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Ecotoxicological assays have traditionally focused on the effects of chemicals at the individual level by exploiting mortality and reproduction as endpoints. Although these two parameters are ecologically relevant, they rarely provide information regarding the elemental toxic mechanisms. Obviously, the number of xenobiotics used has been rapidly increased. Thus, any established measurement that shortens the actual outcome and, simultaneously provides information about toxic mechanisms is desirable. This research focused on the study of oxidative stress response as a biomarker in the eukaryotic model organism, Saccharomyces cerevisiae. For this, yeast cells were exposed to a set of selected environmentally relevant chemicals via different approaches, including cellular diagnostics, gene expression analysis and chemo-genetic screening. The results demonstrated that at the cellular level, model organisms reacted to different chemicals in distinct manner. For each xenobiotic, the correlation between toxic response of molecular and cellular levels are presented. Namely, the expression of target genes after chemical exposure affected the cellular alteration as evidenced by an elevated level of superoxide dismutase and a reduced amount of glutathione. Furthermore, the results derived from chemo-genetic screening, in which mutants lacking of gene of interest were employed, exhibited more susceptibility to test chemicals in comparison to the wildtype. The response of oxidative stress upon chemical exposure in budding yeast from this study is potentially useful for an establishment of a proper bio-test system which can eventually be linked to adverse effects at an individual level in higher eukaryotes. [Display omitted] • Six selected test chemicals induce oxidative stress in yeasts. • The concentration leading to the state of oxidative stress is lower than cytotoxic-induced concentration. • GSH content can be used as a potential oxidative stress biomarker. • Oxidative stress-based testing model is more sensitive than cytotoxicity testing. [ABSTRACT FROM AUTHOR]

The demand for biofuels increases due to concerns regarding greenhouse gas emissions and depletion of fossil oil reserves. Many substances identified as potential biofuels are solvents or already used as flavors or fragrances. Although humans and the environment may be readily exposed little is known regarding their (eco)toxicological effects. In this study, the three potential biofuels ethyl levulinate (EL), 2-methyltetrahydrofuran (2-MTHF) and 2-methylfuran (2-MF) were investigated for their acute embryo toxicity and teratogenicity using the fish embryo toxicity (FET) test to identify unknown hazard potentials and to allow focusing further research on substances with low toxic potentials. In addition, two fossil fuels (diesel and gasoline) and an established biofuel (rapeseed oil methyl ester) were investigated as references. The FET test is widely accepted and used in (eco)toxicology. It was performed using the zebrafish Danio rerio , a model organism useful for the prediction of human teratogenicity. Testing revealed a higher acute toxicity for EL (LC 50 : 83 mg/L) compared to 2-MTHF (LC 50 : 2980 mg/L), 2-MF (LC 50 : 405 mg/L) and water accommodated fractions of the reference fuels including gasoline (LC 50 : 244 mg DOC/L). In addition, EL caused a statistically significant effect on head development resulting in elevated head lengths in zebrafish embryos. Results for EL reduce its likelihood of use as a biofuel since other substances with a lower toxic potential are available. The FET test applied at an early stage of development might be a useful tool to avoid further time and money requiring steps regarding research on unfavorable biofuels. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In a former study, a German lignite extract exhibited toxicity to Danio rerio and Caenorhabditis elegans and was shown to have mutagenic and dioxin-like activity. Besides the comparatively low content of known toxic polycyclic aromatic hydrocarbons (PAH), highly intensive peaks of m/z 274 and m/z 324 were observed during the chromatographic analysis. These compounds are assumed to be alkylated chrysenes and picenes (3,3,7-trimethyl-1,2,3,4-tetrahydrochrysene, 1,2-(1′-isopropylpropano)-7-methylchrysene and an isomer of the latter, 1,2,9-trimethyl-1,2,3,4-tetrahydropicene and 2,2,9-trimethyl-1,2,3,4-tetrahydropicene). These compounds are intermediates in the diagenetic formation of chrysene and picene from triterpenoids. Due to their general high abundance in lignites and the toxicity observed for the lignite extract, the mechanism-specific toxicity and bioavailability of these compounds were investigated in the present study using the approach of effect-directed analysis. After the separation of the compounds from other PAH, their mutagenic activity (Ames Fluctuation test) and dioxin-like activity (EROD activity) were studied. Both, mutation induction factor (up to 2.9 ± 2.7) and dioxin-like activity (Bio-TEQ of 224 ± 75 pg/g; represents the amount (pg) 2,3,7,8-tetrachlorodibenzo- p -dioxin per g coal that would provoke the same toxic effect) were rather low. Bioavailability estimated by the bioaccumulation test with Lumbriculus variegatus was also very limited. Based on the obtained results, the environmental risk of the highly abundant alkylated chrysenes and picenes in lignites is concluded to be low. [ABSTRACT FROM AUTHOR]

Investigations of the bioavailability and toxicity of polycyclic aromatic compounds (PAC) have rarely considered the heterogeneity of coals and the impact of more polar PAC besides polycyclic aromatic hydrocarbons (PAH). Earlier, we investigated the toxicity of eight heterogeneous coals and their extracts. In the present study, the hazard potential with respect to mechanism-specific toxicity of polar fractions of dichloromethane extracts from coals was studied. Polar extract fractions of all coal types except for anthracite induced EROD activity (determined in RTL-W1 cells), independent of coal type (Bio-TEQs between 23 ± 16 and 52 ± 22 ng/g). The polar fractions of all bituminous coal extracts revealed mutagenic activity (determined using the Ames Fluctuation test). No significant mutation induction was detected for the polar extract fractions from the lignite, sub-bituminous coal and anthracite samples, which indicates a higher dependency on coal type for polar PAC here. Additionally, information on bioavailability was derived from a bioaccumulation test using the deposit-feeding oligochaete Lumbriculus variegatus which was exposed for 28 days to ground coal samples. Despite the high toxic potential of most coal extracts and a reduced biomass of Lumbriculus in bituminous coal samples, bioaccumulation of PAH and mortality after 28 days were found to be low. Limited bioaccumulation of PAH (up to 3.6 ± 3.8 mg/kg EPA-PAH) and polar PAC were observed for all coal samples. A significant reduction of Lumbriculus biomass was observed in the treatments containing bituminous coals (from 0.019 ± 0.004 g to 0.046 ± 0.011 g compared to 0.080 ± 0.025 g per replicate in control treatments). We conclude that bioavailability of native PAC from coals including polar PAC is low for all investigated coal types. In comparison to lignite, sub-bituminous coals and anthracite, the bioavailability of PAC from bituminous coals is slightly increased. [ABSTRACT FROM AUTHOR]

As part of the risk assessment process within REACh, prior to manufacturing and distribution of chemical substances their (eco)toxicological impacts have to be investigated. The fish embryo toxicity test (FET) with the zebrafish Danio rerio has gained a high significance as an in vitro alternative to animal testing in (eco)toxicology. However, for hydrophobic organic chemicals it remains a technical challenge to ensure constant freely dissolved concentration at the maximum exposure level during such biotests. Passive dosing with PDMS silicone was thus applied to control the freely dissolved concentration of ten PAHs at their saturation level in the FET. The experiments gave repeatable results, with the toxicity of the PAHs generally increasing with the maximum chemical activities of the PAHs. HPLC analysis confirmed constant exposure at the saturation level. In additional experiments, fish embryos without direct contact to the silicone surface showed similar mortalities as those exposed with direct contact to the silicone. Silicone oil overlaying the water phase as a novel passive dosing phase had no observable effects on the development of the fish embryos until hatching. This study provides further data to support the close relationship between the chemical activity and the toxicity of hydrophobic organic compounds. Passive dosing from PDMS silicone enabled reliable toxicity testing of (highly) hydrophobic substances at aqueous solubility, providing a practical way to control toxicity exactly at the maximum exposure level. This approach is therefore expected to be useful as a cost-effective initial screening of hydrophobic chemicals for potential adverse effects to freshwater vertebrates. [ABSTRACT FROM AUTHOR]

