Fluorescent labeling of micro/nanoplastics for biological applications with a focus on "true-to-life" tracking.

The increased environmental presence of micro-/nanoplastics (MNPLs) and the potential health risks associated with their exposure classify them as environmental pollutants with special environmental and health concerns. Consequently, there is an urgent need to investigate the potential risks associated with secondary MNPLs. In this context, using "true-to-life" MNPLs, resulting from the laboratory degradation of plastic goods, may be a sound approach. These non-commercial secondary MNPLs must be labeled to track their presence/journeys inside cells or organisms. Because the cell internalization of MNPLs is commonly analyzed using fluorescence techniques, the use of fluorescent dyes may be a sound method to label them. Five different compounds comprising two chemical dyes (Nile Red and Rhodamine-B), one optical brightener (Opticol), and two industrial dyes (Amarillo Luminoso and iDye PolyPink) were tested to determine their potential for such applications. Using commercial standards of polystyrene nanoplastics (PSNPLs) with an average size of 170 nm, different characteristics of the selected dyes such as the absence of impact on cell viability, specificity for plastic staining, no leaching, and lack of interference with other fluorochromes were analyzed. Based on the overall data obtained in the wide battery of assays performed, iDye PolyPink exhibited the most advantages, with respect to the other compounds, and was selected to effectively label "true-to-life" MNPLs. These advantages were confirmed using a proposed protocol, and labeling titanium-doped PETNPLs (obtained from the degradation of milk PET plastic bottles), as an example of "true-to-life" secondary NPLs. These results confirmed the usefulness of iDye PolyPink for labeling MNPLs and detecting cell internalization.

Trace cisplatin adsorption by thiol-functionalized sponge (TFS) and Sn/SnO2-coated TFS: Adsorption study and mechanism investigation.

To remove trace cisplatin from aqueous solution, commercial sponges were functionalized by esterification with 3-mercaptopropionic acid, followed by reduction with Na2S·9H2O or SnCl2·2H2O. The resulting thiol-functionalized sponges (TFSs), TFS_1 and TFS_2, were tested for the removal of cisplatin (235 µg L-1) achieving maximum removal of 95.5 ± 0.8% and 99.5 ± 0.1% respectively, which were significantly higher than the non-functionalized counterpart. The successful grafting of thiol groups, verified through FTIR, elemental analysis, SEM-EDS, and XPS characterization, facilitated Pt-S complexation during adsorption. The aqua-derivatives of cisplatin, formed through hydration, complexed with thiol sites through ligand displacement. Additionally, the presence of Sn/SnO2 coating on TFS_2 further enhanced the adsorption process. The rapid adsorption process conformed to pseudo-second-order kinetic model, involving both diffusion and chemisorption. While the Langmuir isotherm model generally described the monolayer adsorption behavior of cisplatin, the aggregation of Sn/SnO2 onto TFS_2 at 343 K introduced surface heterogeneity, rendering the Freundlich model a better fit for the adsorption isotherm. Differential pH dependence and the evaluation of mean free energy, derived from the Dubinin-Radushkevich isotherm model, indicated that cisplatin adsorption onto TFS_1 involved physisorption, including electrostatic attraction, while chemisorption predominated for TFS_2. Increasing the temperature notably promoted adsorption by facilitating the thermal-favored formation of Pt-S bonds.

Validated method for polystyrene nanoplastic separation in aqueous matrices by asymmetric-flow field flow fraction coupled to MALS and UV-Vis detectors.

Plastics with nanosize (nanoplastics, NPLs) must be characterized, since they can be toxic or act as carriers of organic and inorganic pollutants, but there is a lack of reference materials and validated methods in the nanosize range. Therefore, this study has focused on the development and validation of a separation and size characterization methodology of polystyrene latex nanospheres, by using an asymmetric-flow field flow fraction system coupled to multi-angle light scattering and ultraviolet-visible detectors (AF4-MALS-UV). Hence, this work presents a fully validated methodology in the particle size range 30 to 490 nm, with bias between 95 and 109%, precision between 1 and 18%, LOD and LOQ below 0.2 and 0.3 µg respectively, except for 30-nm standard, for both detectors, and showing stable results for 100 analyses.

