supplemental aeration was provided when dissolved oxygen levels could not be determined (due to inclement weather) or were approaching b 4 ppm. Catfish were fed once a day to approximate satiation and the amount of feed consumed was recorded daily. Prior to dosing with medicated feed the fish were allowed to acclimate for 44 days during which they were fed a commercial diet. During acclimation 10 control fish were collected and muscle sampled to check for possible interference with the analytical method and to serve as control matrix for analytical method performance checks. After acclimation the fish were fed medicated feed for 12 days (extended from the originally intended 10 days due to poor feeding during the initial 2 days of medication) and then were fed the prepared control feed (equivalent to a commercial diet) during withdrawal. Twenty fish were arbitrarily collected at 1, 2, 4, 7, 14, and 21 days post-withdrawal of florfenicol-medicated feed and were transported live in ice water to DREC. At DREC collected fish were euthanized via pithing (severing of the brain and spinal cord), sexed via internal examination, and 2 skinless muscle fillets were taken from each. One of the fillets from each fish was then shipped to SPRI for homogenization and residue analysis.
a Nucleosil C18 guard column, 30 3 mm i.d. The flow rate was 0.8 mL/min, the detection wavelength was 225 nm, the injection volume was 20 AL, and no column temperature control was used. The mobile phase was 2:1/0.01 M sodium acetate/acetonitrile (pH = 4.4) with a total run time of 15 min. HPLC analysis of tissue samples was performed using Perkin-Elmer Series 200 Pump and Autosampler, and UV detector set at 220 nm. Perkin-Elmer Turbochrom software (v. 6.1.1.0.0) was used to quantitate residue peaks. The method used was previously reported. 2.5. Residue analysis Skinless catfish fillets were homogenized to a fine powder using a Robot Coupe processor and dry ice. The homogenized samples were transferred to storage bottles and stored at 40 8C. After placement in the freezer the bottles were capped loosely to allow the subliming dry ice to escape. After ~ 24 h the caps were completely tightened. The residue analysis method used has been reported elsewhere (Wrzesinski et al., 2003). Briefly, a 2 g (F 0.2 g) aliquot of catfish muscle homogenate was hydrolyzed with 6 N HCl in a shaking water bath set at 100 8C, for ~ 3 h. The hydrolysate was then extracted with ethyl acetate followed by basification. The extracted basified hydrolysate was applied to a Varian Chem ElutR CE1020 diatomaceous earth sorbent column and allowed to adsorb. Residues were eluted from the column with ethyl acetate, the eluate was taken to dryness, and the residues were resuspended in mobile phase for HPLC analysis. Florfenicol and its related metabolites are converted to FFA during the method’s acid hydrolysis step (US FDA NADA 141-063, 1996; EMEA MRL 251/97-Final, 1997; EMEA MRL 589/ 99-Final, 1999; EMEA MRL 591/99-Final, 1999) and it is FFA that is quantitated.
2.4. Instrumentation 3. Results HPLC analysis of medicated feed was performed using a Varian 2010 HPLC pump, a Varian 2050 UV detector, a manually operated Valco C6W injector valve with 20 AL sample loop, and a Varian 4270 Integrator. Separation was accomplished on a Nucleo˚ , 5 Am), 250 4.6 mm i.d. with sil C18 column (100 A
3.1. Feed analysis results The average florfenicol level found in the 3 medicated feed samples taken prior to the start of dosing (beginning, middle, and end) was 3872F 328 ppm (87.5 F
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7.42% of the 4425 ppm nominal concentration). Drug levels in the non-medicated feed were below the 13 ppm limit of detection. Drug levels in feed storage stability samples taken after the completion of dosing averaged 3906 F 41.3 ppm, in close agreement with the original results and demonstrating the stability of florfenicol in the feed over the course of dosing. Procedural recoveries from fortified control catfish feed analyzed concurrently with the medicated feed were 98.5–106% indicating that the assay was working correctly. 3.2. In-life results In the initial 2 days of treatment, fish consumed less florfenicol-medicated feed than was anticipated and therefore treatment was extended from 10 to 12 days. Over the entire 12-day treatment period, the fish consumed an average of 3.03 lb of feed/day equivalent to a 0.21% feeding rate (based upon the 700 total fish and an average 2.02 lb body weight determined prior to the start of acclimation) and an 8.1 mg florfenicol/kg b.w./ day dosing rate. Over the last 10 days of medication, the fish consumed an average of 3.39 lb of feed/day equivalent to a 0.24% feeding rate and a 9.3 mg florfe-