Coals contain native polycyclic aromatic compounds (PACs), which include polycyclic aromatic hydrocarbons (PAHs), and heterocyclic aromatic compounds (NSO-PACs) in considerably varying amounts up to 2500 mg/kg. Whereas PAC bioavailability and toxicity from coals are generally considered to be low, few studies have considered potential variations arising from the composition of different coal types including native PAC content. In the present study, fine particles of different coal types exhibiting variable properties were systematically investigated regarding their PAC bioavailability. PAH content reached up to 79 mg/ kg EPA-PAH and 865 mg/kg total PAH. Determination of the toxic potential of extracted PACs in bioassays showed inhibition of Caenorhabditis elegans reproduction (up to 94%) and increased mortality of Danio rerio embryos (up to 100%) after exposure to extracts from lignite, sub-bituminous, and bituminous coals. Anthracite extracts showed no effects. Contact assays using whole coal samples revealed no toxicity to D. rerio embryos in any of the coal samples, suggesting low bioavailability of PACs. In contrast, C. elegans reproduction was inhibited by direct coal contact; however, the observed toxicity probably resulted from other coal effects. The results suggest that despite the high toxic potential of PACs present, their bioavailability from different coal types is very limited and independent of coal properties and native PAH content. [ABSTRACT FROM AUTHOR]

Abstract: The zebrafish embryo test is a widely used bioassay for the testing of chemicals, effluents and other types of environmental samples. Oxygen depletion in the testing of sediments and effluents is especially important and may be a confounding factor in the interpretation of apparent toxicity. In order to identify oxygen levels critical to early developmental stages of zebrafish, oxygen consumption of zebrafish embryos between 0 and 96h post-fertilization, minimum oxygen levels required by the embryos for survival as well as the effects of oxygen depletion following exposure to model sediments were determined. No significant effects on zebrafish embryo development were observed for oxygen concentrations between 7.15 and 3.33mg/L, whereas at concentrations between 3.0and 2.0mg/L minor developmental retardations were observed, yet without any pathological consequences. Oxygen concentrations lower than 0.88mg/L were 100% lethal. In the sediment contact tests with zebrafish embryos, native sediments rich in organic materials rapidly developed strongly hypoxic conditions, particularly at the sediment–water interface (0 to 500μm distance to the sediment). [Copyright &y& Elsevier]

Organic total extracts play an important role in soil and sediment risk assessment. Beside a routine application in analytical chemistry, they are used in bio-analytical investigations as a “worst-case scenario” or, e.g., in order to simulate chronic intoxication, and as samples for effect-directed analysis. While theoretically providing highly reliable data and good reproducibility, the whole process of sample handling and extract preparation can lead to extracts that might fail to accurately represent a toxic potential of their corresponding sampling site. This review identifies and discusses the most important possible alterations that have the potential to lead to over and, more often, underestimation of the effectiveness of extracts. Since incorrect data will compromise soil and sediment risk assessment as a whole, results from analytical and bio-analytical investigations of extracts demand cautious interpretation. Reliability of extract testing grows with reproducibility; experiments should therefore be repeated with independent extraction replicates. New or optimized extraction procedures should circumvent the issues mentioned here while being suitable for routine application. [ABSTRACT FROM AUTHOR]

Abstract The usage of pesticides has been steadily increasing over the last decades, and among them herbicides are the most commonly used ones. Despite their main mode of action targeting plant organisms, they can also have adverse effects on non-target animal organisms. In soil ecosystems, earthworms play an important role due to their positive impacts on the soil functioning and they represent good model organisms in soil ecotoxicology. The aim of the present study was to assess the effects of two herbicides on several endpoints at different levels of biological organization in the earthworm Eisenia andrei. Diuron and fluazifop- p -butyl were selected for the investigation and their lethal concentrations were determined: LC 50 48 h: 89.087 μg/cm2 for diuron and 6.167 μg/cm2 for fluazifop- p -butyl. Furthermore, measurements of enzymatic biomarkers (catalase (CAT), acetylcholinesterase (AChE), carboxylesterase (CES) and glutathione S-transferase (GST)), multixenobiotic resistance (MXR) activity and gene expression of antioxidative enzymes (only for fluazifop- p -butyl) were conducted. Enzymatic biomarker responses showed no significant differences compared to the control after the exposure to the investigated herbicides, whereas the MXR activity was significantly inhibited. The gene expression level of superoxide dismutase (sod) and glutathione S-transferase (gst) after fluazifop- p -butyl exposure showed a significant increase. Finally, avoidance behavior in soil was assessed and it was determined that both herbicides caused significant avoidance response. The obtained results show that both investigated herbicides significantly affect earthworms on different levels of biological organization. This emphasizes the importance of comprehensive ecotoxicological assessment of herbicide effects on non-target organisms at all organizational levels. Highlights • Exposure of earthworm Eisenia andrei to herbicides diuron and fluazifop- p -butyl. • Significant effects at both lower and higher level endpoints were observed. • Both herbicides caused a significant inhibition of the MXR activity. • Both herbicides caused a significant avoidance response. • Fluazifop- p -butyl caused an increase in the gene expression level of sod and gst. [ABSTRACT FROM AUTHOR]

The zebrafish as a test organism enables the investigation of effects on a wide range of biological levels from molecular level to the whole-organism level. The use of fish embryos represents an attractive model for studies aimed at understanding toxic mechanisms and the environmental risk assessment of chemicals. In the present study, a zebrafish ( Danio rerio ) in vivo model was employed in order to assess the effects of two commonly used pesticides, the insecticide diazinon and the herbicide diuron, on zebrafish early life stages. Since it was previously established that diazinon and diuron cause effects at the whole-organism level, this study assessed the suborganismic responses to exposure to these pesticides and the enzymatic responses (biochemical level) and the gene expression changes (molecular level) were analyzed. Different exposure scenarios were employed and the following endpoints measured: acetylcholinesterase (AChE), carboxylesterase (CES), ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT) and glutathione peroxidase (GPx) activities; and gene expressions of the corresponding genes: acetylcholinesterase ( ache ), carboxylesterase ( ces2 ), cytochrome P450 ( cyp1a ), glutathione-S-transferase ( gstp1 ), catalase ( cat ), glutathione peroxidase ( gpx1a ) and additionally glutathione reductase ( gsr ). Significant changes at both the biochemical and the molecular level were detected. In addition, different sensitivities of different developmental stages of zebrafish were determined and partial recovery of the enzyme activity 48 h after the end of the exposure was observed. The observed disparity between gene expression changes and alterations in enzyme activities points to the necessity of monitoring changes at different levels of biological organization. Different exposure scenarios, together with a comparison of the responses at the biochemical and molecular level, provide valuable data on the effects of diazinon and diuron on low organizational levels in zebrafish embryos and larvae. [ABSTRACT FROM AUTHOR]

