To Örebro University

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.

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.

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.

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.

PFAS are ubiquitous industrial chemicals with known adverse health effects, particularly on the liver. The liver, being a vital metabolic organ, is susceptible to PFAS-induced metabolic dysregulation, leading to conditions such as hepatotoxicity and metabolic disturbances. In this study, we investigated the phenotypic and metabolic responses of PFAS exposure using two hepatocyte models, HepG2 (male cell line) and HepaRG (female cell line), aiming to define phenotypic alterations, and metabolic disturbances at the metabolite and pathway levels. The PFAS mixture composition was selected based on epidemiological data, covering a broad concentration spectrum observed in diverse human populations. Phenotypic profiling by Cell Painting assay disclosed predominant effects of PFAS exposure on mitochondrial structure and function in both cell models as well as effects on F-actin, Golgi apparatus, and plasma membrane-associated measures. We employed comprehensive metabolic characterization using liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS). We observed dose-dependent changes in the metabolic profiles, particularly in lipid, steroid, amino acid and sugar and carbohydrate metabolism in both cells as well as in cell media, with HepaRG cell line showing a stronger metabolic response. In cells, most of the bile acids, acylcarnitines and free fatty acids showed downregulation, while medium-chain fatty acids and carnosine were upregulated, while the cell media showed different response especially in relation to the bile acids in HepaRG cell media. Importantly, we observed also nonmonotonic response for several phenotypic features and metabolites. On the pathway level, PFAS exposure was also associated with pathways indicating oxidative stress and inflammatory responses. Taken together, our findings on PFAS-induced phenotypic and metabolic disruptions in hepatocytes shed light on potential mechanisms contributing to the broader comprehension of PFAS-related health risks.

The presence of microplastics (MPs) is increasing at a dramatic rate globally, posing risks for exposure and subsequent potential adverse effects on human health. Apart from being physical objects, MP particles contain thousands of plastic-associated chemicals (i.e., monomers, chemical additives, and non-intentionally added substances) captured within the polymer matrix. These chemicals are often migrating from MPs and can be found in various environmental matrices and human food chains; increasing the risks for exposure and health effects. In addition to the physical and chemical attributes of MPs, plastic surfaces effectively bind exogenous chemicals, including environmental pollutants (e.g., heavy metals, persistent organic pollutants). Therefore, MPs can act as vectors of environmental pollution across air, drinking water, and food, further amplifying health risks posed by MP exposure. Critically, fragmentation of plastics in the environment increases the risk for interactions with cells, increases the presence of available surfaces to leach plastic-associated chemicals, and adsorb and transfer environmental pollutants. Hence, this review proposes the so-called triple exposure nexus approach to comprehensively map existing knowledge on interconnected health effects of MP particles, plastic-associated chemicals, and environmental pollutants. Based on the available data, there is a large knowledge gap in regard to the interactions and cumulative health effects of the triple exposure nexus. Each component of the triple nexus is known to induce genotoxicity, inflammation, and endocrine disruption, but knowledge about long-term and inter-individual health effects is lacking. Furthermore, MPs are not readily excreted from organisms after ingestion and they have been found accumulated in human blood, cardiac tissue, placenta, etc. Even though the number of studies on MPs-associated health impacts is increasing rapidly, this review underscores that there is a pressing necessity to achieve an integrated assessment of MPs’ effects on human health in order to address existing and future knowledge gaps.

This study presents a comprehensive chemical and toxicological screening of chemicals extracted from WEEE (waste from electrical and electronic equipment) plastics. Chemical identification was conducted through suspect and target screening methods, revealing a diverse array of hazardous compounds including polycyclic aromatic compounds (PACs), organophosphate flame retardants (OPFRs), phthalates, benzotriazoles, and others. Toxicological endpoints included cell morphological phenotypes, inflammatory response, aryl hydrocarbon receptor (AhR) activation, activation of estrogenic receptor, and anti-androgenic activity. Results demonstrated that WEEE plastic chemicals significantly altered cell morphological phenotypes, particularly affecting the cytoskeleton, endoplasmic reticulum (ER), and mitochondrial measures. Moreover, WEEE chemicals induced inflammatory responses in resting human macrophages and altered ongoing inflammatory responses in lipopolysaccharide (LPS)-primed macrophages. Furthermore, WEEE chemicals exhibited potent AhR agonistic activity, activated estrogen receptor-α (ERα), and inhibited androgen receptor (AR) activation. The findings suggest that WEEE plastic chemicals exert their effects through multiple modes of action, targeting various subcellular sites. Thus, a combined approach utilizing non-target and target screening tools is essential for comprehensively assessing the toxic effects and health hazards associated with WEEE plastic chemicals.

