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- Development and Optimization of an Online SPE-HPLC-FD Method for Quantification of Fluoroquinolones in Wastewater EffluentsPublication . Meira, Bernardo; Maia, Alexandra S.; Gonçalves, Virgínia M. F.; Ribeiro, Ana R.; Tiritan, Maria E.Fluoroquinolones are antimicrobial agents widely found in environmental matrices and extensively studied due to their persistence and implications for multiresistant bacteria. The presence of fluoroquinolones in the environment is mainly due to the incapability of wastewater treatment plants (WWTPs) to completely remove those compounds. The amount of fluoroquinolones released through effluents depends on the type of treatment used by the WWTPs. So, accurate analytical methods to quantify those compounds on WWTPs process and in effluents are crucial. Solid phase extraction (SPE) coupled to liquid chromatography is a straightforward technique that provides analyte extraction, cleanup, separation and detection while providing a good reproducibility and efficiency. The purpose of this work was the establishment of a novel method for quantification of Ofloxacin, Norfloxacin, Ciprofloxacin and Moxifloxacin on WWTPs effluents using on-line SPE. Samples were injected directly on a restricted access material column LichroCart 25-4 Lichrospher® RP-18 ADS (25 μm) and then transferred to an analytical column Luna PFP (2) (150 x 4.6 mm ID, 100 Å, 3 μm) for separation in isocratic mode with a mixture of 0.1% triethylamine in water (acidified to pH = 2.2 with trifluoroacetic acid) and ethanol as mobile phase; column oven was set at 45ºC. The detection was performed by fluorescence with an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The injection volume of 100 μL of previous preconcentrated sample was compared with larger volume injection of only filtered effluent samples. The study was conducted with effluent samples collected from a municipal WWTP in the north of Portugal.
- Enantioselective Degradation of Enantiomers of Fluoxetine Followed by HPLC- FDPublication . Ribeiro, Ana R.; Maia, Alexandra S.; Moreira, Irina S.; Afonso, Carlos; Castro, Paula M.L.; Tiritan, Maria E.Environmental fate assessment of chiral pharmaceuticals is an important issue and little information is known about enantioselectivity in the environment. This kind of information is important for regulamentation of pharmaceutical industry and chiral switching processes. Fluoxetine (FLX), an anti-depressant worldwide used, is a chiral pharmaceutical prescribed in racemic form, and its main metabolite norfluoxetine (NFLX) is also chiral. In this study, enantioselective degradation of rac-FLX and degradation of its enantiomers separately, in a minimal salts medium inoculated by a bacterium consortium was examined both at light and dark conditions. Theassays were performed in a shaker at aerobic and ambient temperature conditions. The analytical method used was an enantioselective HPLC-FD method using a vancomycin-based chiral stationary phase in reversed mode to monitor enantiomers of FLX and NFLX. No degradation of enantiomers of FLX in the abiotic controls was observed. In theall assays (R)-FLX was degraded faster and totally until day 24th while (S)-FLX remained up to 20% of its initial concentration until the end of the experiment (38 days). NFLX wasdetected in all biotic experiments.
- 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.
- Occurrence of chiral bioactive compounds in the aquatic environment: a reviewPublication . Ribeiro, Cláudia; Ribeiro, Ana Rita; Maia, Alexandra S.; Tiritan, Maria ElizabethIn recent decades, the presence of micropollutants in the environment has been extensively studied due to their high frequency of occurrence, persistence and possible adverse effects to exposed organisms. Concerning chiral micropollutants in the environment, enantiomers are frequently ignored and enantiomeric composition often neglected. However, enantioselective toxicity is well recognized, highlighting the need to include enantioselectivity in environmental risk assessment. Additionally, the information about enantiomeric fraction (EF) is crucial since it gives insights about: (i) environmental fate (i.e., occurrence, distribution, removal processes and (bio)degradation); (ii) illicit discharges; (iii) consumption pattern (e.g., illicit drugs, pharmaceuticals used as recreational drugs, illicit use of pesticides); and (iv) enantioselective toxicological effects. Thus, the purpose of this paper is to provide a comprehensive review about the enantioselective occurrence of chiral bioactive compounds in aquatic environmental matrices. These include pharmaceuticals, illicit drugs, pesticides, polychlorinated biphenyls (PCBs) and polycyclic musks (PCMs). Most frequently analytical methods used for separation of enantiomers were liquid chromatography and gas chromatography methodologies using both indirect (enantiomerically pure derivatizing reagents) and direct methods (chiral stationary phases). The occurrence of these chiral micropollutants in the environment is reviewed and future challenges are outlined.