In the past decades, bioassays and whole-organism bioassay have become important tools not only in compliance testing of industrial chemicals and plant protection products, but also in the monitoring of environmental quality. With few exceptions, such test systems are discontinuous. They require exposure of the biological test material in small units, such as multiwell plates, during prolonged incubation periods, and do not allow online read-outs. It is mostly due to these shortcomings that applications in continuous monitoring of, e.g., drinking or surface water quality are largely missing. We propose the use of pipetting robots that can be used to automatically exchange samples in multiwell plates with fresh samples in a semi-static manner, as a potential solution to overcome these limitations. In this study, we developed a simple and low-cost, versatile pipetting robot constructed partly using open-source hardware that has a small footprint and can be used for online monitoring of water quality by means of an automated whole-organism bioassay. We tested its precision in automated 2-fold dilution series and used it for exposure of zebrafish embryos (Danio rerio)–a common model species in ecotoxicology—to cadmium chloride and permethrin. We found that, compared to conventional static or semi-static exposure scenarios, effects of the two chemicals in zebrafish embryos generally occurred at lower concentrations, and analytically verified that the increased frequency of media exchange resulted in a greater availability of the chemical. In combination with advanced detection systems this custom-made pipetting robot has the potential to become a valuable tool in future monitoring strategies for drinking and surface water. [ABSTRACT FROM AUTHOR]

Effluents from wastewater treatment plants (WWTPs) are a major source of estrogenic compounds to the aquatic environment. In the present work, estrogenic activities of effluents from eight municipal WWTPs in Finland were studied. The main objectives of the study were to quantify the concentrations of selected estrogenic compounds, to evaluate their contribution to estrogenic potency and to test the feasibility of the commercial bioassays for wastewater analysis. The effluent samples were analyzed by two in vitro tests, i.e. ERα-CALUX ® and ELISA-E2, and by liquid chromatography mass spectrometry for six estrogenic compounds: estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), 17α-estradiol and bisphenol A (BPA). Estrogenic effects were found in all of the effluent samples with both of the bioassays. The concentrations measured with ELISA-E2 (8.6–61.6 ng/L) were clearly higher but exhibited a similar pattern than those with chemical analysis (E2

Green toxicology is a novel approach increasingly applied for the development of materials and chemicals that are more benign to the environment and human health than their conventional counterparts. It includes predictive eco-toxicological assessments of chemicals during the early developmental process to exclude adverse effects. In the present study, two guanidine zinc catalysts for the ring-opening polymerization of lactide were investigated using eco-toxicological tools. Namely, the fish embryo toxicity assay for teratogenic effects, the ER (α) CALUX assay for endocrine activity and the Ames fluctuation assay for mutagenic potential were applied. Both complexes showed no endocrine activity, mutagenicity or acute aquatic toxicity, however a delayed hatch could be observed, therefore suggesting potential effects on a molecular level. This proof-of-concept study aims to assess the toxicity of guanidine zinc catalysts and is a first step towards the incorporation of toxicological assessments into chemical developmental processes to achieve a sustainable and safe production of catalysts. [Display omitted] • Green Toxicology approach used for assessment of catalysts. • Eco-toxicological tools showed low toxicity of assessed guanidine-zinc catalysts. • No mutagenic potential or endocrine activity observed. • Delayed hatch but no mortality observed in zebrafish larvae. [ABSTRACT FROM AUTHOR]

Pharmaceuticals in surface waters have raised significant concern in recent years for their potential environmental effects. This study identified that at present a total of 477 substances (including 66 metabolites and transformation products) have been analyzed in European surface waters. Around 60% (284) of these compounds belonging to 16 different therapeutic groups were positively detected in one or more of 33 European countries. To conveniently and effectively prioritize potential high-risk compounds, an optimized method that considers the frequency of concentrations above predicted no effects levels was developed on the basis of the traditional method, and it was then used to identify and screen candidate priority pollutants in European surface waters. The results proved the feasibility and advantages of the optimized method. Pharmaceuticals detected in European surface waters were classified into 5 categories (high, moderate, endurable, negligible and safe) depending on their potential environmental effects and the distribution of pharmaceuticals. Circa 9% (45 out of 477) analyzed compounds showed a potential environmental risk to aquatic ecosystems. Among these 45 compounds, 12 compounds were indicated to have high environmental risk in aquatic environments, while 17 and 7 compounds showed moderate and small-scale environmental risks, respectively. Unlabelled Image • An optimized method was developed to identify and screen candidate priority pollutants in surface waters. • Different therapeutic groups were positively detected in many European countries. • 45 compounds showed potential environmental risks in European surface waters. [ABSTRACT FROM AUTHOR]

Abstract Micropollutants, as a serious water pollution issue, raise considerable toxicological concerns, particularly when present as components of complex mixtures. Due to the interactions of environmental pollution components (contaminant), the micropollutant problem is increasingly complex, thus, water quality of organic chemical contamination assessed substance-by-substance might lead to underestimation in aquatic environmental risk assessment. To assess the aquatic environmental risk of micropollutants mixture, a total of 10 organic micropollutants were selected and analysed by an approach of integration of literature data, laboratory experiments and prediction techniques. The experiment results showed that all 10 micropollutants were capable of causing toxicity in zebrafish embryos, aquatic invertebrates and algae with the LC50 (50% lethal concentration) values from 1.14 mg/L to 14.37 mg/L. Triclosan, carbamazepine, diazinon and diuron were the most hazardous compounds in the Danube River and the Rhine River. The artificial mixture presented a strong antagonistic relationship, which demonstrated an independent action (IA) model of the mixture. Based on the observed toxicity data, the risk quotients (RQs) of environmental mixtures of the Danube River and the Rhine River were extrapolated. It can be concluded that the micropollutant mixture may pose a potential risk for aquatic ecosystems with the present environmentally measured concentrations in the Danube River and Rhine River. Mixture risk assessment results suggested that the toxicity of studied chemicals might be induced by dissimilar actions, which is in agreement with the mixture toxicity prediction of the IA model. The observed findings could be useful to establish an overview of the pressures, vision, measures and expectations for hazardous substances pollution, which can help in making to informed decisions to reduce the concentration and bioactive fraction of pollutants. Graphical abstract Unlabelled Image Highlights • All 10 micropollutants were capable of causing toxicity on zebrafish embryos (78characters). • An independent action model of the mixture was found by an integrative approach (81 characters). • The micropollutants pose a potential risk in the Danube River and Rhine River (79 characters). [ABSTRACT FROM AUTHOR]

Abstract Although the effects of pharmaceuticals on aquatic organisms have been widely investigated during the last decades, toxic effects, especially delayed toxicity, during the developmental stage at environmental relevant concentrations were rarely known. In this study, a sensitive assay based on behavioral alterations was used for studying the delayed toxicity during the developmental stage on zebrafish embryos. Eight pharmaceuticals that were frequently detected with concentrations ranging from ng/l to μg/l were screened for this study. Behavioral alterations of zebrafish at 118 hpf (hours post fertilization) after exposing to eight single pharmaceuticals with concentrations in the ranges of environmental detected and their mixtures during embryonic development (2–50 h post fertilization, hpf) were observed. Multiple endpoints, including mortality, hatching rate, swimming speed and angular velocity were evaluated. Results showed that behavioral profile alterations in zebrafish larvae are promising for predicting delayed sublethal effects of chemicals. Delayed hatch was observed at 72 hpf following embryonic exposure to triclosan (1 μg/l) and carbamazepine (100 μg/l) up to 50 hpf. The zebrafish larval locomotor behavior following embryonic exposure to 0.1 μg/l triclosan and 1 μg/l caffeine in the early stages of development (2–50 hpf) was altered. Furthermore, the effects of the mixture of 8 pharmaceuticals each with the highest environmental concentration on larval behavior were observed during embryonic development. Generally, this study showed that the effects of pharmaceuticals singly or their mixtures in surface waters cannot be ignored. Graphical abstract Unlabelled Image Highlights • Behavioral alterations are suitable to show the effects of pharmaceuticals. • 0.1 μg/l triclosan caused accelerated erratic movement in larvae. • Mixture at the environmental-related concentrations caused decreased swimming speed. [ABSTRACT FROM AUTHOR]