Comparative study on removal of platinum cytostatic drugs at trace level by cysteine, diethylenetriamino functionalized Si-gels and polyethyleneimine functionalized sponge: Adsorption performance and mechanisms.

To efficiently remove trace Pt-based cytostatic drugs (Pt-CDs) from aqueous environments, a comparative investigation was conducted on the adsorption behavior of three commercial adsorbents including cysteine-functionalized silica gel (Si-Cys), 3-(diethylenetriamino) propyl-functionalized silica gel (Si-DETA) and open-celled cellulose MetalZorb® sponge (Sponge). The research on the adsorption of cisplatin and carboplatin encompasses investigations of pH dependence, adsorption kinetics, adsorption isotherms, and adsorption thermodynamics. The obtained results were compared with those of PtCl42- to better understand the adsorption mechanisms. The adsorption of cisplatin and carboplatin by Si-Cys was significantly better than Si-DETA and Sponge, which suggested that in chelation-dominated chemisorption, thiol groups provided high-affinity sites for Pt(II) complexation. Adsorption of the anion PtCl42- was more pH dependent and generally superior to that of cisplatin and carboplatin, benefiting from the contribution of ion association with protonated surfaces. The removal process of aqueous Pt(II) compounds occurred by the hydrolysis of complexes in solution and subsequent adsorption, and the specific adsorption process was explained by the synergistic action of ion association and chelation mechanisms. The rapid adsorption processes involving diffusion and chemisorption were well described by pseudo-second-order kinetic model. The isotherm studies suggested monolayer adsorption, consistent with the Langmuir model. Indicated from the adsorption enthalpy results, the chelation of cisplatin and carboplatin with thiol groups was an endothermic reaction, while the adsorption of PtCl42- was exothermic. At 343 K, Si-Cys achieved 98.5 ± 0.1 % (cisplatin) and 94.1 ± 0.1 % (carboplatin) removal. To validate the obtained findings, the described process was applied to urine samples doped with Pt-CDs as analog of hospital wastewaters and the removal was very efficient, ranging from 72 ± 1 % to 95 ± 1 %, when using Si-Cys as adsorbent, although limited matrix effects were observed.

Hazard Assessment of Polystyrene Nanoplastics in Primary Human Nasal Epithelial Cells, Focusing on the Autophagic Effects.

The human health risks posed by micro/nanoplastics (MNPLs), as emerging pollutants of environmental/health concern, need to be urgently addressed as part of a needed hazard assessment. The routes of MNPL exposure in humans could mainly come from oral, inhalation, or dermal means. Among them, inhalation exposure to MNPLs is the least studied area, even though their widespread presence in the air is dramatically increasing. In this context, this study focused on the potential hazard of polystyrene nanoplastics (PSNPLs with sizes 50 and 500 nm) in human primary nasal epithelial cells (HNEpCs), with the first line of cells acting as a physical and immune barrier in the respiratory system. Primarily, cellular internalization was evaluated by utilizing laboratory-labeled fluorescence PSNPLs with iDye, a commercial, pink-colored dye, using confocal microscopy, and found PSNPLs to be significantly internalized by HNEpCs. After, various cellular effects, such as the induction of intracellular reactive oxygen species (iROS), the loss of mitochondrial membrane potential (MMP), and the modulation of the autophagy pathway in the form of the accumulation of autophagosomes (LC3-II) and p62 markers (a ubiquitin involved in the clearance of cell debris), were evaluated after cell exposure. The data demonstrated significant increases in iROS, a decrease in MMP, as well as a greater accumulation of LC3-II and p62 in the presence of PSNPLs. Notably, the autophagic effects did indicate the implications of PSNPLs in defective or insufficient autophagy. This is the first study showing the autophagy pathway as a possible target for PSNPL-induced adverse effects in HNEpCs. When taken together, this study proved the cellular effects of PSNPLs in HNEpCs and adds value to the existing studies as a part of the respiratory risk assessment of MNPLs.

