Markus Brinkmann

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.

Quantitative assessment of the embryotoxic potential of NSO-heterocyclic compounds using zebrafish (Danio rerio)

Heterocyclic aromatic compounds (NSO-HET) have frequently been detected in the environment. Several studies have concluded that NSO-HET pose a threat to organisms in waters, sediments and soils. However, few publications are available assessing the ecotoxicology of NSO-HET. The present study aims to assess the embryo toxicity of heterocycles using Danio rerio. A combination of the Fish Embryo Toxicity Test and analytical quantification should aid to determine the hazard potential. Changes of the total concentrations due to sorption or volatility were quantified by GC/MS. Loss of compounds during the test was observed primarily for volatile or hydrophobic NSO-HET. The LC50 calculated with nominal concentrations underestimates the toxicity by a factor up to 16 (2 h), demonstrating that a chemical analysis for comparing nominal and measured concentrations is essential for such investigations.

Some heterocyclic aromatic compounds are Ah receptor agonists in the DR-CALUX assay and the EROD assay with RTL-W1 cells

PurposeHeterocyclic aromatic compounds containing nitrogen, sulfur, or oxygen heteroatoms (NSO-HET) have been detected in air, soil, marine, and freshwater systems. However, only few publications are available investigating NSO-HET using in vitro bioassays. To support better characterization of environmental samples, selected NSO-HET were screened for dioxin-like activity in two bioassays.MethodsThe present study focuses on the identification and quantification of dioxin-like effects of 12 NSO-HET using the DR-CALUX assay, and the 7-ethoxyresorufin-O-deethylase (EROD) assay with the permanent fish liver cell line RTL-W1. Changes of the total medium compound concentrations during the test procedure due to, e.g., sorption or volatilization were quantified using GC/MS.ResultsThe NSO-HET benzofuran, 2,3-dimethylbenzofuran, dibenzofuran, dibenzothiophen, acridine, xanthene, and carbazole caused a response in the DR-CALUX assay. Only benzofuran and 2,3-dimethylbenzofuran were also positive in the EROD assay. All other compounds were inactive in the EROD assay. Relative potency (REP) values ranged from (2.80 ± 1.32) · 10−8 to (3.26 ± 2.03) · 10−6 in the DR-CALUX and from (3.26 ± 0.91) · 10−7 to (4.87 ± 1.97) · 10−7 in the EROD assay.ConclusionsThe REP values were comparable to those of larger polycyclic aromatic hydrocarbons, e.g., fluoranthene and pyrene. Thus, and because of the ubiquitous distribution of heterocyclic aromatic compounds in the environment, the provided data will further facilitate the bioanalytical and analytical characterization of environmental samples towards these toxicants.

How flood events affect rainbow trout: Evidence of a biomarker cascade in rainbow trout after exposure to PAH contaminated sediment suspensions

Increasing frequency and intensity of flood events are major concerns in the context of climate change. In addition to the direct hydrological implications of such events, potential ecotoxicological impacts are of increasing interest. It is vital to understand mechanisms of contaminant uptake from suspended particulate matter (SPM) and related effects in aquatic biota under realistic conditions. However, little is known about these processes. Due to recent changes in climate, during summer temperatures of German rivers frequently exceed 25°C. Effects of re-suspension of sediments on biota under elevated temperature regimes are likely to differ from those under lower temperature regimes. To elucidate this differential response of aquatic vertebrates, rainbow trout were exposed to suspensions of sediment from the Rhine River that was spiked with a mixture of polycyclic aromatic hydrocarbons (PAH). The experiments were conducted under two different temperature regimes (24°C or 12°C). Physicochemical parameters, including concentration of PAHs in SPM, and biomarkers in fish (biliary PAH metabolites, 7-ethoxyresorufin O-deethylase activity, lipid peroxidation (LPO), mRNA expression of some genes and micronuclei) were measured over the course of a 12d study. Concentrations of pyrene and phenanthrene decreased over time, while no decrease was observed for chrysene and benzo[a]pyrene. The biomarker cascades, more specifically the temporal dynamics of biomarker reactions, did not only show quantitative differences (i.e. different induction intensity or rate of biomarker responses) at the two temperatures but also qualitative differences, i.e. different biomarker responses were observed. A slight significant increase of biliary metabolites in fish was observed in un-spiked sediment at 24°C. In bile of fish exposed to PAH spiked sediment concentrations of 1-hydroxypyrene and 1-hydroxyphenanthrene increased significantly during the first two days, and then decreased. At 12°C uptake of PAHs was slower and maximum metabolite concentrations in bile were less than in fish exposed at 24°C. Following a latency of two days, concentrations of PAH metabolites in bile of fish exposed at 24°C were followed by a peak in LPO. PAHs spiked into sediments under laboratory conditions were significantly more bioavailable than the PAHs that were already present in un-spiked field-collected sediments.

