Chemische Charakterisierung und Spurenanalytik
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Per- and polyfluoroalkyl substances (PFAS) are a large, ever-growing and widely used class of chemicals. Due to the strength of the C-F bond, they do not decompose but accumulate in the environment posing a risk for nature and humans alike. While the use of some PFAS, like perfluorooctanoic acid (PFOA) is already regulated in the EU, new ‘precursor’ substances are used to replace them. However, these precursors may be equally harmful to the environment.
In recent years, many ‘hot spot’ sites with high PFAS contaminations in soil have been distinguished, but no German-wide background values have been determined yet. Knowing these background values is crucial to understand the degree of PFAS contamination, underpinning future regulatory decisions.
Due to the complexity and variety of different PFAS compounds, one needs to apply a broad spectrum of different techniques to capture most of the PFAS content in one sample.
In the framework of this project, 600 soil samples are taken at different sites all over Germany. The concentration of 30 PFAS - 13 carboxylic acids (C4-C18), 5 sulfonic acids (C4-C10) and 12 precursor substances) - is determined in these soil samples using three different sample preparation approaches: 1) ultrasonic extraction of the soil samples with Methanol; 2) preparation of eluates to test the leaching behaviour of the PFAS and 3) TOP assays following the method published by Houtz & Sedlak.
Targeted LC-MS/MS is used to determine PFAS concentration after all three preparation steps.
For the TOP-assay, the concentration of all 30 PFAS is compared prior to and after the oxidation reaction.
Besides the ‘classic’ TOP-Assay, two other TOP-Assay approaches, the dTOP Assay and the photoTOP-Assay, are tested and the results of all three approaches are compared. First, all three TOP-Assay approaches will be tested on a reference soil spiked with a solution including all 30 PFAS measured in the project. Later, soil samples taken in the frame of the priorly described project will be tested.
This poster will focus on the results of the experiments comparing the three different TOP Assay approaches in spiked reference soil. Additionally, some of the results of the other sample preparation methods, extraction and eluate preparation will be presented.
Bisphenol A (BPA) is used in Polycarbonate, Polyacrylic resins, Polysulfones, Epoxy resins, and Polyetherimides. It is also used in recycled Polyvinyl chloride [1–3]. BPA has been classified as a substance of very high concern (SVHC) under REACH [4] due to its endocrine disrupting properties. The German competent authorities want to reduce the content of BPA in the environment. Possible substitutes for BPA are Bisphenol B (BPB), Bisphenol E (BPE),
Bisphenol F (BPF), or Bisphenol S (BPS), as they are similar in structure.
BPA-based materials have a wide area of application especially outside. These outdoor applications are exposed to different external influences, including physical, biological, mechanical and chemical influences. This results in damage and aging of the material with leaching or migration into the environment. There, the substance is transformed by various transformation processes. The emerging metabolites and transformation products (TPs) can have different properties than the parent substance. Understanding the fate and behavior of the emerging pollutants is very important.
Therefore, different transformation products of selected bisphenols will be generated and analyzed:
To investigate the fate of different bisphenols in water treatment plants technical transformation products [5] will be generated by chlorination, ozonization, the Fenton reaction, and UV-radiation.
For the investigation of the fate of Bisphenols in surface water global radiation will be simulated with UVA-lamps
Simulation of phase-I-metabolism with an electrochemical cell coupled to mass spectrometry (EC-MS).
Natural and synthetic estrogens are key endocrine-disrupting chemicals. Despite occurring at ultra-trace levels (below ng L-1), it is believed that they are contributing to an increase in feminized fish and other endocrine disruptive effects, and hence, their inclusion in the Watch list was not unexpected. One of the main sources ofestrogens to surface waters is wastewater effluent. Once in surface waters, they can partition into different compartments, i.e., water and suspended particulate matter. For this reason, there is an urgent need for a methodology to monitor estrogen levels below the environmental quality standards (EQS) set by the Water Framework Directive requirements.
In this study, a precise and accurate gas chromatography-mass spectrometry method (GC-MS/MS) for the analysis of estrone (E1), 17β-estradiol (17β-E2), 17α-estradiol (17α- E2), 17-alpha-ethinylestradiol (EE2), and estriol (E3) in whole water samples with ng L-1 limit of quantification (LOQ) was developed and validated in accordance with CEN/TS 16800:2020 guidelines.
