ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2007, p. 1043–1047 0066-4804/07/$08.00⫹0 doi:10.1128/AAC.01002-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 51, No. 3
Orally Administered Erythromycin in Rainbow Trout (Oncorhynchus mykiss): Residues in Edible Tissues and Withdrawal Time䌤 Annarita Esposito,1 Laura Fabrizi,1 Dario Lucchetti,1 Luigi Marvasi,2 Ettore Coni,1 and Emilio Guandalini1* National Center for Food Quality and Risk Assessment, Istituto Superiore di Sanita `, Rome, Italy,1 and Veterinary Health and Animal Pathology Dept., University of Bologna, Ozzano Emilia (Bologna), Italy2 Received 11 August 2006/Returned for modification 11 October 2006/Accepted 18 December 2006
Aquaculture production has notably increased in the last decades, mainly thanks to intensive farming. Together with market globalization, this gives rise to the spreading of several fish diseases, thus increasing the demand for veterinary drugs for aquatic species. Nonetheless, very few chemicals are registered for use in aquaculture, and fish farmers are often forced to resort to off-label use of drugs authorized for other food-producing animal species. Rainbow trout is the major farmed fish species in Italy and the second one in Europe. Erythromycin is the antibiotic of choice against gram-positive cocci, the major concern for trout farming, but it is not yet registered for aquaculture use in most European countries. The aim of this study was to follow the depletion of erythromycin in rainbow trout (Oncorhynchus mykiss), after its administration at 100 mg kgⴚ1 trout body weight dayⴚ1 for 21 days through medicated feed (water temperature, 11.5°C). Erythromycin residues in fish muscle plus skin in natural proportion were determined by a validated liquid chromatography-electrospray ionization-tandem mass spectrometry method. Interpolation of our data, following European Agency for the Evaluation of Medicinal Products guidelines, gives a withdrawal time of 255°C-days (°C-day ⴝ water temperature ⴛ days), thus showing that the general value (500°C-day) recommended by the Council Directive (EEC) no. 82/2001 for off-label drug use in aquaculture would be too conservative in this case, with excessive costs for the farmers. Our study provides preliminary data for a more prudent use of erythromycin in rainbow trout, suggesting a possible withdrawal time after treatment. to large-animal terrestrial farming. Therefore, the aquaculture market is unable to support the costs of gaining, and often even retaining, marketing approval for many pharmaceuticals (34). In the Mediterranean area, lactococcosis represents the most important risk factor for rainbow trout farming during summer (19). Moreover, the global climatic changes toward a progressive warming of the whole Mediterranean area represent a further favoring factor for the spread of these diseases. Currently, available vaccines confer only limited protection against lactococcosis due to the large number of bacterial species involved. At present, erythromycin, used in both human and veterinary medicine, is the antibiotic of choice against gram-positive cocci such as those causing lactococcosis (33); it is also efficaciously used against bacterial kidney disease (BKD), a systemic infection causing serious mortality in lake trout and Pacific and Atlantic salmon (32). In the European Union (EU), the Council Regulation EEC no. 2377/90 has established for erythromycin a maximum residue limit (MRL) of 200 g/kg for finfish “muscle and skin in natural proportions” (10). However, erythromycin can only be administered off-label, as it is not yet registered for its use in aquaculture in most European countries. The Council Directive (EEC) no. 82/2001 provides a general withdrawal time of 500°C-day for off-label use of drugs in fishes (9). Unfortunately, when specific pharmacological data are lacking, both general EU recommendations and veterinarian prescriptions can be either excessively conservative or unsafe for the consumer (24). Studies that provide data useful for the registration of drugs in veterinary medicine may help reduce the human
According to Food and Agriculture Organization statistics (18) aquaculture’s contribution to global supplies of fish, crustaceans, and mollusks continues to grow, increasing from 3.9% of total production by weight in 1970 to 31.9% in 2003. In the Mediterranean area, the average annual increment of aquaculture production in the last 20 years has passed from 4 to 13%, with a noteworthy diversification of the farmed species (from 18 in 1981 to 40 in 2001). Within farmed fishes, rainbow trout is the major farmed species in Italy (58% of the total production in 2004) and the second one in Europe after Atlantic salmon (2). The great increase in aquaculture production has primarily been due to the adoption of intensive farm plants and integrated fish farming. However, these modern systems of production, together with world trade and market globalization, foster the spread of a large number of fish diseases, thus increasing the demand for veterinary drugs and chemicals in aquaculture. Nonetheless, very few drugs are specifically registered for their use in aquaculture, and so fish farmers are often forced to resort to off-label use of drugs authorized for food-producing animal species different from fish (1, 8, 25). This lack of authorized drugs is due to the fact that, in most European countries, aquaculture is a small market compared
* Corresponding author. Mailing address: National Center for Food Quality and Risk Assessment, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy. Phone: 390649903647. Fax: 390649902712. E-mail:
[email protected]. 䌤 Published ahead of print on 28 December 2006. 1043
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FIG. 1. Chemical structure of erythromycin A.
