Browsing by Author "Afonso, Carlos M."
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- Effect of the metals iron, copper and silver on fluorobenzene biodegradation by Labrys portucalensisPublication . Moreira, Irina S.; Amorim, Catarina L.; Carvalho, Maria F.; Ferreira, António C.; Afonso, Carlos M.; Castro, Paula M. L.Organic and metallic pollutants are ubiquitous in the environment. Many metals are reported to be toxic to microorganisms and to inhibit biodegradation. The effect of the metals iron, copper and silver on the metabolism of Labrys portucalensis F11 and on fluorobenzene (FB) biodegradation was examined. The results indicate that the addition of 1 mM of Fe2+ to the culture medium has a positive effect on bacterial growth and has no impact in the biodegradation of 1 and 2 mM of FB. The presence of 1 mM of Cu2+ was found to strongly inhibit the growth of F11 cultures and to reduce the biodegradation of 1 and 2 mM of FB to ca. 50 %, with 80 % of stoichiometrically expected fluoride released. In the experiments with resting cells, the FB degraded (from 2 mM supplied) was reduced ca. 20 % whereas the fluoride released was reduced to 45 % of that stoichiometrically expected. Ag+ was the most potent inhibitor of FB degradation. In experiments with growing cells, the addition of 1 mM of Ag+ to the culture medium containing 1 and 2 mM of FB resulted in no fluoride release, whereas FB degradation was only one third of that observed in control cultures. In the experiments with resting cells, the addition of Ag+ resulted in 25 % reduction in substrate degradation and fluoride release was only 20 % of that stoichiometrically expected. The accumulation of catechol and 4-fluorocatechol in cultures supplemented with Cu2+ or Ag+ suggest inhibition of the key enzyme of FB metabolism-catechol 1,2-dioxygenase.
- Enantioselective biodegradation of pharmaceuticals, alprenolol and propranolol, by an activated sludge inoculumPublication . Ribeiro, Ana R.; Afonso, Carlos M.; Castro, Paula M. L.; Tritan, Maria E.Biodegradation of chiral pharmaceuticals in the environment can be enantioselective. Thus quantification of enantiomeric fractions during the biodegradation process is crucial for assessing the fate of chiral pollutants. This work presents the biodegradation of alprenolol and propranolol using an activated sludge inoculum, monitored by a validated enantioselective HPLC method with fluorescence detection. The enantioseparation was optimized using a vancomycin-based chiral stationary phase under polar ionic mode. The method was validated using a minimal salts medium inoculated with activated sludge as matrix. The method was selective and linear in the range of 10–800 ng/ml, with a R2>0.99. The accuracy ranged from 85.0 percent to 103 percent, the recovery ranged from 79.9 percent to 103 percent, and the precision measured by the relative standard deviation (RSD) was <7.18 percent for intra-batch and <5.39 percent for inter-batch assays. The limits of quantification and detection for all enantiomers were 10 ng/ml and 2.5 ng/ml, respectively. The method was successfully applied to follow the biodegradation of the target pharmaceuticals using an activated sludge inoculum during a fifteen days assay. The results indicated slightly higher biodegradation rates for the S-enantiomeric forms of both beta-blockers. The presence of another carbon source maintained the enantioselective degradation pattern while enhancing biodegradation extent up to fourteen percent.
- Enantioselective HPLC analysis and biodegradation of atenolol, metoprolol and fluoxetinePublication . Ribeiro, Ana R.; Afonso, Carlos M.; Castro, Paula M. L.; Tiritan, Maria E.The accurate quantification of enantiomers is crucial for assessing the biodegradation of chiral pharmaceuticals in the environment. Methods to quantify enantiomers in environmental matrices are scarce. Here, we used an enantioselective method, high-performance liquid chromatography with fluorescence detection (HPLC-FD), to analyze two beta-blockers, metoprolol and atenolol, and the antidepressant fluoxetine in an activated sludge consortium from a wastewater treatment plant. The vancomycin-based chiral stationary phase was used under polar ionic mode to achieve the enantioseparation of target chiral pharmaceuticals in a single chromatographic run. The method was successfully validated over a concentration range of 20–800 ng/mL for each enantiomer of both beta-blockers and of 50–800 ng/mL for fluoxetine enantiomers. The limits of detection were between 5 and 20 ng/mL and the limits of quantification were between 20 and 50 ng/mL, for all enantiomers. The intra- and inter-batch precision was lower than 5.66 and 8.37 %, respectively. Accuracy values were between 103.03 and 117.92 %, and recovery rates were in the range of 88.48–116.62 %. Furthermore, the enantioselective biodegradation of atenolol, metoprolol and fluoxetine was followed during 15 days. The (S)-enantiomeric form of metoprolol was degraded at higher extents, whereas the degradation of atenolol and fluoxetine did not show enantioselectivity under the applied conditions.
- 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.