Abstract Water pollution risks to human health and the environment are emerging as serious concerns in the European Union and worldwide. With the aim to achieve good ecological and chemical status of all European water bodies, the "European Water Framework Directive" (WFD) was enacted. With the framework, bioanalytical techniques have been recognized as an important aspect. However, there are limitations to the application of bioassays directly for water quality assessment. Such approaches often fail to identify pollutants of concern, since the defined priority and monitored pollutants often fail to explain the observed toxicity. In this study, we integrated an effect-based risk assessment with a zebrafish-based investigation strategy to evaluate water sample extracts and fractions collected from the Danube. Four tiered bioassays were implemented, namely RNA-level gene expression assay, protein-level ethoxyresorufin- O -deethylase (EROD) assay, cell-level micronucleus assay and organism-level fish embryo test (FET). The results show that teratogenicity and lethality during embryonic development might be induced by molecular or cellular damages mediated by the aryl hydrocarbon receptor (AhR) -mediated activity, estrogenic activity and genotoxic activity. With the combination of high-throughput fractionation, this effect-based strategy elucidated the major responsible mixtures of each specific toxic response. In particularly, the most toxic mixture in faction F4, covering a log Kow range from 2.83 to 3.42, was composed by 12 chemicals, which were then evaluated as a designed mixture. Our study applied tiered bioassays with zebrafish to avoid interspecies differences and highlights effect-based approaches to address toxic mixtures in water samples. This strategy can be applied for large throughput screenings to support the main toxic compounds identification in water quality assessment. Graphical abstract Image 1 Highlights • Bioassays at different level of biological complexity. • Minimum sample consumption for large throughput screenings of water samples. • Zebrafish-based bioassays were implemented avoiding interspecies differences. • Toxicity-based assessment of mixtures in surface water. [ABSTRACT FROM AUTHOR]

Abstract In the present study, both bioanalytical and instrumental tools were employed to examine the endocrine-disruptive potentials of water samples, cyanobloom samples, and sediment samples collected from in the northern region of Taihu Lake (China) during cyanobloom season. Results from cell-based bioassays suggested the occurrence of estrogenic, anti-estrogenic, anti-androgenic, and anti-glucocorticogenic activities, while no androgenic and glucocorticogenic activities were observed in the collected samples. Using an UPLC-MS/MS system, 29 endocrine disrupting compounds including seven estrogens, seven androgens, six progestogens, and five adrenocortical hormones and four industrial pollutants were simultaneously detected. 17, 20 and 12 chemicals were detected at least in one of the water samples, cyanobloom samples and sediment samples, respectively. Since both agonistic and antagonistic endocrine-disruptive activities were detected in the present study, commonly used receptor-based in vitro bioassays resulted in net effects, suggesting that the hormone receptor agonistic potentials might be underestimated with this practice. The EDCs detected in cyanobloom samples also highlight the necessity to consider the phytoplankton matrix for understanding the mass fluxes of endocrine disruptors in eutrophic freshwaters and to consider it in monitoring strategies. Graphical abstract Unlabelled Image Highlights • Endocrine disruptors in multiple matrices were systematically examined. • Results from bioassays suggest estrogenic but not androgenic and glucocorticogenic agonists were detected. • Antagonistic effects were widely observed in all the matrices. • Endocrine disruptors were pronouncedly detected in cyanoblooms. [ABSTRACT FROM AUTHOR]

Abstract Identification of hazardous compounds, as the first step of water protection and regulation, is still challenged by the difficulty to establish a linkage between toxic effects and suspected contaminants. Genotoxic compounds are one type of highly relevant toxicants in surface water, which may attack the DNA and lead to cancer in individual organism, or even damaged germ cells to be passed on to future generations. Thus, the establishment of a linkage between genotoxic effects and genotoxicant is important for environmental toxicologists and chemists. For this purpose, in the present study in silico methods were integrated with bioassays, chemical analysis and literature information to identify genotoxicants in surface water. Large volume water samples from 22 sampling sites of the Danube were collected and subjected to biological and chemical analysis. Samples from the most toxic sites (JDS32, JDS44 and JDS63) induced significant genotoxic effects in the micronucleus assay, and two of them caused mutagenicity in the Ames fluctuation assay. Chemical analysis showed that 68 chemicals were detected in these most toxic samples. Literature findings and in silico techniques using the OECD QSAR Toolbox and the ChemProp software package revealed genotoxic potentials for 29 compounds out of 68 targeted chemicals. To confirm the integrative technical data, the micronucleus assay and the Ames fluctuation assay were applied with artificial mixtures of those compounds and the raw water sample extracts. The results showed that 18 chemicals explained 48.5% of the genotoxicity in the micronucleus assay. This study highlights the capability of in silico techniques in linking adverse biological effect to suspicious hazardous compounds for the identification of toxicity drivers, and demonstrates the genotoxic potential of pollutants in the Danube. Graphical abstract Unlabelled Image Highlights • Bioassays of Ames fluctuation and micronucleus cover essential genotoxic endpoints. • In silico techniques reduced complexity of aquatic mixture with low workload. • An artificial mixture explained 48.5% of genotoxicity in the micronucleus assay. [ABSTRACT FROM AUTHOR]

Lake ecosystems are sensitive recorders of environmental changes that provide continuous archives at annual to decadal resolution over thousands of years. The systematic investigation of land use changes and emission of pollutants archived in Holocene lake sediments as well as the reconstruction of contamination, background conditions, and sensitivity of lake systems offer an ideal opportunity to study environmental dynamics and consequences of anthropogenic impact that increasingly pose risks to human well-being. This paper discusses the use of sediment and other lines of evidence in providing a record of historical and current contamination in lake ecosystems. We present a novel approach to investigate impacts from human activities using chemical-analytical, bioanalytical, ecological, paleolimnological, paleoecotoxicological, archeological as well as modeling techniques. This multi-time slice weight-of-evidence (WOE) approach will generate knowledge on conditions prior to anthropogenic influence and provide knowledge to (i) create a better understanding of the effects of anthropogenic disturbances on biodiversity, (ii) assess water quality by using quantitative data on historical pollution and persistence of pollutants archived over thousands of years in sediments, and (iii) define environmental threshold values using modeling methods. This technique may be applied in order to gain insights into reference conditions of surface and ground waters in catchments with a long history of land use and human impact, which is still a major need that is currently not yet addressed within the context of the European Water Framework Directive. [ABSTRACT FROM AUTHOR]

