Results of an international interlaboratory study on dioxin-like activities in drinking-, river surface- and wastewater using DR CALUX bioassay.

Aquatic animals and consumers of aquatic animals are exposed to increasingly complex mixtures of known and as-yet-unknown chemicals with dioxin-like toxicities in the water cycle. Effect- and cell-based bioanalysis can cover known and yet unknown dioxin and dioxin-like compounds as well as complex mixtures thereof but need to be standardized and integrated into international guidelines for environmental testing. In an international laboratory testing (ILT) following ISO/CD 24295 as standard procedure for rat cell-based DR CALUX un-spiked and spiked extracts of drinking-, surface-, and wastewater were validated to generate precision data for the development of the full ISO-standard. We found acceptable repeatability and reproducibility ranges below 36 % by DR CALUX bioassay for the tested un-spiked and spiked water of different origins. The presence of 17 PCDD/Fs and 12 dioxin-like PCBs was also confirmed by congener-specific GC-HRMS analysis. We compared the sum of dioxin-like activity levels measured by DR CALUX bioassay (expressed in 2,3,7,8-TCDD Bioanalytical Equivalents, BEQ; ISO 23196, 2022) with the obtained GC-HRMS chemical analysis results converted to toxic equivalents (TEQ; van den Berg et al., 2013).

Immunotoxic, genotoxic, and endocrine disrupting impacts of polyamide microplastic particles and chemicals.

Due to their exceptional properties and cost effectiveness, polyamides or nylons have emerged as widely used materials, revolutionizing diverse industries, including industrial 3D printing or additive manufacturing (AM). Powder-based AM technologies employ tonnes of polyamide microplastics to produce complex components every year. However, the lack of comprehensive toxicity assessment of particulate polyamides and polyamide-associated chemicals, especially in the light of the global microplastics crisis, calls for urgent action. This study investigated the physicochemical properties of polyamide-12 microplastics used in AM, and assessed a number of toxicity endpoints focusing on inflammation, immunometabolism, genotoxicity, aryl hydrocarbon receptor (AhR) activation, endocrine disruption, and cell morphology. Specifically, microplastics examination by means of field emission scanning electron microscopy revealed that work flow reuse of material created a fraction of smaller particles with an average size of 1-5 µm, a size range readily available for uptake by human cells. Moreover, chemical analysis by means of gas chromatography high-resolution mass spectrometry detected several polyamide-associated chemicals including starting material, plasticizer, thermal stabilizer/antioxidant, and migrating slip additive. Even if polyamide particles and chemicals did not induce an acute inflammatory response, repeated and prolonged exposure of human primary macrophages disclosed a steady increase in the levels of proinflammatory chemokine Interleukin-8 (IL-8/CXCL-8). Moreover, targeted metabolomics disclosed that polyamide particles modulated the kynurenine pathway and some of its key metabolites. The p53-responsive luciferase reporter gene assay showed that particles per se were able to activate p53, being indicative of a genotoxic stress. Polyamide-associated chemicals triggered moderate activation of AhR and elicited anti-androgenic activity. Finally, a high-throughput and non-targeted morphological profiling by Cell Painting assay outlined major sites of bioactivity of polyamide-associated chemicals and indicated putative mechanisms of toxicity in the cells. These findings reveal that the increasing use of polyamide microplastics may pose a potential health risk for the exposed individuals, and it merits more attention.

NLRP3 inflammasome as a sensor of micro- and nanoplastics immunotoxicity.

Micro- and nanoplastics (MNPs) are emerging pollutants with scarcely investigated effects on human innate immunity. If they follow a similar course of action as other, more thoroughly investigated particulates, MNPs may penetrate epithelial barriers, potentially triggering a cascade of signaling events leading to cell damage and inflammation. Inflammasomes are intracellular multiprotein complexes and stimulus-induced sensors critical for mounting inflammatory responses upon recognition of pathogen- or damage-associated molecular patterns. Among these, the NLRP3 inflammasome is the most studied in terms of activation via particulates. However, studies delineating the ability of MNPs to affect NLRP3 inflammasome activation are still rare. In this review, we address the issue of MNPs source and fate, highlight the main concepts of inflammasome activation via particulates, and explore recent advances in using inflammasome activation for assessment of MNP immunotoxicity. We also discuss the impact of co-exposure and MNP complex chemistry in potential inflammasome activation. Development of robust biological sensors is crucial in order to maximize global efforts to effectively address and mitigate risks that MNPs pose for human health.