A new source of representative secondary PET nanoplastics. Obtention, characterization, and hazard evaluation.

Micro and nanoplastics (MNPLs) are emergent environmental pollutants requiring urgent information on their potential risks to human health. One of the problems associated with the evaluation of their undesirable effects is the lack of representative samples, matching those resulting from the environmental degradation of plastic wastes. To such end, we propose an easy method to obtain polyethylene terephthalate nanoplastics from water plastic bottles (PET-NPLs) but, in principle, applicable to any other plastic goods sources. An extensive characterization indicates that the proposed process produces uniform samples of PET-NPLs of around 100 nm, as determined by using AF4 and multi-angle and dynamic light scattering methodologies. An important point to be highlighted is that to avoid the metal contamination resulting from methods using metal blades/burrs for milling, trituration, or sanding, we propose to use diamond burrs to produce metal-free samples. To visualize the toxicological profile of the produced PET-NPLs we have evaluated their ability to be internalized by cells, their cytotoxicity, their ability to induce oxidative stress, and induce DNA damage. In this preliminary approach, we have detected their cellular uptake, but without the induction of significant biological effects. Thus, no relevant increases in toxicity, reactive oxygen species (ROS) induction, or DNA damage -as detected with the comet assay- have been observed. The use of representative samples, as produced in this study, will generate relevant data in the discussion about the potential health risks associated with MNPLs exposures.

Discriminating the origin of calcium oxalate monohydrate formation in kidney stones via synchrotron microdiffraction.

Nephrolithiasis is a multifactor disease that produces nephrolites in the kidneys. Calcium oxalate hydrate (dihydrated, COD, or monohydrated, COM) stones are the most common ones with more than sixty percent incidence worldwide. They are related to different pathologies, COD with hypercalciuria and COM with hyperoxaluria. COD is an unstable species and transforms into COM (herein named TRA to distinguish the origin of the monohydrated species). TRA and COM are chemically and crystallographically identical leading to misdiagnosis and recurrence increase. In the current study, the composition and crystalline structures of several calcium oxalate stones, classified by morpho-constitutional analysis, were examined by IR and synchrotron through-the-substrate micro-X-ray diffraction (tts-µXRD). Both IR and linear diffractogram studies were able to distinguish between the monohydrated and dihydrated phases but not between COM and TRA, as expected. The analysis of 2D diffraction patterns revealed that TRA showed a lower degree of crystallinity and less texture with respect to COM which can be used as a signature to distinguish between the two. This study confirms that despite the subtle differences between COM and TRA, the origin of the monohydrate oxalates can be unraveled using tts-µXRD. This valuable information should be taken into account in order to improve patients' diagnosis and reduce recurrence by considering and treating the origin of the formed stones.

Comparative evaluation of chemical composition and biological activities of tropical fruits consumed in Manaus, central Amazonia, Brazil.

Fruits are widely recognized as sources of biologically active metabolites, such as antioxidant compounds. In this context, fruits commonly consumed in the central Amazonia, especially in its biggest metropolis (Manaus - AM/Brazil), are attractive as potential sources of antioxidant compounds related to biological activities. Most of such fruits are still poorly studied and/or remain unknown outside the Amazon region. Therefore, this study aims to investigate nine fruits (abiu, cubiu, biribá, breadfruit, genipap, peach palm, murici, soursop, and umari) regarding their chemical composition (fixed and volatile), reducing capacity, antioxidant activity, enzyme inhibition, and cytotoxicity. Determination of small organic acids, hydroxycinnamic acids, flavan-3-ols and flavonoid aglycones was done by HPLC-MS/MS, whereas determination of volatile organic compounds (VOCs) was done by HS-SPME/GC-MS. Reducing capacity was determined by the Folin-Ciocalteu method, and antioxidant activities were evaluated by DPPH, ABTS, and H-ORACFL assays. In vitro activities regarding inhibition of enzymes were tested for α-glucosidase, lipase, and α-amylase, and anti-glycation activities were evaluated for methylglyoxal and fructose. Cytotoxicity of fruit extracts was evaluated by cell viability of human fibroblast cell line (MRC-5). A total of 16 antioxidant compounds and 139 VOCs were determined, whose profiles were unique for each studied fruit. Total phenolic contents as well as antioxidant activities found herein were similar or even higher than those reported for several traditional fruits. Some of fruit extracts were able to inhibit α-glucosidase and glycation in methylglyoxal and fructose models, whereas none of them was active for lipase and α-amylase. All of the fruit extracts showed to be non-cytotoxic to MRC-5 cell line.

