Progress, applications, and challenges in high-throughput effect-directed analysis for toxicity driver identification - is it time for HT-EDA?

The rapid increase in the production and global use of chemicals and their mixtures has raised concerns about their potential impact on human and environmental health. With advances in analytical techniques, in particular, high-resolution mass spectrometry (HRMS), thousands of compounds and transformation products with potential adverse effects can now be detected in environmental samples. However, identifying and prioritizing the toxicity drivers among these compounds remain a significant challenge. Effect-directed analysis (EDA) emerged as an important tool to address this challenge, combining biotesting, sample fractionation, and chemical analysis to unravel toxicity drivers in complex mixtures. Traditional EDA workflows are labor-intensive and time-consuming, hindering large-scale applications. The concept of high-throughput (HT) EDA has recently gained traction as a means of accelerating these workflows. Key features of HT-EDA include the combination of microfractionation and downscaled bioassays, automation of sample preparation and biotesting, and efficient data processing workflows supported by novel computational tools. In addition to microplate-based fractionation, high-performance thin-layer chromatography (HPTLC) offers an interesting alternative to HPLC in HT-EDA. This review provides an updated perspective on the state-of-the-art in HT-EDA, and novel methods/tools that can be incorporated into HT-EDA workflows. It also discusses recent studies on HT-EDA, HT bioassays, and computational prioritization tools, along with considerations regarding HPTLC. By identifying current gaps in HT-EDA and proposing new approaches to overcome them, this review aims to bring HT-EDA a step closer to monitoring applications.

Dataset on target chemical and bioassay analysis-Exploring contaminants of emerging concern in a low mountain river of central Germany.

Chemical pollution of the aquatic environment is nowadays characterised by increasing levels of anthropogenic organic compounds at low concentrations and is recognised as one of the main drivers of the deteriorated ecological state of European waterbodies. To improve the understanding of the impact of chemical pollution in surface waters, a combined approach of chemical and bioanalytical testing is considered necessary for effective ecologically oriented water management. For this dataset, six 25-L water samples were collected at six sampling sites along the Holtemme River in Central Germany using large-volume solid phase extraction. All samples were analysed by targeted high-resolution liquid chromatography-mass spectrometry (LC-MS) and a selected bioanalytical test battery using effect-based methods. These methods included cytotoxicity assessment, several mechanism-specific CALUXⓇ tests to identify endocrine and oxidative stress-related effects and the fish embryo acute toxicity test to investigate (sub)lethal effects in the model species Danio rerio. This approach provided a dataset that offers a longitudinal characterisation of the chemical pollution and ecotoxicological impacts. The combination of chemical analysis and effect-based analysis is valuable for future studies as it will help researchers, risk assessors and authorities to identify hot spots of chemical pollution, monitor environmental quality standards and recommend mitigation strategies.

Impact of chemical pollution on threatened marine mammals: A systematic review.

Marine mammals, due to their long life span, key position in the food web, and large lipid deposits, often face significant health risks from accumulating contaminants. This systematic review examines published literature on pollutant-induced adverse health effects in the International Union for Conservation of Nature (IUCN) red-listed marine mammal species. Thereby, identifying gaps in literature across different extinction risk categories, spatial distribution and climatic zones of studied habitats, commonly used methodologies, researched pollutants, and mechanisms from cellular to population levels. Our findings reveal a lower availability of exposure-effect data for higher extinction risk species (critically endangered 16%, endangered 15%, vulnerable 66%), highlighting the need for more research. For many threatened species in the Southern Hemisphere pollutant-effect relationships are not established. Non-destructively sampled tissues, like blood or skin, are commonly measured for exposure assessment. The most studied pollutants are POPs (31%), metals (30%), and pesticides (17%). Research on mixture toxicity is scarce while pollution-effect studies primarily focus on molecular and cellular levels. Bridging the gap between molecular data and higher-level effects is crucial, with computational approaches offering a high potential through in vitro to in vivo extrapolation using (toxico-)kinetic modelling. This could aid in population-level risk assessment for threatened marine mammals.

A plea for the integration of Green Toxicology in sustainable bioeconomy strategies - Biosurfactants and microgel-based pesticide release systems as examples.