Present and future challenges for the investigation of transgenerational epigenetic inheritance.

Epigenetic pathways are essential in different biological processes and in phenotype-environment interactions in response to different stressors and they can induce phenotypic plasticity. They encompass several processes that are mitotically and, in some cases, meiotically heritable, so they can be transferred to subsequent generations via the germline. Transgenerational Epigenetic Inheritance (TEI) describes the phenomenon that phenotypic traits, such as changes in fertility, metabolic function, or behavior, induced by environmental factors (e.g., parental care, pathogens, pollutants, climate change), can be transferred to offspring generations via epigenetic mechanisms. Investigations on TEI contribute to deciphering the role of epigenetic mechanisms in adaptation, adversity, and evolution. However, molecular mechanisms underlying the transmission of epigenetic changes between generations, and the downstream chain of events leading to persistent phenotypic changes, remain unclear. Therefore, inter-, (transmission of information between parental and offspring generation via direct exposure) and transgenerational (transmission of information through several generations with disappearance of the triggering factor) consequences of epigenetic modifications remain major issues in the field of modern biology. In this article, we review and describe the major gaps and issues still encountered in the TEI field: the general challenges faced in epigenetic research; deciphering the key epigenetic mechanisms in inheritance processes; identifying the relevant drivers for TEI and implement a collaborative and multi-disciplinary approach to study TEI. Finally, we provide suggestions on how to overcome these challenges and ultimately be able to identify the specific contribution of epigenetics in transgenerational inheritance and use the correct tools for environmental science investigation and biomarkers identification.

A Novel Nanosafety Approach Using Cell Painting, Metabolomics, and Lipidomics Captures the Cellular and Molecular Phenotypes Induced by the Unintentionally Formed Metal-Based (Nano)Particles.

Additive manufacturing (AM) or industrial 3D printing uses cutting-edge technologies and materials to produce a variety of complex products. However, the effects of the unintentionally emitted AM (nano)particles (AMPs) on human cells following inhalation, require further investigations. The physicochemical characterization of the AMPs, extracted from the filter of a Laser Powder Bed Fusion (L-PBF) 3D printer of iron-based materials, disclosed their complexity, in terms of size, shape, and chemistry. Cell Painting, a high-content screening (HCS) assay, was used to detect the subtle morphological changes elicited by the AMPs at the single cell resolution. The profiling of the cell morphological phenotypes, disclosed prominent concentration-dependent effects on the cytoskeleton, mitochondria, and the membranous structures of the cell. Furthermore, lipidomics confirmed that the AMPs induced the extensive membrane remodeling in the lung epithelial and macrophage co-culture cell model. To further elucidate the biological mechanisms of action, the targeted metabolomics unveiled several inflammation-related metabolites regulating the cell response to the AMP exposure. Overall, the AMP exposure led to the internalization, oxidative stress, cytoskeleton disruption, mitochondrial activation, membrane remodeling, and metabolic reprogramming of the lung epithelial cells and macrophages. We propose the approach of integrating Cell Painting with metabolomics and lipidomics, as an advanced nanosafety methodology, increasing the ability to capture the cellular and molecular phenotypes and the relevant biological mechanisms to the (nano)particle exposure.

Extensive chemical and bioassay analysis of polycyclic aromatic compounds in a creosote-contaminated superfund soil following steam enhanced extraction.