Calcium oxalate kidney stones, where is the organic matter?: A synchrotron based infrared microspectroscopy study.

Kidney stones are collections of microcrystals formed inside the kidneys, which affect 6% to 12% of the population worldwide, with an increasing recurrence (50%-72%) after the first episode. The most abundant type is calcium oxalate (66%), described as monohydrated (COM) and dihydrated (COD). An issue in their chemistry is the transformation process of the metastable specie (COD) into the stable one, which is chemically, and in appearance, monohydrated. Since the origin of these species is different, it is important to differentiate between the transformation stage (and what stabilize COD) to understand the physiopathology and prevent the patients' recurrence. This work focuses on the organic matter distribution along these nephroliths by synchrotron radiation-based infrared microspectroscopy. Differences in the asymmetric stretching of the aliphatic hydrocarbons suggest that lipids may participate in the stabilization of COD and as inhibitors of COM formation/development; however, the presence of proteins in the nucleus could indicate a promoting role.

Characterization of Calcium Oxalate Hydrates and the Transformation Process.

Calcium oxalate can be found in humans as kidney stones and in cultural heritage as films in two crystallographic species, dihydrate (COD/weddellite) and/or monohydrate (COM/whewellite). Due to its instability, COD is transformed into COM. Studying this crystalline conversion provides information about the origin of the monohydrated species, which will help in the assessment of prevention measurements to avoid their formation. In the present study, the synthesis of calcium oxalate hydrate microcrystals has been carefully performed to avoid mixture of phases in the final products; the long and short range order structure of both species have been studied by X-ray diffraction (XRD) and X-ray absorption spectroscopy (XAS), respectively. This structural information was considered in the density functional theory (DFT) computational study performed to assign the characteristic vibrational IR and Raman frequencies found. This detailed characterization allows an unambiguous assignment of the vibrations, thus providing the appropriate parameters required to monitor and characterize the transformation process.

Heavy metal availability assessment using portable X-ray fluorescence and single extraction procedures on former vineyard polluted soils.

The periodic application of copper-based fungicides (Bourdeaux mixture) to vineyards of the Mediterranean region has generated an important pollution source that in some cases requires a quick intervention due to the high bioavailable copper content measured. Despite some vineyards were abandoned 40 years ago, noticeable amounts of Cu and other man-related metals are still nowadays detected in soils. In the present work, the development of a mobility test for the available heavy metal (Cu, Pb, Zn and As) content in soil has been performed using portable X-ray fluorescence (FP-XRF) combined with single leaching test, and was applied to a calcareous soil of a former vineyard area in Catalonia (NE Spain). The combined methodology has provided useful information for fast and detailed risk assessment, in terms of mobility and bioavailability of metals. The anthropogenic contribution was evaluated by means of the Concentration Enrichment Ratios (CER) in soil. The results reflect a clear anthropogenic contribution for Cu, a partial anthropogenic contribution for Pb proceeding from an external pollution source, and a non-significant external contribution for As and Zn. The topsoil concentration ranges for Cu (70-128 mg kg-1) were found to be above the background level and several samples above the regional governmental limits (Generic Reference Levels or GRL values) for soil ecosystem protection and for human health (90 mg kg-1). The present study reveals that the use of FP-XRF equipment constitute a highly valid option for quick decision making during the field location, characterization and quantitative elemental analysis of soil samples for screening of potential pollutants such as heavy metals.

