Mechthild Schmitt-Jansen

Mixture Toxicity Revisited from a Toxicogenomic Perspective

The advent of new genomic techniques has raised expectations that central questions of mixture toxicology such as for mechanisms of low dose interactions can now be answered. This review provides an overview on experimental studies from the past decade that address diagnostic and/or mechanistic questions regarding the combined effects of chemical mixtures using toxicogenomic techniques. From 2002 to 2011, 41 studies were published with a focus on mixture toxicity assessment. Primarily multiplexed quantification of gene transcripts was performed, though metabolomic and proteomic analysis of joint exposures have also been undertaken. It is now standard to explicitly state criteria for selecting concentrations and provide insight into data transformation and statistical treatment with respect to minimizing sources of undue variability. Bioinformatic analysis of toxicogenomic data, by contrast, is still a field with diverse and rapidly evolving tools. The reported combined effect assessments are discussed in the light of established toxicological dose-response and mixture toxicity models. Receptor-based assays seem to be the most advanced toward establishing quantitative relationships between exposure and biological responses. Often transcriptomic responses are discussed based on the presence or absence of signals, where the interpretation may remain ambiguous due to methodological problems. The majority of mixture studies design their studies to compare the recorded mixture outcome against responses for individual components only. This stands in stark contrast to our existing understanding of joint biological activity at the levels of chemical target interactions and apical combined effects. By joining established mixture effect models with toxicokinetic and -dynamic thinking, we suggest a conceptual framework that may help to overcome the current limitation of providing mainly anecdotal evidence on mixture effects. To achieve this we suggest (i) to design studies to establish quantitative relationships between dose and time dependency of responses and (ii) to adopt mixture toxicity models. Moreover, (iii) utilization of novel bioinformatic tools and (iv) stress response concepts could be productive to translate multiple responses into hypotheses on the relationships between general stress and specific toxicity reactions of organisms.

What contributes to the sensitivity of microalgae to triclosan

Differential sensitivities of microalgae to triclosan have been reported, which may have significant implications for environmental risk assessment of this widely used biocide. Therefore, the aim of this study was to derive a mechanistic understanding of varying microalgal sensitivity to this substance. The toxicity of triclosan was evaluated using microalgal systems varying in biological complexity, exposure time and systematic position (a synchronized culture of the chlorophyte Scenedesmus vacuolatus, a diatom Nitzschia palea cultivated in suspension as well as attached to surfaces and periphyton communities). The results revealed (1) differences in sensitivity of the selected microalgal systems of three orders of magnitude and (2) highest sensitivity of the chlorophyte to triclosan in the range of environmental concentrations. To investigate algal sensitivity to triclosan in more detail, bioavailability was considered by investigating suspended and attached living algae. Differences in the generation time (in comparison to test duration) of the species were addressed by evaluating and modeling concentration-time-effect relationships. However, varying sensitivities of the selected microalgal systems remained unexplained. Comparison of species-specific toxic responses to calculated effect concentrations, derived from quantitative relationships for narcosis and uncoupling mode-of-action, leads us to the conclusion that triclosan may address multiple target sites in different microalgal species.

How to confirm identified toxicants in effect-directed analysis

AbstractDue to the production and use of a multitude of chemicals in modern society, waters, sediments, soils and biota may be contaminated with numerous known and unknown chemicals that may cause adverse effects on ecosystems and human health. Effect-directed analysis (EDA), combining biotesting, fractionation and chemical analysis, helps to identify hazardous compounds in complex environmental mixtures. Confirmation of tentatively identified toxicants will help to avoid artefacts and to establish reliable cause–effect relationships. A tiered approach to confirmation is suggested in the present paper. The first tier focuses on the analytical confirmation of tentatively identified structures. If straightforward confirmation with neat standards for GC–MS or LC–MS is not available, it is suggested that a lines-of-evidence approach is used that combines spectral library information with computer-based structure generation and prediction of retention behaviour in different chromatographic systems using quantitative structure–retention relationships (QSRR). In the second tier, the identified toxicants need to be confirmed as being the cause of the measured effects. Candidate components of toxic fractions may be selected based, for example, on structural alerts. Quantitative effect confirmation is based on joint effect models. Joint effect prediction on the basis of full concentration–response plots and careful selection of the appropriate model are suggested as a means to improve confirmation quality. Confirmation according to the Toxicity Identification Evaluation (TIE) concept of the US EPA and novel tools of hazard identification help to confirm the relevance of identified compounds to populations and communities under realistic exposure conditions. Promising tools include bioavailability-directed extraction and dosing techniques, biomarker approaches and the concept of pollution-induced community tolerance (PICT). FigureToxicity confirmation in EDA as a tiered approach

