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- HPLC-FD method to follow biodegradation of four fluoroquinolonesPublication . Maia, Alexandra; Duque, Anouk F.; Ribeiro, Ana L.; Castro, Paula M. L.; Tiritan, Maria E.
- Biodegradation of fluoroquinolones by single bacteriaPublication . Amorim, Catarina L.; Moreira, Irina S.; Maia, Alexandra; Tiritan, Maria E.; Castro, Paula M. L.
- Pharmaceuticals biodegradation by activated sludge and pure culturesPublication . Bessa, Vânia S.; Moreira, Irina S.; Maia, Alexandra S.; Tiritan, Maria E.; Castro, Paula M. L.The occurrence of pharmaceuticals in the environment have been a topic of increasing concern. Most of the pharmaceuticals are not completely mineralized in the human body and are released on the sewage systems as the pharmaceutical itself and as their “biologically active” metabolites through excretion, as well as by improper elimination and disposal. Once current wastewater treatment plants (WWTPs) are not designed to remove these emerging pollutant, they are easily released into the environment.The effects of halogen on biological properties of molecules have had a marked impact on various fields such as pharmacology. It can improve metabolic stability, bioavailability and interactions with the biological target. However, it also increases the recalcitrance of the molecule to biotic and abiotic degradation. Ciprofloxacin (CPF) and Diclofenac (DCF) are two widely used halogenated pharmaceuticals, commonly found in the environment in concentrations that can range ng L−1 to mg L−1. CPF is a common human and veterinary broad-spectrum fluoroquinolone antibiotic. DCF is a non-steroidal anti-inflammatory drug (NSAID), also used as analgesic and antithermic treatment. In the present study, biodegradation of CPF and DCF was assessed by selective enrichements with actived sludge and with pure cultures of three different strains - Labrys portucalensis (F11) and two Rhodococcus spp. (FP1 and S2) – which had previously demonstrated capacity to degrade a range of halogenated compounds. For CPF selective enrichments, a degradation of 100% was achived after 206 days, although without fluoride release. These findings indicate that these selective enrichments are good candidates to find a bacterial strain able to biodegrade CPF. Regarding the pure cultures, F11 exhibited the highest degradation capacity (57%) after 62 days, but also without fluoride release. Concerning DCF selective enrichments, it was released 68% of the stoichiometrically expected cloride ion behind 131 days. In the experiments with pure cultures, F11 and S2, was released the totality of the stoichiometrically expected cloride, elapsed 131 days. These results indicate that these two strains are promising for DCF biodegradation.
- Biodegradation of ofloxacin, norfloxacin, and ciprofloxacin as single and mixed substrates by Labrys portucalensis F11Publication . Amorim, Catarina L.; Moreira, Irina S.; Maia, Alexandra S.; Tiritan, Maria E.; Castro, Paula M. L.Fluoroquinolone (FQ) antibiotics are extensively used both in human and veterinary medicine, and their accumulation in the environment is causing an increasing concern. In this study, the biodegradation of the three most worldwide used FQs, namely ofloxacin, norfloxacin, and ciprofloxacin, by the fluoroorganic-degrading strain Labrys portucalensis F11 was assessed. Degradation occurred when the FQs were supplied individually or as mixture in the culture medium, in the presence of an easily degradable carbon source. Consumption of individual FQs was achieved at different extents depending on its initial concentration, ranging from 0.8 to 30 μM. For the lowest concentration, total uptake of each FQ was observed but stoichiometric fluoride release was not achieved. Intermediate compounds were detected and identified by LC-MS/MS with a quadrupole time of flight detector analyzer. Biotransformation of FQs by L. portucalensis mainly occurred through a cleavage of the piperazine ring and displacement of the fluorine substituent allowing the formation of intermediates with less antibacterial potency. FQ-degrading microorganisms could be useful for application in bioaugmentation processes towards more efficient removal of contaminants in wastewater treatment plants.
- Biodegradation of water emerging pharmaceutical contaminants by two microbial consortia from different originsPublication . Carvalho, M.F.; Ribeiro, A.R.; Gonçalves, V.; Maia, A.; Maranhão, C.; Soares, M.T.; Tiritan, M.E.; Castro, Paula M. L.
