Eggs are the main product generated from backyard poultry production systems (BPS) because they can quickly be consumed and sold to meet essential family needs. Nevertheless, antimicrobial residues can accumulate in this product. The objective of this study was to evaluate the presence of antimicrobial residues in eggs produced by poultry kept in BPS in central Chile. To assess this, eggs were obtained from 83 BPS and analysed to evaluate the presence of antibiotic residues (families: tetracyclines, beta-lactams, aminoglycosides and macrolides), using a Four-Plate Test screening method for the detection, based on a bacterial growth inhibition method. Results show a lack of biosecurity procedures at BPS level, making these systems susceptible to the dissemination of antimicrobial residues. These include intensive animal production units in the proximity, and the presence of shared watercourses with other farms. Furthermore, 66% of the surveyed owners are indicated as giving pharmacological treatments to their chickens. Eggs from 61 BPS were positive for at least one antimicrobial. Fifty-three BPS were positive for more than one antimicrobial, and one BPS was positive for all four antimicrobials tested. Consequently, there is a risk that poultry eggs produced in BPS in central Chile carry residues of different families of antimicrobials.

Tetracyclines, sulphonamides, and quinolones are families of antimicrobials (AMs) widely used in the poultry industry and can excrete up to 90% of AMs administrated, which accumulate in poultry litter. Worryingly, poultry litter is widely used as an agriculture fertilizer, contributing to the spread AMs residues in the environment. The aim of this research was to develop a method that could simultaneously identify and quantify three AMs families in poultry litter by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Samples of AMs free poultry litter were used to validate the method according to 657/2002/EC and VICH GL49. Results indicate that limit of detection (LOD) ranged from 8.95 to 20.86 μg kg⁻¹, while limits of quantitation (LOQ) values were between 26.85 and 62.58 µg kg⁻¹ of tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline, enrofloxacin, ciprofloxacin, flumequine, sulfachloropyridazine, and sulfadiazine. Recoveries obtained ranged from 93 to 108%. The analysis of field samples obtained from seven commercial poultry flocks confirmed the adequacy of the method since it detected means concentrations ranging from 20 to 10,364 μg kg⁻¹. This provides us an accurate and reliable tool to monitor AMs residues in poultry litter and control its use as agricultural fertilizer.

Tetracyclines are important antimicrobial drugs for poultry farming that are actively excreted via feces and urine. Droppings are one of the main components in broiler bedding, which is commonly used as an organic fertilizer. Therefore, bedding becomes an unintended carrier of antimicrobial residues into the environment and may pose a highly significant threat to public health. For this depletion study, 60 broiler chickens were treated with 20% chlortetracycline (CTC) under therapeutic conditions. Concentrations of CTC and 4-epi-CTC were then determined in their droppings. Additionally, this work also aimed to detect the antimicrobial activity of these droppings and the phenotypic susceptibility to tetracycline in E. coli isolates, as well as the presence of tet(A), tet(B), and tet(G) resistance genes. CTC and 4-epi-CTC concentrations that were found ranged from 179.5 to 665.8 µg/kg. Based on these data, the depletion time for chicken droppings was calculated and set at 69 days. All samples presented antimicrobial activity, and a resistance to tetracyclines was found in bacterial strains that were isolated from these samples. Resistance genes tet(A) and tet(B) were also found in these samples.

