J. Cornejo Kelly^1,2; E. Pokrant^1,2; A. Maddaleno¹

¹Laboratory of Veterinary Pharmacology FARMAVET, Faculty of Veterinary and Livestock Sciences FAVET; ²Laboratory of Food Safety LIA, Faculty of Veterinary and Livestock Sciences FAVET, La Pintana, Santiago, Chile

With billions of food animals produced globally, as much as 37% of broiler chickens, 44% of pigs, and 49% of cattle are inedible to humans (1). Along with animal production centres and slaughterhouses there are rendering plants that turn inedible by-products (blood, bones, head, feet, feathers, etc.) into animal feed and fertilizer. Chemical constituents of the by-products—including antimicrobial drugs—that are not inactivated during the rendering process will persist in the by-product as residues. These drug residues may be of public health concern given their potential to recycle antimicrobial drugs and other chemicals into the food chain as animals consume them (2), however, they also represent an opportunity to monitor food systems for antimicrobial administered during production when surveillance data are limited.

The use of antibiotics is common practice to treat bacterial infections in the poultry sector. Antibiotic treatments, mostly orally administered through drinking water, should be carried out according to registration in order to prevent excessive antibiotic residues in food products meant for human consumption.

The quantity of AMs used in all animals as reported by the World Organization for Animal Health (WHO) in the 2017 was 85,330 tonnes (3). By 2030, the global use of antimicrobials for animal production is expected to increase 67%, from 63 tons to 105 tons, mainly because of the growing demand for animal protein in low- and middle-income countries (4,5). At global level the antimicrobial agents most used in food production animals are tetracyclines, macrolides, sulfonamides and penicillins (6,7).

Following different food safety regulations presence of antibiotics in food of animal are strictly monitor on the basis maximum residue limits (MRL), which are establishes for matrices such as muscle, liver, and eggs, among others edible tissues. Nevertheless, since feathers and feather meals are not considered edible products, no MRLs have been established. Scarce regulations exist worldwide considering feather meal is used as an animal feed constituent and presence of antimicrobials in this by product. For example, in EU the use of feather meal in animal feed falls within the scope of 2009/767/EC (8). This regulation refers to 2002/32/EC (9) with regard to undesired substances in feed. This regulation covers the presence of anticoccidials but does not mention any antibiotics.

Feathers make up 5%–6% of the weight of poultry and, when ground and hydrolysed, create feather meal (1,10). Feather meal is a low-cost feed ingredient that is high in protein but not easily digestible by animals, therefore, this by-product a minor ingredient in complete feeds for aquaculture, cattle, pig and poultry (1). In Europe, feather meal is a Category 3 material with no restrictions on where it can be used (11), similarly no use restrictions exist in the U.S. Feather meal is also used in agriculture as a slow release nitrogen fertilizer (12). Europe produces about 175,000 metric tons of feather meal annually (10). Annual reporting of feather meal production in the U.S. is not available, however by extrapolating from the 8.7 billion poultry slaughter each year and average yield data, it was estimated that 64,400 metric tons of feather meal were produced in 2015 (13). In 2015, 478,000 metric tons of feather meal were consumed in the U.S. and 87,000 metric tons were exported to other countries (13). The largest importers of U.S. feather meal were Indonesia, Chile Canada, Vietnam, China, Peru, and Ecuador (13). No data was available on European feather meal consumption or trade. Feather meal production is expected to increase in the next 10 years in the U.S. at roughly the same 15% growth rate as poultry production, the animals it is derived from (13). Feather meal production in other countries depends on the capacity and development of a rendering industry in those countries.

Considering the antimicrobial sales, the estimated consume of antimicrobials in poultry is 148 mg per kg of animal weight produced (14), the feather meal incorporation rates into diets, and the lack of surveillance of antimicrobial in this ingredient, made poultry feather an interest matrix to study antimicrobial behavior.