Monitoring programs should generate high-quality data on the concentrations of substances and other pollutants in the aquatic environment to enable reliable risk assessment. Furthermore, the need for comparability over space and time is critical for analysis of trends and evaluation of restoration of natural environment. Additionally, research work and exercises at the European level have highlighted that reliable measurements of estrogenic substances at the PNEC level are still challenging to achieve.
The project EDC-WFD Metrology for monitoring endocrine disrupting compounds under the EU Water Framework Directive aims to develop traceable analytical methods for determining endocrine disrupting compounds and their effects, with a specific focus on three estrogens of the first watch list (17-beta-estradiol (17βE2), 17-alpha-ethinylestradiol (EE2), and estrone (E1)). Estrogens 17-alpha-estradiol (17E2) and estriol (E3) will be included to demonstrate the reliability of the developed methods and to support the requirements of Directive 2013/39/EC, Directive 2009/90/EC and Commission Implementation Decision (EU) 2018/840, hence improving the comparability and compatibility of measurement results within Europe. During the EDC-WFD project four selected effect-based methods (EBM) will be deeply investigated in order to improve their rationale use and their support in water quality assessment. In particular, the EBM sensitivity, specificity and accuracy on reference materials with single or mixture solutions of the five substances at a concentration of EQS values will be explored.
This contribution will present the objectives and methods applied within the EDC-WFD project.
Contamination of the environment with antibiotics is of great concern as it promotes the evolution of antimicrobial resistances. In case of amoxicillin (AMX) in the aquatic environment, further risk arises from hydrolysis products (HPs) which can cause allergy. To assess these risks, a comprehensive investigation and understanding of the degradation of AMX is necessary. We investigated the hydrolysis rate of AMX in different types of water as well as the influence of temperature and irradiation. The content of the heavy metal ions copper and zinc was found to be crucial for the hydrolysis rate of AMX and stability of HPs. Eventually, a new degradation pathway for AMX could be elaborated and confirmed by tandem mass spectrometry (LC-MS/MS).
Steps Forward in the Application of 3,3’,5,5’- Tetramethylbenzidine (TMB) in Amperometric Assays
(2022)
Electrochemical methods make great promise to meet the demand for user-friendly on-site devices for monitoring important parameters. Food industry often runs own lab procedures, e.g. for mycotoxin analysis, but it is a major goal to simplify analysis, linking analytical methods with miniaturized technologies. Enzyme-linked immunosorbent assays, with photometric detection of the horseradish peroxidase (HRP) substrate, 3,3’,5,5’-tetramethylbenzidine (TMB), form a good basis for sensitive detection. To provide a straight-forward approach for the miniaturization of the detection step, we have studied the pitfalls of the electrochemical TMB detection. By cyclic voltammetry it was found that the TMB electrochemistry is strongly dependent on the pH and the electrode material.
It was found that screen-printed gold electrodes and a highly acidic pH value (pH 1) are well-suited to perform the electrochemical detection of TMB, due to the reversible character of the redox reaction under these conditions. This set-up provides a good signal stability over several measuring cycles, providing the basis for analysing multiple samples. In contrast to this, for carbon screen-printed electrodes, it was found that the signal response has changed after the electrochemical reaction with TMB at pH 1. At a weakly acidic pH value (pH 4), neither with carbon nor with gold electrodes a reproducible electrochemical detection of TMB could be achieved [1].
Based on these findings we created a smartphone-based, electrochemical, immunomagnetic assay for the detection of ochratoxin A and ergometrine in real samples. Therefore, a competitive assay was performed on magnetic beads using HRP and TMB/H2O2 to generate the signal. Enzymatically oxidized TMB was quantified after addition of H2SO4 by amperometry with screen-printed gold electrodes in a custom-made wall-jet flow cell. The results are in good correlation with the established photometric detection method, providing a solid basis for sensing of further analytes in HRP-based assays using the newly developed miniaturized smartphone-based, electrochemical, immunomagnetic assay.
The general understanding has been that PEGs are biologically inert, meaning they pass the human body without any relevant reaction with anything. But recent studies have shown that migth not be correct. Here a retrospective analysis of LC-MS data was performed to determine correlations between blood PEG levels in humans and different experimental factors.