Chemicals in the environment occur in mixtures rather than as individual entities. Environmental quality monitoring thus faces the challenge to comprehensively assess a multitude of contaminants and potential adverse effects. Effect-based methods have been suggested as complements to chemical analytical characterisation of complex pollution patterns. The regularly observed discrepancy between chemical and biological assessments of adverse effects due to contaminants in the field may be either due to unidentified contaminants or result from interactions of compounds in mixtures. Here, we present an interlaboratory study where individual compounds and their mixtures were investigated by extensive concentration-effect analysis using 19 different bioassays. The assay panel consisted of 5 whole organism assays measuring apical effects and 14 cell- and organism-based bioassays with more specific effect observations. Twelve organic water pollutants of diverse structure and unique known modes of action were studied individually and as mixtures mirroring exposure scenarios in freshwaters. We compared the observed mixture effects against component-based mixture effect predictions derived from additivity expectations (assumption of non-interaction). Most of the assays detected the mixture response of the active components as predicted even against a background of other inactive contaminants. When none of the mixture components showed any activity by themselves then the mixture also was without effects. The mixture effects observed using apical endpoints fell in the middle of a prediction window defined by the additivity predictions for concentration addition and independent action, reflecting well the diversity of the anticipated modes of action. In one case, an unexpectedly reduced solubility of one of the mixture components led to mixture responses that fell short of the predictions of both additivity mixture models. The majority of the specific cell- and organism-based endpoints produced mixture responses in agreement with the additivity expectation of concentration addition. Exceptionally, expected (additive) mixture response did not occur due to masking effects such as general toxicity from other compounds. Generally, deviations from an additivity expectation could be explained due to experimental factors, specific limitations of the effect endpoint or masking side effects such as cytotoxicity in in vitro assays. The majority of bioassays were able to quantitatively detect the predicted non-interactive, additive combined effect of the specifically bioactive compounds against a background of complex mixture of other chemicals in the sample. This supports the use of a combination of chemical and bioanalytical monitoring tools for the identification of chemicals that drive a specific mixture effect. Furthermore, we demonstrated that a panel of bioassays can provide a diverse profile of effect responses to a complex contaminated sample. This could be extended towards representing mixture adverse outcome pathways. Our findings support the ongoing development of bioanalytical tools for (i) compiling comprehensive effect-based batteries for water quality assessment, (ii) designing tailored surveillance methods to safeguard specific water uses, and (iii) devising strategies for effect-based diagnosis of complex contamination. [ABSTRACT FROM AUTHOR]

The modes of action of pollutants are diverse, and a common consequences to pollutant exposure is oxidative stress. This phenomenon is caused by an imbalance or disurption in the control of Reactive Oxygen Species (ROS) resulting in an accumulation of free radicals. Oxidative stress may cause damages to the DNA, phospholipids and proteins, and lead to cell death. Due to the possible contribution of oxidative stress to pollutant toxicity, it is valuable to assess its occurrence, role and mechanism. Detection of oxidative stress at low concentrations soon after the onset of exposure can be a sensitive, general marker for contamination. This study aimed at developing and benchmarking a set of novel fluorescence-based procedures to assess the occurrence of oxidative stress in zebrafish larvae (96 hpf) by measuring the antioxidant glutathione (GSH) and general ROS. Zebrafish larvae were exposed to tert -butyl hydroperoxide ( t -BHP). ROS and GSH were made visible by means of specific fluorescent molecular probes in different experimental scenarios. The induction was qualified using microscopy and quantified through photometric measurement. For quantitative assessment, an approach based on homogenized larvae and a non-invasive plate assay were developed. The novel procedures proved suitable for oxidative stress detection. Comparisons of qualitative to quantitative data showed that the orientation of the larvae in the well can influence fluorescence data evaluation. The non-invasive quantitative assay proved robust against any influence of the orientation of the larvae. The developed protocols promise to be useful tools for the detection of oxidative stress in zebrafish larvae. [ABSTRACT FROM AUTHOR]

Among all the nuclear-receptor mediated endocrine disruptive effects, antiandrogenicity is frequently observed in aquatic environments and may pose a risk to aquatic organisms. Linking these effects to responsible chemicals is challenging and a great share of antiandrogenic activity detected in the environment has not been explained yet. To identify drivers of this effect at a hot spot of antiandrogenicity in the German river Holtemme, we applied effect-directed analysis (EDA) including a parallel fractionation approach, a downscaled luciferase reporter gene cell-based anti-AR-CALUX assay and LC-HRMS/MS nontarget screening. We identified and confirmed the highly potent antiandrogen 4-methyl-7-diethylaminocoumarin (C47) and two derivatives in the active fractions. The relative potency of C47 to the reference compound flutamide was over 5.2, whereas the derivatives were less potent. C47 was detected at a concentration of 13.7 μg/L, equal to 71.4 ±g flutamide equivalents per liter (FEq/L) in the nonconcentrated water extract that was posing an antiandrogenic activity equal to 45.5 (±13.7 SD) FEq/L. Thus, C47 was quantitatively confirmed as the major cause of the measured effect in vitro. Finally, the antiandrogenic activity of C47 and one derivate was confirmed in vivo in spiggin-gfp Medaka. An endocrine disrupting effect of C47 was observed already at the concentration equal to the concentration in the nonconcentrated water extract, underlining the high risk posed by this compound to the aquatic ecosystem. This is of some concern since C47 is used in a number of consumer products indicating environmental as well as human exposure. [ABSTRACT FROM AUTHOR]

Growing concern about the adverse environmental and human health effects of a wide range of micropollutants requires the development of novel tools and approaches to enable holistic monitoring of their occurrence, fate and effects in the aquatic environment. A European-wide demonstration program (EDP) for effect-based monitoring of micropollutants in surface waters was carried out within the Marie Curie Initial Training Network EDA-EMERGE. The main objectives of the EDP were to apply a simplified protocol for effect-directed analysis, to link biological effects to target compounds and to estimate their risk to aquatic biota. Onsite large volume solid phase extraction of 50 L of surface water was performed at 18 sampling sites in four European river basins. Extracts were subjected to effect-based analysis (toxicity to algae, fish embryo toxicity, neurotoxicity, (anti-)estrogenicity, (anti-)androgenicity, glucocorticoid activity and thyroid activity), to target analysis (151 organic micropollutants) and to nontarget screening. The most pronounced effects were estrogenicity, toxicity to algae and fish embryo toxicity. In most bioassays, major portions of the observed effects could not be explained by target compounds, especially in case of androgenicity, glucocorticoid activity and fish embryo toxicity. Estrone and nonylphenoxyacetic acid were identified as the strongest contributors to estrogenicity, while herbicides, with a minor contribution from other micropollutants, were linked to the observed toxicity to algae. Fipronil and nonylphenol were partially responsible for the fish embryo toxicity. Within the EDP, 21 target compounds were prioritized on the basis of their frequency and extent of exceedance of predicted no effect concentrations. The EDP priority list included 6 compounds, which are already addressed by European legislation, and 15 micropollutants that may be important for future monitoring of surface waters. The study presents a novel simplified protocol for effect-based monitoring and draws a comprehensive picture of the surface water status across Europe. [ABSTRACT FROM AUTHOR]

Surface waters can contain a diverse range of organic pollutants, including pesticides, pharmaceuticals and industrial compounds. While bioassays have been used for water quality monitoring, there is limited knowledge regarding the effects of individual micropollutants and their relationship to the overall mixture effect in water samples. In this study, a battery of in vitro bioassays based on human and fish cell lines and whole organism assays using bacteria, algae, daphnids and fish embryos was assembled for use in water quality monitoring. The selection of bioassays was guided by the principles of adverse outcome pathways in order to cover relevant steps in toxicity pathways known to be triggered by environmental water samples. The effects of 34 water pollutants, which were selected based on hazard quotients, available environmental quality standards and mode of action information, were fingerprinted in the bioassay test battery. There was a relatively good agreement between the experimental results and available literature effect data. The majority of the chemicals were active in the assays indicative of apical effects, while fewer chemicals had a response in the specific reporter gene assays, but these effects were typically triggered at lower concentrations. The single chemical effect data were used to improve published mixture toxicity modeling of water samples from the Danube River. While there was a slight increase in the fraction of the bioanalytical equivalents explained for the Danube River samples, for some endpoints less than 1% of the observed effect could be explained by the studied chemicals. The new mixture models essentially confirmed previous findings from many studies monitoring water quality using both chemical analysis and bioanalytical tools. In short, our results indicate that many more chemicals contribute to the biological effect than those that are typically quantified by chemical monitoring programs or those regulated by environmental quality standards. This study not only demonstrates the utility of fingerprinting single chemicals for an improved understanding of the biological effect of pollutants, but also highlights the need to apply bioassays for water quality monitoring in order to prevent underestimation of the overall biological effect. [ABSTRACT FROM AUTHOR]