Pharmacokinetic characteristics of different antibiotics used in the poultry industry, and the growth and molting characteristics of feathers, have studied since 2007 (15). Different authors have demonstrated the persistence of antibiotic residues in feathers versus concentrations in tissues as muscle and liver. First report about this issue was provided by our research group. San Martin et al. (15) performed a depletion study with laying hens treated intramuscularly with 5% enrofloxacin at 10 mg/kg body weight over 3 days. Thirty-three birds were treated and slaughtered at different times between 6 and 216 h after treatment; samples of muscle plus skin, liver, kidney, and feathers were collected by LC MS–MS. Authors found high concentrations of enrofloxacin and its metabolite ciprofloxacin in feathers from treated animals when compared with edible tissues (muscle, liver and kidney), through all sampling points.

Similarly, Cornejo et al. (16) found higher concentrations of flumequine in feathers when compared with liver and muscle samples. In both cases, Enrofloxacin and flumequine, the presence of residues in feathers can be explained by the pharmacokinetic properties of quinolones, such as large volume of distribution, low affinity to plasma proteins and high lipophilicity. Because of these characteristics, residues can reach high concentrations in peripheric tissues, as skin and in the case of birds, feathers (17). Afterwards the high affinity and bioaccumulation of fluoroquinolones in feathers has recently been confirmed in works by Mestorino et al. (18) and Jansen et al. (19).

Also, the behavior and bioaccumulation of oxytetracycline in feathers has been assessed. In 2013, Berendsen et al. (20) as in previous studies, they concluded that the accumulation in feathers was higher than in edible tissues. More recently, in 2017, depletion of oxytetracycline (OTC) and its metabolite 4-epi-oxytetracycline (4-epi-OTC) in muscle, liver, and feathers of broiler chickens was studied by Cornejo et al. (21), to compare residues concentrations in the three different tissues using LC–MS/MS. Feathers show affinity for this antibiotic, as OTC and 4-epi-OTC were found in feathers for 46 days, whereas they were found in the muscle and liver for only 12 and 6 days, respectively. Results prove that the analytes remain in feathers in higher concentrations than they do in edible tissues after oxytetracycline treatment.

Florfenicol (FF) and its active metabolite florfenicol amine (FFA) transfer to feathers was evaluated (22). Feathers were analyzed by LC–MS/MS. 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 concentrations of 221.8 and 28.8 μg kg⁻¹ for FF and FFA, respectively, were detected by the authors.

Tylosin one of the most used antimicrobial drugs from the macrolide family and in broiler chickens, was also studied to characterize the depletion behavior in feather, while considering its depletion in muscle and liver tissue samples as a reference point. This drug was quantified in muscle, liver, and feathers. High concentrations of the analyte were detected in feather samples over the whole experimental period, with concentration ranging between 9246.53–356.59 μg kg⁻¹ (23).

Sulfachloropyridazine (SCP) is another antimicrobial studied in feathers. Pokrant et al. (24) determined that this antibiotic remained in feathers for a long period of time, with average concentrations of 18.9 μg kg⁻¹ detected 32 days after treatment. SCP persistence in this matrix was projected to 55 days, with a confidence level of 95% and a LOD of the analytical technique of 10 μg kg⁻¹ as a cut-off point. Furthermore, the same group of researchers determined that lincomycin residues could persist for an even longer period of up to 98 days, considering the LOQ (62 μg kg⁻¹) established for the methodology as the cut-off value for the calculations (25).

Enough evidence is published showing that antimicrobials accumulate in broilers' feathers at higher levels and for longer periods than in edible tissues demonstrating that when poultry had been treated with therapeutic doses of antimicrobials, drug persist in higher concentration levels in their feathers than in their edible tissues (muscle and liver). suggesting that the withdrawal times estimated do not guarantee the absence of this drug in chicken nonedible tissues such as feathers (15,16,18–27).

Also, considering the lack of accurate data on antimicrobial uses on farms is a challenge to regulatory groups tasked with surveillance of antimicrobial uses in food production, and the interesting concentrations that have been proved to be found, different authors have suggested the use of poultry feathers a non-invasive matrix of assessing antimicrobial use on farms by testing animal by-products (26,28,29). This is how feathers can be tested using analytical methods to evaluate antimicrobial use policies in animal production. As well as the analysis of poultry feather meal, considering that the reintroduction of this by-product into the food chain can become a risk for the feed supply.

Conclusion: Feathers has a potential as “biomarker of compliance”, considering they feasibility of monitoring and that analytical methods exist and are feasible to use. Nevertheless, would need to validate sensitivity and specificity of assay based on data on drug persistence according to drug/drug class.