health risk through limiting off-label use and may help reduce economic losses due to disease. Few pharmacokinetic studies with erythromycin have been conducted in fish species other than salmon. The aim of the present study was to follow the uptake and depletion time of erythromycin in rainbow trout (Oncorhynchus mykiss) muscle plus skin in natural proportion after oral administration of the drug given by medicated feed. Moreover, a tentative withdrawal time is interpolated. In previous studies different dosages and times of administration were tested; the results showed that salmon treated with medicated feed at 100 mg/kg body weight/day for 21 consecutive days had the best protection against BKD (27). The same treatment protocol was thus adopted in the present study. MATERIALS AND METHODS Chemicals. Erythromycin A dihydrate (Fig. 1), purity 99.1%, and tricaine methanesulfonate were purchased from Sigma (Milan, Italy) at 99.1% stated purity. An internal standard [N,N-dimethyl-13(C2) erythromycin A; chemical purity, 96.8%; isotopic purity, 92.1%] was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA) at 96.8% stated purity. Ammonium acetate, n-hexane, and acetic acid were of analytical-reagent grade and were purchased from J. T. Baker (Florence, Italy). Acetonitrile of high-performance liquid chromatograpy grade was purchased from J. T. Baker (Florence, Italy). Water was purified in a Milli-Q system (Millipore, Milan, Italy). Animals and diet. One hundred twenty rainbow trout, with an average weight of 200 ⫾ 11 g, supplied by Ichthyologic Institute (Rome, Italy), were used for the investigation. The trout were reared in a round tank (1,000 liters) with water (flowthrough) at pH 7.5, an O2 concentration of 9.5 mg liter⫺1, and an average temperature of 11.5 ⫾ 0.5°C. Water flow into the tank was set at 20 liter min⫺1, and temperature was monitored continuously and recorded every 12 h by means of two digital thermometer probes situated at two sampling sites in the tank (corresponding to inflow and outflow water). Housing conditions were suitably representative of the real situation of fish farming sites. Procedures for trout care and management complied with those required by Italian laws, and they adhered to ethical standards for humane treatment of experimental animals established by the ethical committee of the Istituto Superiore di Sanita` (11). Two different diets were prepared in house for the experimental trial, as follows: (i) a standard diet for fish (diet A) and (ii) a standard diet for fish supplemented with 7.2% (wt/wt) veterinary formulation of 20% erythromycin on a dextrose support (DOX-AL Italia S.p.A.) (diet B). The drug concentration was selected according to a rainbow trout feed rate and to therapeutic doses sug-
gested for fish disease treatment (about 100 mg kg⫺1 trout body weight day⫺1). Hendrix S.p.A. (Mozzecane, Verona, Italy) supplied the diet ingredients (fish meal and fish oil). According to the protocol, Hendrix fish meal (54%), Hendrix fish oil (5.4%), and water (40.6%) were thoroughly mixed and homogenized; an adequate pellet (about 0.3-cm diameter) was then produced by means of a bench food mixer-extruder. In diet B (medicated feed), 7.2% water was replaced by the veterinary formulation described above. Finally, wet pellets were divided in aliquots (100 g) and dried in an oven at 35°C for about 12 h to achieve the final moisture content (about 10%). Dry pellets were stored at⫺20°C until they were administrated to the fishes. The amount of erythromycin in the dry pellets was confirmed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI–MS-MS) analysis. Before starting the experiment, rainbow trout were acclimatized for a period of 7 days, during which they all received the diet A (standard feed). After one fasting day, rainbow trout were fed the diet B (medicated feed) for 21 days. A low feed rate was chosen (i.e., 0.7% body weight per day) for feeding trout without leftovers. This feed rate assured the administration, on average, of the selected therapeutic dose. The tank was carefully cleaned during the trial to avoid the possibility that some trout would reingest fecal matter containing the drug. On the other hand, round tanks have the advantage of a naturally self-cleaning action. As the water swirls around the tank, solids are drawn toward the middle, where the outlet is situated. No adverse effects were observed during this trial. Sample collection and preparation. A control group of 10 trout was euthanized before drug administration was started. Subsequently, 10 trout were randomly sampled and euthanized at 3, 12, 24, 48, 96, 168, 240, 480, and 720 h after the pharmacological treatment. Trout were killed through an overdose of anesthetic (tricaine methanesulfonate, administered by bath). Muscle and skin in natural proportion (half trout fillets) were collected in situ. Samples were each placed into polyethylene bags and transferred to the laboratory on dry ice, where they were stored at ⫺80°C for no longer than 2 weeks before analysis. Erythromycin stability has been estimated previously (23) at different temperatures and light conditions. Analytical procedures. The methodology used for the determination of erythromycin in fish muscle and skin in natural proportion was based on a rapid and simple LC-ESI–MS-MS method developed and validated by the authors according to the European Commission Decision 2002/657/EC (7) and previously published (23).
