Sebastian Buchinger

The European technical report on aquatic effect-based monitoring tools under the water framework directive

The Water Framework Directive (WFD), 2000/60/EC, requires an integrated approach to the monitoring and assessment of the quality of surface water bodies. The chemical status assessment is based on compliance with legally binding Environmental Quality Standards (EQSs) for selected chemical pollutants (priority substances) of EU-wide concern. In the context of the mandate for the period 2010 to 2012 of the subgroup Chemical Monitoring and Emerging Pollutants (CMEP) under the Common Implementation Strategy (CIS) for the WFD, a specific task was established for the elaboration of a technical report on aquatic effect-based monitoring tools. The activity was chaired by Sweden and co-chaired by Italy and progressively involved several Member States and stakeholders in an EU-wide drafting group. The main aim of this technical report was to identify potential effect-based tools (e.g. biomarkers and bioassays) that could be used in the context of the different monitoring programmes (surveillance, operational and investigative) linking chemical and ecological status assessment. The present paper summarizes the major technical contents and findings of the report.

In vitro bioassays for detecting dioxin-like activity--application potentials and limits of detection, a review.

Use of in vitro assays as screening tool to characterize contamination of a variety of environmental matrices has become an increasingly popular and powerful toolbox in the field of environmental toxicology. While bioassays cannot entirely substitute analytical methods such as gas chromatography-mass spectrometry (GC-MS), the increasing improvement of cell lines and standardization of bioassay procedures enhance their utility as bioanalytical pre-screening tests prior to more targeted chemical analytical investigations. Dioxin-receptor-based assays provide a holistic characterization of exposure to dioxin-like compounds (DLCs) by integrating their overall toxic potential, including potentials of unknown DLCs not detectable via e.g. GC-MS. Hence, they provide important additional information with respect to environmental risk assessment of DLCs. This review summarizes different in vitro bioassay applications for detection of DLCs and considers the comparability of bioassay and chemical analytically derived toxicity equivalents (TEQs) of different approaches and various matrices. These range from complex samples such as sediments through single reference to compound mixtures. A summary of bioassay derived detection limits (LODs) showed a number of current bioassays to be equally sensitive as chemical methodologies, but moreover revealed that most of the bioanalytical studies conducted to date did not report their LODs, which represents a limitation with regard to low potency samples.

Direct Coupling of Thin-Layer Chromatography with a Bioassay for the Detection of Estrogenic Compounds: Applications for Effect-Directed Analysis

The present study investigated the hypothesis that the coupling of high-performance thin-layer chromatography with the yeast estrogen screen (planar-YES, p-YES) can be used as a screening tool for effect-directed analysis. Therefore, the proposed method was challenged for the first time with several real samples from various origins such as sediment pore water, wastewater, and sunscreens. It was possible to detect and quantify estrogenic effects in all investigated sample types, even in the presence of demanding matrixes. Furthermore, the specific agonistic effect of the estrogen receptor activation could be detected in samples exhibiting cytotoxic effects and at cytotoxic levels of analyzed estrogenic compounds, which is not possible with the classic YES. The analysis of samples by the p-YES results in profiles of estrogenic activity. By means of this profiles samples can be compared qualitatively and quantitatively with respect to different compositions of bioactive compounds in mixtures. In conclusion, the p-YES approach seems to have a high potential to be used as a valuable screening tool for various applications in effect-directed analysis.

Bacterial genotoxicity bioreporters.

Ever since the introduction of the Salmonella typhimurium mammalian microsome mutagenicity assay (the ‘Ames test’) over three decades ago, there has been a constant development of additional genotoxicity assays based upon the use of genetically engineered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test, or on promoter–reporter fusions that generate a quantifiable dose‐dependent signal in the presence of potential DNA damaging compounds and the induction of repair mechanisms; the latter group is the subject of the present review. Some of these assays were only briefly described in the scientific literature, whereas others have been developed all the way to commercial products. Out of these, only one, the umu‐test, has been fully validated and ISO‐ and OECD standardized. Here we review the main directions undertaken in the construction and testing of bacterial‐based genotoxicity bioassays, including the attempts to incorporate at least a partial metabolic activation capacity into the molecular design. We list the genetic modifications introduced into the tester strains, compare the performance of the different assays, and briefly describe the first attempts to incorporate such bacterial reporters into actual genotoxicity testing devices.

Integrated biological-chemical approach for the isolation and selection of polyaromatic mutagens in surface waters

AbstractMany environmental mutagens, including polyaromatic compounds are present in surface waters, often in complex mixtures and at low concentrations. The present study provides and applies a novel, integrated approach to isolate polyaromatic mutagens in river water using a sample from the River Elbe. The sample was taken downstream of industrial discharges using blue rayon (BR) as a passive sampler that selectively adsorbs polyaromatic compounds and was subjected to effect-directed fractionation in order to characterise the compounds causing the detected effect(s). The procedure relies on three complementary fractionation steps, the Ames fluctuation assay with strains TA98, YG1024 and YG1041 with and without S9 activation and analytical screening. Several mutagenic fractions were isolated by combining mutagenicity testing with fractionation. The enhanced mutagenicity in the nitroreductase and/or O-acetyltransferase overexpressing strains YG1024 and YG1041 strains suggested amino- and/or nitro-compounds causing mutagenicity in several fractions. Analytical screening of mutagenic fractions with LC-HRMS/MS provided a list of molecular formulas typically containing one to ten nitrogen and at least two oxygen atoms supporting the presence of amino and nitro-compounds in the mutagenic fractions. Figureᅟ