Comparison of six sewage effluents treated with different treatment technologies--population level responses in the harpacticoid copepod Nitocra spinipes.

Since conventional treatment technologies may fail in removing many micro-pollutants, there is currently a focus on the potential of additional treatment technologies for improved sewage treatment. The aim of the present study was to evaluate six different effluents from Henriksdal Sewage Treatment Plant in Stockholm, Sweden. The effluents were; conventionally treated effluent (chemical phosphorous removal in combination with an activated sludge process, including biological nitrogen removal and a sand filter), with additional treatments individually added to the conventional treatment; active carbon filtration, ozonation at 5 mg l(-1), ozonation at 15 mg l(-1), ozonation at 5 mg l(-1)+moving bed biofilm reactor and irradiation with ultraviolet radiation+hydrogen peroxide. The evaluation was done by characterizing and comparing the effluents using a Lefkovitch matrix model based on a life cycle test with the harpacticoid copepod Nitocra spinipes, combined with analysis of juvenile development and survival over time. The conventionally treated effluent resulted in the most negative effects, leading to the conclusion that all additional treatments in the present study created effluents with less negative impacts on the copepod populations. The ozone treatments with the low dose treatment in particular, resulted in the overall least negative effects. Moving bed biofilm reactor combined with ozone did not improve the quality of the effluent in the sense that slightly more negative effects on the population abundance were seen for this treatment technology compared to ozonation alone. The active carbon treatment had more negative effects than the ozone treatments, most of which could possibly be explained by removal of essential metal ions. The effluent which was treated with ultraviolet radiation+hydrogen peroxide resulted in few developmental and survival effects over time, but still showed negative effects on the population level. Matrix population modeling proved a useful tool for biologically characterizing and comparing the effluents. Basing the assessment either on the individual level data (development and survival over time or total reproductive output) or the population level data (lambda values and projected population abundances) would not have resulted in the same conclusions as combining both analyses. The juvenile development and survival over time allowed for closer monitoring of the important molting process, whereas the population modeling provided an integrated measure of potential effects at the population level. If the dilution of the effluent in the recipient is considered, the biological effects recorded in the present study were not of substantial significance for the copepod populations, regardless of treatment technology.

Heterocyclic Aromatic Hydrocarbons Show Estrogenic Activity upon Metabolization in a Recombinant Transactivation Assay

Heterocyclic aromatic hydrocarbons (hetero-PAHs) are increasingly studied at contaminated sites; especially at former industrial facilities where coal tar-oil was handled, e.g., wood treatment plants, high concentrations of hetero-PAHs are frequently detected in groundwater plumes. In previous studies, fractions of groundwater with high estrogenic activity contained hetero-PAHs and their hydroxylated metabolites. To evaluate this preliminary evidence, selected hetero-PAHs were screened for their estrogenic activity in lyticase yeast estrogen screen (LYES) and ER CALUX. All tested substances were inactive in the LYES. Hetero-PAHs such as acridine, xanthene, indole, 2-methylbenzofuran, 2,3-dimethylbenzofuran, dibenzofuran, dibenzothiophene, quinoline, and 6-methylquinoline were positive in the ER CALUX, with estradiol equivalence factors (EEFs) from 2.85 × 10(-7) to 3.18 × 10(-5). The EEF values of these substances were comparable to those of other xenoestrogens (e.g., alkylphenols or bisphenol A) that are sometimes found in surface water. Chemical analyses revealed that T47Dluc cells could metabolize most of the substances. Among the metabolites (tentatively) identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were hydroxides and their keto tautomers, sulfates, sulfoxides, and N-oxides. Because of their high concentrations measured in groundwater, we conclude that hetero-PAHs and metabolites may be a potential risk and should be the subject of further research.