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nicol/kg b.w./day dosing rate (93% of target). The total florfenicol consumed over the entire 12-day treatment period was 97.2 mg/kg which is 97.2% of the 100 mg/ kg therapeutic dose (10 mg/kg/day over 10 days). Water temperature averaged 20.7 8C and 17.9 8C during the treatment and withdrawal periods, respectively, and remained below 28 8C for the entire course of treatment and withdrawal. At the end of the in-life phase the test pond was drained and the remaining fish were counted and weighed. The total number of fish accounted for in the pond (fish remaining plus fish removed) was 709, in very close agreement to the original determination of 707 fish. The average weight of the remaining fish was 2.10 lb, approximately 4% greater than the initial 2.02 lb average weight. This growth rate could be expected of catfish maintained at the cooler temperatures observed over the course of this study. 3.3. Tissue analysis results All control tissue samples were analyzed one time prior to analysis of tissues from treated fish. There were no apparent residues greater than or equal to the method
Table 2 Florfenicol amine residue level ranges, average residue levels, and quartile values in catfish fillet by withdrawal day and by sex Withdrawal day/sex
n
Residue level range (ppb)
Average
Standard deviation
%CV
Median (ppb)
Q1 (ppb)
Q3 (ppb)
Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day
20 15 5 20 12 8 20 12 8 20 10 10 20 11 9 20 15 5
bLODa–27,650 bLODa–27,650 523–10,832 bLOQb–11,151 bLOQb–11,151 329–1900 250–2051 250–2051 254–715 bLODa–424 93–424 bLODa–335 bLODa–317 bLODa–317 bLOQb–173 bLOQb–285 bLOQb–230 112–285
5378 5785 4238 2303 3362 847 876 1196 533 232 228 238 168 165 147 169 163 183
7014 7872 4218 2959 3560 482 537 722 187 109 121 97 80 73 38 50 41 72
130.42 136.08 99.53 128.48 105.89 56.91 61.30 60.37 35.08 46.98 53.07 40.76 47.62 44.24 25.85 29.59 25.15 39.34
2684 2669 4526 831 2043 766 702 1104 592 185 211 181 142 146 137 151 149 167
620 757 541 543 533 550 516 623 355 102 111 67 76 75 38 128 130 119
5620 9110 7791 2784 3989 957 1344 1857 702 300 320 216 173 195 173 200 199 256
1/All 1/Male 1/Female 2/All 2/Male 2/Female 4/All 4 /Male 4/Female 7/All 7/Male 7/Female 14/All 14/Male 14/Female 21/All 21/Male 21/Female
Values b LOQ and b LOD were not used in calculating average. a Less than the 44 ppb limit of detection. b Less than the 75 ppb limit of quantitation.
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A FFA
mAU
150.00
10.97 min
100.00
50.00
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
4
6
8
10
12
14
16
18
20
22
24
26
28
14
16
18
20
22
24
26
28
B
mAU
200.00
150.00
100.00
50.00 0
2
C
mAU
200.00
FFA 11.01 min
150.00
100.00
50.00 0
2
4
6
8
10
12
Time (minutes) Fig. 2. Representative chromatograms of a 2000 ng/mL florfenicol amine standard (A), extract from control catfish muscle sample (B), and extract from a Withdrawal Day 2 catfish muscle sample (C).
C. Wrzesinski et al. / Aquaculture 253 (2006) 309–316
315
8000 catfisha troutb salmonc
7000 6000 5000 4000 3000 2000 1000 0 0
5
10
15
20
Withdrawal Day Fig. 3. Residue depletion curve for florfenicol amine in catfish, trout, and salmon muscle following administration in feed. All 3 species dosed at a nominal 10 mg/kg b.w./day rate in feed for 10 days. (a) 20 fish per time point. Catfish maintained in outside pond. Water temperature during treatment and withdrawal averaged 20.7 8C and 17.9 8C, respectively. (b) 10 fish per time point. Trout maintained in tanks at 10 8C (Pinault et al., 1997). (c) 6 fish per time point. Salmon maintained in tanks at 10 8C. For all time points after 7 days, average residue levels were below the 300 ppb limit of quantitation (EMEA MRL 251/97-Final, 1997).