Polycyclic aromatic compounds (PACs) are organic compounds commonly found in contaminated soil. Previous studies have shown the removal of polycyclic aromatic hydrocarbons (PAHs) in creosote-contaminated soils during steam enhanced extraction (SEE). However, less is known about the removal of alkyl-PAHs and heterocyclic compounds, such as azaarenes, and oxygen- and sulfur-heterocyclic PACs (OPACs and PASHs, respectively). Further, the impact of SEE on the freely dissolved concentration of PACs in soil as well as the soil bioactivity pre- and post-SEE have yet to be addressed. To fulfil these research gaps, chemical and bioanalytical analysis of a creosote-contaminated soil, collected from a U.S. Superfund site, pre- and post-SEE were performed. The decrease of 64 PACs (5-100%) and increase in the concentrations of nine oxygenated-PAHs (OPAHs) (150%) during SEE, some of which are known to be toxic and can potentially contaminate ground water, were observed. The freely dissolved concentrations of PACs in soil were assessed using polyoxymethylene (POM) strips and the concentrations of 66 PACs decreased post-SEE (1-100%). Three in vitro reporter gene bioassays (DR-CALUX®, ERα-CALUX® and anti-AR CALUX®) were used to measure soil bioactivities pre- and post-SEE and all reporter gene bioassays measured soil bioactivity decreases post-SEE. Mass defect suspect screening tentatively identified 27 unique isomers of azaarenes and OPAC in the soil. As a remediation technique, SEE was found to remove alkyl-PAHs and heterocyclic PACs, reduce the concentrations of freely dissolved PACs, and decrease soil bioactivities.

Particle Safety Assessment in Additive Manufacturing: From Exposure Risks to Advanced Toxicology Testing.

Additive manufacturing (AM) or industrial three-dimensional (3D) printing drives a new spectrum of design and production possibilities; pushing the boundaries both in the application by production of sophisticated products as well as the development of next-generation materials. AM technologies apply a diversity of feedstocks, including plastic, metallic, and ceramic particle powders with distinct size, shape, and surface chemistry. In addition, powders are often reused, which may change the particles' physicochemical properties and by that alter their toxic potential. The AM production technology commonly relies on a laser or electron beam to selectively melt or sinter particle powders. Large energy input on feedstock powders generates several byproducts, including varying amounts of virgin microparticles, nanoparticles, spatter, and volatile chemicals that are emitted in the working environment; throughout the production and processing phases. The micro and nanoscale size may enable particles to interact with and to cross biological barriers, which could, in turn, give rise to unexpected adverse outcomes, including inflammation, oxidative stress, activation of signaling pathways, genotoxicity, and carcinogenicity. Another important aspect of AM-associated risks is emission/leakage of mono- and oligomers due to polymer breakdown and high temperature transformation of chemicals from polymeric particles, both during production, use, and in vivo, including in target cells. These chemicals are potential inducers of direct toxicity, genotoxicity, and endocrine disruption. Nevertheless, understanding whether AM particle powders and their byproducts may exert adverse effects in humans is largely lacking and urges comprehensive safety assessment across the entire AM lifecycle-spanning from virgin and reused to airborne particles. Therefore, this review will detail: 1) brief overview of the AM feedstock powders, impact of reuse on particle physicochemical properties, main exposure pathways and protective measures in AM industry, 2) role of particle biological identity and key toxicological endpoints in the particle safety assessment, and 3) next-generation toxicology approaches in nanosafety for safety assessment in AM. Altogether, the proposed testing approach will enable a deeper understanding of existing and emerging particle and chemical safety challenges and provide a strategy for the development of cutting-edge methodologies for hazard identification and risk assessment in the AM industry.

Observed and predicted embryotoxic and teratogenic effects of organic and inorganic environmental pollutants and their mixtures in zebrafish (Danio rerio).