Degradation of the 16 US EPA priority PAHs in soil subjected to bioremediation is often achieved. However, the PAH loss is not always followed by a reduction in soil toxicity. For instance, bioanalytical testing of such soil using the chemical-activated luciferase gene expression (CALUX) assay, measuring the combined effect of all Ah receptor (AhR) activating compounds, occasionally indicates that the loss of PAHs does not correlate with the loss of Ah receptor-active compounds in the soil. In addition, standard PAH analysis does not address the issue of total toxicant bioavailability in bioremediated soil.

To address these questions, we have used the CALUX AhR agonist bioassay and the Comet genotoxicity bioassay with RTL-W1 cells to evaluate the toxic potential of different extracts from a PAH-contaminated soil undergoing large-scale bioremediation. The extracts were also chemically analyzed for PAH16 and PCDD/PCDF. Soil sampled on five occasions between day 0 and day 274 of biological treatment was shaken with n-butanol with vortex mixing at room temperature to determine the bioavailable fraction of contaminants. To establish total concentrations, parts of the same samples were extracted using an accelerated solvent extractor (ASE) with toluene at 100A degrees C. The extracts were tested as inducers of AhR-dependent luciferase activity in the CALUX assay and for DNA breakage potential in the Comet bioassay.

The chemical analysis of the toluene extracts indicated slow degradation rates and the CALUX assay indicated high levels of AhR agonists in the same extracts. Compared to day 0, the bioavailable fractions showed no decrease in AhR agonist activity during the treatment but rather an up-going trend, which was supported by increasing levels of PAHs and an increased effect in the Comet bioassay after 274 days. The bio-TEQs calculated using the CALUX assay were higher than the TEQs calculated from chemical analysis in both extracts, indicating that there are additional toxic PAHs in both extracts that are not included in the chemically derived TEQ.

The response in the CALUX and the Comet bioassays as well as the chemical analysis indicate that the soil might be more toxic to organisms living in soil after 274 days of treatment than in the untreated soil, due to the release of previously sorbed PAHs and possibly also metabolic formation of novel toxicants.

Our results put focus on the issue of slow degradation rates and bioavailability of PAHs during large-scale bioremediation treatments. The release of sorbed PAHs at the investigated PAH-contaminated site seemed to be faster than the degradation rate, which demonstrates the importance of considering the bioavailable fraction of contaminants during a bioremediation process.

It has to be ensured that soft remediation methods like biodegradation or the natural remediation approach do not result in the mobilization of toxic compounds including more mobile degradation products. For PAH-contaminated sites this cannot be assured merely by monitoring the 16 target PAHs. The combined use of a battery of biotests for different types of PAH effects such as the CALUX and the Comet assay together with bioavailability extraction methods may be a useful screening tool of bioremediation processes of PAH-contaminated soil and contribute to a more accurate risk assessment. If the bioremediation causes a release of bound PAHs that are left undegraded in an easily extracted fraction, the soil may be more toxic to organisms living in the soil as a result of the treatment. A prolonged treatment time may be one way to reduce the risk of remaining mobile PAHs. In critical cases, the remediation concept might have to be changed to ex situ remediation methods.

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).

Toxicity of polyethylene microplastics (PE-MP) of size ranges similar to their natural food to zooplanktonic organisms representative of the main taxa present in marine plankton, including rotifers, copepods, bivalves, echinoderms and fish, was evaluated. Early life stages (ELS) were prioritized as testing models in order to maximize sensitivity. Treatments included particles spiked with benzophenone-3 (BP-3), a hydrophobic organic chemical used in cosmetics with direct input in coastal areas. Despite documented ingestion of both virgin and BP-3 spiked microplastics no acute toxicity was found at loads orders of magnitude above environmentally relevant concentrations on any of the invertebrate models. In fish tests some effects, including premature or reduced hatching, were observed after 12 d exposure at 10 mg L^-1 of BP-3 spiked PE-MP. The results obtained do not support environmentally relevant risk of microplastics on marine zooplankton. Similar approaches testing more hydrophobic chemicals with higher acute toxicity are needed before these conclusions could be extended to other organic pollutants common in marine ecosystems. Therefore, the replacement of these polymers in consumer products must be carefully considered.