Predicting and observing responses of algal communities to photosystem ii‐herbicide exposure using pollution‐induced community tolerance and species‐sensitivity distributions

Various test strategies are in use in ecotoxicology to assess the potential risks of toxicants on aquatic communities. The species-sensitivity distribution concept (SSD) works by arranging single-species laboratory test data in a cumulative frequency distribution. The pollution-induced community tolerance concept (PICT) uses observable community responses by measuring increases in community tolerance caused by the replacement of sensitive species after exposure. The aim of this study was to compare these two concepts in assessing the effects of three herbicides. Atrazine, prometryn, and isoproturon were found to increase community tolerance by a factor up to six. Atrazine increased community tolerance only at higher test concentrations (0.125 mg L(-1)). Species-sensitivity distributions correspond well to community responses: The median effective concentrations (EC50s) of untreated periphyton communities tested covered 55 to 65% of affected species represented in the SSD. The sensitivities of tolerant algal communities shifted to the right end of the SSDs. In the microcosm experiments, higher test concentrations affected biomass, species numbers, and community structure. Community tolerance could not be induced any further, suggesting that these concentrations represent a maximum of functional redundancy of a functional group. At higher concentrations, even the least-sensitive species are affected. These results can be interpreted as a confirmation of the SSD concept by observed algal community responses, when applied to photosystem II (PSII)-inhibiting herbicides.

Assessing the Impact of Multiple Stressors on Aquatic Biota: The Receptor’s Side Matters

Aquatic ecosystems are confronted with multiple stress factors. Current approaches to assess the risk of anthropogenic stressors to aquatic ecosystems are developed for single stressors and determine stressor effects primarily as a function of stressor properties. The cumulative impact of several stressors, however, may differ markedly from the impact of the single stressors and can result in nonlinear effects and ecological surprises. To meet the challenge of diagnosing and predicting multiple stressor impacts, assessment strategies should focus on properties of the biological receptors rather than on stressor properties. This change of paradigm is required because (i) multiple stressors affect multiple biological targets at multiple organizational levels, (ii) biological receptors differ in their sensitivities, vulnerabilities, and response dynamics to the individual stressors, and (iii) biological receptors function as networks, so that actions of stressors at disparate sites within the network can lead via indirect or cascading effects, to unexpected outcomes.

Active bio-monitoring of contamination in aquatic systems—An in situ translocation experiment applying the PICT concept

The environmental risk assessment of toxicants is often derived from chemical monitoring, based on single species tests performed in the laboratory. However, to provide ecologically relevant information, community approaches are required. The aim of this study was to causally link prometryn exposure to community-level effects in complex field situations and to identify response times of adaptation to pollution and recovery from pollution. For this reason sensitivity shifts in communities were detected and related to structural changes within the periphyton community. Furthermore, it was intended to illustrate the possibility of a combined approach of community translocation and sensitivity assessment for active monitoring of polluted sites. Periphyton was grown at a reference (R) and at a polluted (P) site of the river Elbe basin for 26 days, was subsequently transferred from the polluted site to the reference site and vice versa. Sensitivity of communities to prometryn was determined according to the pollution-induced community tolerance (PICT)-concept in short-term tests by measuring photosynthesis inhibition and was related to structural changes in algal class and diatom species composition. Exposure to prometryn was determined using polar organic integrative samplers (POCIS), giving time-weighted average concentrations. Environmental concentrations of prometryn were significantly higher at the polluted site compared to the reference site. Communities grown at the polluted site showed a higher tolerance to prometryn in comparison to the reference site. 17 Days after the translocation to the reference site, EC(50) decreased 2-fold compared to the non-translocated P-community of the same age. By contrast, EC(50) of the community grown at the reference site was 5 times higher after 17 days exposure at the polluted site. Furthermore, P-R communities were less sensitive to prometryn (higher EC(50)) than R-P communities, 24 days after translocation. These changes in sensitivity to prometryn were consistent with changes in species composition and clearly indicate that the exposure history of communities is defining the time-response of recovery and adaptation. In conclusion, the PICT-concept is shown to be a suitable tool for analysis of recovery and adaptation processes of communities under natural conditions. Therefore, it improves the link between cause and effect in field situations. In situ translocation studies provide an ecological relevant assessment of pesticide effects under field conditions and could be used as a diagnostic tool in active monitoring for decision-making frameworks as used in the implementation of the European Water Framework Directive (WFD).