It is imperative to establish monitoring programmes for antimicrobial residue analysis, in order to re-establish the fate of feathers with measurable residues and not be used in the formulation of diets for food-producing animals. Other opportunities may exist with other by-products that could be used as biomarkers of compliance for various food-producing animal species.

References: 1. Meeker, D. L., editor. (2006). Essential rendering: all about the animal by-products industry. Alexandria, Va.: National Renderers Association: Fats and Proteins Research Foundation: Animal Protein Producers Industry; 302.

2. Love, D. C., Halden, R. U., Davis, M. F., Nachman, K. E. (2012). Feather meal: A previously unrecognized route for reentry into the food supply of multiple pharmaceuticals and personal care products (PPCPs). Environ Sci Technol. 3 de abril de, 46(7), 3795–3802.

3. World Organization for Animal Health. (2021). Fifth Report: OIE Annual Report on Antimicrobial Agents Intended for Use in Animals [Internet]. Available online: https://www.woah.org/fileadmin/Home/eng/Our_scientific_expertise/docs/pdf/AMR/A_Fifth_Annual_Report_AMR.pdf

4. Van Boeckel, T. P., Glennon, E. E., Chen, D., Gilbert, M., Robinson, T. P., Grenfell, B. T., et al. (2017). Reducing antimicrobial use in food animals. Science. 29 de septiembre de, 357(6358), 1350–1352.

5. Xiong, W., Sun, Y., Zeng, Z. (2018). Antimicrobial use and antimicrobial resistance in food animals. Environ Sci Pollut Res. julio de, 25(19),18377–18384.

6. FAO. (2020). Sources of Meat [Internet]. [citado 15 de septiembre de 2020]. Available online: http://www.fao.org/ag/againfo/themes/en/meat/backgr_sources.html

7. Mulchandani, R., Wang, Y., Gilbert, M., Van Boeckel, T. P. (2023). Global trends in antimicrobial use in food-producing animals: 2020 to 2030. Odetokun IA, editor. PLOS Glob Public Health. 1 de febrero de, 3(2), e0001305.

8. Regulation (EC) No 767/2009 of the European Parliament and of the Council of 13 July 2009 on the placing on the market and use of feed, amending European Parliament and Council Regulation (EC) No 1831/2003 and repealing Council Directive 79/373/EEC, Commission Directive 80/511/EEC, Council Directives 82/471/EEC, 83/228/EEC, 93/74/EEC, 93/113/EC and 96/25/EC and Commission Decision 2004/217/ECText with EEA relevance.

9. Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed. Official Journal of the European Communities. (2002).

10. Adler, S., Honkapää, K., Slizyte,R., Løes, A. K. (2012). Feather meal production in Europe. 2014.

11. Canadian Food Inspection Agency. Definition of Categories 1, 2, and 3 Animal Products and By-Products According to the European Legislation (EC) 1069/2009 [Internet]. Available online: https://inspection.canada.ca/animal-health/terrestrial-animals/exports/export-policies/2009-9/eng/1321202144060/1321202222038

12. Hadas, A., Kautsky, L. (1994). Feather meal, a semi-slow-release nitrogen fertilizer for organic farming. Fertil Res, 38(2), 165–170.

13. Swisher, K. (2016). Market Report: Prices are Down butDemand Remains Strong. Render: The International Magazine of Rendering. abril de, 10–15.

14. Van Boeckel, T. P., Brower, C., Gilbert, M., Grenfell, B. T., Levin, S. A., Robinson, T. P., et al. (2015). Global trends in antimicrobial use in food animals. Proc Natl Acad Sci. 5 de mayo de , 112(18), 5649–5654.

15. San Martín, B., Cornejo, J., Iragüen, D., Hidalgo, H., Anadón, A. (2007). Depletion Study of Enrofloxacin and Its Metabolite Ciprofloxacin in Edible Tissues and Feathers of White Leghorn Hens by Liquid Chromatography Coupled with Tandem Mass Spectrometry. J Food Prot. agosto de , 70(8), 1952–1957.

