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This study comprises the development of enzyme-linked immunosorbent assays (ELISAs) for the quantification of 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) in complex aqueous matrices without any sample clean-up procedures. Salinity and dissolved organic matter were selected as potential interfering agents in the analysis of E2 and EE2. The optimization was performed in order to (i) overcome matrix effects, and to (ii) increase sensitivity. The addition of a sample buffer containing bovine serum albumin (BSA) prior to the sample was found to decrease the influence of matrix effects. Moreover, adjustments of this buffer's pH together with the optimization of tracer (T) dilution and incubation time were undertaken in order to lower the quantification range. The optimized methods allowed the quantification of E2 and EE2 in the ranges 0.03200 µg L-1 and 0.0210 µg L-1, respectively. The assays were applied to real aqueous samples. It was possible to do a first approach to the levels of E2 in Portuguese surface and waste waters; however, it was not feasible to detect EE2 in the samples tested.
The occurrence of carbamazepine (CBZ) and its metabolites in German and Portuguese wastewater was investigated. A total of 46 samples from influent and effluent wastewater were analyzed by liquid-chromatography (LC) tandem mass spectrometry. The five metabolites 10,11-dihydro-10,11-dihydroxy-CBZ (DiOH-CBZ), 10,11-dihydro-10-hydroxy-CBZ (10-OH-CBZ), 10,11-epoxy-10,11-dihydro-CBZ, 2-hydroxy-CBZ and 3-hydroxy-CBZ were very persistent with little to no removal during wastewater treatment. The highest concentrations were found for CBZ, DiOH-CBZ, and 10-OH-CBZ, with up to 5.0, 4.8 and 1.1 µg/L, respectively. Furthermore, the related pharmaceutical oxcarbazepine and the metabolites 9-hydroxymethyl-10-carbamoylacridan, 1-hydroxy-CBZ (1-OH-CBZ) and 4-hydroxy-CBZ (4-OH-CBZ) were detected. Explicit care was taken to achieve a good chromatographic separation of the numerous isomers that were difficult to distinguish by mass spectrometry alone. A phenylether stationary phase provided the best separation. In combination with high resolution mass spectrometry and hydrogen-deuterium exchange, this LC column enabled us to identify 1-OH-CBZ and 4-OH-CBZ in wastewater for the first time.
The use of magnetic materials as heterogeneous catalysts has attracted increasing attention in the last years since they proved to be promising candidates for water treatment. In the present study, two types of surface-modified magnetite (Fe3O4) nanoparticles, coated with non-hazardous naturally occurring agents—either tannic acid (TA) or dissolved natural organic matter—were evaluated as magnetic heterogeneous catalysts. Chemical synthesis (co-precipitation) was chosen to yield the nanocatalysts due to its well-established simplicity and efficiency. Subsequently, the properties of the final products were fully assessed by various characterization techniques. The catalytic activity in heterogeneous oxidation of aqueous solutions containing a model pollutant, Bisphenol A (BPA), was comparatively studied. The effect of operational parameters (catalyst loading, H2O2 dosage, and UV light irradiation) on the Degradation performance of the oxidation process was investigated. The optimum experimental parameters were found to be 1.0 g/L of catalysts and 10 mM H2O2, under UV irradiation. The highest mineralization rates were observed for Fe3O4-TA catalyst. More than 80 % of BPA was removed after 30 min of reaction time under the specified experimental conditions. The obtained results showed that the two catalysts studied here are suitable candidates for the removal of pollutants in wastewaters by means of heterogeneous reaction using a green sustainable treatment method.