Combination of high-performance thin-layer chromatography with a specific bioassay - A tool for effect-directed analysis

The objective of this study was to combine the Yeast Estrogen Screen (YES) for the detection of estrogenic compounds with thinlayer chromatography. Such an approach might serve as an effective tool in effect-directed analysis, i.e., the identification of bioactive compounds in mixtures. The proposed method (planar-YES) allows the detection of estrogenic effects on the surface of a TLC plate after an exposure time of 2–3 h instead of the usual 18 h which are used for the traditional microplate-YES. The estimated limits of quantification for the estrogenic model compound 17α-ethinylestradiol (EE2) were 0.8 pg without and 1.6 pg with a prior chromatographic development of the TLC plate. The hormones 17α- ethinylestradiol (EE2), 17β-estradiol (E2) and estrone (E1) could be separated by thin-layer chromatography and were detected by means of the specific bioassay subsequently. Finally, the applicability of the method on real samples was demonstrated with the detection of estrogenic effects in sediment extracts from sediments of the river Elbe.

Identification of mutagens in freshwater sediments by the Ames‐fluctuation assay using nitroreductase and acetyltransferase overproducing test strains

Extracts of sediments from an area of concern in the Elbe river basins (Spittelwasser creek) were analyzed with the Ames‐fluctuation test and in parallel with gas chromatography/mass spectrometry for compound identification. The standard test strains TA 98 and TA 100 showed mutagenicity mainly in medium‐polar fractions of the sediment extracts. PAHs contribute to the overall mutagenic potential of the sample. Especially, cyclopenta[c,d]pyrene that was previously not defined as a priority hazardous substance has to be considered as well. The addition of metabolically competent test strains, which overexpress nitroreductase and acetyltransferase (e.g., YG1041 and YG1042) to the test battery, increased significantly the sensitivity of the Ames test for medium polar to polar genotoxins. The increased mutagenicity that was found in these bacterial strains indicates the presence of nitroarenes and/or aromatic amines. In fact, a number of heterocyclic and nitrogen‐substituted aromatic compounds were identified in the sediments of the Spittelwasser creek of which methyl parathion, 1‐naphthylamine, and N‐phenyl‐2‐naphthylamine are mutagenic. Environ. Mol. Mutagen., 2011.

Evaluation of chrono-amperometric signal detection for the analysis of genotoxicity by a whole cell biosensor

Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alternative to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl beta-D-galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the standard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.

Roles of human sulfotransferases in genotoxicity of carcinogens using genetically engineered umu test strains

Human sulfotransferase (SULT) 1A1, 1A2, and 1A3 cDNA genes were subcloned separately into the pTrc99AKM vector. The generated plasmids were introduced into the Salmonella typhimurium O‐acetyltransferase‐deficient strain NM6000 (TA1538/1,8‐DNP/pSK1002), resulting in the new strains NM7001, NM7002, and NM7003. We compared the sensitivities of these three strains with the parental strain NM7000 against 51 chemicals including aromatic amines, nitroarenes, alkenylbenzenes, estrogens‐like chemicals, and other compounds with and without S9 mix by making use of the umu test system that is based on the bacterial SOS induction. 2‐Amino‐6‐methyl‐dipyrido[1,2‐α:3′,2′‐d]imidazole, 3‐methoxy‐4‐aminoazobenzene, 3‐nitrobenzanthrone, 5‐nitroacenaphthene, and 3,9‐dinitrofluoranthene caused high genotoxicity in the NM7001 strain. The genotoxic effects of 2‐aminofluorene, 2‐acetylaminofluorene, 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine, 2‐nitrofluorene, 1‐nitropyrene, and 2‐nitropropane were stronger in the NM7002 strain compared with the NM7001 and NM7003 strains. Among the tested benzylic and allylic compounds, 1‐hydroxymethylpyrene was detected in the NM7001 strain with the highest sensitivity. Estragole and 1′‐hydroxysafrole exhibited strong genotoxicity in the NM7003 strain. The estrogen‐like chemicals such as bisphenol A, genistein, p,n‐nonylphenol, and 4‐hydroxytamoxifen were not detected as genotoxins in any strain used. Collectively, the present results suggest that the generated test strains are valuable tools in order to elucidate the role of SULT enzymes in the bioactivation of chemicals to environmental carcinogens. Environ. Mol. Mutagen. 2012.

Determination of the CYP1A-inducing potential of single substances, mixtures and extracts of samples in the micro-EROD assay with H4IIE cells

This protocol describes a quantitative and robust 96-well-plate-reader–based assay for the measurement of ethoxyresorufin-O-deethylase (EROD) activity using the rat hepatoma cell line H4IIE. The assay can be used to determine the cytochrome P450 subfamily 1A (CYP1A)-inducing potential of single substances, as well as of mixtures and extracts of samples. It is based on the aryl hydrocarbon receptor (AhR)–mediated induction of cytochrome P450 enzymes (subfamily 1A) in cells after exposure to dioxins and dioxin-like compounds. One enzymatic reaction catalyzed by CYP1A is the deethylation of the exogenous substrate 7-ethoxyresorufin to the fluorescent product resorufin, which is measured as EROD activity in the assay. The CYP1A-inducing potential of a sample can be reliably quantified by comparing the EROD activity with the concentration-response curve of the standard substance 2,3,7,8-tetrachlorodibenzo-p-dioxin, which can be detected at concentrations down to the picogram per liter range. A researcher familiar with the procedure can process up to 160 samples with four wells each within 3 d. The series described uses four plates with three concentrations per sample, which can be easily scaled to accommodate different sample sizes.