IIIA   26586
INSTITUTO DE INVESTIGACION E INGENIERIA AMBIENTAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Screening of Transformation Products and Intermediates from the Aerobic Degradation of Oxytetracycline by HPLC-MS
Autor/es:
IVANIC, FEDERICO M.; CANDAL, ROBERTO J.; BUTLER, MATIAS
Lugar:
Online Meeting
Reunión:
Conferencia; 68th ASMS Conference on Mass Spectrometry and Allied Topics; 2020
Institución organizadora:
American Society for Mass Spectrometry
Resumen:
Introduction Emerging contaminants (ECs) are chemical compounds and metabolites that are currently not regulated by the existent water quality legislations, and therefore are usually ignored, but still represent a harmful threat to the ecosystems and human health. Once they reach natural environments, the ECs experience different types of processes, through which they can transform into new metabolites. These processes include biodegradation, chemical oxidation and reduction, hydrolysis and photolysis, as well as adsorption in sediments and soils. One example of ECs are pharmaceutical compounds, which not only are used to prevent and treat human illnesses, but also are employed in large quantities in areas of intense animal husbandry worldwide. Oxytetracycline (OTC) is a broad-spectrum antibiotic used in veterinary medicine to treat bacterial infections and as growth promoter, and represents a good example to be used as model contaminant. As it is poorly absorbed in the body of animals once it is consumed, it tends to accumulate in animal wastes ending in natural water bodies, such as lakes and lagoons. The chemical structure of OTC is shown in Figure 1, where it can be seen that OTC presents multiples reactive sites, such as aromatic rings, olefins, carbonyl and hydroxyl groups, prone to suffer different types of transformations and sensitive to the pH of the solution. Mass spectrometry coupled to HPLC arises as a powerful technique to track in time the degradation of OTC in water samples, as well as to separate and identify possible intermediates and products that originate from the previously mentioned processes. Moreover, as these reactions lead to a wide complexity of products, their spectra analysis benefit usually from the use of a non-targeted metabolomic screening approach.Lab simulated experiments may aid in the study of degradation of ECs in natural environments, provided these conditions are correctly modelled. In this type of experiments, multiple variables need to be fixed or controlled such as pH, redox potential, agitation rate and water/sediment proportions. Biotic and abiotic degradation simulations for different types of organic molecules have been widely discussed in previous works and guidelines (Baginska et al., 2014; Shrestha et al., 2016, OECD 308, 309), including degradation of OTC (Doi et al., 2000), although the focus was mainly set on the disappearance of the studied contaminant. On the other hand, forced-degradation pathways for OTC and other tetracyclines as a result of specific remediation techniques have been studied (Han et al., 2020; Liu et al., 2016), yet the environmental conditions in which these pollutants may be found are not considered. Lastly, some recent publications have reviewed the main pathways in which OTC is naturally degraded but fail to discriminate between environmental conditions (Li et al., 2019). This work aims to present an approach combining simulated natural degradation experiments with mass spectrometry as a tool to follow the evolution of OTC and its transformation products. Four main conditions, including two different pH values and presence/absence of microorganisms, were studied and contrasted.MethodsDegradation test design. Glass bottles of 1 L of capacity were filled with deionized water and artificial sediment (mixture of sand-clay-peat 93:5:2) in proportions 8:1. Prior to this step, the sediment was thoroughly sieved, mixed and hydrated. Once the water/sediment system reached equilibrium, pH was fixed to two desired ranges: 5.5-5.7 and 7.3-7.5 (pHA and pHB respectively). After a week of stabilization, the biotic systems were spiked with a microbial inoculum (0.22 % v/v), obtained from a pig farm effluent. Biotic growth in the abiotic experiments was avoided by adding NaN3 (5 % m/v) as biocide. At this stage, all bottles were covered in aluminum foil and connected to an air flow system boosted by an air pump which bubbled air directly into the solution. After another week of stabilization, OTC was added (final concentration 200 µM) in all flasks except blank.Batch experiment. 8 experiments were carried out simultaneously under agitation on an orbital shaker at 80 rpm at room temperature. Experiments included biotic and abiotic conditions, each at pHA and pHB, and OTC blank. Air was pumped continuously and pH, redox potential and dissolved O2 were measured periodically. For this purpose, each glass bottle possessed a working Pt electrode, a reference Ag/AgCl electrode, temperature sensor and an entrance for pH and dissolved O2 electrodes. Samples were taken each day during the first week and then once a week for approximately 1 month, with a glass syringe through a septum.Samples analysis. Extracted samples were filtered through a 0.45 µm cellulose acetate membrane, collected in glass vials and then injected into the HPLC, employing a Dionex UltiMate 3000 RS LC system (Thermo Scientific) for solvent injection. Chromatography separation was achieved using a Kinetex C18 column (100 mm x 2.1 mm, 2.6 µm, Phenomenex). A mixture of acetonitrile and water containing formic acid (0,1% /v/v) was used for the elution gradient, setting the flow rate at 0.2 mL min-1. Mass spectra were acquired using a