Antimicrobial Agents and Chemotherapy, October 2004, p . 3912-3917, Vol . 48, No . 10

The use of antibiotics and chemotherapeutic agents in animals reared for human consumption should be based on toxicological and pharmacokinetic data obtained for the specific animal species considered . However, the present knowledge of antibacterial drug pharmacokinetics in different fish species is very poor . The availability of adequate data on the pharmacokinetics and bioavailabilities of antibacterial drugs in farmed fish is also important in order to minimize the environmental impacts of the drugs used in aquaculture .

The fish farming industry is rapidly expanding in Italy, and as in other countries, it has been accompanied by recurrent problems with bacterial infectious diseases . Among the several aquaculture species produced in Italy, farmed trout represent 80% of the fish produced by the nation's aquaculture production system (30) . Trout is a so-called minor species (15), but its level of consumption in some European countries is quite high .

Quinolones are effective antibacterial drugs widely used both in human and in veterinary medicine for therapy for various systemic bacterial infections (6, 23, 34) . During the last few years, enrofloxacin, a molecule belonging to the quinolone antimicrobial family, has received growing attention because of its potential efficacy for the treatment of diseases in fish (5-7, 9, 11, 26, 33) . This drug is a fluorinated quinolone carboxylic acid derivative (Fig . 1a), and it has been extensively used in veterinary medicine because of its broad spectrum of activity (12) . In both mammalian and nonmammalian species, enrofloxacin is dealkylated to ciprofloxacin, a metabolite that contributes to the activity of enrofloxacin and which is itself a potent antimicrobial agent (Fig . 1b) . Some microbiological, metabolism and pharmacokinetic aspects of enrofloxacin have been reported from previous studies with fishes (3, 4, 11, 25, 26, 29, 33); however, very few data on enrofloxacin depletion in fishes reared under field conditions are available (11, 32) .

 
Depletion of drugs from minor species of fishes must be assessed in order to determine the time needed before the antibiotic disappears from animal tissue and to assess in a definite way when the treated animal can be safely consumed . In this framework, there is a demand for suitable and sensitive analytical methods that can be used to monitor the levels of quinolone residues in foods and to establish drug withdrawal times in fishes after pharmacological treatments .

The Commission of the European Union establishes and periodically reviews drug maximum residue levels (MRLs) in foods . An MRL is defined (19) as the maximum concentration of residue resulting from the use of a veterinary medicinal product (expressed in milligrams per kilogram of body weight or micrograms per kilogram on a fresh weight basis) that may be accepted by the European Union to be legally permitted or recognized as acceptable in or on a food . It is based on the type and amount of residue considered to be without any toxicological hazard for human health, as expressed by the acceptable daily intake (ADI), or is based on a temporary ADI that uses an additional safety factor . It also takes into account other relevant public health risks as well as food technology aspects . The Commission of the European Union has established an MRL of 100 µg kg^–1 as the sum of the levels of enrofloxacin and ciprofloxacin in tissues (muscle plus skin in natural proportions) from fishes in general (19) . Since the pharmacokinetic properties of antibacterial agents vary significantly between species, the aim of this study was to determine the depletion times of enrofloxacin and its metabolite, ciprofloxacin, in rainbow trout (Oncorhynchus mykiss) after the administration of medicated feed . In this investigation we have considered for the analysis fish muscle plus skin in natural proportions because the MRLs for fishes refer to this edible tissue only; moreover, we have investigated the fish bones, as a previous study on the depletion of flumequine in sea bream (27) reported a remarkable bioaccumulation of the drug in this tissue . It is clear that during cooking of the fish, which is usually done with the fish bones in place, the release of drug from bone to muscle might cause the MRLs to be exceeded .

The analytical determination of enrofloxacin and ciprofloxacin levels was carried out by a high-performance liquid chromatography (HPLC) method, developed on the basis of previously published HPLC methods (31, 36), with minor modifications . The modified method has been validated in-house for determination of the levels of enrofloxacin and ciprofloxacin residues in fish tissues, following the requests of European Economic Commission (EEC) Decision No . 657/2002 (16) .

Animals and diet. One hundred thirty trout (weight, about 250 g) supplied by Rio Piavesella S.p.A . (Treviso, Italy) were used in the investigation . On their arrival at the fish farm, the animals were transferred into a cage that was immersed in a river . The trout were reared under field conditions at temperatures ranging from 13.25°C (for the first 20 days posttreatment) to 12°C (from the 21st day posttreatment) . The temperature was monitored continuously and was recorded every 12 h by two digital thermometer probes situated at two sampling sites in the tank (corresponding to the inflow water and the outflow water, respectively) . Procedures for trout care and management complied with those required by Italian laws (10) .