A key aspect of the transformation of the economic sector towards a sustainable bioeconomy is the development of environmentally friendly alternatives for hitherto used chemicals, which have negative impacts on environmental health. However, the implementation of an ecotoxicological hazard assessment at early steps of product development to elaborate the most promising candidates of lowest harm is scarce in industry practice. The present article introduces the interdisciplinary proof-of-concept project GreenToxiConomy, which shows the successful application of a Green Toxicology strategy for biosurfactants and a novel microgel-based pesticide release system. Both groups are promising candidates for industrial and agricultural applications and the ecotoxicological characterization is yet missing important information. An iterative substance- and application-oriented bioassay battery for acute and mechanism-specific toxicity within aquatic and terrestrial model species is introduced for both potentially hazardous materials getting into contact with humans and ending up in the environment. By applying in silico QSAR-based models on genotoxicity, endocrine disruption, skin sensitization and acute toxicity to algae, daphnids and fish, individual biosurfactants resulted in deviating toxicity, suggesting a pre-ranking of the compounds. Experimental toxicity assessment will further complement the predicted toxicity to elaborate the most promising candidates in an efficient pre-screening of new substances.

Comparative toxicity assessment of in situ burn residues to initial and dispersed heavy fuel oil using zebrafish embryos as test organisms.

In situ burning (ISB) is discussed to be one of the most suitable response strategies to combat oil spills in extreme conditions. After burning, a highly viscous and sticky residue is left and may over time pose a risk of exposing aquatic biota to toxic oil compounds. Scientific information about the impact of burn residues on the environment is scarce. In this context, a comprehensive ISB field experiment with approx. 1000L IFO 180 was conducted in a fjord in Greenland. The present study investigated the toxicity of collected ISB residues to early life stages of zebrafish (Danio rerio) as a model for potentially exposed pelagic organisms. The toxicity of ISB residues on zebrafish embryos was compared with the toxicity of the initial (unweathered) IFO 180 and chemically dispersed IFO 180. Morphological malformations, hatching success, swimming behavior, and biomarkers for exposure (CYP1A activity, AChE inhibition) were evaluated in order to cover the toxic response on different biological organization levels. Across all endpoints, ISB residues did not induce greater toxicity in zebrafish embryos compared with the initial oil. The application of a chemical dispersant increased the acute toxicity most likely due to a higher bioavailability of dissolved and particulate oil components. The results provide insight into the adverse effects of ISB residues on sensitive life stages of fish in comparison with chemical dispersant application.

Receptor-mediated estrogenicity of native and chemically dispersed crude oil determined using adapted microscale reporter gene assays.

Endocrine disrupting compounds (EDCs) emerged as a major concern for water quality in the last decade and have been studied extensively since. Besides typical natural and synthetic estrogens also petroleum product compounds such as some PAHs have been identified as potential EDCs, revealing endocrine disruption to be a relevant mode of action for crude oil toxicity. Hence, in the context of a comprehensive retro- or prospective risk assessment of oil spills the implementation of mechanism-specific toxicity such as endocrine disruption is of high importance. To evaluate the exposure risk for the aquatic biota, research focuses on water-soluble fractions underlying an oil slick that could be simulated via water-accommodated fractions (WAF). Against this background human (ERα-CALUX®) and yeast based (A-YES®) reporter gene bioassays were successfully optimized for the application in estrogenicity evaluation of the water-accommodated fraction (WAF) from a crude oil. Combining different approaches, the estrogenicity of the WAFs from a naphthenic North Sea crude oil was tested with and without the addition of a chemical dispersant addressing specific aspects of estrogenicity including the influence of biotransformation capacities and different salinity conditions. Both the WAF free from droplets (LEWAF) as well as the chemically dispersed WAF (CEWAF) gave indications of an ER-mediated estrogenicity with much stronger ERα agonists in the CEWAF treatment. Resulting estradiol equivalents of the WAFs were above the established effect-based trigger values for both bioassays. Results indicate that the dispersant rather increased the fraction of ER-activating crude oil compounds instead of interacting with the receptor itself. Only slight changes in estrogenic responses were observed when cells capable of active metabolism (T47D) were used instead of cells without endogenous metabolism (U2-OS) in the recombinant ER transactivation CALUX assay. With the yeast cells a higher estrogenic activity was observed in the experiments under elevated salinity conditions (6‰), which was in contrast to previous expectations due to typical decrease in dissolved PAH fraction with increasing salinity (salting-out effect) but might be related to increased cell sensitivity.

Combining Different In Vitro Bioassays to Evaluate Genotoxicity of Water-Accommodated Fractions from Petroleum Products.