Use of alternating current for colored water purification by anodic oxidation with SS/PbO2 and Pb/PbO2 electrodes.

This paper suggests a new alternative for the acceleration of dye removal by adopting alternating current instead of direct current in the treatment of methylene blue solutions and industrials effluents, using anodic oxidation on Pb/PbO2 and stainless steel (SS)/PbO2 anodes. A comparative study of the influence of electrolyte support (NaCl, NaNO3, and Na2SO4) on the anodic oxidation performance and the anode stability was performed. The best results were obtained in presence of NaCl where the color removal percentage reached about 80.13% and 55.8%, for Pb/PbO2 anodes, and 89.5% and 60.4% for the SS/PbO2 anodes for alternating and direct current, respectively. Treatment in alternating current conditions enhanced the removal speed. Atomic absorption analysis confirmed the decrease of the release of (Pb2+) ions to much lower values compared with direct current and to those allowed by the Standards. LC/MS and phytotoxicity analyses confirmed the non-toxicity of the generated by-products during the anodic oxidation of methylene blue and the possibility of the reuse of the treated water.

Molecularly imprinted polymer for the removal of diclofenac from water: Synthesis and characterization.

Contaminants of Emerging Concerns (CECs) have been introduced as one type of recalcitrant pollutant sources in water. In this study, the non-steroidal anti-inflammatory drug diclofenac (DCF) has been removed from water solutions using Molecularly Imprinted Polymer (MIP), synthetized via bulk polymerization with allylthiourea (AT) as the functional monomer and using DCF as template (MIP-DCF). DCF detection has been performed by UV spectrophotometer. From the kinetic study in batch mode, approximately 100% of removal is observed by using 10mg of MIP-DCF, with an initial concentration of 5mg/L of DCF at pH7, within 3min and agitated at 25°C. In continuous flow mode study, using a cartridge pre-packed with 10mg of MIP-DCF, a high adsorption capacity of 160mgDCF/g MIP was obtained. To study the porosity of MIPs, scanning electron microscopy (SEM) has been used. In order to characterize the chemical interaction between monomer and template, the pre-polymerization mixture for MIP and DCF has also been studied by 1H NMR. One of the chemical shift observed has been related to the formation of a complex between amine protons of thiourea group of AT with carboxylic acid on DCF. In conclusion, the developed MIP works as a good adsorbent for DCF removal, and is selective to DCF in the presence of indomethacin and ibuprofen.

Hollow fibre supported liquid membrane extraction for BTEX metabolites analysis in human teeth as biomarkers.

The use of human teeth as biomarkers has been previously applied to characterize environmental exposure mainly to metal contamination. Difficulties arise when the contaminants are volatile or its concentration level is very low. This study presents the development of a methodology based on the transport through hollow fibre membrane liquid-phase microextraction (HF-LPME), followed by HPLC-UV measurement, to determine three different metabolites of BTEX contaminants, mandelic acid (MA), hyppuric acid (HA), and methylhippuric acid (4mHA). The driving force for the liquid membrane has been studied by using both non-facilitated (pH gradient 2-12) and facilitated transport (ionic and non-ionic carriers). Enrichment factors of several hundreds were accomplished. Different ionic and non-ionic water insoluble compounds were used as metabolite carriers for the facilitated transport at HF-LPME. Three organic solvents were used to constitute the liquid membrane, dodecane, dihexyl ether and n-decanol. Other parameters affecting the extraction process, such as extraction time, stirring speed, acceptor buffer and salt content were optimised in spiked solutions and selected those that presented the best enrichment factors for all analytes. Final conditions were established for donor solution as 20mL, pH2 of 0.5M NaCl, the OLM (Organic Liquid Membrane) as n-decanol and the acceptor solution as 40µL of 1M NaOH. The selected extraction time was 20h with stirring speed of 500rpm. Validation of the optimised method included the determination of individual linearity range (MA: 0.002-5.7µg; HA: 0.01-7.9µg; 4mHA 0.002-5.3µg), limits of detection (MA: 1.6ng; HA: 0.2ng; 4mHA 0.2ng), repeatability (RSD 7-10%) and reproducibility (5-8%). The developed method was applied to the analysis of MA, HA and 4mHA in teeth samples of 8 workers exposed to BTEX.

