Aldo Maddaleno

Occurrence, variability and human exposure to Polychlorinated Dibenzo-p-dioxins (PCDDs), Polychlorinated Dibenzofurans (PCDFs) and Dioxin-Like Polychlorinated Biphenyls (DL-PCBs) in dairy products from Chile during the 2011–2013 survey

Levels, congener profiles of PCDD/Fs, DL-PCBs and human exposure for these xenobiotics never have been reported in Chile. For that purpose 102 raw cow milk samples were collected from seven different regions of Chile during 2011 until 2013. The highest mean level for PCDD/Fs, corresponds to 0.32 pg WHO-TEQ2005 g(-1) fat (2012) and for DL-PCBs 0.17 pg WHO-TEQ2005 g(-1) fat (2011), using the upper bound approach. Penta and tetra chlorinated congeners dominated PCDD/Fs profiles in a WHO-TEQ2005 basis during the survey. In the case of DL-PCBs, PCB 126 dominated the profiles with 89%. Statistical analysis showed significant difference among years only in DL-PCBs residues. Also dietary intake was estimated, and the highest level for total sum of PCDD/Fs and DL-PCBs for adult was 0.16 pg WHO-TEQ kg(-1) b.w d(-1) (2011) and for children correspond to 0.65 pg WHO-TEQ kg(-1) b.wd(-1) (2011). Concentrations and dietary intake for the studied compounds in milk and butter samples were below international and national regulations.

Single-laboratory validation of an LC-MS/MS method for determining florfenicol (FF) and florfenicol amine (FFA) residues in chicken feathers and application to a residue-depletion study

ABSTRACT A suitable analytical method is required to study the behaviour of florfenicol (FF) and its metabolite florfenicol amine (FFA) in broiler’s feathers. An LC-MS/MS method was developed, assessed and intra-laboratory-validated for FF and FFA analyses. We chose cloramphenicol-d5 as an internal standard, acetone as a solvent for the extraction of the analytes and dichloromethane for the clean-up. Through LC-MS/MS analysis, we established a detection limit of 20 μg kg–1, as well as calculated quantification limits of 24.4 and 24.5 μg kg–1 for FF and FFA, respectively. Validation parameters such as linearity, recovery and precision were calculated following Commission Decision 2002/657/EC. For linearity, all standard curves showed a standard coefficient greater than 0.99. Recoveries ranged from 99% to 102% for all studied concentrations. The results show that this analytical method is precise and reliable. For the depletion study, 64 Ross 308 broilers were treated with a therapeutic dosage of 10% FF during 5 consecutive days and their feathers were then analysed. Samples were drawn on days 5, 10, 15, 20, 25, 30, 35 and 40 post-treatments. As for the control group, 16 broiler chickens were raised under the same regime. Throughout the whole study, the detected concentrations of FF and FFA in feather samples were above 100 µg kg–1. In fact, even on day 30 post-treatment we detected concentrations of 221.8 and 28.8 µg kg–1 for FF and FFA, respectively. Based on these results, we conclude that these analytes will persist for a long time and will deplete slowly in feathers of treated broiler chickens. GRAPHICAL ABSTRACT

Withdrawal Times of Oxytetracycline and Tylosin in Eggs of Laying Hens after Oral Administration

Antimicrobials administered to laying hens may be distributed into egg white or yolk, indicating the importance of evaluating withdrawal times (WDTs) of the pharmaceutical formulations. In the present study, oxytetracycline and tylosins WDTs were estimated. The concentration and depletion of these molecules in eggs were linked to their pharmacokinetic and physicochemical properties. Twenty-seven Leghorn hens were used: 12 treated with oxytetracycline, 12 treated with tylosin, and 3 remained as an untreated control group. After completion of therapies, eggs were collected daily and drug concentrations in egg white and yolk were assessed. The yolk was used as the target tissue to evaluate the WDT; the results were 9 and 3 days for oxytetracycline and tylosin, respectively. In particular, oxytetracycline has a good oral bioavailability, a moderate apparent volume of distribution, a molecular weight of 460 g/mol, and is lightly liposoluble. Tylosin, a hydrosoluble compound, with a molecular weight of 916 g/mol, has a low oral bioavailability and a low apparent volume of distribution, too. Present results suggest that the WDTs of the studied antimicrobials are strongly influenced by their oral bioavailability, the distribution, and the molecular weight and solubility, and that these properties also influence the distribution between the egg yolk and white.