Risk assessment of chemicals is still primarily focusing on single compound evaluation, even if environmental contamination consists of a mixture of pollutants. The concentration addition (CA) and independent action (IA) models have been developed to predict mixture toxicity. Both models assume no interaction between the components, resulting in an additive mixture effect. In the present study, the embryo toxicity test (OECD TG no. 236) with zebrafish embryos (Danio rerio) was performed to investigate whether the toxicity caused by binary, ternary, and quaternary mixtures of organic (Benzo[a]pyrene, perfluorooctanesulfonate, and 3,3´,4,4´,5-pentachlorobiphenyl 126) and inorganic (arsenate) pollutants can be predicted by CA and IA. The acute toxicity and sub-lethal alterations such as lack of blood circulation were investigated. The models estimated the mixture toxicity well and most of the mixtures were additive. However, the binary mixture of PFOS and PCB126 caused a synergistic effect, with almost a ten-fold difference between the observed and predicted LC50-value. For most of the mixtures, the CA model was better in predicting the mixture toxicity than the IA model, which was not expected due to the chemicals' different modes of action. In addition, some of the mixtures caused sub-lethal effects not observed in the single compound toxicity tests. The mixture of PFOS and BaP caused a division of the yolk and imbalance was caused by the combination of PFOS and As and the ternary mixture of PFOS, As, and BaP. Interestingly, PFOS was part of all three mixtures causing the mixture specific sub-lethal effects. In conclusion, the present study shows that CA and IA are mostly resulting in good estimations of the risks that mixtures with few components are posing. However, for a more reliable assessment and a better understanding of mixture toxicity, further investigations are required to study the underlying mechanisms.

Examination of aryl hydrocarbon receptor (AhR), estrogenic and anti-androgenic activities, and levels of polyaromatic compounds (PACs) in tire granulates using in vitro bioassays and chemical analysis.

Tire granulates recovered from end-of-life tires contain a complex mixture of chemicals, amongst them polyaromatic compounds (PACs), of which many are recognized to be toxic and persistent in the environment. Only a few of these PACs are regularly monitored. In this study a combined approach of chemical analysis and a battery of CALUX® in vitro bioassays was used to determine PAC concentrations and estrogenic, (anti)-androgenic and aryl hydrocarbon receptor (AhR) activities in tire granulates. Tire granulates from a recycling company was analyzed for PAHs, alkyl-PAHs, oxy-PAHs and heterocyclic PACs (NSO-PACs), in total 85 PACs. The concentrations of PACs were between 42 and 144 mg/kg, with major contribution from PAHs (74-88%) followed by alkyl-PAHs (6.6-20%) and NSO-PACs (1.8-7.0%). The sum of eight priority PAHs were between 2.3 and 8.6 mg/kg, contributing with 4.7-8.2% of ∑PACs. Bioassay analysis showed presence of AhR agonists, estrogen receptor (ERα) agonists, and androgen receptor (AR) antagonists in the tire granulate samples. Only 0.8-2.4% of AhR-mediated activities could be explained by the chemical analysis. Benzo[k+j]fluoranthenes, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, 2-methylchrysene, and 3-methylchrysene were the major contributors to the AhR-mediated activities. The high contribution (98-99%) of unknown bioactive compounds to the bioassay effects in this study raises concerns and urges for further investigations of toxicants identification and source apportionment.

Predicting Chemical-Induced Liver Toxicity Using High-Content Imaging Phenotypes and Chemical Descriptors: A Random Forest Approach.

Hepatotoxicity is a major reason for the withdrawal or discontinuation of drugs from clinical trials. Thus, better tools are needed to filter potential hepatotoxic drugs early in drug discovery. Our study demonstrates utilization of HCI phenotypes, chemical descriptors, and both combined (hybrid) descriptors to construct random forest classifiers (RFCs) for the prediction of hepatotoxicity. HCI data published by Broad Institute provided HCI phenotypes for about 30 000 samples in multiple replicates. Phenotypes belonging to 346 chemicals, which were tested in up to eight replicates, were chosen as a basis for our analysis. We then constructed individual RFC models for HCI phenotypes, chemical descriptors, and hybrid (chemical and HCI) descriptors. The model that was constructed using selective hybrid descriptors showed high predictive performance with 5-fold cross validation (CV) balanced accuracy (BA) at 0.71, whereas within the given applicability domain (AD), independent test set and external test set prediction BAs were equal to 0.61 and 0.60, respectively. The model constructed using chemical descriptors showed a similar predictive performance with a 5-fold CV BA equal to 0.66, a test set prediction BA within the AD equal to 0.56, and an external test set prediction BA within the AD equal to 0.50. In conclusion, the hybrid and chemical descriptor-based models presented here should be considered as a new tool for filtering hepatotoxic molecules during compound prioritization in drug discovery.