- Biodegradation of fluoroquinolones by a bacterial consortiumPublication . Maia, Alexandra S.; Duque, Anouk F.; Ribeiro, Ana R.; Tiritan, Maria E.; Castro, Paula M. L.The present concern in pharmaceuticals in the environment is well known and research studies in this area have been regularly reported. Pharmaceuticals reach the environment by several ways but mostly due to their incorrect disposal and the incomplete elimination during the treatment processes in Wastewater Treatment Plants (WWTP). These residues continuously enter aquatic environments and many of them are resistant to degradation, being so called as pseudo-persistent pollutants. In aquatic compartments, pharmaceutical residues reach concentrations in the ng L-1 to µg L-1 range. This work describes the biodegradation of four fluoroquinolones, Ofloxacin (OFL), Norfloxacin (NOR), Ciprofloxacin (CPF) and Moxifloxacin (MOX), by a bacterial consortium constituted by three bacterial species isolated in our labs, namely F11, FP1 and S2, known to degrade different aromoatic fluorinated compounds. The experiments were conducted in batch mode using a mineral medium supplemented with acetate and 10 mg l-1 of each compound or 10 mg l-1 of a mix of the compounds. The bacterial consortium was capable of aerobic biodegradation of OFL, NOR and CPF during successive feedings of the compounds to the medium, as measured by monitoring removal of the compounds by HPLC-FD and fluoride release by potenciometry. Degradation profile of the fluoroquinolones used in this study indicated that intermediate metabolites were accumulated. Two of the initial constituting strains, F11, belonging to the α-proteobacteria group, and S2, belonging to the Actinobacteria group, were recovered from the medium, F11 predominating in cultures fed with moxifloxacin whereas S2 was mostly found in the remaining cultures. Degradation by single bacteria is under evaluation.
- Enantiomeric fraction evaluation of pharmaceuticals in environmental matrices by liquid chromatography-tandem mass spectrometryPublication . Ribeiro, Ana Rita; Santos, Lúcia H. M. L. M.; Maia, Alexandra S.; Delerue-Matos, Cristina; Castro, Paula M. L.; Tiritan, Maria ElizabethThe interest for environmental fate assessment of chiral pharmaceuticals is increasing and enantioselective analytical methods are mandatory. This study presents an enantioselective analytical method for the quantification of seven pairs of enantiomers of pharmaceuticals and a pair of a metabolite. The selected chiral pharmaceuticals belong to three different therapeutic classes, namely selective serotonin reuptake inhibitors (venlafaxine, fluoxetine and its metabolite norfluoxetine), beta-blockers (alprenolol, bisoprolol, metoprolol, propranolol) and a beta2-adrenergic agonist (salbutamol). The analytical method was based on solid phase extraction followed by liquid chromatography tandem mass spectrometry with a triple quadrupole analyser. Briefly, Oasis® MCX cartridges were used to preconcentrate 250 mL of water samples and the reconstituted extracts were analysed with a Chirobiotic™ V under reversed mode. The effluent of a laboratory-scale aerobic granular sludge sequencing batch reactor (AGS-SBR) was used to validate the method. Linearity (r2 > 0.99), selectivity and sensitivity were achieved in the range of 20–400 ng L−1 for all enantiomers, except for norfluoxetine enantiomers which range covered 30–400 ng L−1. The method detection limits were between 0.65 and 11.5 ng L−1 and the method quantification limits were between 1.98 and 19.7 ng L−1. The identity of all enantiomers was confirmed using two MS/MS transitions and its ion ratios, according to European Commission Decision 2002/657/EC. This method was successfully applied to evaluate effluents of wastewater treatment plants (WWTP) in Portugal. Venlafaxine and fluoxetine were quantified as non-racemic mixtures (enantiomeric fraction ≠ 0.5). The enantioselective validated method was able to monitor chiral pharmaceuticals in WWTP effluents and has potential to assess the enantioselective biodegradation in bioreactors. Further application in environmental matrices as surface and estuarine waters can be exploited.