Determination of Oxytetracycline and 4-Epi-Oxytetracycline Residues in Feathers and Edible Tissues of Broiler Chickens Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Antibiotics have been widely used in poultry production for the treatment of bacterial diseases. However, drug residues can remain in products derived from animals after the cessation of the drug therapies. Feathers, in particular, have shown an affinity for antibiotics such as tetracycline, suggesting the persistence of these drugs in nonedible tissue. After the birds are slaughtered, feathers are ground into feather meals, which are used as organic fertilizer or an ingredient in animal diets, thereby entering into the food chain and becoming a potential risk for public health. To evaluate the depletion of oxytetracycline (OTC) and its metabolite 4-epi-oxytetracycline (4-epi-OTC) in the muscles, liver, and feathers, 64 broiler chickens, bred under controlled conditions, were treated orally with a commercial formulation of 10% OTC for 7 days. The analytes were quantified using liquid chromatography-tandem mass spectrometry. OTC and 4-epi-OTC were found in the feathers for 46 days, whereas they were found in the muscle and liver for only 12 and 6 days, respectively. These results prove that the analytes remain in feathers in higher concentrations than they do in edible tissues after treatment with tetracyclines. Thus, feather meals represent a potential source of antimicrobial residue contamination in the food chain.

Residue depletion of oxytetracycline (OTC) and 4-epi-oxytetracycline (4-epi-OTC) in broiler chicken’s claws by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

ABSTRACT Antibiotics are widely used in poultry production for the treatment of bacterial diseases. However, residues may remain in products and by-products destined for human consumption or animal feeding. The claws of chickens, which are a by-product of the poultry industry, can directly or indirectly enter the food chain as meals destined to feed other productive animals. Thus, it becomes necessary to determine and quantify antimicrobial residues present in this matrix. The objective of the study was to assess the depletion of oxytetracycline (OTC) and its metabolite 4-epi-OTC in broiler chicken’s claws. Claws of 32 broilers treated with a therapeutic dosage of 10% OTC during 7 days were analysed. Samples were taken at days 3, 9, 15 and 19 post-treatment. As for the control group, eight broiler chickens were raised under the same conditions. Extraction was carried out through EDTA-McIlvaine buffer, and clean-up employed a SPE C-18 Sep-Pak®. Instrumental analysis was performed through LC-MS/MS. The concentrations of both analytes were determined in claw samples until day 19 post-treatment. Average concentrations were within the LOD (20 μg kg–1) and LOQ (22 µg kg–1) for OTC and 84 μg kg–1 for 4-epi-OTC. Withdrawal times (WDTs) of 39 days for OTC and 54 days for 4-epi-OTC were established in claws based on 95% confidence. These findings demonstrate that claws can be a source of antimicrobial residue entry into the food chain, since the results showed that OTC and its metabolite can be found in chicken’s claws for long periods, even exceeding the average lifespan of a broiler chicken. Graphical Abstract

In-House validation of HPLC-MS/MS methods for detection and quantification of tetracyclines in edible tissues and feathers of broiler chickens

For the detection of tetraciclines in feathers, muscle and liver, an internal protocol was designed for in-house validation of two methods, through high performance liquid chromatography with tandem mass spectrometric (HPLC-MS/MS) techniques. This protocol was based on the recommendation of the decision 2002/657/EC from the European Community as well as the food and drug administration (FDA) VICHGL49. Limit of detection (LOD) was set at 20 μg kg and the limit of quantification (LOQ) ranged from 21.5 to 24.2, 21.2 to 21.6, and 25.0 to 27.7 μg kg in feahers, muscle and liver samples, respectively, for all analytes. The calibrations curves show a coefficient of determination (R) above 0.98, 0.99 and 0.96 for feathers, muscle and liver samples, respectively. Analyte recovery ranged from 92 to 108%. In conclusion, these methods can be deemed accurate and reliable, and their validation is a fundamental step to be performed in depletion studies on these matrices.

