Pesticides such as organothiophosphates (OTPs) are neurotoxically active and enter the aquatic environment. Bioassays, using acetylcholinesterase (AChE), a suitable substrate and reactant, can be ...applied for the photometric detection of AChE-inhibiton (AChE-I) effects. The oxidized forms of OTPs, so-called oxons, have higher inhibition potentials for AChE. Therefore, a higher sensitivity is achieved for application of oxidized samples to the AChE assay. In this study, the oxidation of malathion, parathion, and chlorpyrifos by n-bromosuccinimide (NBS) was investigated in an approach combining high-performance thin-layer chromatography (HPTLC) with an AChE-I assay. Two AChE application approaches, immersion and spraying, were compared regarding sensitivity, precision, and general feasibility of the OTP effect detection. The oxidation by NBS led to an activation of the OTPs and a strong increase in sensitivity similar to the oxons tested. The sensitivity and precision of the two application techniques were similar, although the spray method was slightly more sensitive to the oxidized OTPs. The 10% inhibition concentrations (IC
10
) for the spray approach were 0.26, 0.75, and 0.35 ng/spot for activated malathion, parathion, and chlorpyrifos, respectively. AChE-I effect recoveries in samples from a stormwater retention basin and receiving stream were between 69 and 92% for malathion, parathion, and chlorpyrifos. The overall workflow, including sample enrichment by solid-phase extraction, HPTLC, oxidation of OTPs, and AChE-I assay, was demonstrated to be suitable for the detection of AChE-I effects in native water samples. An effect of unknown origin was found in a sample from a stormwater retention basin.
Effect-based methods including cell-based bioassays, reporter gene assays and whole-organism assays have been applied for decades in water quality monitoring and testing of enriched solid-phase ...extracts. There is no common EU-wide agreement on what level of bioassay response in water extracts is acceptable. At present, bioassay results are only benchmarked against each other but not against a consented measure of chemical water quality. The EU environmental quality standards (EQS) differentiate between acceptable and unacceptable surface water concentrations for individual chemicals but cannot capture the thousands of chemicals in water and their biological action as mixtures. We developed a method that reads across from existing EQS and includes additional mixture considerations with the goal that the derived effect-based trigger values (EBT) indicate acceptable risk for complex mixtures as they occur in surface water. Advantages and limitations of various approaches to read across from EQS are discussed and distilled to an algorithm that translates EQS into their corresponding bioanalytical equivalent concentrations (BEQ). The proposed EBT derivation method was applied to 48 in vitro bioassays with 32 of them having sufficient information to yield preliminary EBTs. To assess the practicability and robustness of the proposed approach, we compared the tentative EBTs with observed environmental effects. The proposed method only gives guidance on how to derive EBTs but does not propose final EBTs for implementation. The EBTs for some bioassays such as those for estrogenicity are already mature and could be implemented into regulation in the near future, while for others it will still take a few iterations until we can be confident of the power of the proposed EBTs to differentiate good from poor water quality with respect to chemical contamination.
Display omitted
•Effect-based triggers (EBTs) for bioassays discriminate good from poor water quality.•EBTs can be derived by read across from existing water quality guideline values.•Mixture factor warranted for bioassays responding to many different chemicals.•EBT derivation method applicable to every bioassay subject to data availability•Here we derived preliminary EBTs for 32 bioassays and discuss many more.
Effect-directed analysis (EDA) that combines effect-based methods (EBMs) with high-performance thin-layer chromatography (HPTLC) is a useful technique for spatial, temporal, and process-related ...effect evaluation and may provide a link between effect testing and responsible substance identification. In this study, a yeast multi endocrine-effect screen (YMEES) for the detection of endocrine effects is combined with HPTLC. Simultaneous detection of estrogenic, androgenic, and gestagenic effects on the HPTLC plate is achieved by mixing different genetically modified
Arxula adeninivorans
yeast strains, which contain either the human estrogen, androgen, or progesterone receptor. Depending on the yeast strain, different fluorescent proteins are formed when an appropriate substance binds to the specific hormone receptor. This allows to measure hormonal effects at different wavelengths. Two yeast cell application approaches, immersion and spraying, are compared. The sensitivity and reproducibility of the method are shown by dose-response investigations for reference compounds. The spraying approach indicated similar sensitivities and higher precisions for the tested hormones compared to immersion. The EC
10
s for estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2), 5α-dihydrotestosterone (DHT), and progesterone (P4) were 95, 1.4, 10, 7.4, and 15 pg/spot, respectively. Recovery rates of E1, E2, EE2, DHT, and P4 between 88 and 120% show the usability of the general method in combination with sample enrichment by solid phase extraction (SPE). The simultaneous detection of estrogenic, androgenic, and gestagenic effects in wastewater and surface water samples demonstrates the successful application of the YMEES in such matrices. This promising method allows us to identify more than one endocrine effect on the same HPTLC plate, which saves time and material. The method could be used for comparison, evaluation, and monitoring of different river sites and wastewater treatment steps and should be tested in further studies.