Influence of a mixture of metals on PAHs biodegradation processes in soils.

In order to assess the effect of mixed pollutants, the influence of different concentration levels of a mixture of metals (Cr, Co, Pb, Mn, Ni, Cu, Zn) on the biodegradation of some PAHs (phenanthrene, fluoranthene, pyrene, benzo[b]fluoranthene and benzo[a]pyrene) in soil samples was evaluated. To do so, groups of microcosms of a natural soil from the region of Sabadell (Barcelona, Spain) were prepared as a reproduction of the native environment at laboratory scale, under controlled conditions. Mixtures of PAHs and metals were carefully selected, according to soil characterization and microbiological growth preliminary assays, and were added to microcosms. These microcosms were analyzed at various times, along two months, to obtain PAHs dissipation time-courses. A first-order kinetic modelling allowed obtaining different rate constants and DT50 values as a function of the metal levels introduced in microcosms. As a general observation, the higher the concentration of metals, the lower the biodegradation of PAHs of 3-4 rings (phenanthrene, fluoranthene and pyrene). On the other hand, no important effect on the biodegradation of higher molecular weight PAHs (benzo[b]fluoranthene and benzo[a]pyrene) was observed at the different concentration levels of metals tested.

Simultaneous determination of BTEX and their metabolites using solid-phase microextraction followed by HPLC or GC/MS: An application in teeth as environmental biomarkers.

Applications of benzene, toluene, ethylbenzene, and o-, m-, and p-xylenes (BTEX) release them into the environment exposing living organism. These endocrine disruptors are toxic, highly volatile and easily absorbed by the lungs and can cause adverse consequences for the human health as neurological diseases and cancer. A method for the analysis of BTEX and its metabolites (phenols and aromatic acids) in teeth is presented. The method consists in a one-step simple extraction procedure from spiked tooth using NaOH solution followed by SPME-HPLC or HS-SPME-GC/MS determination. Optimization of both, spiking procedure and extraction step of these analytes from tooth, was carried out. Two fibers CAR/PDMS for BTEX and PA for BTEX metabolites were used for the SPME and variables were optimized for analytes at 30°C using spiked solutions. The optimized adsorption times were 30, 75 and 30min and desorption times were 10, 40 and 30min for BTEX, phenols and aromatic acids, respectively. Linearity for SPME-HPLC method was established using spiked solutions with both, BTEX and metabolites, at 2.5, 5.0, 10.0, 25.0µg/mL. The obtained results indicated a good linearity (r2 above 0.994) for all analytes. Triplicate analyses were performed with RSD lower than 15%. LODs were in the range 0.2-33.3ng/mL for SPME-HPLC and 0.06-0.09pg/mL for HS-SPME-GC/MS methods in spiking solutions. Once the method was optimized, bovine teeth were used as biological matrix model for the tuning of spiking and extraction steps. Optimal adsorption and desorption times were 4h for both procedures. Micrograms per tooth gram of BTEX and phenols were quantified in ten human teeth and aromatic acids were not identified. The developed method for BTEX and metabolites analyses using SPME-HPLC or HS-SPME-GC/MS shows good precision, linearity and sensitivity. The method was successfully applied in human teeth as environmental biomarker of BTEX and metabolites.

Fast determination of bioactive phytic acid and pyrophosphate in walnuts using microwave accelerated extraction.

Bioactive compounds phytic acid (IP6) and pyrophosphate (PPi) are minor components of walnuts with the ability of being inhibitors of urolithiasis, among others. Since simultaneous analysis of IP6 and PPi have known drawbacks, a new method to determine their content in walnuts has been developed with emphasis on their extraction from walnuts by microwave-assisted extraction (MAE). Acid content of extracting solvent, extraction time and temperature were optimized. After extraction, compounds were purified by selective adsorption/desorption on an anion exchange solid phase extraction and analyzed by inductive coupled plasma/mass spectrometry. A mixture of H2SO4 and HCl as solvent to extract both, IP6 and PPi, provided results slightly higher than those determined by conventional extraction with no statistical difference. The possible hydrolysis of phytic acid by MAE was analyzed. Compared with the conventional acid extraction method, significant improvement is achieved by the MAE method reducing extraction time from 3h to 10min.