Two types of surface modified magnetite (Fe3O4) nanoparticles, coated with either tannic acid (TA) or dissolved natural organic matter (NOM), were evaluated as magnetic heterogeneous catalysts. Simple and efficient procedures for the synthesis of the magnetic catalysts were employed, their properties being fully assessed by various characterization techniques. The catalytic activity in heterogeneous oxidation of aqueous solutions containing Bisphenol A (BPA) over the catalysts was comparatively studied. The optimum experimental parameters were: 1g/L of catalysts, 10 mM H2O2, under UV irradiation. The highest mineralization rates were observed for Fe3O4-TA catalyst. More than 80% of BPA were removed after 30 minutes of reaction time under the specified experimental conditions. The results showed that the obtained catalysts are suitable candidates for the removal of pollutants in wastewaters by means of heterogeneous reaction
A bead suspension array for measuring emerging pollutants and anthropogenic markers in wastewater
(2016)
Background: The assessment of water quality demands for high-throughput and multi-target compatible analytical methods. On the one hand, the influence of sewage on natural surface waters must be routinely monitored - according to the European Water Framework Directive. On the other hand, estimation of removal efficiencies of pollutants, such as drug residues, is in the focus of industrial and public wastewater treatment. Immunoassays, such as ELISA, are often applied in this area owing to their cost-efficiency and high-throughput capability. However, it is only possible to measure a single analyte in one measurement. In contrast, array technology is capable for measuring multiple substances in parallel. Here we present a four-plex bead-based flow cytometric assay for measuring three drugs (carbamazepine, an anti-epileptic drug, diclofenac, an antiphlogistic and caffeine, a psychoactive substance) and the bile acid isolithocholic acid, which is proposed to be used as a fecal marker. The drugs can be detected from 1 to 4 µg/L and the bile acid can be detected up to 72 µg/L in wastewater, requiring a highly sensitive detection method.
Methods: We use core-shell particles as the spherical platform, consisting of a polystyrene core and a silica shell. Such beads can easily be encoded with an organic fluorophore in the core and functionalized with amino groups on the silica shell surface for creating binding sites for antibodies: Therefore, the marker substances are bound to the amino groups via NHS chemistry. The hybrid particles were synthesized in house. Polystyrene was chosen as core material because of its low density and high refractive index, both important requirements for the use in suspension arrays. Easy surface modifications using silane chemistry are accessible on the silica shell. For the determination of the pollutants, the beads are incubated with a mixture of the analyte-specific antibodies. The antibodies undergo a competitive reaction to bind the immobilized molecules on the surface of the beads or the free analyte in the solution, respectively. Bound antibodies can then be visualized via fluorophore-labelled secondary antibodies. For read-out a flow cytometer is used to virtually separate the different dye encoded beads and to measure the resulting Signal intensity of the immunoassay. A four-parametric function was used as calibration curve.
Results: In order to obtain highly selective binding of the antibodies, we investigated different types of silica shell surface modifications to overcome unspecific binding, finding that a PEG-modified surface is suitable to support our immunoassay format. Multiplexed small molecule detection can therefore be realized in a simple mix and read procedure without washing steps.
Conclusion: We presented a multiplexing suspension array for the detection of four marker substances in wastewater samples, based on an indirect immunoassay format with polystyrene core-silica shell microbeads. The resulting multiplexing assay is appropriate to detect the marker substances in the low µg/L range. Therefore, wastewater samples can be measured without further preconcentration.
A bead suspension array for measuring emerging pollutants and anthropogenic markers in wastewater.
(2016)
The assessment of water quality demands for high-throughput and multi-target compatible analytical methods. On the one hand, the influence of sewage on natural surface waters must be routinely monitored - according to the European Water Framework Directive. On the other hand, estimation of removal efficiencies of pollutants, such as drug residues, is in the focus of industrial and public wastewater treatment. Immunoassays, such as ELISA, are often applied in this area owing to their cost-efficiency and high-throughput capability. However, it is only possible to measure a single analyte in one measurement.
In contrast, array technology is capable for measuring multiple substances in parallel. Here we present a four-plex bead-based flow cytometric assay for measuring three drugs (carbamazepine, an anti-epileptic drug, diclofenac, an antiphlogistic and caffeine, a psychoactive substance) and the bile acid isolithocholic acid, which is proposed to be used as a fecal marker.