RESULTS Tissue depletion. Results of erythromycin depletion at different times in rainbow trout muscle plus skin are shown in Table 1. In order to consider the influence of water temperature on fish metabolism and, consequently, on the drug pharmacokinetics, the time parameter is also expressed as °C-day. Degree-days are calculated by multiplying the mean daily water temperature by the total number of days at which the temperature was measured to that point. Determination of withdrawal time. Fig. 2 shows some of the data reported in Table 1, displayed here on a semilogarithmic
TABLE 1. Erythromycin depletion at different times in rainbow trout muscle plus skin in natural proportion
Day
°C-days
Erythromycin concn in muscle plus skin in natural proportion (mg kg⫺1)a
0.13 0.50 1.00 2.00 4.00 7.00 10.00 20.00 30.00
1.44 5.75 11.50 23.00 46.00 80.50 115.00 230.00 345.00
45.37 ⫾ 6.46 50.00 ⫾ 21.56 34.96 ⫾ 10.50 17.50 ⫾ 9.56 7.14 ⫾ 6.68 0.56 ⫾ 2.25 0.33 ⫾ 1.16 0.10 ⫾ 0.10 0.07 ⫾ 0.03
Time
a
Values shown are concentration means ⫾ standard deviations from 10 fish.
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FIG. 2. Linear regression line and upper one-sided tolerance limit (95%) linear regression line, with a confidence of 95%, of erythromycin concentrations in muscle plus skin in natural proportion from trout treated with erythromycin for 21 days (100 mg kg⫺1 trout body weight day⫺1) versus time. Degree-days are calculated by multiplying the mean daily water temperatures by the total number of days measured.
graph, where singular animal data are plotted. On the y axis, erythromycin concentration (g/kg) is plotted, while on the x axis, time posttreatment (days and degree-days) is shown. The MRL value for erythromycin is set at 200 g kg⫺1, as reported by Council Regulation (EEC) no. 2377/1990 (10). The regression line and the upper, one-sided tolerance limit (95%) regression line with a confidence of 95% are also traced. This graph has been obtained using the statistical program recommended by the European Agency for the Evaluation of Medicinal Products (EMEA) and is downloadable from the same EMEA web site (13). Using this statistical method, a withdrawal time of 255°C-days has been interpolated for rainbow trout treatment, with 21 days of 100 mg kg⫺1 trout body weight day⫺1 erythromycin. DISCUSSION To the best of our knowledge, no data about the depletion of erythromycin residues after therapeutic treatment in rainbow trout were available. Pharmacokinetic studies with erythromycin in cow, guinea pig, horse, rat, foal, pigeon, and human have been carried out (6, 12, 16, 20, 22, 36, 37). With regard to aquatic species treated with erythromycin orally, Moffit et al. (26) followed the accumulation and depletion of erythromycin thiocyanate in Chinook salmon (Oncorhynchus tshawytscha) and found that the drug was not detectable in the fish muscle 10 days after the last erythromycin feeding (using the same dosage as in our study, i.e., 100 mg kg⫺1 trout body weight day⫺1 for 21 days). Fairgrieve et al. (17) compared the accumulation and clearance of erythromycin and azithromycin in juvenile fall Chinook salmon (Oncorhynchus tshawytscha)
treated with 100 mg kg⫺1 trout body weight day⫺1 for 28 days. They found that erythromycin was not detectable after 21 days posttreatment. These last data are in accordance with our results, where we calculated a withdrawal time of 255°C-days (corresponding to about 22 days). However, differences in fish species, erythromycin salts, and dosing times do not allow an easy comparison among the three studies. Moffitt et al. (28) reported deleterious changes in kidney and liver tissues of Chinook salmon when erythromycin is administered by injection. Fairgrieve et al. (17) did not confirm these results with erythromycin given orally and found that erythromycin, compared to azithromycin and other macrolides, shows a much lower penetration and persistence in fish tissues. This could be a reason that explains the very high dosage requested to treat salmonids with erythromycin and, at the same time, the relatively rapid elimination of these drugs from fish tissues. This relatively rapid elimination has been confirmed also in our study (Table 1). As shown in Fig. 2, an interpolation of our experimental data produces