Microplastics in sea-surface waters surrounding Sweden sampled by manta trawl and in-situ pump.

Microplastics were sampled in open surface waters by using a manta trawl and an in-situ filtering pump. A total of 24 trawl samples and 11 pump samples were taken at 12 locations around Sweden. Overall, the concentration of microplastic particles was higher in pump samples compared to trawl samples. The median microplastic particle concentration was 0.04 particles per m-3 for manta trawl samples and 0.10 particles per m-3 in pump samples taken with a mesh size of 0.3 mm. The highest concentrations were recorded on the west coast of Sweden. Fibers were found in all samples and were also more frequent in the pump samples. Even higher concentrations of fibers and particles were found on the 0.05 mm pump filters. Using near-infrared hyperspectral imaging the majority of the particles were identified as polyethylene followed by polypropylene.

Aryl hydrocarbon receptor-mediated potencies in field-deployed plastics vary by type of polymer.

Plastic is able to sorb environmental pollutants from ambient water and might act as a vector for these pollutants to marine organisms. The potential toxicological effects of plastic-sorbed pollutants in marine organisms have not been thoroughly assessed. In this study, organic extracts from four types of plastic deployed for 9 or 12 months in San Diego Bay, California, were examined for their potential to activate the aryl hydrocarbon receptor (AhR) pathway by use of the H4IIE-luc assay. Polycyclic aromatic hydrocarbons (PAH), including the 16 priority PAHs, were quantified. The AhR-mediated potency in the deployed plastic samples, calculated as bio-TEQ values, ranged from 2.7 pg/g in polyethylene terephthalate (PET) to 277 pg/g in low-density polyethylene (LDPE). Concentrations of the sum of 24 PAHs in the deployed samples ranged from 4.6 to 1068 ng/g. By use of relative potency factors (REP), a potency balance between the biological effect (bio-TEQs) and the targeted PAHs (chem-TEQs) was calculated to 24-170%. The study reports, for the first time, in vitro AhR-mediated potencies for different deployed plastics, of which LDPE elicited the greatest concentration of bio-TEQs followed by polypropylene (PP), PET, and polyvinylchloride (PVC).

Effect of perfluorooctanesulfonic acid (PFOS) on the liver lipid metabolism of the developing chicken embryo.

Perfluorooctanesulfonate (PFOS) is a well-known contaminant in the environment and it has shown to disrupt multiple biological pathways, particularly those related with lipid metabolism. In this study, we have studied the impact of in ovo exposure to PFOS on lipid metabolism in livers in developing chicken embryos using lipidomics for detailed characterization of the liver lipidome. We used an avian model (Gallus gallus domesticus) for in ovo treatment at two levels of PFOS. The lipid profile of the liver of the embryo was investigated by ultra-high performance liquid chromatography combined with quadrupole-time-of-flight mass spectrometry and by gas chromatography mass spectrometry. Over 170 lipids were identified, covering phospholipids, ceramides, di- and triacylglycerols, cholesterol esters and fatty acid composition of the lipids. The PFOS exposure caused dose dependent changes in the lipid levels, which included upregulation of specific phospholipids associated with the phosphatidylethanolamine N-methyltransferase (PEMT) pathway, triacylglycerols with low carbon number and double bond count as well as of lipotoxic ceramides and diacylglycerols. Our data suggest that at lower levels of exposure, mitochondrial fatty acid ß-oxidation is suppressed while the peroxisomal fatty acid ß -oxidation is increased. At higher doses, however, both ß -oxidation pathways are upregulated.

