Determination of antibiotics and other veterinary drugs in the solid phase of pig manure
Introduction
The consumption of pork meat is growing annually worldwide. Consequently, the number of pig farms is gradually increasing. The European Union (EU) is the world’s second biggest producer of pork, only surpassed by China. In particular, Germany, Spain and France are leading the European ranking, and between them they represent half of the EU’s total production.
In industrial intensive swine farming, pigs are usually raised in stables. In order to comply with health and hygiene controls, pigsties are regularly cleaned by using high volumes of water pressure. The resulting piggery wastewater (PWW) mainly contains pig urine and feces, in addition to other components such as rests of feed and straw bed. PWW is made up to 98% of water and high concentrations of inorganic nutrients and organic matter (Makara and Kowalski, 2015).
For many years, PWW has been commonly discharged as fertilizer directly onto the agricultural land surrounding the farms. It was the simplest and most inexpensive way to dispose this kind of wastewater (Khatri and Tyagi, 2015). However, underlying aquifers were becoming contaminated by nutrient leaching. Thus, in 1991 the EU issued the Directive 91/676/EEC in order to protect the waters against pollution caused by nitrates from agricultural sources, and limited the discharge of total nitrogen to 170 kg ha−1 y−1.
At the same time, since 2006 the use of antibiotics as growth promoters in livestock is only authorized under prescription in EU, and its use has been severely restricted in the US since 2016. However, little or no control is enforced in many other countries around the world. Furthermore, as it happens for other types of drugs, many antibiotics are poorly absorbed in the digestive system and are scarcely metabolized. Therefore, they are mainly excreted and incorporated into the manure (Ezzariai et al., 2018) (Spielmeyer, 2018). Stunning antimicrobial loads of up to 100 mg L−1 have been reported in the literature revision published by (Van Epps and Blaney, 2016). In fact, it is very difficult to find residues-free manure (Ezzariai et al., 2018) (Spielmeyer, 2018). Consequently, when PPW is discharged, antibiotics are probably released into the environment too, which enhances the risk of developing antibiotic resistance in pathogenic bacteria, as consistently reported in the last years (Peeples, 2015).
Highest amounts of nitrogen are found in the liquid phase pig manure. In addition, transportation costs drastically decrease when the liquid phase is removed from the pig manure. Hence, latest management approaches tend to separate both phases. Liquid phase is usually further treated as sewage and solid phase manure is commonly processed to be used as fertilizer in agriculture.
To date, only a few methods intended to determine pharmaceuticals in pig slurry (Patyra et al., 2020) (López-Serna et al., 2019) (Bourdat-Deschamps et al., 2014), and only one tailor-made it for its solid phase (Marti et al., 2020). The latter used of pressurized liquid extraction as technique to extract the analytes from the matrix, which is expensive and not often available in analytical laboratories. In addition, its efficiency to extract the analytes is often depleted by its associated matrix effect, as it also shows a high efficiency extracting the matrix (Pérez-Lemus et al., 2019). It is paramount to assess the feasibility of a wider range of techniques, including the most commonly present in routine-analysis laboratories, which would enlarge analytical capacity for this type of analysis. This study aimed at developing and optimizing a creative analytical method consisting of Ultrasound-assisted-extraction (UAE) with in-situ dispersive solid phase extraction (d-SPE) followed by compact-solid-phase-extraction (c-SPE) and Ultra-High-Performance-Liquid-Chromatography (UHPLC) coupled to Mass spectrometry in tandem (MS/MS) for 21 veterinary drugs in the solid phase of pig slurry, using statistical experimental design. To the authors’ knowledge, this is the first time statistical design has been specifically tailor-made to find the optimum conditions to analyze veterinary drugs of different chemical properties and medical indications in swine manure solid-phase.
Section snippets
Standards and reagents
Twenty-one veterinary pharmaceuticals, including 18 antibiotics (5 sulfonamides, 4 fluoroquinolones, 3 tetracyclines, 2 β-lactams, 1 macrolide, 1 pleuromutilins and 2 others), as well as 1 anti-parasitic, 1 hormone and 1 analgesic were selected according to their high prescription in veterinary farms in Spain, particularly in Castilla y León, region where the farm providing with samples for this study was located (AEMPS, 2011). Drug standards of high purity grade (>95%) were obtained from
Preliminary experiments
Some preliminary experiments were carried out in triplicate in a one-factor-at-a-time approach to optimize the sample size, type of cleanup agent and extraction technique. These qualitative discrete variables were not tested within the experimental design described in section 3.1.2 as they included more than two levels per factor, which would complicate the design. The method protocol described in section 2.2.1 was used as starting point and only the tested parameter was modified in each case.
Conclusions
The development of a tailor-made method for the analysis of 21 veterinary drugs in the very complex matrix of the fresh solid phase pig slurry has been conducted. The resulting methodology based on UAE with in situ d-SPE followed by c-SPE and UHPLC-MS/MS was intra-laboratory validated and subsequently applied to real samples. The main conclusions drawn were as follows:
- 1.
After some preliminary assays, aliquots of 0.3 g of freeze-dried sample, activated Al2O3 and UAE were selected as the best
CRediT author statement
Manuel Argüeso-Mata - Investigation · Validation. Data curation · Writing - Original draft. Silvia Bolado - Funding acquisition · Project administration. Juan José Jiménez - Conceptualization · Methodology · Formal Analysis · Supervision. Rebeca López-Serna - Methodology · Investigation · Data curation· Resources, Writing - Review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the Regional Government of Castilla y León, the EU-FEDER programme (CLU 2017 – 09, UIC 071, INFRARED2018-UVA3, VA080G18) and the Spanish Ministry of Science, Innovation and Universities (CTQ2017-84006-C3-1-R project). It was partially carried out in the Laboratory of Instrumental Techniques from the University of Valladolid (UVa). The UVa is also recognized for the postdoctoral contract held by Rebeca López Serna (2018POSTDOC UVA12).
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