Two different diets were prepared in-house for the experimental trial: (i) a standard diet for fish (diet A) and (ii) a standard diet for fish to which the 1% (wt/wt) veterinary drug formulation 10% Baytril was added (diet B) . The drug concentration was selected according to trout feed consumption data and the therapeutic doses suggested for fish disease treatment (about 10 mg kg of trout body weight^–1 day^–1) . The diet ingredients (fish meal and fish oil) were supplied by Hendrix S.p.A . (Mozzecane, Verona, Italy) . According to the protocol, Hendrix fish meal (58%), Hendrix fish oil (5.8%), and water (36.2%) were thoroughly mixed and homogenized; an adequate pellet was then produced with a bench food mixer-extruder . In diet B (medicated feed), 1% water was replaced by the veterinary formulation described above . Finally, the wet pellets were dried in an oven at 35°C for about 12 h to achieve the final moisture content (about 10%) . The dry pellets were stored at –20°C until they were administered to the fishes . The enrofloxacin concentration in the feed provided by diet B was verified by HPLC by the same method used for the fish tissues and was 1,006 ± 38 mg kg^–1 .

Before the start of drug administration, the trout were acclimatized for a period of 7 days, during which they all received diet A (standard feed) . After 1 day of fasting, the trout were fed diet B (medicated feed) for 5 days . At the end of this drug administration period, the trout were again fed diet A until they were killed for sample analysis .

The trout appeared healthy during the whole experimental trial, and the drug had no effect on their feed intake or weight gain .

Sample collection and preparation. A control group of 10 trout was killed before the start of drug administration . Subsequently, 10 trout were randomly sampled at 3, 12, 24, 48, 96, 168, 240, 480, 720, 960, 1,152, and 1,416 h after the start of the pharmacological treatment and killed . The trout were killed by freezing them in melting ice .

Muscle and skin in natural proportions and fish bones were collected in situ . Each sample was placed in a polyethylene bag, and the bags were transferred to the analysis laboratory in containers with dry ice . They were stored in the bags at –80°C until they were analyzed .

Analytical procedures. The methodology used to determine the levels of enrofloxacin and ciprofloxacin in the muscle and skin in natural proportions and fish bone was based on the extraction of these drugs from the matrix, successive purification, and final separation by HPLC with fluorescence detection . The sample pretreatment procedure and the chromatographic conditions were similar to those described by Plakas et al . (31) and Yorke and Froc (36), respectively, except for some modifications both to the extraction procedure and to the chromatographic conditions .

Briefly, 5 ml of methanol-acetic acid (98:2 [vol/vol]) was added to the homogenized tissue sample (about 1 g) . After agitation on a vortex mixer for 2 min and sonication for 10 min at room temperature, the sample was centrifuged at 4,000 x g for 10 min at 5°C . The supernatant was then transferred into a 50-ml flask, and the residue was again extracted with a fresh portion of methanol-acetic acid (98:2 [vol/vol]; 5 ml) . After centrifugation, the resulting supernatant was added to the first one, and the combined extracts were evaporated at 50°C on a rotary evaporator to about 2 ml . Water-acetic acid (98:2 [vol/vol]; 10 ml) was added to the sample solution . SPE cartridges (500 mg, 6 ml; Spe-ed SPE C₁₈; Applied Separations), previously conditioned with methanol (6 ml) and then with water (6 ml), were used to purify the quinolone drugs . The whole sample solution was deposited on top of the cartridge, which was subsequently washed with water-1 M phosphoric acid (3:2 [vol/vol]; 6 ml) . The drug residues were eluted from the SPE column with methanol-1 M phosphoric acid (9:1 [vol/vol]; 6 ml), followed by methanol (3 ml) . The collected fractions were evaporated to dryness on a rotary evaporator at 50°C, and the dried pellet was reconstituted with Tris buffer solution (pH 9.1; 1 ml) . After filtration through 0.45-µm-pore-size nylon filters, 20 µl of the final solution was injected into an HPLC system, assembled as follows: a pump with an autosampler device (model Alliance 2690; Waters S.p.A., Vimodrone, Milan, Italy) fitted with a PLRP-S column (100 Å, 150 by 4.6 mm, 5 µm) and an RP18-E guard cartridge (40 by 4 mm), both of which were packed by Polymer Laboratories Inc . (Church Stretton, United Kingdom); a Croco-cil oven heated to 40°C; and a fluorescence detector (model 474; Waters) set at 280 and 450 nm as the excitation and the emission wavelengths, respectively . The eluents were 25 mM ortho-phosphoric acid (eluent A), acetonitrile (eluent B), and tetrahydrofuran (eluent C) . The elution gradient consisted of the following steps: at 0 min, A-B-C at 85:15:0; at 13 min, A-B-C at 80:15.5:4.5; at 20 min, A-B-C at 69:17:14; at 30 min, A-B-C at 69:17:14; at 35 min, A-B-C at 85:15:0; and at 40 min, A-B-C at 85:15:0 for the equilibration step . The flow rate was 1 ml/min .