Occurrence and leachability of polycyclic aromatic compounds in contaminated soils: Chemical and bioanalytical characterization.

An important concern regarding sites contaminated with polycyclic aromatic compounds (PACs) is the risk of groundwater contamination by release of the compounds from soils. The goal of this study was to investigate the occurrence and leachability of 77 PACs including polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic compounds (NSO-PACs) among total aryl hydrocarbon receptor (AhR) agonists in soils from historical contaminated sites. A novel approach combining chemical and bioanalytical methods in combination with characterization of leachability by use of a column leaching test was used. Similar profiles of relative concentrations of PACs were observed in all soils, with parent PAHs accounting for 71 to 90% of total concentrations in soils. Contribution of oxy-PAHs, alkyl-PAHs and N-PACs ranged from 2 to 9%, 3 to 9% and 1 to 14%, respectively. Although the contributions of groups of PACs were small, some compounds were found in similar or greater concentrations than parent PAHs. Leachable fractions of 77 PACs from soils were small and ranged from 0.002 to 0.54%. Polar PACs were shown to be more leachable than parent PAHs. The contribution of analyzed PACS to overall AhR-mediated activities in soils and leachates suggests presence of other AhR agonists in soils, and a potential risk. Only a small fraction of AhR agonists was available in soils, indicating an overestimation of the risk if only total initial concentrations in soils would be considered in risk assessment. The results of the study strongly support that focus on 16US EPA PAHs may result in inadequate assessment of risk and hazard of PACs in complex environmental samples.

Effects of perfluorooctane sulfonate on genes controlling hepatic fatty acid metabolism in livers of chicken embryos.

Per- and polyfluoroalkyl substances (PFAS) are synthetic surfactants with a wide variety of applications; however, due to their stability, they are particularly resistant to degradation and, as such, are classed as persistent organic pollutants. Perfluorooctane sulfonate (PFOS) is one such PFAS that is still detectable in a range of different environmental settings, despite its use now being regulated in numerous countries. Elevated levels of PFOS have been detected in various avian species, and the impact of this on avian health is of interest when determining acceptable levels of PFOS in the environment. Due to its similarities to naturally occurring fatty acids, PFOS has potential to disrupt a range of biological pathways, particularly those associated with lipid metabolism, and this has been shown in various species. In this study, we have investigated how in ovo exposure to environmentally relevant levels of PFOS affects expression of genes involved in lipid metabolism of developing chicken embryos. We have found a broad suppression of transcription of genes involved in fatty acid oxidation and PPAR-mediated transcription with more significant effects apparent at lower doses of PFOS. These results highlight the need for more research investigating the biological impacts of low levels of PFAS to properly inform environmental policy governing their regulation.

Methylated PACs are more potent than their parent compounds: A study of aryl hydrocarbon receptor-mediated activity, degradability, and mixture interactions in the H4IIE-luc assay.

Twenty-six polycyclic aromatic compounds (PACs; including native polycyclic aromatic hydrocarbons [PAHs], hydroxylated PAHs, alkylated and oxygenated PAHs, and [alkylated] heterocyclic compounds) were investigated for their aryl hydrocarbon receptor (AhR)-mediated potencies in the H4IIE-luc bioassay. Potential degradabilities of PACs were investigated by use of various durations of exposure (24, 48, or 72 h), and various mixtures of PACs including PAHs, alkylated and oxygenated PAHs, and heterocyclic compounds were tested for their joint AhR-mediated potency. Additive behaviors of PACs in mixtures were studied by comparing observed mixture potencies with mixture potencies predicted by use of the concentration addition model. Methylated derivatives were more potent than their parent compounds in the H4IIE-luc assay. A time-dependent decrease in relative potency was observed for all AhR-active compounds, which may be indicative of in vitro biotransformation. Monomethylated compounds seemed to be more rapidly transformed than analogous unsubstituted compounds. In addition, the results showed that the predictive power of the concentration addition model increased with the number of compounds, suggesting additivity in multicomponent mixtures. Due to the greater potency of methylated derivatives and their ubiquitous occurrence, there is a need for further research on the toxicity and mixture behavior of these environmentally and toxicologically relevant compounds. Environ Toxicol Chem 2018;37:1409-1419. © 2018 SETAC.