Identification of a phytotoxic photo-transformation product of diclofenac using effect-directed analysis

The pharmaceutical diclofenac (DCF) is released in considerably high amounts to the aquatic environment. Photo-transformation of DCF was reported as the main degradation pathway in surface waters and was found to produce metabolites with enhanced toxicity to the green algae Scenedesmus vacuolatus. We identified and subsequently confirmed 2-[2-(chlorophenyl)amino]benzaldehyde (CPAB) as a transformation product with enhanced toxicity using effect-directed analysis. The EC(50) of CPAB (4.8 mg/L) was a factor of 10 lower than that for DCF (48.1 mg/L), due to the higher hydrophobicity of CPAB (log K(ow) = 3.62) compared with DCF (log D(ow) = 2.04) at pH 7.0.

Flow cytometry as a tool to study phytotoxic modes of action

The objective of the present investigation was to provide a diagnostic tool for the analysis of phytotoxic interactions between environmental contaminants and algae by application of flow cytometry. Therefore, an experimental design was developed consisting of synchronized Scenedesmus vacuolatus cell populations at defined cell-cycle stages, short-term exposure against different inhibitors with known molecular targets, and fluorochrome labeling of different metabolic processes. To discriminate cells with compromised metabolic processes from intact and metabolically inactive cells, references for every fluorochrome were defined using control and heat-treated populations. The experimental results showed that fluorescence markers are able to detect disturbance of specific cellular characteristics, such as membrane integrity, chlorophyll synthesis, and degradation. A differentiation of impacts on specific metabolic process caused by the reference inhibitors in concentration-dependent patterns could be seen using flow-cytometric fluorochrome analysis. These findings were compared with effects observed for N-phenyl-2-naphthylamine (PNA), a sediment contaminant of high phytotoxicity but unclear mode of action. Rhodamine 123 and cyano-ditolyl tetrazolium chloride detected significant metabolic changes for relevant exposures against PNA, thus pointing to compromised mitochondrial activity and changes in membrane potential as causes of phytotoxicity.

In situ cage experiments with Potamopyrgus antipodarum - a novel tool for real life exposure assessment in freshwater ecosystems.

In situ experiments are an important tool within ecotoxicological research but there is a lack of suitable methodologies especially for freshwater invertebrate species. Within this study, a novel in situ methodology with Potamopyrgus antipodarum was developed. Snails were inserted into cages, made of Plexiglas measuring 7 × 9 × 7 cm(3) and fixed with stainless steel pins into the sediment at the relevant sampling sites. During the experiment physico-chemical properties of the water and concentrations of metals, PAHs and PCBs were measured in the sediment. The growth and survival of the snails was not affected, but the reproduction increased significantly at one of the most polluted sites. The increase in reproduction was neither correlated with physico-chemical parameters, nor with the concentrations of the different compounds, but maybe related to certain groups of estrogenic compounds. The study demonstrates the excellent applicability of this novel in situ test.

Bioassays with Unicellular Algae: Deviations from Exponential Growth and Its Implications for Toxicity Test Results

Growth assays with unicellular green algae are an established tool in ecotoxicological effect assessment for chemicals and environmental samples. From an ecological perspective it seems appropriate to use the growth rate as a process variable rather than a measure of biomass gain for calculating inhibitory effects of contaminants. The notion of simple exponential growth for the description of the population increase in undisturbed suspension cultures of unicellular green algae, however, seems to be an oversimplification. Experimental findings describe the increase in biomass, cell number, the development of cell volume distributions of populations, and the relationship between cell size and chlorophyll content for individual cells over one generation at a time resolution of 2-h intervals. It was observed that algal populations of Desmodesmus subspicatus show a time pattern of cell size growth; the average cell volume increases about sixfold, without corresponding increase in population size. This is followed by a distinct cell division phase with little gain in biomass. This synchronous growth behavior despite continuous illumination may be explained by the multiple fission characteristic of unicellular green algae which is an adaptation to cyclic light-dark changes in the environment. It might be controlled by an independent cell cycle clock. For routine regulatory testing fluorescence-based measurements rather than cell counting minimizes the confounding effect on toxicity determination. For investigations of time-dependent effects, e.g., by pulsed exposure, an alternative mechanistically based growth function for unicellular algae is proposed that accommodates for the observed growth pattern.