In this suspension array core-shell particles, consisting of a polystyrene core and a silica shell are used. They can be easily encoded with a fluorophore which is introduced to the core and functionalized with amino groups on the surface for creating binding sites for antibodies: Therefore, the marker substances are bound to the amino groups via NHS chemistry.
For the determination of the pollutants, the beads are incubated with a mixture of the analyte-specific antibodies. The antibodies undergo a competitive reaction to bind the immobilised molecules on the surface of the beads or the free analyte in the solution, respectively. Bound antibodies can then be visualized via fluorophore-labelled secondary antibodies. For read-out a flow cytometer is used to virtually separate the different encoded beads and to measure the resulting signal intensity of the immunoassay. In order to obtain highly selective binding of the antibodies, we investigated different types of surface modifications to overcome unspecific binding, finding that a PEG-based surface is suitable to support our immunoassay format. The resulting multiplexing assay is appropriate to detect the marker substances in the low µg/L range.
The annual demand of phosphorus (P) for fertilizer use is more than 550,000 t in Germany. Several of the input streams like mineral fertilizer produced from phosphate rock or sewage sludge are problematic due to contamination with heavy metals and organic pollutants and/or supply risks. Phosphorus recovery from the wastewater stream might help to diminish these problems. Several procedures have been developed to recover P from either sewage sludge ash (SSA) or wastewater via precipitation or crystallization as struvite. This study determined the elemental composition and trace metal mass fractions of 252 SSA samples and 17 struvite samples from wastewater treatment plants (WWTP) to see whether there are differences related to the source of the sludge (municipal/industrial) or the stage of the WWTP where the struvite is produced. Results indicated that there is no clear trend of the elemental mass fractions related to the source of the sludge. All struvite samples show very low heavy metal contents, even though the struvite precipitated from the sludge shows slightly elevated heavy metal mass fractions compared to struvite crystallized from sludge liquor. Struvite is a clean and plant available material for fertilizer production from wastewater streams and has some advantages for the wastewater treatment plant. However, since the P recovery rate of struvite processes is low (7–11%) compared to that from SSA processes (90%), recovery from SSA is necessary for a substantial P recovery from the wastewater stream.
Enzyme-linked immunosorbent assay (ELISA) for the anthropogenic marker isolithocholic acid in water
(2016)
Bile acids are promising chemical markers to assess the pollution of water samples with fecal material.
This study describes the optimization and validation of a direct competitive enzyme-linked immunosorbent assay for the bile acid isolithocholic acid (ILA). The quantification range of the optimized Assay was between 0.09 and 15 mg/L. The assay was applied to environmental water samples. Most studies until now were focused on bile acid fractions in the particulate phase of water samples. In order to avoid tedious sample preparation, we undertook to evaluate the dynamics and significance of ILA levels in the aqueous phase. Very low concentrations in tap and surface water samples made a pre-concentration step necessary for this matrix as well as for wastewater treatment plant (WWTP) effluent. Mean recoveries for spiked water samples were between 97% and 109% for tap water and WWTP influent samples and between 102% and 136% for WWTP effluent samples. 90th percentiles of intra-plate and inter-plate coefficients of variation were below 10% for influents and below 20% for effluents and surface water. ILA concentrations were quantified in the range of 33-72 mg/L in influent, 21-49 ng/L in effluent and 18-48 ng/L in surface water samples. During wastewater Treatment the ILA levels were reduced by more than 99%. ILA concentrations of influents determined by ELISA and LC-MS/MS were in good agreement.
However, findings in LC-ELISA experiments suggest that the true ILA levels in concentrated samples are lower due to interfering effects of matrix compounds and/or cross-reactants. Yet, the ELISA will be a valuable tool for the performance check and comparison of WWTPs and the localization of fecal matter input into surface waters.