Oxytetracycline (OTC), enrofloxacin (EFX), and sulfachloropyridazine (SCP) are critically important antimicrobials (AMs) in both human and veterinary medicine, where they are widely used in farm animals. Lettuce has become a matrix of choice for studying the presence of residues of these AMs in plants, as the concentrations of residues detected in lettuce can range from ng to mg. While several analytical methodologies have been developed for the purpose of detecting AMs in lettuce, these currently do not detect both the parent compound and its active metabolites or epimers, such as in the case of ciprofloxacin (CFX) and 4-epi-oxitetracycline (4-epi-OTC), which also pose a risk to public health and the environment due to their AM activity. In light of this situation, this work proposes an analytical method that was developed specifically to allow for the detection of OTC, 4-epi-OTC, EFX, CFX, and SCP in a lettuce matrix. This method uses acetonitrile, methanol, 0.5% formic acid, and McIlvaine-EDTA buffer as extraction solvents, and dispersive solid-phase extraction (dSPE) for the clean-up. The analytes were detected using a liquid chromatography technique coupled to mass spectrometry (HPLC-MS/MS). Parameters such as the specificity, linearity, recovery, precision, limit of detection, and limit (LOD) of quantification (LOQ) were calculated according to the recommendations established in the European Union decision 2021/808/EC and VICH GL2: Validation of analytical procedures. The LOQ for the analytes OTC, 4-epi-OTC, CFX, and SCP was 1 μg·kg⁻¹, whereas for EFX, it was 5 μg·kg⁻¹ dry weight. All calibration curves showed a coefficient of determination (R²) of >0.99. The recovery levels ranged from 93.0 to 110.5% and the precision met the acceptance criteria, with a coefficient of variation of ≤14.02%. Therefore, this methodology allows for the precise and reliable detection and quantification of these analytes. The analysis of commercial samples confirmed the suitability of this method.

Oxytetracycline (OTC) is widely used in broiler chickens. During and after treatment a fraction of OTC is excreted in its original form and as its epimer, 4-epi-OTC in droppings. To address the transfer of OTC into the environment, we evaluated the dissemination of OTC and 4-epi-OTC from treated birds to the environment and sentinels, through the simultaneous analysis of broiler droppings and litter. Male broiler chickens were bred in controlled conditions. One group was treated by orogastric tube with 80 mg kg⁻¹ of OTC and two groups received no treatment (sentinels). OTC+4-epi-OTC were analyzed and detected by a HPLC-MS/MS post the end of treatment. The highest concentrations of OTC+4-epi-OTC were detected in the droppings of treated birds 14-days following the end of treatment (2244.66 µg kg⁻¹), and one day following the end of treatment in the litter (22,741.68 µg kg⁻¹). Traces of OTC+4-epi-OTC were detected in the sentinels’ droppings and litter (<12.2 µg kg⁻¹). OTC+4-epi-OTC can be transferred from treated birds to the environment and to other untreated birds. The presence and persistence of OTC+4-epi-OTC in litter could contribute to the selection of resistant bacteria in the environment, increasing the potential hazard to public and animal health.

Recent studies have detected different antimicrobial residues in broiler chicken feathers, where they persisted for longer periods of time and at greater concentrations than in edible tissues. However, until today, lincomycin behaviour in this nonedible tissue has not been assessed yet. Considering this, an analytical methodology to detect and quantify this antibiotic concentration in feathers, muscle, and liver tissues from broiler chickens was implemented and in-house validated. The methodology will allow the determination of the bioaccumulation of this highly persistent antibiotic in feathers of treated birds. For this purpose, 98% lincomycin and 95% lincomycin D3 standards were used. Methanol was selected as the extraction solvent, and Chromabond® Florisil® cartridges were used for the clean-up stage. The separation of analytes was performed through the analytical column SunFire C18 with a running time of 4 minutes, and the instrumental analysis was performed through an LC-MS/MS, with a liquid chromatograph Agilent® 1290 Infinity, coupled to an AB SCIEX® API 5500 mass spectrometer. An internal protocol for an in-house validation was designed based on recommendations from Commission Decision 2002/657/EC and the Guidance document on the estimation of limit of detection and limit of quantification for measurements in the field of contaminants in feed and food. The average retention time for lincomycin was 2.255 min (for quantifier ion, 126.0). The calibration curves showed a coefficient of determination (r²) greater than 0.99 for all matrices, while recovery levels ranged between 98% and 101%. The limit of detection (LOD) calculated was of 19, 22, and 10 μg·kg⁻¹, and the limit of quantification (LOQ) was of 62, 73, and 34 μg·kg⁻¹ in feathers, muscle, and liver, respectively. This method detects lincomycin in the studied matrices, confidently and accurately, as it is required for designing analytical studies of drug residues in edible and nonedible tissues, such as feathers.