In this study, we examined 31 samples of varying chemical composition, including samples of soils from gasworks, coke production sites, and sites where wood preservatives were heavily used; ash and soot from municipal solid waste incinerators; antiskid sand; and dust from areas with heavy road traffic. The samples were comprehensively chemically characterized, especially their polycyclic aromatic compound contents, using gas chromatography-time-of-flight mass spectrometry, whereas their biological effects were assessed using dehydrogenase activity, root growth (Hordeum vulgare), reproduction of springtails (Folsomia candida), algal growth (Desmodesmus subspicatus), germinability (Sinapis alba), Vibrio fischeri, DR-CALUX, and Ames Salmonella assays. The number of compounds detected in the samples ranged from 123 to 527. Using the multivariate regression technique of partial-least-squares projections to latent structures, it was possible to find individual compounds that exhibited strong correlations with the different biological responses. Some of the results, however, indicate that a broader chemical characterization may be needed to identify all the compounds that may cause the measured biological responses.

A technique for studying 7-ethoxyresorufin O-deethylase (EROD) induction in chick embryo liver in vitro was developed. Livers from 8-d-old embryos were cultured in rotating vials at 37 degrees C for 48 h in a medium to which DMSO-dissolved test compounds had been added. This bioassay proved to be highly sensitive to dioxin-like compounds, and its usefulness for assessing the toxic potency of such compounds in environmental samples was demonstrated. Concentration-response curves were determined for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5-penlachlorobiphenyl (PCB IUPAC no. 126), 3,3',4,4'-tetrachlorobiphenyl (PCB 77), 2,3,3',4,4'-pentachlorobiphenyl (PCB 105), and benzo[k]fluoranthene (BkF). TCDD induced EROD in a concentration-dependent manner, having an EC50 of 5.0 x 10(-12) M. The cultured embryo livers were extremely sensitive to TCDD, and about 30 fg of this compound per liver (2 x 10(-13) M) was enough to significantly induce EROD. The EC50 values obtained for PCBs 126, 77, 105, and BkF were 4,4 x 10(-11) M, 9.2 x 10(-9) M, 1.6 x 10(-8) M, and 6.2 x 10(-7) M, respectively. The maximal induction levels obtained for three different preparations of polychlorinated naphthalenes (PCNs) were less than those of the other compounds tested. When the technical PCN mixture Halowax 1014 was coadministered with TCDD, the induction was lower than that caused by TCDD alone. An organic extract of fly ash from a municipal waste combustion plant was very potent. Considering its contents of polychlorinated dibenzo-p-dioxins/furans, expressed as TCDD equivalents, the EC50 obtained was close to that for TCDD.

Four bioassays were used in this study for the hazard assessment of sediments from sediment traps and several ponds in a treatment wetland for landfill leachate at Atleverket, Sweden. In the 6-day solid phase microbiotest with the sediment-dwelling crustacean Heterocypris incongruens both acute and chronic effects were observed with a gradual decrease and loss of toxicity with treatment in the wetland system. Some samples showed a low toxicity in porewater and only one sample was weakly toxic in the whole sediment test when assessed with Aliivibrio fischeri (Vibro fischeri). No genotoxicity was detected in the umu test. The toxicity response in the H4IIE- luc test evaluating the presence of dioxin-like compounds was considerably higher in the samples from the sediment traps. The hazard of the sediment therefore appears to be highest in the sediment traps and pond 1 with the methods employed. The result indicates that the wetland system has a design supporting the concentration and sequestration of toxic substances in the first part of the wetland. Based upon the results we suggest that hazard assessment of sediments from other treatment wetlands for landfill leachate should be conducted.

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.

The Partnership for Chemicals Risk Assessment (PARC) is currently under development as a joint research and innovation programme to strengthen the scientific basis for chemical risk assessment in the EU. The plan is to bring chemical risk assessors and managers together with scientists to accelerate method development and the production of necessary data and knowledge, and to facilitate the transition to next-generation evidence-based risk assessment, a non-toxic environment and the European Green Deal. The NORMAN Network is an independent, well-established and competent network of more than 80 organisations in the field of emerging substances and has enormous potential to contribute to the implementation of the PARC partnership. NORMAN stands ready to provide expert advice to PARC, drawing on its long experience in the development, harmonisation and testing of advanced tools in relation to chemicals of emerging concern and in support of a European Early Warning System to unravel the risks of contaminants of emerging concern (CECs) and close the gap between research and innovation and regulatory processes. In this commentary we highlight the tools developed by NORMAN that we consider most relevant to supporting the PARC initiative: (i) joint data space and cutting-edge research tools for risk assessment of contaminants of emerging concern; (ii) collaborative European framework to improve data quality and comparability; (iii) advanced data analysis tools for a European early warning system and (iv) support to national and European chemical risk assessment thanks to harnessing, combining and sharing evidence and expertise on CECs. By combining the extensive knowledge and experience of the NORMAN network with the financial and policy-related strengths of the PARC initiative, a large step towards the goal of a non-toxic environment can be taken.