Before sample analysis, the HPLC method described above was validated in-house according to the recommendations of Commission Decision (EEC) No . 657/2002 (16) .

Validation of in-house method. Since no certified reference material is available for enrofloxacin, the trueness of the method was evaluated on the basis of the levels of recoveries from standard materials used in-house (fortified blank matrix) . Precision, expressed as repeatability, was calculated by repeated analyses of the same sample sets used for the recovery tests .

In order to determine trueness and precision, blank tissue samples (muscle plus skin in natural proportions and fish bone) were fortified with enrofloxacin at six concentrations, ranging from 15 to 400 µg kg^–1, and with ciprofloxacin at five concentrations, ranging from 25 to 400 µg kg^–1 . Tissue samples fortified at each level were analyzed twice daily for 3 days . Recoveries and interday repeatability were calculated as described in Commission Decision (EEC) No . 657/2002 (16) .

The limit of detection (LOD) and the limit of quantification (LOQ) for muscle plus skin in natural proportions and for fish bones were calculated according to the requirements of the International Conference on Harmonization (20, 24) . Briefly, 20 blank tissue samples (double replicates from 10 different trout) for each tissue and for each quinolone were fortified with an amount of standard quinolone able to produce signal-to-noise ratios ranging from 2.5 to 5 . In our case the suitable concentrations were 5 and 8 µg kg^–1 for enrofloxacin and ciprofloxacin, respectively . LODs were determined by multiplying the standard deviations (SDs) calculated for the 20 blank tissue samples fortified with low drug concentrations by the Student t-test value (n – 1, 1 – = 0.99) . The exact equation of the calculation is as follows: LOD = SD x Student t-test value (n – 1, 1 – = 0.99), where the SD is for the 20 blank tissue samples fortified with low concentrations . In our case n was equal to 20; therefore, the Student t-test value was 2.543 . The LOQs were estimated to be 10 times the same SD (i.e., LOQ = SD x 10, where SD is the standard deviation for the 20 blank tissue samples fortified with low drug concentrations) .

 
Recovery data were also satisfactory, with values varying from 89.5 to 102.0% and 84.0 to 103.0% for enrofloxacin and ciprofloxacin, respectively . Indeed, these values fall within the guideline range (–20 to +10%) for mass fractions 10 µg kg^–1 reported in the EEC Commission Decision cited above (16) .

For enrofloxacin, the LODs were 1.3 and 1.0 µg kg^–1 in muscle plus skin in natural proportions and fish bones, respectively . For ciprofloxacin, the LODs were 3.8 and 1.3 µg kg^–1 for muscle plus skin in natural proportions and fish bones, respectively . The calculated LOQs in muscle plus skin in natural proportions and fish bones were 5.0 and 4.0 µg kg^–1, respectively, for enrofloxacin and 15.0 and 5.0 µg kg^–1, respectively, for ciprofloxacin .

Calibration curves corrected for recoveries in fish tissue samples were used for the quantification of enrofloxacin (for muscle plus skin in natural proportions, y = 3 x 10^–5x + 2.1211 [R² = 0.9992]; for fish bones, y = 2 x 10^–5x + 1.000 [R² = 0.9990]) and ciprofloxacin (for muscle plus skin in natural proportions, y = 5 x 10^–5x – 9.8987 [R² = 0.9995]; for fish bones, y = 3 x 10^–5x + 11.794 [R² = 0.9966]) . For samples with enrofloxacin concentrations above the highest value of the calibration curve, samples were diluted so that the concentrations were within the proven linear range .

Depletion from tissue. The results for enrofloxacin and ciprofloxacin depletion from fish tissues at different times are shown in Table 3 . In order to consider the influence of water temperature on fish metabolism and, consequently, on the pharmacokinetics of the drugs, the time parameter was also expressed as degree-days . Degree-days are calculated by multiplying the mean daily water temperatures (in degrees Celsius) by the total number of days measured .

 
The MRL for enrofloxacin was set at 100 µg kg^–1 and includes the sum of the level of enrofloxacin and the level of its metabolite, ciprofloxacin . Therefore, the concentration-time relation of the depletion of this sum is also shown in Fig . 2 .