The demand for miniaturized analytical devices monitoring important parameters in the food and medical industry has increased strongly in the past decades. With fast progress, smart technologies are finding their way into our everyday life. For the future, it is, therefore, a major goal to also link analytical methods with smart technologies to create user-friendly on-site devices. In food industry the monitoring of harmful substances such as dioxins, heavy metals or mycotoxins plays a key role, since the European Commission prescribes legal limits for various food products and beverages[1]. Therefore, companies often have their own laboratories and trained personnel. For one of the most abundant and toxic mycotoxins, Ochratoxin A (OTA) we want to present an electrochemical detection system in which the read-out can be performed with a smartphone connected via Bluetooth to a miniaturized potentiostat. The recognition of OTA is performed with specific antibodies in a competitive assay format. Anti-OTA-antibodies were captured on magnetic beads on which the competitive binding between OTA and an OTA horseradish peroxidase (HRP) tracer was performed. To quantify OTA, the enzymatic reaction of the tracer with 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2 is employed. Oxidized TMB, which is enzymatically produced by the reduction of H2O2, is quantified by amperometry with screen-printed electrodes in a custom-made flow system. Since it is well-known that oxidized TMB can precipitate on electrode surfaces[2], we have studied pitfalls of the electrochemical detection of TMB. By cyclic voltammetry we have compared the stability of the electrochemistry of TMB at different electrode materials (gold and carbon) and pH values (pH 1 and pH 4). It was found that a stable response of the electrode could be achieved at pH 1 on gold electrodes. Thus, we applied these reaction conditions for amperometric detection of TMB in the OTA assay. The results of the electrochemical detection method are in good correlation with the photometric detection of TMB. To demonstrate the applicability, we tested our system with OTA-spiked beer and performed the measurement via smartphone.
In addition to previously reported results on the accelerated weathering of polystyrene samples (PS) containing 1 wt.% hexabromocyclododecane (HBCD), we present the first results of our investigations of polypropylene samples (PP) containing 0.1 wt.% BDE-209. All studied polymer samples were exposed to a defined weathering schedule in a climate chamber in accordance to regulation EN ISO 4892-3:2006. For the determination of BDE-209 in the collected raining water the samples were prepared in accordance to a validated protocol. Before the analyses each sample was spiked with isotopically labeled BDE-209. Subsequently the samples were extracted with isooctane. The obtained extracts were concentrated, and the resulting solutions were analyzed by GC/MS. Additionally, the total bromine content was monitored for the weathered and untreated samples using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and X-ray fluorescence analysis (XRF) as a non-destructive and rapid method. In general, the resulting data from the accelerated weathering will be compared to those from the natural weathering experiments. Here, the surfaces of the test pieces were analyzed by LA-ICP-MS and XRF as well. Moreover, soil bed tests were conducted in a well characterized model soil. This soil (boulder-clay, sand with 12 % loam, particle size in total 0.2-4 mm) is filled in a free-draining basin inside of an air-conditioned cellar. In this manner, TOC, water capacity and humidity are recorded parameters. To induce a leaching process from the samples by the raining water, the target water content is calculated to 8%. The actual humidity is measured by a tensiometer, assuring the duration of the raining period. A defined humidity is a fundamental parameter for a biological activity in the soil. The test polymers were placed up to the half in the soil. Microbial activity of the soil is monitored by a reference polymer (polyurethane) and should induce the release of HBCD and BDE-209 out of the test materials. These released analytes will be captured by passive samplers (silicone tubes) placed in a distinct distance to the polymer samples in the soil. The soil bed experiments are complementary to the weathering experiments due to the biological activity in the soil.
Warfarin, also known as Coumadin is one of the most popular anticoagulant drugs used as a therapeutic in humans to prevent thrombosis, atrial fibrosis, and fibrillation since the 1950s. Because of its ability to hinder blood from clotting by blocking vitamin K-depending carboxylation of blood clotting precursors, it is also used as a rodenticide worldwide. Until today it has been partially substituted by far more potent anticoagulant rodenticides (ARs), so-called superwarfarins. There are numerous references confirming secondary and tertiary poisoning in non-target-animals and wildlife like mammals, birds, and fish associated with ARs. Up to now relatively little is known about persistence and toxicity of transformation products of those AR in the environment and food chain. Herein, warfarin was chosen as a model compound to determine andelucidate TPs which are generated technically and under environment-like conditions. Such as the simulation of the oxidative phase I metabolism by implementing electrochemical flow cells coupled to mass spectrometry (MS). Obtained results are being compared to TPs generated by UVB-irradiation and ozonation.
Analysis and separation of degradation products and TPs were achieved by a variety of gas- and liquid chromatographic techniques coupled to (high resolution) MS.