The use of zebrafish for aquatic vertebrate (eco)toxicity testing allows the assessment of effects on a wide range of biological levels – from enzymes to sensory organs and behavioral endpoints. The present study investigated the effects of the insecticide diazinon and the herbicide diuron regarding the acute toxicity and behavior of zebrafish embryos and larvae. After conducting the fish embryo toxicity test, three concentrations (1, 2 and 3.5 mg L −1 for diazinon and 1, 2 and 3.8 mg L −1 for diuron) were evaluated for effects on embryonic spontaneous movement and heartbeat, larval light-dark transition response, and thigmotaxis. Although the modes-of-action are different, both pesticides proved to be moderately toxic to early life stages of zebrafish with 96 h LC 50 of approximately 6.5 mg L −1 and similar EC 50 values of approximately 4 mg L −1 . Changes in behavioral endpoints were detected 24 h of exposure, suggesting that behavioral measurements can serve as sensitive and early indicators of pesticide exposure. Changes in behavior, such as decrease in spontaneous coiling movements of embryos and reduction of thigmotaxis in larvae, were pronounced for diuron, indicating the usefulness of the application of behavioral endpoints to assess the effects of other herbicides. In the case of diazinon, the effects were less prominent, but the detected changes in ratios between activity in light and darkness also point to the possibility of using behavioral changes for evaluation of insecticide effects. The obtained results support the usage of behavioral endpoints in zebrafish embryos and larvae for the detection of early effects of pesticides. [ABSTRACT FROM AUTHOR]

Effect-directed analysis (EDA) is a powerful strategy to identify biologically active compounds in environmental samples. However, in current EDA studies, fractionation and handling procedures are laborious, consist of multiple evaporation steps, and thus bear the risk of contamination and decreased recoveries of the target compounds. The low resulting throughput has been one of the major bottlenecks of EDA. Here, we propose a high-throughput EDA (HT-EDA) work-flow combining reversed phase high-performance liquid chromatography fractionation of samples into 96-well microplates, followed by toxicity assessment in the micro- EROD bioassay with the wild-type rat hepatoma H4IIE cells, and chemical analysis of bioactive fractions. The approach was evaluated using single substances, binary mixtures, and extracts of sediment samples collected at the Three Gorges Reservoir, Yangtze River, China, as well as the rivers Rhine and Elbe, Germany. Selected bioactive fractions were analyzed by highly sensitive gas chromatography-atmospheric pressure laser ionization-time-of-flight-mass spectrometry. In addition, we optimized the work-flow by seeding previously adapted suspension- cultured H4IIE cells directly into the microplate used for fractionation, which makes any transfers of fractionated samples unnecessary. The proposed HT-EDA work-flow simplifies the procedure for wider application in ecotoxicology and environmental routine programs. [ABSTRACT FROM AUTHOR]

The implementation of targeted and nontargeted chemical screening analysis in combination with in vitro and organism-level bioassays is a prerequisite for a more holistic monitoring of water quality in the future. For chemical analysis, little or no sample enrichment is often sufficient, while bioanalysis often requires larger sample volumes at a certain enrichment factor for conducting comprehensive bioassays on different endpoints or further effect-directed analysis (EDA). To avoid logistic and technical issues related to the storage and transport of large volumes of water, sampling would benefit greatly from onsite extraction. This study presents a novel onsite large volume solid phase extraction (LVSPE) device tailored to fulfill the requirements for the successful effect-based and chemical screening of water resources and complies with available international standards for automated sampling devices. Laboratory recovery experiments using 251 organic compounds in the log D range from − 3.6 to 9.4 (at pH 7.0) spiked into pristine water resulted in acceptable recoveries and from 60 to 123% for 159 out of 251 substances. Within a European-wide demonstration program, the LVSPE was able to enrich compounds in concentration ranges over three orders of magnitude (1 ng L − 1 to 2400 ng L − 1 ). It was possible to discriminate responsive samples from samples with no or only low effects in a set of six different bioassays (i.e. acetylcholinesterase and algal growth inhibition, androgenicity, estrogenicity, fish embryo toxicity, glucocorticoid activity). The LVSPE thus proved applicable for onsite extraction of sufficient amounts of water to investigate water quality thoroughly by means of chemical analysis and effect-based tools without the common limitations due to small sample volumes. [ABSTRACT FROM AUTHOR]

Surface waters can contain a range of micropollutants from point sources, such as wastewater effluent, and diffuse sources, such as agriculture. Characterizing the source of micropollutants is important for reducing their burden and thus mitigating adverse effects on aquatic ecosystems. In this study, chemical analysis and bioanalysis were applied to assess the micropollutant burden during low flow conditions upstream and downstream of three wastewater treatment plants (WWTPs) discharging into small streams in the Swiss Plateau. The upstream sites had no input of wastewater effluent, allowing a direct comparison of the observed effects with and without the contribution of wastewater. Four hundred and five chemicals were analyzed, while the applied bioassays included activation of the aryl hydrocarbon receptor, activation of the androgen receptor, activation of the estrogen receptor, photosystem II inhibition, acetylcholinesterase inhibition and adaptive stress responses for oxidative stress, genotoxicity and inflammation, as well as assays indicative of estrogenic activity and developmental toxicity in zebrafish embryos. Chemical analysis and bioanalysis showed higher chemical concentrations and effects for the effluent samples, with the lowest chemical concentrations and effects in most assays for the upstream sites. Mixture toxicity modeling was applied to assess the contribution of detected chemicals to the observed effect. For most bioassays, very little of the observed effects could be explained by the detected chemicals, with the exception of photosystem II inhibition, where herbicides explained the majority of the effect. This emphasizes the importance of combining bioanalysis with chemical analysis to provide a more complete picture of the micropollutant burden. While the wastewater effluents had a significant contribution to micropollutant burden downstream, both chemical analysis and bioanalysis showed a relevant contribution of diffuse sources from upstream during low flow conditions, suggesting that upgrading WWTPs will not completely reduce the micropollutant burden, but further source control measures will be required. [ABSTRACT FROM AUTHOR]

The Joint Danube Survey 3 (JDS3; the biggest river expedition in 2013) had offered the unique opportunity for a large-scale monitoring approach for biomarker response in feral fish collected along a Danube stretch from Kehlheim (DE) to Sulina (RO). The advantage of genotoxicity as a marker for pollution exposure in fish is the early detection of possible long-term effects such as cancer. Therefore, genotoxicity was in the focus of the biomarker investigations in fish during the expedition. Blood samples of common bleak ( Alburnus alburnus ) for the investigation of the micronucleus frequency and comet tail intensity of fragmented DNA material in erythrocytes were collected at 18 and 12 sampling sites, respectively. For 9 sampling sites same samples were used to compare the in-situ data for the comparable genotoxic endpoint in the micronucleus (MN) and comet assay (CM). The data of both in-situ assays showed a significant correlation, indicating the strength and comparability of the data sets. Significant variation in DNA damage in fish along the longitudinal profile of the Danube was demonstrated for both assays compared to reference sites. The results suggest that DNA damage in erythrocytes of fish was mainly affected by wastewater of highly populated regions. No linkage between the results and the general health/dietary status of the fish were revealed, whereas correlation with some genotoxicity drivers in the water phase, suspended particulate matter and sediments could be demonstrated. [ABSTRACT FROM AUTHOR]