- Enantioselective quantification of fluoxetine and norfluoxetine by HPLC in wastewater effluentsPublication . Ribeiro, Ana R.; Maia, Alexandra S.; Moreira, Irina S.; Afonso, Carlos M.; Castro, Paula M.L.; Tiritan, Maria E.Microbial degradation is the most important process to remove organic pollutants in Waste Water Treatment Plants. Regarding chiral compounds this process is normally enantioselective and needs the suitable analytical methodology to follow the removal of both enantiomers in an accurate way. Thus, this paper describes the development and validation of an enantioselective High Performance Liquid Chromatography with Fluorescence Detection (HPLC-FD) method for simultaneous analysis of fluoxetine (FLX) and norfluoxetine (NFLX) in wastewater effluents. Briefly, this method preconcentrated a small volume of wastewater samples (50 mL) on 500 mg Oasis MCX cartridges and used HPLC-FD with a vancomycin-based chiral stationary phase under reversed mode for analyses. The optimized mobile phase was EtOH/aqueous ammonium acetate buffer (92.5/7.5, v/v) at pH 6.8. The effect of EtOH percentage, buffer concentration, pH, column oven temperature and flow rate on chromatographic parameters was systematically investigated. The developed method was validated within the wastewater effluent used in microcosms laboratory assays. Linearity (R2 > 0.99), selectivity and sensitivity were achieved in the range of 4.0–60 ng mL 1 for enantiomers of FLX and 2.0–30 ng mL 1 for enantiomers of NFLX. The limits of detection were between 0.8 and 2.0 ng mL 1 and the limits of quantification were between 2.0 and 4.0 ng mL 1 for both enantiomers of FLX and the enantiomers of its demethylated metabolite NFLX. The validated method was successfully applied and proved to be robust to follow the degradation of both enantiomers of FLX in wastewater samples, during 46 days.
- Strategies to enhance the removal of FluoroquinolonesPublication . Amorim, Catarina L.; Maia, Alexandra S.; Moreira, Irina S.; van Loosdrecht, Mark C.M.; Tiritan, Maria E.; Castro, Paula M.L.Fluoroquinolones (FQs) are broad-spectrum antibiotics that play an important role in the treatment of serious bacterial infections. Currently, several FQs are available but ciprofloxacin (CPF), ofloxacin (OFL) and norfloxacin (NOR) are amongst the most worldwide prescribed antibiotics. Antibiotics can reach wastewater treatment plants (WWTP) from different routes. Thus removal of these contaminants during the biotreatment process is of major importance in order to avoid their release to other environmental matrices. Granular sludge sequencing batch reactors (SBR) constitute a novel biofilm technology for wastewater treatment extremely promising for the treatment of effluents containing toxic compounds. Therefore, in this study a granular sludge SBR, established with activated sludge from a WWTP, was operated for the treatment of an aqueous stream containing FQs. No evidence of FQ biodegradation followed by HPLC with Fluorescence Detection was observed but FQs adsorbed to the aerobic granular sludge, being gradually released into the medium after withdrawal of the FQs in the inlet stream. In a previous study, Labrys portucalensis F11 demonstrated to be able to degrade FQs, namely OFL, NOR and CPF, when supplied individually or as a mixture, in the presence of an easy degradable carbon source. Different removal extents were obtained for the tested concentrations (ranging from 0.8 to 30 μM), but overall the uptake capacity of strain F11 for individual FQs decreased with increasing the initial FQ concentration. When supplied with a mixture FQs, strain F11 concomitantly removed each target antibiotic but a decrease on the biodegradability of FQs was observed which could be explained by competition mechanisms. The ability of Labrys portucalensis F11 to grow using the readily available carbon source while maintain its ability to degrade FQs reinforce the potential of this strain in bioaugmentation processes. As the indigenous microbial communities in biotreatment processes rarely are able to remove such contaminants, using this promising FQ-degrading strain, bioaugmentation strategies such as inoculation of the degrading strain, as a suspension or immobilized on carrier material, or using a plasmid donor strain carrying the degradative genes, could be assessed to improve FQ removal. Acknowledgments: C.L. Amorim, A.S. Maia and I.S. Moreira wish to acknowledge the research grants from Fundação para a Ciência e Tecnologia (FCT), Portugal (Ref. SFRH/BD/47109/2008, SFRH/BD/86939/2012 and SFRH/BPD/87251/2012, respectively) and Fundo Social Europeu (Programa Operacional Potencial Humano (POPH), Quadro de Referência Estratégico Nacional (QREN))). This work was supported by FCT through the projects PTDC/EBB-EBI/111699/2009 and PEst-OE/EQB/LA0016/2011.
- 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.
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