Methylated polycyclic aromatic hydrocarbons and/or their metabolites are important contributors to the overall estrogenic activity of polycyclic aromatic hydrocarbon-contaminated soils.

In the present study 42 polycyclic aromatic compounds (PACs) were investigated for their estrogenic potential using the VM7Luc4E2 transactivation assay. Relative potencies were determined for mass-balance analysis. In addition, compounds were tested in combination with the estrogen receptor (ER) antagonist ICI182,780 (ICI) and the aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor α-naphthoflavone. Luciferase induction and CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity were measured to assess whether the estrogenic activity was elicited by the compound itself and/or by its metabolites. Relative potencies ranged between 10-7 and 10-4 . The ability of ICI to decrease luciferase activity stimulated by all compounds indicated that the induction responses were ER-dependent. The aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor α-naphthoflavone decreased luciferase induction and EROD activity by several compounds, including the methylated chrysenes, suggesting that metabolites of these chemicals contributed to ER activation. Several PACs, such as acridine and its derivatives, appear to directly activate the ER. Furthermore, extracts of soils from industrial areas were examined using this bioassay, and estrogenic activity was detected in all soil samples. Mass-balance analysis using a combination of relative potencies and chemical analysis of the samples suggested that polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs, such as 1- and 3-methylchrysene, are important contributors to the overall estrogenic activity. However, these results revealed that a considerable proportion of the estrogenic activity in the soil remained unexplained, indicating the presence of other significant estrogenic compounds. Environ Toxicol Chem 2018;37:385-397. © 2017 SETAC.

Developmental toxicity of PFOS and PFOA in great cormorant (Phalacrocorax carbo sinensis), herring gull (Larus argentatus) and chicken (Gallus gallus domesticus).

Perfluoroalkyl acids (PFAAs) are found globally in environmental samples and have been studied in various species. In this study, we compare the sensitivity of three avian species to the toxic effects of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA). Eggs of great cormorant (Phalacrocorax carbo sinensis), herring gull (Larus argentatus) and the domestic White Leghorn chicken (Gallus gallus domesticus) were exposed in ovo by injection into the air sac. Effects on embryo survival were observed following exposure to PFOS and PFOA in chicken and herring gull. Chicken was found to be the most sensitive species with 50 % reduced embryo survival at 8.5 µg/g egg for PFOS and 2.5 µg/g egg for PFOA. Cormorant was shown to be the least sensitive species. The difference in sensitivity between chicken and herring gull was a factor of 2.7 for PFOS and 3.5 for PFOA. Between chicken and great cormorant, the sensitivity difference was 2.6 for PFOS and 8.2 for PFOA. Effects on embryo survival were seen at egg injection doses of PFOS close to levels found in environmental samples from wild birds, indicating that PFOS could be having effects in highly exposed populations of birds. This study also shows that there are differences in species sensitivity to PFOS and PFOA that should be taken into consideration in avian wildlife risk assessment.

Does perfluorooctane sulfonate (PFOS) act as chemosensitizer in zebrafish embryos?