- Solid Phase Extraction of Fluoroquinolone Antibiotics from Wastewaters – Assessment of Different Commercial SorbentsPublication . Maia, Alexandra S.; Ribeiro, Ana R.; Castro, Paula M. L.; Tiritan, Maria E.Microbial degradation of fluorinated pharmaceuticals during wastewater treatment processes remains inadequate in most situations. Due to incomplete elimination, these residues are continually being introduced into the aquatic environments in which they settle throughout time since many of them are resistant to degradation. Fluoroquinolone antibiotics due to its persistence and implication on resistant-bacteria development, pose special interest in environmental analysis. Due to their zwitterionic characteristics, the extraction/pre-concentration process of fluoroquinolones prior analyses is an unquestionable challenge. This work compares the solid phase extraction efficiency of four fluoroquinolones (Ofloxacin, Norfloxacin, Ciprofloxacin and Moxifloxacin) from wastewater effluents by different commercial sorbents. Prior to wastewater analysis, preliminary tests were conducted in distilled water with a larger number of sorbents. Different experimental protocols and sorbents, namely OASIS® HLB, OASIS® WAX, OASIS® WCX (500 mg) and the molecularly imprinted polymer SupelMIP TM were applied to wastewater samples collected from a municipal wastewater treatment plant from the north of Portugal. The extracts were analyzed by a HPLC withFluorescence Detection validated method using a Luna PFP (2) 3µm column. Despite good results obtained with the molecularly imprinted polymer in distilled water, these cartridges did not perform efficiently when applied to wastewater effluents, probably due to the sample high complexity especially since their specific design for biological samples. Regarding OASIS® considered sorbents; HLB 500mg and WAX 500mg presented the best recovery rates of the fourstudied antibiotics, between 84-75% and 64-94%, respectively. Although the recoveries achievedwere not that dissimilar between the two mentioned sorbents, chromatograms of WAX extracts appear much cleaner in the antibiotics retention times while chromatograms of HLB extractsclearly show the presence of strong polar substances, probably matrix humic and fulvic acids,that behave as resilient interferences in the analysis, disturbing a proper identification of target compounds and reducing chromatographic resolution.
- Dispersive liquid–liquid microextraction and HPLC to analyse fluoxetine and metoprolol enantiomers in wastewatersPublication . Ribeiro, Ana R.; Gonçalves, Virgínia M. F.; Maia, Alexandra S.; Ribeiro, Cláudia; Castro, Paula M. L.; Tiritan, Maria E.Sample extraction is a major step in environmental analyses due both to the high complexity of matrices and to the low concentration of the target analytes. Sample extraction is usually expensive, laborious, time-consuming and requires a high amount of organic solvents. Actually, there is a lack of miniaturized methodologies for sample extraction and chiral analyses. Here, we developed a dispersive liquid-liquid microextraction (DLLME) to extract the pharmaceuticals fluoxetine and metoprolol, as models of basic chiral compounds, from wastewater samples. Compounds were then analysed by enantioselective high-performance liquid chromatography. We monitored the influence of sample pH, extracting and dispersive solvent and respective volumes, salt addition, extracting and vortexing time. The DLLME method was validated within the range of 1-10 A mu g L-1 for fluoxetine enantiomers and 0.5-10 A mu g L-1 for metoprolol enantiomers. Accuracy ranged from 90.6 to 106 % and recovery rates from 54.5 to 81.5 %. Relative standard deviation values lower than 7.84 and 9.00 % were obtained for intra- and inter-batch precision, respectively.
- HPLC-FD Method Validation for Determination of Several Fluoroquinolones in Biodegradation AssaysPublication . Maia, Alexandra S.; Ribeiro, Ana R.; Castro, Paula M. L.; Tiritan, Maria E.The present concern in pharmaceuticals in the environment is well known and research studies in this area have been regularly reported [1]. Pharmaceuticals reach the environment by several ways but mostly due to their incorrect disposal and the incomplete elimination during the treatment processes in Wastewater Treatment Plants (WWTP). These residues continuously enter aquatic environments and many of them are resistant to degradation, being so called as pseudo-persistent pollutants [2]. In aquatic compartments, pharmaceutical residues reach concentrations in the ng/L to µg/L range [3]. Antibiotics are a class extensively studied due to its implications on development of multi-resistant bacteria. This work describes a validated HPLC (High Performance Liquid Chromatography) method with Fluorescence Detection for monitorization of biodegradation of four fluoroquinolones: Ofloxacin (OFL), Norfloxacin (NOR), Ciprofloxacin (CPF) and Moxifloxacin (MOX). The separation of the target pharmaceuticals was performed at 38ºC using a Luna 3µ PFP(2) 100 A (150 x 4.60 mm) column in isocratic mode with a mixture of 0.1% triethylamine (TEA) in water acidified to pH = 2.2 with trifluoroacetic acid (TFA) and ethanol as mobile phase. The validated method demonstrated selectivity, linearity (r2>0.999) and precision (intra-day: 0.71>RSD>2.51; inter-day: 1.11>RSD>2.74) in the range of 0.5 µg/mL – 12 µg/mL. The quantification limits were 5 ng/mL for OFL, NOR and CPF and 20 ng/mL for MOX. The biodegradation study was performed during 15 days using a microbial consortia constituted by three bacterial species known to degrade fluorinated compounds. The OFL presented higher extent of degradation than CPF and MOX.