Determination of Oxalate Content in Herbal Remedies and Dietary Supplements Based on Plant Extracts.

Lifestyle, especially diet, is a prominent risk factor that affects the formation of calcium oxalate stones. Urinary oxalate excretion is directly related to the amount of oral intake and intestinal absorption rate of oxalate. This work evaluated the possibility of increasing oxalate ingestion, which could lead to secondary hyperoxaluria, associated with the intake of herbal remedies and dietary supplements containing plant extracts. A wide variety of 17 commercially available drugs and dietary supplements were analyzed using ion chromatography. The results showed remarkable differences in oxalate contents of the extracts. Total oxalate concentrations ranged from 0.03 to 2.2 mg/g in solid samples and from 0.005 to 0.073 mg/mL in liquid samples. The selected herbal remedies and dietary supplements containing plant extracts represent only a low risk for calcium oxalate stone formers, if the recommended daily dose is not exceeded.

Relevance of Toxicity Assessment in Wastewater Treatments: Case Study-Four Fenton Processes Applied to the Mineralization of C.I. Acid Red 14.

Fenton and Fenton-like processes, both in homogeneous and heterogeneous phases, have been applied to an aqueous solution containing the dye AR 14 in order to study the mineralization and toxicity of the solutions generated after color elimination. The mineralization of AR 14 occurred slower than the decolorization. The Microtox analysis of the treated solutions showed low toxicity intrinsic to the chemicals used in the process rather than the degradation products obtained after the treatment of the dye solution. The dye degradation for the Fenton oxidation process was initially faster than for the Fenton-like process but after a short time, the four processes showed similar degradation yields. All processes have shown good results being the heterogeneous process the most convenient since the pH adjustment is not necessary, the catalyst is recovered and reused and the generation of contaminated sludge is avoided.

Hollow fibre liquid phase micro-extraction by facilitated anionic exchange for the determination of flavonoids in faba beans (Vicia faba L.).

INTRODUCTION: Flavonoids are polyphenolic compounds found ubiquitously in foods of plant origin. They are commonly extracted from plant materials with ethanol, methanol, water, their combination or even with acidified extracting solutions. The disadvantages of these methods are the use of high quantity of organic solvent, the possible loss of analytes in the different steps and the laborious process of the techniques. In addition, the complexity of the phenolic mixtures present in plant materials requires a preliminary clean-up and fractionation of the crude extracts. OBJECTIVE: To develop a hollow fibre liquid phase micro-extraction (HF-LPME) method for a one step clean-up and pre-concentration of flavonoids. METHODOLOGY: Two flavonoids (catechin and rutin) has been extracted by HF-LPME and analysed by HPLC. The related driving force for the liquid membrane has been studied by means of facilitated and non-facilitated transport. Different ionic and non-ionic water insoluble compounds [trioctylamine (TOA), tributyl phosphate (TBP), trioctylphosphine oxide (TOPO) and methyltrioctylammonium chloride (aliquat 336)] were used as carriers. The liquid membrane was constituted by a solution of n-decanol in the presence or absence of carriers. RESULTS: Maximum enrichment factors were obtained with n-decanol/aliquat 336 (20%) as organic liquid membrane, sodium hydroxide (NaOH) (0.1 M) as donor solution, sodium chloride (NaCl) (2 M) as acceptor solution and 3 h as extraction time. Under these conditions, good results for validation parameters were obtained [for linearity, limit of detection (LOD), limit of quantitation (LOQ) and repeatability]. CONCLUSIONS: The developed method is simple, effective and has been successfully applied to determine catechin and rutin in ethanolic extracts of faba beans.