Determination of sulfachloropyridazine residue levels in feathers from broiler chickens after oral administration using liquid chromatography coupled to tandem mass spectrometry

Several antimicrobials are routinely used by the poultry farming industry on their daily operations, however, researchers have found for some antimicrobials that their residues persist for longer periods in feathers than they do in edible tissues, and at higher concentrations, as well. But this information is not known for other classes of antimicrobials, such as the sulfonamides. Therefore, this work presents an accurate and reliable analytical method for the detection of sulfachloropyridazine (SCP) in feathers and edible tissues from broiler chickens. This method was also validated in-house and then used to study the depletion of sulfachloropyridazine in those matrices. The experimental group comprised 54 broiler chickens, who were raised under controlled conditions and then treated with a commercial formulation of 10% sulfachloropyridazine for 5 days. Samples were analyzed via LC-MS/MS, using 13C6-sulfamethazine (SMZ-13C6) as an internal standard. Aromatic sulfonic acid solid phase extraction (SPE) cartridges were used to clean up the samples. The Limit of Detection (LOD) for this method was set at 10 μg kg-1 on feathers and liver; and at 5 μg kg-1 on muscle. Within the range of 10–100 μg kg-1, the calibration curves for all matrices presented a determination coefficient greater than 0.96. Our results show, with a 95% confidence level, that sulfachloropyridazine persisted in feathers for up to 55 days after ceasing treatment, and its concentrations were higher than in edible tissues. In consequence, to avoid re-entry of antimicrobial residues into the food-chain, we recommend monitoring and inspecting animal diets that contain feather derivatives, such as feathers meals, because they could be sourced from birds that might have been medicated with sulfachloropyridazine.

Depletion of tylosin residues in feathers, muscle and liver from broiler chickens after completion of antimicrobial therapy

ABSTRACT Tylosin is one of the most commonly used antimicrobial drugs from the macrolide family and in broiler chickens it is used specially for the treatment of infectious pathologies. The poultry industry produces several by-products, among which feathers account for up to 7% of a chicken’s live weight, thus they amount to a substantial mass across the whole industry. Feathers have been repurposed as an animal feed ingredient by making them feather meal. Therefore, the presence of high concentrations of residues from antimicrobial drugs in feathers might pose a risk to global public health, due to re-entry of these residues into the food chain. This work aimed to characterise the depletion behaviour of tylosin in feather samples, while considering its depletion in muscle and liver tissue samples as a reference point. To achieve this goal, we have implemented and validated an analytical methodology suitable for detecting and quantifying tylosin in these matrices. Sixty broiler chickens, raised under controlled conditions, received an oral dose of 32 mg kg-1 of tylosin for 5 days. Tylosin was quantified in muscle, liver and feathers by liquid chromatography coupled with a photodiode array detector (HPLC-DAD). High concentrations of tylosin were detected in feather samples over the whole experimental period after completing both the therapy and the recommended withdrawal time (WDT). On the other hand, tylosin concentrations in muscle and liver tissue samples fell below the limit of detection of this method on the first sampling day. Our results indicate that the WDT for feather samples is 27 days, hence using feather meal for the formulation of animal diets or for other agricultural purposes could contaminate with antimicrobial residues either other livestock species or the environment. In consequence, we recommend monitoring this matrix when birds have been treated with tylosin, within the context of poultry farming.

A four-year survey in the farming region of Chile, occurrence and human exposure to polychlorinated dibenzo-p-dioxins and dibenzofurans, and dioxin -like polychlorinated biphenyls in different raw meats

For the first time in South America, a four-year survey (2011-2014) was conducted to assess the occurrence of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) in different raw meats (bovine, pork, ovine, chicken, and turkey) sampled from ten of the fifteen regions of Chile. When expressed as pg World Health Organization Toxic Equivalent (WHO-TEQ2005)g-1 fat, the highest PCDD/F values for each species were 0.54 (bovine-2012), 0.27 (pork-2013), 0.23 (ovine-2011), 0.61 (chickens-2013), and 0.34 (turkey-2012). The highest mean dl-PCBs levels were 0.18 (bovine-2011), 0.05 (pork-2014), 0.13 (ovine-2011), 0.1 (chicken-2014), and 0.21 (turkey-2013). Penta- and tetra-chlorinated congeners dominated PCDD/F WHO-TEQ2005 profiles during the survey, while PCB 126 dominated dl-PCBs profiles. Statistically significant interspecies differences were found. Dietary intake was also estimated, and the highest total PCDD/F and dl-PCBs values, found in poultry meat, were 0.09pgWHO-TEQ2005kg-1bwd-1 (2013) for adults and 0.36pgWHO-TEQ2005kg-1bwd-1 (2013) for children. The concentrations and dietary intakes for the studied compounds in raw meat were below international and national maximum permitted limits.

Determination of Chlortetracycline Residues, Antimicrobial Activity and Presence of Resistance Genes in Droppings of Experimentally Treated Broiler Chickens

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.