Graphical abstract
Display omitted
The combination of a representative microplastic sampling method and a fast-quantitative analysis using Pyrolysis-GC–MS (Py-GC–MS) for investigation of the microplastic load and mass ...balances is presented in this work. A representative microplastic filtration requires a method allowing quick extraction of the sample. The developed steel based cascadic microplastic filtration uses steel basket filters with mesh sizes of 100 μm, 50 μm and 10 μm and a mean recovery of 86 % without cross contamination was achieved. Thermoanalytical methods have the advantage of minimal sample preparation with short analysis times. The presented platinum filament-based Py-GC–MS method requires little sample preparation and quantification limits for polystyrene (PS) and polyethylene (PE) were 0.03 μg and 1 μg absolute, respectively. The relative standard deviation of the analytical method is 11 %. The combined method allows representative sampling and analysis of MP from water bodies and waste water treatment plants within 48 h.
•Presentation of a validated steel based cascadic microplastic filtration plant.•Fast and reproduceable Py-GC–MS analysis method for microplastic.•Py-GC–MS allows microplastic analysis with little sample preparation.
Display omitted
•EU-wide diclofenac and steroidal estrogen monitoring was performed with LC-MS/MS and biotests.•Low estrogenicity and partly high COX-inhibition were found in the sampled surface ...waters.•Bioassays showed good screening function and applicability for investigative monitoring.•LC-MS/MS detection of EE2 remains challenging at its predicted no-effect level of 35 pg/L.•Standard addition tackled LC-MS/MS detection challenges.
Three steroidal estrogens, 17α-ethinylestradiol (EE2), 17β-estradiol (E2), estrone (E1), and the non-steroidal anti-inflammatory drug (NSAID), diclofenac have been included in the first Watch List of the Water Framework Directive (WFD, EU Directive 2000/60/EC, EU Implementing Decision 2015/495). This triggered the need for more EU-wide surface water monitoring data on these micropollutants, before they can be considered for inclusion in the list of priority substances regularly monitored in aquatic ecosystems. The revision of the priority substance list of the WFD offers the opportunity to incorporate more holistic bioanalytical approaches, such as effect-based monitoring, alongside single substance chemical monitoring. Effect-based methods (EBMs) are able to measure total biological activities (e.g., estrogenic activity or cyxlooxygenase COX-inhibition) of specific group of substances (such as estrogens and NSAIDs) in the aquatic environment at low concentrations (pg/L). This makes them potential tools for a cost-effective and ecotoxicologically comprehensive water quality assessment. In parallel, the use of such methods could build a bridge from chemical status assessments towards ecological status assessments by adressing mixture effects for relevant modes of action. Our study aimed to assess the suitability of implementing EBMs in the WFD, by conducting a large-scale sampling and analysis campaign of more than 70 surface waters across Europe. This resulted in the generation of high-quality chemical and effect-based monitoring data for the selected Watch List substances. Overall, water samples contained low estrogenicity (0.01–1.3 ng E2-Equivalent/L) and a range of COX-inhibition activity similar to previously reported levels (12–1600 ng Diclofenac-Equivalent/L). Comparison between effect-based and conventional analytical chemical methods showed that the chemical analytical approach for steroidal estrogens resulted in more (76%) non-quantifiable data, i.e., concentrations were below detection limits, compared to the EBMs (28%). These results demonstrate the excellent and sensitive screening capability of EBMs.
Regarding endocrine disrupting chemicals, an impact on aquatic organisms at already very low concentrations is observed. Consequently, there is a need for sensitive analytical methods. Currently, no ...instrumental analysis methods exist that fulfil this requirement. To overcome these limitations, effect-based methods (EBMs) are one option for screening and quantification. Determination of agonistic effects caused by estrogens are the focus of current research activities due to environmental impacts and regulatory measures. However, antagonistic effects have to be considered to be able to correlate effects to single compounds, which is still necessary due to regulatory requirements. Since not much is known about relevant antagonists, this review highlights the perspectives and limitations of EBMs. The relevance of antagonistic acting compounds shows the potential of a systematic identification by effect-directed analysis. Collected data of already known antagonists are presented and discussed in a harmonized fashion as basis for future studies.
Display omitted
•Overview of most sensitive instrumental methods for analysis of estrogens.•Application of effect based methods under the EU WFD as screening tool.•Relevance of antagonists within mixture-effects and correlation analysis.•Summary of known anti-estrogenic/anti-androgenic compounds in a harmonized fashion.•Potential of effect based methods if applying effect based trigger values.