16. Cornejo, J., Lapierre, L., Iragüen, D., Pizarro, N., Hidalgo, H., Martín, B. S. (2011). Depletion study of three formulations of flumequine in edible tissues and drug transfer into chicken feathers: Depletion of flumequine in chicken edible tissues and feathers. J Vet Pharmacol Ther. abril de, 34(2), 168–175.

17. Martinez, M., McDermott, P., Walker, (2006). R. Pharmacology of the fluoroquinolones: A perspective for the use in domestic animals. Vet J. julio de, 172(1), 10–28.

18. Mestorino, N., Marchetti, M. L., Lucas, M. F., Modamio, P., Zeinsteger, P., Fernández Lastra, C., et al. (2016). Bioequivalence study of two long-acting formulations of oxytetracycline following intramuscular administration in bovines. Front Vet Sci [Internet]. [citado 11 de enero de 2021];3. Available Online: 10.3389/fvets.2016.00050/full

19. Jansen, L. J. M., Bolck, Y. J. C., Berendsen, B. J. A. (2016). Feather segmentation to discriminate between different enrofloxacin treatments in order to monitor off-label use in the poultry sector. Anal Bioanal Chem. enero de, 408(2), 495–502.

20. Berendsen, B. J. A., Bor, G., Gerritsen, H. W., Jansen, L. J. M., Zuidema, T. (2013). The disposition of oxytetracycline to feathers after poultry treatment. Food Addit Contam Part A. diciembre de, 30(12), 2102–2107.

21. Cornejo, J., Pokrant, E., Krogh, M., Briceño, C., Hidalgo, H., Maddaleno, A., et al. (2017). Determination of oxytetracycline and 4-Epi-oxytetracycline residues in feathers and edible tissues of broiler chickens using liquid chromatography coupled with tandem mass spectrometry. J Food Prot. 1 de abril de, 80(4), 619–25.

22. Cornejo, J., Pokrant, E., Riquelme, R., Briceño, C., Maddaleno, A., Araya-Jordán, C., et al. (2017). 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. Food Addit Contam Part A. 3 de abril de, 34(4), 469–76.

23. Cornejo, J., Pokrant, E., Carvallo, C., Maddaleno, A., San Martín, B. (2018). Depletion of tylosin residues in feathers, muscle and liver from broiler chickens after completion of antimicrobial therapy. Food Addit Contam Part A. 4 de marzo de, 35(3), 448–57.

24. Pokrant, E., Medina, F., Maddaleno, A., San Martín, B., Cornejo, J. (2018). Determination of sulfachloropyridazine residue levels in feathers from broiler chickens after oral administration using liquid chromatography coupled to tandem mass spectrometry. Sant‘Ana A de S, editor. PLoS One. 5 de julio de, 13(7), e0200206.

25. Pokrant, E., Maddaleno, A., Lobos, R., Trincado, L., Lapierre, L., San Martín, B., et al. (2019). Assessing the depletion of lincomycin in feathers from treated broiler chickens: a comparison with the concentration of its residues in edible tissues. Food Addit Contam Part A. 2 de noviembre de, 36(11), 1647–1653.

26. Chiesa, L. M., Nobile, M., Panseri, S., Arioli, F. (2018). Suitability of feathers as control matrix for antimicrobial treatments detection compared to muscle and liver of broilers. Food Control. septiembre de, 91, 268–275.

27. Jansen, L. J. M., Bolck, Y. J. C., Rademaker, J., Zuidema, T., Berendsen, B. J. A. (2017). The analysis of tetracyclines, quinolones, macrolides, lincosamides, pleuromutilins, and sulfonamides in chicken feathers using UHPLC–MS/MS in order to monitor antibiotic use in the poultry sector. Anal Bioanal Chem. agosto de, 409(21), 4927–4941.

28. Dréano, E., Miquel, D., Taillandier, J. F., Laurentie, M., Hurtaud-Pessel, D., Mompelat, S. (2023). Antimicrobial residues along the broiler feathers: Analysis of sulfadiazine, trimethoprim and oxytetracycline in feather segments over time. Food Control. junio de, ;148, 109674.

29. Gajda, A., Nowacka-Kozak, E., Gbylik-Sikorska, M., Posyniak, A. (2019). Feather analysis as a non-invasive alternative to tissue sampling for surveillance of doxycycline use on poultry farms. Poult Sci. noviembre de, 98(11), 5971–5980.