 
Studies on the pharmacokinetics of enrofloxacin in a variety of fish species, mainly salmonids, treated by the oral or the parenteral route at doses ranging from 5 to 50 mg kg^–1 have been published (4, 25, 26, 29, 33) . In contrast, very few studies on the pharmacokinetics of the quinolones in fishes treated with multiple doses of medicated feed and reared under field conditions have been carried out (27, 32, 35) . Moreover, previous studies (11) have very seldom taken into consideration the muscle plus the skin matrix and/or the sum of the levels of the enrofloxacin and ciprofloxacin residues in fish tissues . Indeed, the European Union Commission has only recently established the MRLs for enrofloxacin and marker residues in this target matrix (17) . For these reasons, it is difficult to compare our results with other data available in the scientific literature . However, our results indicate that enrofloxacin depletion in rainbow trout follows a much slower trend compared to that observed in other studies carried out under field conditions with fishes treated with multiple doses of different quinolones and/or with different fishes (14, 22, 27, 32, 35) .

Our data indicate a slow depletion of enrofloxacin from edible trout tissue; moreover, its metabolite, ciprofloxacin, is no longer detectable in the same samples at 265.00°C-day . In particular, the enrofloxacin concentration reaches its highest level (15.45 mg kg^–1) shortly after the last administration of drug (1.66°C-day) . This concentration remains approximately constant until 26.50°C-day, when it starts to decrease rapidly and reach 0.40 mg kg^–1 at 265.00°C-day . The rate of further enrofloxacin depletion is very low: at 708.00°C-day the residue levels are about 0.10 mg kg^–1, which is the value corresponding to the MRL . Ciprofloxacin residue levels reach their maximum values (about 0.78 mg kg^–1) at 13.25°C-day after the end of treatment; they decrease below the LOD (0.015 mg kg^–1) at 265°C-day .

The trend of depletion observed in the fish bones was similar to that observed in edible tissues . The only significant differences were the following: the initial rate of decrease in the enrofloxacin concentration in fish bones was higher, and the maximum ciprofloxacin concentration was absent after the end of treatment . These results are in contrast to the data reported for the depletion of flumequine in sea bream (27) and Atlantic salmon (13), in which flumequine showed a notable accumulation in fish skin and vertebrae compared to that in muscle tissues . However, they confirm the outcomes of another published study (32), which showed the presence of possible reservoirs of quinolone residues in salmon . In particular, this investigation has provided evidence that oxolinic acid and flumequine are especially bound to salmon bones, enrofloxacin is especially bound to salmon skin, and sarafloxacin is bound to both salmon skin and bones .

To our knowledge no drug depletion studies have previously been performed with rainbow trout to which enrofloxacin was administered through multiple-dose regimens in medicated feed .

In a pharmacokinetic study (4), a single dose of 10 mg of enrofloxacin kg^–1 was administered orally to rainbow trout reared at temperatures similar to those used in our study . The maximum concentration of drug in plasma (about 1.3 mg kg^–1) measured in that pharmacokinetic study was much lower than the maximum level measured in tissue (about 15 mg kg^–1) in the present study . This is probably due to the different dosage regimens (dosing over five consecutive days versus administration of a single dose) and the different routes of drug administration (through medicated feed versus by gavage) carried out in the two studies and is also probably due to the fact that quinolones tend to accumulate in edible tissues, evidence for which has been provided previously (32) . In the previous study (32), in which salmon in seawater at a temperature of 6°C were treated with enrofloxacin at 10 mg kg^–1 for 10 days, muscle tissues contained 6 µg of drug kg^–1 60 days after the end of treatment . In our study, at 59 days after the end of treatment (which lasted 5 days and which was provided in freshwater at an average temperature of 13.3°C), the trout still contained 100 µg of drug kg^–1 . We can conclude that enrofloxacin depletion from muscle tissues is much slower in rainbow trout than in salmon . Therefore, our study confirms that drug depletion in diverse fish species can show significant differences, as has already been shown for flumequine in sea bream (27) and Atlantic salmon (13, 14, 32) .

In two studies (8, 28), flumequine given to rainbow trout through medicated feed for 5 days showed short times of depletion (48 to 120 h) from muscle tissues . Our conclusions indicate that enrofloxacin depletion in rainbow trout is much slower than flumequine depletion in the same fish species .

Pharmaceutical companies do not advise any specific withdrawal time for enrofloxacin in fish treatments . Commission Directive (EEC) No . 82/2001 (18) provides a minimum withdrawal period of 500°C-day for any type of product that is administered off label and for which no other information is available . In the present study with trout, we obtained an experimental withdrawal time of 708°C-day, a value much higher than the one suggested by EEC legislation . Tentative data extrapolation shows that at 500°C-day edible trout tissues might still contain about 0.17 mg of enrofloxacin kg^–1, whereas the MRL for enrofloxacin plus ciprofloxacin is set at 0.10 mg kg^–1 .

Evaluating the data from the present study on the basis of a pragmatic approach, we would suggest a more appropriate withdrawal time of 816°C-day for the sum of enrofloxacin and ciprofloxacin in rainbow trout muscle plus skin tissues .

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