To reduce the discharge of micropollutants, advanced wastewater treatment methods were investigated in the last years. Estrogenic effects were found to be reduced by ozonation. These activities are ...usually measured using genetically modified cell-based tests. As these bioassays are representing a sum parameter, also inhibitory effects such as antagonistic effects need to be further investigated as they are potentially reducing the detected activities. Therefore, a direct comparison of chemical target analysis and biological equivalent concentrations measured by bioassays is often difficult. To investigate the fate of antagonistic activities and their role in mixtures with agonistic activities, two hospital wastewater treatment plants were studied after different treatment steps. Thereby highly enriched samples were analyzed by a combination of bioassays with chemical target and non-target analyses. In order to achieve an in-depth characterization of the antagonistic activities a fractionation of the enriched samples was performed. To identify relevant compounds an effect directed identification approach was used by combining high-resolution mass spectrometry and bioassays. The results showed a high reduction for estrogene and androgene activities. However, a constant antagonistic activity after membrane bioreactor and ozone treatment was observed. A reduction of the antagonistic activity was observed after passing an activated carbon filter. The fractionation approach showed a specific finger-print of each sample of the different treatment steps. Hereby we could show that the composition of agonistic and antagonistic active compounds is changing after each treatment step while the overall measured activity stays the same. Using fractionation and the combination of bioassays the number of relevant features detected by chemical non-target screening could be reduced by >85%. As a result the phosphorous flame retardant TCEP could be identified as anti-estrogene active. Future research should be done to identify more antagonistic active compounds and potentially active transformation products after ozone treatment.
Display omitted
•Reduction of estrogene and androgene activity during Hospital wastewater treatment using ozone•Behaviour of agonistic and antagonistic endocrine activities during ozonation using Arxula Adeninivorans based yeast assays•Fractionation in combination with effect based analysis reduces the amount of relevant features•Reduction of antagonistic activities by granular activated carbon filter as post-treatment
In this study, pharmaceutically active compounds (PhACs) were analyzed in the influent and effluent of a domestic wastewater treatment plant in Turkey and a municipal wastewater treatment plant in ...Germany and the toxicity of these wastewaters were estimated using a toxic unit (TU) approach. A total of 21 and 32 PhACs were detected in the domestic wastewater and the municipal wastewater, respectively. The TUs estimated for PhACs in municipal wastewater were higher than the TUs estimated for PhACs present in domestic wastewater. The levels of the anti-anxiety drug, oxazepam were estimated to be in the high risk category (HQ > 10) in both wastewaters. In bench-scale tests with ozonation, the removals of four PhACs in the municipal wastewater were investigated. At a dose of 2 mg/L ozone, 97%–98% of diclofenac and carbamazepine were removed. The lowest removal rate at 71% was observed for metoprolol.
The drugs used for treatment during chemotherapy are manufactured individually for each patient in specialised pharmacies. Thorough quality control to confirm the identity of the delivered active ...pharmaceutical ingredient and the final concentration of the prepared application solution is not standardized yet except for optical or gravimetric testing. However, solution stability problems, counterfeit drugs, and erroneous or deliberate underdosage may occur and negatively influence the quality of the product and could cause severe health risks for the patient. To take a step towards analytical quality control, an on-site analytical instrument using Raman and UV absorption spectroscopy was employed and the results were compared to high-performance liquid chromatography coupled to diode array detection. Within the scope of the technology evaluation, the uncertainty of measurement was determined for the analysis of the five frequently used cytostatic drugs 5-fluorouracil, cyclophosphamide, gemcitabine, irinotecan and paclitaxel. The Raman/UV technique (2.0–3.2% uncertainty of measurement; level of confidence: 95%) achieves a combined uncertainty of measurement comparable to HPLC-DAD (1.7–3.2% uncertainty of measurement; level of confidence: 95%) for the substances 5-fluorouracil, cyclophosphamide and gemcitabine. However, the uncertainty of measurement for the substances irinotecan and paclitaxel is three times higher when the Raman/UV technique is used. This is due to the fact that the Raman/UV technique analyses the undiluted sample; therefore, the sample has a higher viscosity and tendency to foam. Out of 136 patient-specific preparations analysed within this study, 96% had a deviation of less than 10% from the target content.
Graphical abstract
Antibiotics, most notably sulfonamides and tetracyclines, are frequently used veterinary pharmaceuticals in animal husbandry. A new field of application for animal manure is in biogas plants for ...generating environmentally friendly energy. As a result, antibiotics contained in manure may still reach the environment as fermentation residues are also used on agricultural fields as fertilizers. Therefore, in fermentation tests seven sulfonamides and trimethoprim were investigated regarding their elimination behavior during a five-week fermentation process. Sulfadiazine, sulfamerazine, sulfamethoxazole, sulfadimethoxine, and trimethoprim were nearly completely eliminated while sulfathiazole, sulfamethazine, and sulfamethoxypyridazine showed persistence. For sulfadiazine it was possible by means of mass spectrometry to identify and partly quantify a metabolite, emerging from a hydroxylation at the pyrimidine ring, 4-OH-sulfadiazine. Furthermore, a microbial inhibition test showed a substantial reduction in the antimicrobial activity of the metabolite compared to the parent compound. Thus, the fermentation process may be an efficient way to reduce the load of selected veterinary antibiotics finding their way into the environment. Degradable drugs such as sulfadiazine may therefore, at least in the aspect of residual antibiotic activity of metabolites, be considered as environmentally friendly drugs.
PDF