a withdrawal time of 255°C-days (corresponding to about 22 days), which derives from the intercept between the time axis and the upper one-sided tolerance limit (95%) linear regression line with a confidence of 95%. The interpolation has been performed following the recommendations described in the EMEA guidelines “Approach towards Harmonisation of Withdrawal Periods for Meat” (13). The statistical analysis was carried out using the downloadable program from the EMEA web site (http://www.emea.europa.eu/index/indexv1.htm). According to EMEA guidelines, in order to build up a graph useful to interpolate a correct withdrawal time, only data points that provide a linear relationship must be used. For
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example, early time points that produce an obvious deviation from linearity should be excluded because they may indicate that the drug distribution process has not yet ended. Therefore, in our study, time points at 3, 12, and 24 h have been excluded. Moreover, as reported within the guidelines, it is justified to exclude late time points, which produce deviations from linearity, as they may be due to concentrations below the limit of detection. In our study, no data points were below our detection limit (CC␣, ⫽ 220 g kg⫺1), but the time point at 30 days was close to our limit of detection (20 g kg⫺1); therefore, this data point has been excluded. As reported within the guidelines, a minimum of one time point where the concentrations of residues for all animals fall below the respective MRL is sufficient to interpolate a withdrawal time. In our study, we could include two of these time points, i.e., 10 and 20 days, while we had to exclude the time point at 30 days, at which the value was too close to the detection limit and also produced an obvious deviation from the plot linearity. Salmonids are considered major species in the EU (14). However, they are a very broad family of species, including different fishes such as salmon and rainbow trout. Most studies aimed at registering drugs for salmonids are carried out on salmons, but often rainbow trout and salmon drug pharmacokinetics are quite different, depending on fish species, water temperature and salinity, dosage, and size (3, 4, 21, 29, 31, 35, 37). Thus, specific studies of rainbow trout are needed in order to determine appropriate drug doses and withdrawal times. Our study provides a proposal for an adequate withdrawal time of erythromycin in rainbow trout after a commonly used therapeutic treatment for streptococcosis, lactococcosis, or BKD. Our results indicate that the general withdrawal time of 500°Cdays, recommended by the Council Directive (EEC) no. 82/ 2001 (9) for off-label drug use in aquaculture, may be too conservative, in the case of erythromycin in rainbow trout, and may result in unnecessary costs to the farmers. Moreover, the measured concentrations of erythromycin in muscle plus skin samples at 3 h after the end of drug administration (i.e., 45.37 mg kg⫺1) are higher than erythromycin minimum bactericidal concentrations verified by Bandı´n et al. (5) with 11 Renibacterium salmoninarum strains (i.e., 21.87 mg kg⫺1 for 90% of isolates). This outcome leads us to believe that it is possible to lower the therapeutic dose of erythromycin in the case of rainbow trout. In conclusion, our study provides preliminary data for a prudent use of the antimicrobial drug erythromycin in rainbow trout, in order to guarantee safety in foods for the consumers and to improve fish farming management. However, it is opportune to underline the fact that, as a general policy, the use of antimicrobials which have an importance in human medicine, like erythromycin, should be limited to the strictly necessary circumstances in veterinary medicine. The potential selection of erythromycin-resistant bacteria in aquaculture settings and the possible dissemination of such resistant clones and/or erythromycin resistance genes to humans may be hazardous for human health (30). ACKNOWLEDGMENTS We thank the Ichthyologic Institute of Rome (Arsial) for its technical assistance and kind support during the experimental phase of the study with live fishes. We thank Hendrix S.p.A. (Mozzecane, Verona,
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