LOD of 44 ppb (data not shown). FFA residue level ranges are presented in Table 2 for all treated fish and also by sex. See Fig. 2 for example analytical standard and control and treated tissue sample chromatograms. Residue levels of the FFA marker residue in catfish muscle averaged 5378 ppb 1 day after dosing. They declined rapidly during the first week of withdrawal to an average of 2303 ppb, 876 ppb, and 232 ppb at 2, 4, and 7 days, respectively, after dosing. Muscle residue levels plateaued at 2 weeks after dosing, at an average of 168 ppb. A residue depletion curve for this study (average FFA residue level vs. withdrawal day) is presented in Fig. 3. 3.4. QC sample analysis As a part of each specimen assay set quality control (QC) samples were analyzed. Duplicate aliquots of control tissue were fortified at 250 ppb and 2000 ppb and processed along with the treated fish tissue samples and an unfortified control sample. At the 250 ppb fortification level, recoveries ranged from 88.6% to 127.6% (102.1 F11.0% average recovery). At the 2000 ppb fortification level, recoveries ranged from
79.2% to 104.8% (94.1 F 6.1% average recovery). For every set analyzed, recoveries from at least 3 out of the 4 fortified QC samples were z 80% and V 110%. For every sample set analyzed, residue levels for the unfortified control samples were all b LOQ (75 ppb) (data not shown).
4. Discussion Individual muscle residue levels varied considerably within each interval during the initial week of withdrawal. On day 1, levels ranged from b LOD (44 ppb) to 27,650 ppb, an approximate 600-fold difference. This high variability is to be expected in a study conducted under field conditions as not all fish consume the same amount of feed and not all fish feed every day. However, average residue levels declined rapidly so that by 4 days the mean concentration of FFA was below the 1000 ppb proposed tolerance level and by 7 days all individual fish were below it (Table 2). Pinault et al. (1997) reported rapidly declining mean residue levels in rainbow trout maintained in
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freshwater tanks at 10 8C and treated with florfenicolmedicated feed by essentially the same dosing regimen. Average residue levels were 3.37 F 1.17 ppm, 1.08 F 0.54 ppm, 0.21 F 0.11 ppm, and 0.15 F 0.05 ppm at 1, 3, 8, and 15 days post withdrawal (10 fish per time point). In Atlantic salmon held in seawater tanks maintained at 10 8C and treated with florfenicolmedicated feed by essentially the same dosing regimen, muscle residue levels of FFA were 7.59 F 1.39 ppm, 7.90 F 2.23 ppm, 6.71 F 0.69 ppm, 2.24 F 0.78 ppm, and 1.31 F1.25 ppm at 3 h, 12 h, and 1, 3, and 7 days after withdrawal, respectively (EMEA MRL 251/ 97-Final, 1997) (values for 3 and 12 h, 3 days, and for standard deviations are personal communication, A. Bova). At 15 days post withdrawal and beyond, average residue levels were below the 300 ppb limit of quantitation (6 fish per time point). No information on the sex of the trout or salmon was provided. A comparison of the depletion curves for catfish, trout and salmon demonstrate they are closely correlated for the 3 species (Fig. 3). By 7 days after treatment the average FFA muscle residue levels were below 1000 ppb for both catfish and trout and only slightly above this level for salmon. By 14 days the residue levels were significantly below 1000 ppb for all 3 species of fish. As with catfish, residue levels in salmon and in trout varied considerably during the first week of withdrawal.
References Bjo¨rklund, H., Bylund, G., 1990. Temperature-related absorption and excretion of oxytetracycline in rainbow trout (Salmo gairdneri R.). Aquaculture 84, 363 – 372. Bjo¨rklund, H.V., Eriksson, A., Bylund, G., 1992. Temperaturerelated absorption and excretion of oxolinic acid in rainbow trout. Aquaculture 102, 17 – 27. EMEA MRL 251/97-FINAL (1997) http://www.emea.eu.int/pdfs/ vet/mrls/025197en.pdf. EMEA MRL 589/99-FINAL (1999) http://www.emea.eu.int/pdfs/ vet/mrls/058999en.pdf. EMEA MRL 591/99-FINAL (1999) http://www.emea.eu.int/pdfs/ vet/mrls/059199en.pdf. FDA NADA 141-063 FOI Summary (1996) http://www.fda.gov/ cvm/efoi/section2/141063053196.html.