Bioactivity screening studies often face sample amount limitation with respect to the need for reliable, reproducible and quantitative results. Therefore approaches that minimize sample use are needed. Low-volume exposure and chemical dilution procedures were applied in an androgen receptor reporter gene human cell line assay to evaluate environmental contaminants and androgen receptor modulators, which were the agonist 5α-dihydrotestosterone (DHT); and the antagonists flutamide, bisphenol A, 1-hydroxypyrene and triclosan. Cells were exposed in around 1/3 of the medium volume recommended by the protocol (70 μL/well). Further, chemical losses during pipetting steps were minimized by applying a low-volume method for compound dilution in medium (250 μL for triplicate wells) inside microvolume glass inserts. Simultaneously, compounds were evaluated following conventional procedures (200 μL/well, dilution in 24-well plates) for comparison of results. Low-volume exposure tests produced DHT EC 50 (3.4–3.7 × 10 − 10 M) and flutamide IC 50 (2.2–3.3 × 10 − 7 M) values very similar to those from regular assays (3.1–4.2 × 10 − 10 and 2.1–3.3 × 10 − 7 M respectively) and previous studies. Also, results were within assay acceptance criteria, supporting the relevance of the downscaling setup for agonistic and antagonistic tests. The low-volume exposure was also successful in determining IC 50 values for 1-hydroxypyrene (2.1–2.8 × 10 − 6 M), bisphenol A (2.6–3.3 × 10 − 6 M), and triclosan (1.2–1.9 × 10 − 6 M) in agreement with values obtained through high-volume exposure (2.3–2.8, 2.5–3.4 and 1.0–1.3 × 10 − 6 M respectively). Finally, experiments following both low-volume dosing and exposure produced flutamide and triclosan IC 50 values similar to those from regular tests. The low-volume experimental procedures provide a simple and effective solution for studies that need to minimize bioassay sample use while maintaining method reliability. The downscaling methods can be applied for the evaluation of samples, fractions or chemicals which require minimal losses during the steps of pipetting, transference to medium and exposure in bioassays. [ABSTRACT FROM AUTHOR]

Only few information on the potential toxic effectiveness of biofuels are available. Due to increasing worldwide demand for energy and fuels during the past decades, biofuels are considered as a promising alternative for fossil fuels in the transport sector. Hence, more information on their hazard potentials are required to understand the toxicological impact of biofuels on the environment. In the German Cluster of Excellence “Tailor-made Fuels from Biomass” design processes for economical, sustainable and environmentally friendly biofuels are investigated. In an unique and interdisciplinary approach, ecotoxicological methods are applied to gain information on potential adverse environmental effects of biofuels at an early phase of their development. In the present study, three potential biofuels, ethyl levulinate, 2-methyltetrahydrofuran and 2-methylfuran were tested. Furthermore, we investigated a fossil gasoline fuel, a fossil diesel fuel and an established biodiesel. Two in vitro bioassays, one for assessing cytotoxicity and one for aryl hydrocarbon receptor agonism, so called dioxin-like activity, as measured by Ethoxyresorufin-O-Deethylase, were applied using the permanent fish liver cell line RTL-W1 (Oncorhynchus mykiss). The special properties of these fuel samples required modifications of the test design. Points that had to be addressed were high substance volatility, material compatibility and low solubility. For testing of gasoline, diesel and biodiesel, water accommodated fractions and a passive dosing approach were tested to address the high hydrophobicity and low solubility of these complex mixtures. Further work has to focus on an improvement of the chemical analyses of the fuel samples to allow a better comparison of any effects of fossil fuels and biofuels. [ABSTRACT FROM AUTHOR]

Surface water can contain countless organic micropollutants, and targeted chemical analysis alone may only detect a small fraction of the chemicals present. Consequently, bioanalytical tools can be applied complementary to chemical analysis to detect the effects of complex chemical mixtures. In this study, bioassays indicative of activation of the aryl hydrocarbon receptor (AhR), activation of the pregnane X receptor (PXR), activation of the estrogen receptor (ER), adaptive stress responses to oxidative stress (Nrf2), genotoxicity (p53) and inflammation (NF-B) and the fish embryo toxicity test were applied along with chemical analysis to water extracts from the Danube River. Mixture-toxicity modeling was applied to determine the contribution of detected chemicals to the biological effect. Effect concentrations for between 0 to 13 detected chemicals could be found in the literature for the different bioassays. Detected chemicals explained less than 0.2% of the biological effect in the PXR activation, adaptive stress response, and fish embryo toxicity assays, while five chemicals explained up to 80% of ER activation, and three chemicals explained up to 71% of AhR activation. This study highlights the importance of fingerprinting the effects of detected chemicals. [ABSTRACT FROM AUTHOR]

Anthropogenic Trace Compounds (ATCs) that continuously grow in numbers and concentrations are an emerging issue for water quality in both natural and technical environments. The complex web of exposure pathways as well as the variety in the chemical structure and potency of ATCs represents immense challenges for future research and policy initiatives. This review summarizes current trends and identifies knowledge gaps in innovative, effective monitoring and management strategies while addressing the research questions concerning ATC occurrence, fate, detection and toxicity. We highlight the progressing sensitivity of chemical analytics and the challenges in harmonization of sampling protocols and methods, as well as the need for ATC indicator substances to enable cross-national valid monitoring routine. Secondly, the status quo in ecotoxicology is described to advocate for a better implementation of long-term tests, to address toxicity on community and environmental as well as on human-health levels, and to adapt various test levels and endpoints. Moreover, we discuss potential sources of ATCs and the current removal efficiency of wastewater treatment plants (WWTPs) to indicate the most effective places and elimination strategies. Knowledge gaps in transport and/or detainment of ATCs through their passage in surface waters and groundwaters are further emphasized in relation to their physico-chemical properties, abiotic conditions and biological interactions in order to highlight fundamental research needs. Finally, we demonstrate the importance and remaining challenges of an appropriate ATC risk assessment since this will greatly assist in identifying the most urgent calls for action, in selecting the most promising measures, and in evaluating the success of implemented management strategies. [ABSTRACT FROM AUTHOR]