linear ion trap mass spectrometer (Thermo LTQ XL) equipped with an electrospray source in positive-ion mode. Degradation of OTC was monitored by following ion of m/z 461 in time. Spectra analysis was executed using a non-target screening approach, via MatLab software (Gorrochategui et al., 2016). The search of possible transformation products involved the scrutiny of regions of interest (ROI), i.e. time intervals containing significant intensity values (above a defined threshold) for given ions of m/z. Comparison of the results with bibliography allowed the suggestion of multiple potential degradation pathways and chemical structures.Results and discussionOTC degradation followed a typical exponential decay (Fig. 2), reaching at least 1% of the initial concentration in all of the experiments within the screened time. OTC?s half-life (t1/2) was similar in all conditions: for abiotic conditions, t1/2 was 1.6±0.7 days and 2.0±0.4 days for pHA and pHB respectively, and for biotic conditions t1/2 was 2.2±0.2 days and 2.2±0.4 days for pHA and pHB respectively. Nevertheless, biotic conditions and a lower pH accelerated the total degradation of the contaminant, reaching 1% of initial OTC concentration by day 22. On the contrary, experiments with abiotic conditions and higher pH achieved the same OTC concentration by day 36. Moreover, changes in chromatographic retention time of OTC have been observed in all experiments: from a retention time of 8.5 min in day 0 to a final 8.7 min at the end of the experiment. This can be attributed to isomerization of OTC to the already reported epimer 4-α-epi-OTC, EOTC (Loke et al., 2003) where the configuration of the C bonded to the dimethylamine group changes, as shown in Figure 3. Analysis of transformation products provided various ions which could be identified as possible transformation products candidates. In Figure 4, a scheme summarizing the most important degradation pathways observed for OTC in all studied conditions is presented. The connection between structures was possible due to the timewise measurement of degradation evolution. Multiple reactions were involved in the degradation process, for example decarbonylation, alcohol oxidation, demethylation, etc. As OTC can be degraded through several pathways, numerous ions of different m/z have been found and their interconnections become complex to compile. It is worth noting that ions of m/z 433 and 447 can be generated by two different degradation pathways and therefore two structures are plausible. Evidence of the presence of both structures in each case relies on the observation of two retention times for each m/z value in the chromatographic runs. In general, transformation products common to the discussed degradation reactions were detected in all the studied conditions, although different rates and prevalence were observed, proving that the evolution of ions in time depends on the condition. For example, ions coming from the demethylation reaction of OTC (m/z 447 followed by m/z 433) were more abundant in biotic conditions than in abiotic conditions. Another example involves ions of m/z 495 and 511, which were only observed under pHA-biotic conditions. Lastly, ions of m/z 479 were only formed at pHB, and under abiotic conditions its abundance was 5 times higher than under biotic conditions. ConclusionsThe methodology hereby described has been successfully applied to identify possible transformation products and intermediates involved in the degradation process of OTC. The simulation tests coupled to HPLC-MS allow the obtention of relevant environmental information that may become useful in future remediation strategies and monitoring of potential contaminated sites. Furthermore, the versatility of the approach allows its application to a wide range of pollutants in different conditions (pH, O2 concentration/presence, light, temperature, etc.), and its performance may be improved by adding new analyses such as the study of the metabolites adsorbed in the sediments.AcknowledgmentsThis work was developed in Instituto de Investigación e Ingeniería Ambiental, Universidad Nacional de San Martín. We thank ANPCYT for the financial support through the program Proyectos de Investigación Científica y Tecnológica (PICT 2014 2386; PICT 2016 2940 and PICT 2018 2788) and CONICET for the PhD fellowship to F.M.I (2019). M.B. and R.J.C. are members of CONICET. BibliographyBaginska E.; Haiß A.; Kümmerer K.; Chemospehere, 2015, Volume 119, 1240-1246.Doi, A. M.; Stoskopf, M. K.; Journal of Aquatic Animal Health, 2000, Volume 12, Issue 3, 246-253.Gorrochategui E.; Jaumot J.; Lacorte S.; Tauler R.; Trends in Analytical Chemistry, 2016, Volume 82, 425-442.Han, C.-H.; Park, H.-D.; Kim S.-B.; Yargeau V.; Choi J.-W.; Lee S.- H.; Park J.-A.; Water Research, 2020, Volume 172.Li, Z.-J-; Qi, W.-N.; Feng, Y.; Liu, Y.-W.; Ebrahim, S.; Long, J.; Journal of Integrative Agriculture, 2019, Volume 18, 1953-1960.Liu, Y.; He, X.; Fu, Dionysiou, D. D.; Chemical Engineering Journal, 2016, 284, 1317-1327.Loke, M.L; Jespersen, S.; Vreeken, R.; Halling-Sørensen, B.; Tjørnelund, J.; Journal of Chromatography B, 2003, Volume 783, Issue 1, 11-23OECD guidelines for Testing of Chemicals; 308, Aerobic and Anaerobic Transformation in Aquatic Sediment Systems; Organization for Economic Co-operation and Development (OECD), 2002.OECD. OECD Guidelines for Testing of Chemicals; 309, Aerobic Mineralisation in Surface Water: Simulation Biodegradation Test; Organization for Economic Co-operation and Development (OECD), 2004.Shrestha, P.; Junker, T.; Fenner, K.; Hahn, S.; Honti, M.; Bakkour, R.; Díaz, C.; Hennecke, D.; Environmental Science and Technology, 2016, Volume 50, Issue 13, 6856?6864.