Gaikowski, M.P., Wolf, J.C., Endris, R.G., Gingerich, W.H., 2003. Safety of Aquaflor (Florfenicol 50% Type A Medicated Article), Administered in Feed to Channel Catfish, Ictalurus punctatus. Toxicol. Pathol. 31, 689 – 697. Gaunt, P.S., Khoo, L., Howard, R., McGinnis, A.L., Santucci, T.D., Katz, T., Endris, R.G., 2004. Determination of dose rate and duration of administration of florfenicol in feed for control of mortality in channel catfish, Ictalurus punctatus (Rafinesque), infected with Edwardsiella ictaluri, etiological agent of enteric septicemia. J. World Aquac. Soc. 35, 279 – 289. Hawke, J.P., McWhorter, A.C., Steigerwalt, A.G., Brenner, D.J., 1981. Edwardsiella ictaluri sp. Nov., the causative agent of enteric septicemia in catfish. Int. J. Syst. Bacteriol. 31, 396 – 400. Hormazabel, V., Steffenak, I., Yndestad, M., 1993. Simultaneous determination of residues of Florfenicol and the metabolite Florfenicol Amine in fish tissues by high performance liquid chromatography. J. Chromatogr. 616, 161 – 165. Horsberg, T.E., Martinsen, B., Varma, K.J., 1994. The disposition of 14 C-Florfenicol in atlantic salmon (Salmo salar). Aquaculture 122, 97 – 106. Horsberg, T.E., Hoff, K.A., Nordmo, R., 1996. Pharmacokinetics of Florfenicol and its Metabolite Florfenicol Amine in Atlantic Salmon. J. Aquat. Anim. Health 8, 292 – 301. Nagabhusahan, T., Miller, G.H., Varma, K.J., 1992. Chloramphenicol and analogues. In: Howe-Grant, Mary (Ed.), Encyclopedia of Chemical Technology, Vol. 2. John Wiley & Sons, Hoboken, NJ, pp. 961 – 978. Namdari, R., Abedini, S., Law, F.C.P., 1996. Tissue distribution and elimination of oxytetracycline in seawater chinook and coho salmon following medicated feed treatment. Aquaculture 144, 27 – 38. Nordmo, R., Holth Riseth, J.M., Varma, K.J., Sutherland, I.H., Brokken, E.S., 1998. Evaluation of florfenicol in Atlantic salmon, Salmo salar L.: efficacy against furunculosis due to Aeromonas salmonicida and cold water vibriosis due to Vibro salmonicida. J. Fish Dis. 21, 289 – 297. Pinault, L.P., Millot, L.K., Sanders, P.J., 1997. Absolute oral bioavailability and residues of Florfenicol in the rainbow trout (Oncorhynchus mykiss). J. Vet. Pharmacol. Ther. 20 (Suppl. 1), 297 – 298. Samuelson, O.B., Hjeltnes, B., Glette, J., 1998. Efficacy of orally administered florfenicol in the treatment of furunculosis in Atlantic salmon. J. Aquat. Anim. Health 10, 56 – 61. Sohlberg, S., Aulie, A., Soli, N.E., 1994. Temperature-dependent absorption and elimination of Flumequine in rainbow trout in fresh water. Aquaculture 119, 1 – 10. Thune, R.L., 1991. Major infectious and parasitic diseases of channel catfish. Vet. Hum. Toxicol. 33 (Supplement 1), 14 – 18. Waltman, W.D., Shotts, E.B., 1986. Antimicrobial susceptibility of Edwardsiella ictaluri. J. Wildl. Dis. 22, 173 – 177. Wrzesinski, C.L., Crouch, L.S., Endris, R., 2003. Determination of Florfenicol Amine in channel catfish muscle by liquid chromatography. JAOC 86, 515 – 520.