Earlier studies have shown that perfluorooctane sulfonate (PFOS) increases the toxicity of other chemicals by enhancing their uptake by cells and tissues. The present study aimed at testing whether the underlying mechanism of enhanced uptake of chemicals by zebrafish (Danio rerio) embryos in the presence of PFOS is by interference of this compound with the cellular efflux transporter Abcb4. Modifications of uptake/clearance and toxicity of two Abcb4 substrates, the fluorescent dye rhodamine B (RhB) and vinblastine, by PFOS were evaluated using 24 and 48h post-fertilization (hpf) embryos. Upon 90min exposure of 24hpf embryos to 1µM RhB and different PFOS concentrations (3-300µM) accumulation of RhB in zebrafish was increased by up to 11.9-fold compared to controls, whereas RhB increases in verapamil treatments were 1.7-fold. Co-administration of PFOS and vinblastine in exposures from 0 to 48hpf resulted in higher vinblastine-caused mortalities in zebrafish embryos indicating increased uptake of this compound. Interference of PFOS with zebrafish Abcb4 activity was further studied using recombinant protein obtained with the baculovirus expression system. PFOS lead to a concentration-dependent decrease of the verapamil-stimulated Abcb4 ATPase activity; at higher PFOS concentrations (250, 500µM), also the basal ATPase activity was lowered indicating PFOS to be an Abcb4 inhibitor. In exposures of 48hpf embryos to a very high RhB concentration (200µM), accumulation of RhB in embryo tissue and adsorption to the chorion were increased in the presence of 50 or 100µM PFOS. In conclusion, the results indicate that PFOS acts as inhibitor of zebrafish Abcb4; however, the exceptionally large PFOS-caused effect amplitude of RhB accumulation in the 1µM RhB experiments and the clear PFOS effects in the experiments with 200µM RhB suggest that an additional mechanism appears to be responsible for the potential of PFOS to enhance uptake of Abcb4 substrates.

Investigations on sediment toxicity of German rivers applying a standardized bioassay battery.

River sediments may contain a huge variety of environmental contaminants and play a key role in the ecological status of aquatic ecosystems. Contaminants adsorbed to sediments and suspended solids may contribute directly or after remobilization to an adverse ecological and chemical status of surface water. In this subproject of the joint research project DanTox, acetonic Soxhlet extracts from three German river sediments from the River Rhine (Altrip and Ehrenbreitstein with moderate contamination) and River Elbe (Veringkanal Hamburg heavily contaminated) were prepared and redissolved in dimethyl sulfoxide (DMSO). These extracts were analyzed with a standard bioassay battery with organisms from different trophic levels (bacteria, algae, Daphnia, fish) as well as in the Ames test and the umuC test for bacterial mutagenicity and genotoxicity according to the respective OECD and ISO guidelines. In total, 0.01% (standard) up to 0.25% (only fish embryo test) of the DMSO sediment extract was dosed to the test systems resulting in maximum sediment equivalent concentrations (SEQ) of 2 up to 50 g l(-1). The sediment of Veringkanal near Hamburg harbor was significantly more toxic in most tests compared to the sediment extracts from Altrip and Ehrenbreitstein from the River Rhine. The most toxic effect found for Veringkanal was in the algae test with an ErC50 (72 h) of 0.00226 g l(-1) SEQ. Ehrenbreitstein and Altrip samples were about factor 1,000 less toxic. In the Daphnia, Lemna, and acute fish toxicity tests, no toxicity at all was found at 2 g l(-1) SEQ. corresponding to 0.01% DMSO. Only when increasing the DMSO concentration the fish embryo test showed a 22-fold higher toxicity for Veringkanal than for Ehrenbreitstein and Altrip samples, while the toxicity difference was less evident for the Daphnia test due to the overlaying solvent toxicity above 0.05% dimethyl sulfoxide (DMSO). The higher toxicities observed with the Veringkanal sample are supported by the PAH and PCB concentrations analyzed in the sediments. The sediment extracts of Altrip and Veringkanal were mutagenic in the Ames tester strain TA98 with metabolic activation (S9-mix). The findings allow a better ecotoxicological characterization of the sediments extensively analyzed in all subprojects of the DanTox project (e.g., Garcia-Kaeufer et al. Environ Sci Pollut Res. doi: 10.1007/s11356-014-3894-4 , 2014; Schiwy et al. Environ Sci Pollut Res. doi: 10.1007/s11356-014-3185-0 , 2014; Hollert and Keiter 2015). In the absence of agreed limit values for sediment extracts in standard tests, further data with unpolluted reference sediments are required for a quantitative risk assessment of the investigated polluted sediments.