Genotoxicity of Heterocyclic PAHs in the Micronucleus Assay with the Fish Liver Cell Line RTL-W1

Heterocyclic aromatic hydrocarbons are, together with their un-substituted analogues, widely distributed throughout all environmental compartments. While fate and effects of homocyclic PAHs are well-understood, there are still data gaps concerning the ecotoxicology of heterocyclic PAHs: Only few publications are available investigating these substances using in vitro bioassays. Here, we present a study focusing on the identification and quantification of clastogenic and aneugenic effects in the micronucleus assay with the fish liver cell line RTL-W1 that was originally derived from rainbow trout (Oncorhynchus mykiss). Real concentrations of the test items after incubation without cells were determined to assess chemical losses due to, e.g., sorption or volatilization, by means of gas chromatography-mass spectrometry. We were able to show genotoxic effects for six compounds that have not been reported in vertebrate systems before. Out of the tested substances, 2,3-dimethylbenzofuran, benzothiophene, quinoline and 6-methylquinoline did not cause substantial induction of micronuclei in the cell line. Acridine caused the highest absolute induction. Carbazole, acridine and dibenzothiophene were the most potent substances compared with 4-nitroquinoline oxide, a well characterized genotoxicant with high potency used as standard. Dibenzofuran was positive in our investigation and tested negative before in a mammalian system. Chemical losses during incubation ranged from 29.3% (acridine) to 91.7% (benzofuran) and may be a confounding factor in studies without chemical analyses, leading to an underestimation of the real potency. The relative potency of the investigated substances was high compared with their un-substituted PAH analogues, only the latter being typically monitored as priority or indicator pollutants. Hetero-PAHs are widely distributed in the environment and even more mobile, e.g. in ground water, than homocyclic PAHs due to the higher water solubility. We conclude that this substance class poses a high risk to water quality and should be included in international monitoring programs.

Contribution of Priority PAHs and POPs to Ah Receptor-Mediated Activities in Sediment Samples from the River Elbe Estuary, Germany

The estuary of the River Elbe between Hamburg and the North Sea (Germany) is a sink for contaminated sediment and suspended particulate matter (SPM). One major concern is the effect of human activities on the hydrodynamics, particularly the intensive dredging activities in this area that may result in remobilization of sediment-bound pollutants. The aim of this study was to identify pollutants contributing to the toxicological risk associated with re-suspension of sediments in the Elbe Estuary by use of an effect-directed analysis that combines chemical and biological analyses in with specific fractionation techniques. Sediments were collected from sites along the Elbe Estuary and a site from a small harbor basin of the Elbe Estuary that is known to be polluted. The sixteen priority EPA-PAHs were quantified in organic extracts of sediments. In addition, dioxin equivalents of sediments were investigated by use of the 7-ethoxyresorufin O-deethylase assay with RTL-W1 cells and the Ah receptor-mediated luciferase transactivation assay with H4IIE-luc cells. Quantification of the 16 priority PAHs revealed that sediments were moderately contaminated at all of the sites in the Elbe River Estuary (<0.02–0.906 µg/g dw). Sediments contained relatively small concentrations of dioxin equivalents (Bio-TEQ) with concentrations ranging from 15.5 to 322 pg/g dw, which were significantly correlated with dioxin equivalents calculated based on toxicity reference values and concentrations of PAH. The concentration of Bio-TEQ at the reference site exceeded 200,000 pg/g dw. In a potency balance the 16 PAHs explained between 47 and 118% of the Bio-TEQ in the luciferase assay, which can be explained by the constant input of PAHs bound to SPM from the upper course of the Elbe River into its estuary. Successful identification of a significant portion of dioxin-like activity to priority PAHs in complex environmental samples such as sediments has rarely been reported.

A new approach to investigate the interactions between sediment transport and ecotoxicological processes during flood events

Extreme hydrodynamic events such as flood events or dredging activities bear the risk of eroding sediments in rivers, reservoirs, harbour basins or estuaries. One of the key concerns associated with these erosion processes is the re-mobilisation of sediment-bound pollutants in highly contaminated sediments. To date, much research has been conducted to characterise flow and sediment processes associated with hydrological events such as floods. Furthermore, there is a large body of literature describing the interaction of contaminants associated with particulate matter to aquatic biota. However, there is little knowledge regarding interactions between hydro-sedimentological and ecotoxicological processes. Understanding of the ecotoxicological consequences and associated risks to aquatic wildlife associated with hydraulic events can provide critical information to regulatory bodies or managing authorities. Specifically, it will aid in assessing risks associated with current management practices and will aid in developing more sustainable future management practices for waterways or harbours. Therefore, a combined experimental methodology between hydraulic engineers and ecotoxicologists was developed to investigate the ecological and toxicological relevance of sediment re-suspension and transport during erosion. An overview of this methodology is given in the present paper.

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.