Depletion of enrofloxacin and ciprofloxacin in broilers

Enrofloxacin (ENR) is a second generation fluoroquinolone with bactericidal activity against Gram-negative bacteria and some Gram-positive cocci (Martinez et al. 2005; Otero et al., 2001a, 2001b). After oral application, is well absorbed and distributed at tissue level. It is metabolized in the liver, generating its major active metabolite, ciprofloxacin (CIP) (Otero et al., 2001b). Fluoroquinolones can cause phototoxicity in humans (Klecak et al., 1997) and in young animals condrotoxic effects and tendon rupture (Pierfitte, 1995). Ho et al. (2003) also observed allergic reactions to CIP. Obtaining safe and innocuous food is an essential public health objective in any production type; therefore antimicrobial residual levels in edible tissues should be below the Maximum Residue Limit (MRL) allowed according to harmonized protocols. The European Community established an MRL for the sum of enrofloxacin and ciprofloxacin of 100 ng/g (0.1 mg/g) in muscle tissue, 200 ng/g in liver and 300 ng/g in kidney of chickens. Considering pharmacokinetics characteristics of enrofloxacin and ciprofloxacin, accumulation of both molecules in non-edible tissues, as feathers, is highly probable. The feather meal is often incorporated as a protein source in diets of other animals, such as calves, pigs, or fishes (trout, salmon) (Bertsch & Coello, 2005); however there is little information available concerning the presence of antimicrobial residues in feathers.

A group of 90 chickens with 20 days of age were conventionally kept and fed, with access to balanced food and water "ad libitum". The food was controlled for determining absence of substances with antimicrobial power. The animals were kept in groups of 10 individuals in wire cages.

A group of 60 experimental animals were treated for 5 days with the formulation of enrofloxacin of Carval S.A. at 10 mg/kg of live weight orally administrated with drinking water. The remaining 20 animals were kept for obtainment of antimicrobial-free tissues to perform the corresponding calibration curves.

After the 5 days of treatment, the experimental animals were sacrificed in groups of 10 individuals at the following intervals post-administration: 1, 2, 3, 4, 5, 7 and 9 days. At slaughter, samples were obtained from liver, fat/skin, kidney, muscle and feathers, which were stored individually at -20°C until analysis. Concentrations of ENR and its metabolite CIP were determined by high resolution liquid chromatography (HPLC) with fluorescence detection (excitation at 278 nm and emission at 446 nm) after liquid/liquid extraction. A column Luna C18, 5μm (150 x 4.6 mm) with C18 column guard and mobile phase was utilized, and the mobile phase was water:acetonitrile:triethylamine (80:19:1) pH 3.

The waiting period (withdrawal time, WT) was calculated using statistical program WT1.4 of EMEA.

Specificity of the analytical methods used was confirmed by the analysis of control tissue samples, since at the retention time of each analyte there was no interference. Recovery percentage was 80 to 109% for ENR and CIP, while the accuracy of the methods expressed by variation coefficient (CV) was 0.77 to 11.95% for ENR and 0.88 to 10.32% for CIP.

Both the parent drug (ENR) and its active metabolite (IPF) were distributed in all tissues tested achieving relatively high and measurable concentrations until 7 days post-administration Fig. 1). The highest concentrations were detected at 24 h post-administration in feathers, liver, kidney, muscle and finally skin/fat.

The waiting time (withdrawal time, WT) to consume these animals treated with enrofloxacin, applying the statistical program WT1.4 of EMEA for the different tissues tested was of 6.34 days for muscle, 6.14 d for skin/fat, 6.02 d for kidney, 6.94 d for liver and 10.94 d for feathers.

Figure 1. Average tissue concentrations obtained by the sum of both analytes in the different tissues tested after oral administration, together with drinking water, of enrofloxacin de Carval at 10 mg/kg for 5 days.

Enrofloxacin showed a good distribution in the tissues tested, reaching a higher concentration in the liver, from where it is slowly eliminated. Similar results were published by other authors (Anadón, 1995; Schneider, 2001, Mestorino et al., 2009). Enrofloxacin is metabolized in the liver into its main metabolite, ciprofloxacin, and some other minor metabolites, however the levels of transformation of enrofloxacin into ciprofloxacin are not very high.

We found that the highest proportion of residual levels measured in the liver and other tissues tested was of enrofloxacin. These findings are consistent with Knoll et al. (1999), who determined that ciprofloxacin concentrations in chicken organs is about 4% of enrofloxacin concentrations, except in liver and kidney, where it reaches levels of 38% and 11% respectively. An interesting finding was the high level of enrofloxacin achieved in feathers, even at concentrations far higher than those measured in liver tissue. It is possible that during the growth of feathers, blood circulation helps to deposit various molecules administered to broiler chickens, which in this case was enrofloxacin. Once feathers stop growing and blood does not circulate as widely throughout them, they become true reservoirs of antimicrobial or xenobiotics in general. Similar results were obtained by San Martín et al. (2007).

According to previously presented and the results obtained in this study, we can conclude that after treating chickens with enrofloxacin made by Carval orally at 10 mg/kg for 5 days, a waiting time of 7 days post-treatment should be considered. It is also important to mention that as the feathers are used as a protein source to supplement food for different food producing species (bovines, pigs, salmons, trouts) they must be considered potential reservoirs of chemical residues that can reach man through the chain food; so it would be advisable to establish a waiting period. In this case the waiting time should be 11 days post-treatment.

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