Environmental quality monitoring of water resources is challenged with providing the basis for safeguarding the environment against adverse biological effects of anthropogenic chemical contamination from diffuse and point sources. While current regulatory efforts focus on monitoring and assessing a few legacy chemicals, many more anthropogenic chemicals can be detected simultaneously in our aquatic resources. However, exposure to chemical mixtures does not necessarily translate into adverse biological effects nor clearly shows whether mitigation measures are needed. Thus, the question which mixtures are present and which have associated combined effects becomes central for defining adequate monitoring and assessment strategies. Here we describe the vision of the international, EU-funded project SOLUTIONS, where three routes are explored to link the occurrence of chemical mixtures at specific sites to the assessment of adverse biological combination effects. First of all, multi-residue target and non-target screening techniques covering a broader range of anticipated chemicals co-occurring in the environment are being developed. By improving sensitivity and detection limits for known bioactive compounds of concern, new analytical chemistry data for multiple components can be obtained and used to characterise priority mixtures. This information on chemical occurrence will be used to predict mixture toxicity and to derive combined effect estimates suitable for advancing environmental quality standards. Secondly, bioanalytical tools will be explored to provide aggregate bioactivity measures integrating all components that produce common (adverse) outcomes even for mixtures of varying compositions. The ambition is to provide comprehensive arrays of effect-based tools and trait-based field observations that link multiple chemical exposures to various environmental protection goals more directly and to provide improved in situ observations for impact assessment of mixtures. Thirdly, effect-directed analysis (EDA) will be applied to identify major drivers of mixture toxicity. Refinements of EDA include the use of statistical approaches with monitoring information for guidance of experimental EDA studies. These three approaches will be explored using case studies at the Danube and Rhine river basins as well as rivers of the Iberian Peninsula. The synthesis of findings will be organised to provide guidance for future solution-oriented environmental monitoring and explore more systematic ways to assess mixture exposures and combination effects in future water quality monitoring. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Interdisciplinary project (GreenToxiConomy) on new bioeconomic high-value products. • Proof-of-concept study on the applicability of Green Toxicology in bioeconomy. • Development of an application-oriented bioassay battery for early hazard assessment. • QSAR-based toxicity prediction differentiates compounds of deviating toxicity. • Experimental toxicity assessment will identify most promising candidates. [ABSTRACT FROM AUTHOR]

Abstract: To evaluate the effect of crop residue burning of agriculturally tilled soils, the soil''s sorption of polycyclic aromatic hydrocarbons (PAHs) is studied using naphthalene as a representative model contaminant in the soil. Soils from agriculturally tilled fields (Dorobantu farm, Calarasi, Romania), either burned or unburned for clearance, were examined for their toxicity and sorption capacity (fields being burned for cleansing experience an increase in nanoparticles because soot particles enter the soil). The burned and unburned soils were separated into several fractions on the basis of size using membranes with different pore sizes. Batch experiments were conducted to test the impact of the following operating parameters: the initial concentration of naphthalene, initial concentration of soil, pH, soil structure and composition and soil characterization. The toxic effectiveness of the various soils was also investigated. [Copyright &y& Elsevier]

A sediment contact test (SCT) battery consisting of five ecotoxicological test systems was applied to 21 native freshwater sediments characterized by a broad variety of geochemical properties and anthropogenic contamination. Higher plants ( Myriophyllum aquaticum), nematodes ( Caenorhabditis elegans), oligochaetes ( Lumbriculus variegatus), zebrafish embryos ( Danio rerio), and bacteria ( Arthrobacter globiformis), representing various trophic levels and exposure pathways, were used as test organisms. The test battery detected sediment toxicity caused by anthropogenic pollution, whereas the various tests provided site-specific, nonredundant information to the overall toxicity assessment. Based on the toxicity pattern derived from the test battery, the sediments were classified according to a newly proposed classification system for sediment toxicity assessment. The SCT-derived classification generally agreed well with the application of consensus-based sediment quality guidelines (SQGs), especially with regard to sediments with high toxic potential. For sediments with low to medium toxic potential, the SQGs often underestimated the toxicity that was detected by the SCTs, underpinning the need for toxicity tests in sediment quality assessment. Environ. Toxicol. Chem. 2013;32:144-155. © 2012 SETAC [ABSTRACT FROM AUTHOR]

Bioanalytical equivalents (BEQs) of mixtures and environmental samples are widely used to reflect the potential threat of pollutants in the environment and can be obtained by bioassays or using chemical analysis combined with relative potencies (REPs). In this study, the relationships between bioassay-detected BEQs (Bio-BEQs) and chemically analyzed BEQs (Chem-BEQs) were studied. BEQs and REPs are correlated with effect level and the concentration-response curves of the reference standard and sample. Thus, effect level (e.g., EC 10 , EC 25 and EC 50) should be addressed for the BEQ values obtained from bioassays or chemical analyses. The previous prerequisites for REPs application (i.e., curves that are parallel and have the same maximum response) are redundant, and the use of REPs for the calculation of BEQs or in risk assessment should instead be based on the same effect level. For a complex mixture with many components, all active components can be regarded as dilutions of a standard compound for inducing a specific effect. Relative toxicity estimates based on EC 50 ignore the contribution of weak-active components with maximum response below EC 50 of the reference standard, especially in complex mixtures or environmental samples. REPs based on an effect level EC 10 that can be clearly discriminated from background response are recommended for BEQ calculation. As an example, the aryl hydrocarbon receptor (AhR)-mediated activity of US EPA priority polycyclic aromatic hydrocarbons (PAHs) in RTL-W1 cells was used to assess the reliability of REPs for mixture toxicity prediction based on the effect level EC 10. Unlabelled Image • Effect level should be addressed for bioanalytical equivalents. • Components can be viewed as dilutions of a compound for inducing a specific effect. • Toxicity estimates at a high effect level ignore weak-active components. • Relative potencies at EC 10 can be used to calculate bioanalytical equivalents. [ABSTRACT FROM AUTHOR]

• Microplastics accumulated in GIT and gill, but not found in brain and muscle. • Microplastic exposure led to the thinning of muscularis layer in the gut. • Zebrafish became hyperactivity after microplastic exposure. • Energy-supplying substances shortage occurred after microplastic exposure. Microplastic pollution has drawn the attention of both scientists and the public regarding their potential ecotoxicological risks. In the present study, we carried out aqueous exposure experiments to adult zebrafish with polystyrene microplastics (5 μm) at a wide range of concentrations (0.001-20 mg/L, equals to 14.5∼2.9 × 105 particles/mL). Our results showed the gastrointestinal tract (GIT) was the dominant microplastic accumulation site in zebrafish, followed by the gill, whereas no microplastics were detected in the brain or muscle. Microplastic accumulation in GIT did not cause obvious damages to intestinal villi in general. However, the thickness of muscularis layer in the foregut reduced by 32% after 1 mg/L (1.45 × 104 particles/mL) microplastic exposure. As there were no signs of oxidative stress or other histological changes found in the fish, we further investigated the energy-supplying influential factors. We found that the zebrafish became hyperactive after microplastic exposure, whose swimming distance had increased to 1.3-2.4 folds than that of control, and also stayed at manic and active states much longer. The fish behavioural alteration is probably attributed to the particulate matter stimulation and the up-regulation of estrogen contents. Results also showed that the excessive movements of zebrafish also led to decreased glucose and acetaldehyde metabolite contents and increased amino acid amounts, which further proved the shortage of energy-supplying substances. Therefore, the present study suggests that micro-sized microplastics can induce obvious behavioural abnormality at concentrations that some other toxicological endpoints may not warn effects. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • Oil-specific (sub-)lethal effect concentrations for oil spill risk assessment were established. • Oil types induced different strength of embryo toxicity not correlated with common PAHs. • Sublethal exposure concentrations resulted in CYP1A induction but no AChE inhibition. • Larvae showed strong behavioral impairment already at low exposure concentrations. • Dispersant contributes to higher toxicity of chemically dispersed oils compared to native oils. [ABSTRACT FROM AUTHOR]

• Validated microscale assays on estrogenicity were optimized for petroleum toxicity testing. • Bioassays were highly sensitive towards WAF exposure in brackish and freshwater conditions. • Native and chemically dispersed crude oil WAFs did activate the ERα. • The dispersant itself had a limited influence on high CEWAF activity. • Metabolization of WAF compounds did not change the estrogenic activity. 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. [ABSTRACT FROM AUTHOR]