Genotoxicity assessment is of high relevance for crude and refined petroleum products, since oil compounds are known to cause DNA damage with severe consequences for aquatic biota as demonstrated in long-term monitoring studies. This study aimed at the optimization and evaluation of small-scale higher-throughput assays (Ames fluctuation, micronucleus, Nrf2-CALUX®) covering different mechanistic endpoints as first screening tools for genotoxicity assessment of oils. Cells were exposed to native and chemically dispersed water-accommodated fractions (WAFs) of three oil types varying in their processing degree. Independent of an exogenous metabolic activation system, WAF compounds induced neither base exchange nor frame shift mutations in bacterial strains. However, significantly increased chromosomal aberrations in zebrafish liver (ZF-L) cells were observed. Oxidative stress was indicated for some treatments and was not correlated with observed DNA damage. Application of a chemical dispersant increased the genotoxic potential rather by the increased bioavailability of dissolved and particulate oil compounds. Nonetheless, the dispersant induced a clear oxidative stress response, indicating a relevance for general toxic stress. Results showed that the combination of different in vitro assays is important for a reliable genotoxicity assessment. Especially, the ZF-L capable of active metabolism and DNA repair seems to be a promising model for WAF testing.

Differences in biomarker and behavioral responses to native and chemically dispersed crude and refined fossil oils in zebrafish early life stages.

Petroleum products including crude oils and refined distillates are unique environmental pollutants consisting of thousands of compounds with varying physical-chemical properties and resulting toxicity for aquatic biota. Hence, for a reliable risk assessment individual petroleum product toxicity profiles are needed. Furthermore, the influence of oil spill response strategies like the application of chemical dispersants has to be implemented. The present study addressed the toxicity of water-accommodated fractions (WAFs) of two different oil types on fish early life stages on different biological organization levels in the laboratory model species Danio rerio. Experiments with a 3rd generation dispersant used in loading rated resembling the exposure in experiments with chemically dispersed oils were included, enabling a direct comparability of results. This approach is of high importance as especially the investigation of dispersant toxicity in relevant exposure concentrations is rather scarce. Zebrafish embryos were exposed to different WAFs shortly after and up to 120 hour post fertilization (hpf). Besides phenotypic effects including edema and spine deformations, reduced responses to dark stimuli, increased CYP1A activity and marginal AChE inhibition were observed in sublethal effect concentrations. Both oil types had varying strength of toxicity, which did not correlate with corresponding chemical analysis of target PAHs. Chemically dispersed oils induced stronger acute toxicity in zebrafish embryos compared to native (initial) oil exposure, which was further reflected by very low exposure concentrations for biomarker endpoints. Based on a comparison to the dispersant alone, a higher toxicity of dispersed oils was related to a combination of dispersant toxicity and an elevated crude oil compound bioavailability, due to dispersion-related partitioning kinetics. In contrast to LEWAF and CEWAF neither typical morphological effects nor mechanism-specific toxicity were observed for the dispersant alone, indicating narcosis as the responsible cause of effects.

Mechanism-specific and whole-organism ecotoxicity of mono-rhamnolipids.

Biosurfactants like rhamnolipids are promising alternatives to chemical surfactants in a range of applications. A wider use requires an analysis of their environmental fate and their ecotoxicological potential. In the present study mono-rhamnolipids produced by a recombinant Pseudomonas putida strain were analyzed using the Green Toxicology concept for acute and mechanism-specific toxicity in an ecotoxicological test battery. Acute toxicity tests with the invertebrate Daphnia magna and with zebrafish embryos (Danio rerio) were performed. In addition, microbial and fungicidal effectiveness was investigated. Mutagenicity of the sample was tested by means of the Ames fluctuation assay. A selected mono-rhamnolipid was used for model simulations regarding mutagenicity and estrogenic activity. Our results indicate that mono-rhamnolipids cause acute toxicity to daphnids and zebrafish embryos comparable to or even lower than chemical surfactants. Rhamnolipids showed very low toxicity to the germination of Aspergillus niger spores and the growth of Candida albicans. No frameshift mutation or base substitutions were observed using the Ames fluctuation assay with the two tester strains TA98 and TA100. This result was confirmed by model simulations. Likewise it was computed that rhamnolipids have no estrogenic potential. In conclusion, mono-rhamnolipids are an environmental friendly alternative to chemical surfactants as the ecotoxicological potential is low.