Introduction: Given the complex interactions of re-suspension processes and bioavailability of sediment-bound pollutants such as dioxin-like chemicals, there is need for a better integrative understanding of the cause-effect relationship of these pollutants. Currently, the majority of studies investigating potential risks of these chemicals only focus on characterizing sediment extracts via in vitro bioassays, thereby disregarding bioavailability, uptake, metabolism, and elimination rates of these compounds in vivo. To determine to which extent mechanism-specific effects in vitro reflect possible adverse effects in vivo, the research project dioRAMA, involving partnership between the Institute for Environmental Research of RWTH Aachen University and the Department Biochemistry/Ecotoxicology of the German Federal Institute of Hydrology, was established.

Methods: Animals from an elevated trophic level-common roach (Rutilus rutilus)-will be exposed to sediments from two major German rivers. Exposure will be performed in a system that enables a concurrent monitoring of environmental parameters. In parallel, in vitro studies will be conducted to determine dioxin-like potentials of sediment and fish extracts from the in vivo exposure experiments using different cell lines with varying endpoints. Moreover, extract fractionation procedures, using the strategy of effect-directed analysis, will enable the detection of specific contaminant groups responsible for the biological activity observed.

Conclusion: A closer interconnection between applied ecotoxicological science and regulatory needs will facilitate the improved assessment of dioxin-like compounds in sediment and biota. Consequently, this will enable their application in sediment management programs, which is one of the main goals of the dioRAMA project.

Use of in vitro assays as screening tool to characterize contamination of a variety of environmental matrices has become an increasingly popular and powerful toolbox in the field of environmental toxicology.

While bioassays cannot entirely substitute analytical methods such as gas chromatography-mass spectrometry (GC-MS), the increasing improvement of cell lines and standardization of bioassay procedures enhance their utility as bioanalytical pre-screening tests prior to more targeted chemical analytical investigations. Dioxin-receptor-based assays provide a holistic characterization of exposure to dioxin-like compounds (DLCs) by integrating their overall toxic potential, including potentials of unknown DLCs not detectable via e.g. GC-MS. Hence, they provide important additional information with respect to environmental risk assessment of DLCs.

This review summarizes different in vitro bioassay applications for detection of DLCs and considers the comparability of bioassay and chemical analytically derived toxicity equivalents (TEQs) of different approaches and various matrices. These range from complex samples such as sediments through single reference to compound mixtures. A summary of bioassay derived detection limits (LODs) showed a number of current bioassays to be equally sensitive as chemical methodologies, but moreover revealed that most of the bioanalytical studies conducted to date did not report their LODs, which represents a limitation with regard to low potency samples.

Extracts of bottom sediment and settling particulate matter (SPM) samples collected in the recipient water body of a bleached pulp mill effluent were separated into three fractions: monoaromatic/aliphatic compounds, diaromatic compounds (e.g., polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans), and polyaromatic compounds assessment of dioxinlike potency were an in vitro assay, based on the induction of 7-ethoxyresorufin-O-deethylase (EROD) in chicken embryo livers, a test for EROD induction in ovo using chicken embryos, and an immunotoxicity test measuring inhibition of lymphoid cell development in cultured fetal mouse thymuses. The samples collected closest to the mill were the most potent. There was a time-dependent decrease in dioxinlike potency in the SPM samples collected near the mill, which coincided with the cessation of chlorine gas bleaching at the mill. The bioassays in general, and the in vitro chicken embryo liver bioassay in particular, proved useful in the assessment of dioxinlike compounds in the sediment and SPM samples. The polyaromatic fractions of the samples were generally more potent than the diaromatic fractions. Only a small part of the effects caused by the polyaromatic fractions could be explained by 15 analyzed PAHs, indicating the presence of unquantified polyaromatic compounds with dioxinlike effects. This investigation indicates that the cessation of chlorine bleaching in the pulp mill resulted in a reduced load of diaromatic dioxinlike compounds in the recipient water body.

Sediments and settling particulate matter (SPM) were sampled in a polychlorinated biphenyl (PCB)-contaminated lake, Lake Jarnsjon, and in lakes located up-/and downstream from Lake Jarnsjon. The 7-ethoxyresorufin O-deethylase (EROD)-inducing potencies of lipophilic extracts from sediment and SPM were investigated in organ cultures of chicken embryo livers (denoted in vitro chicken embryo liver bioassay). The extract from sediments collected in Lake Jarnsjon had the greatest EROD-inducing potency of the extracts studied, and it also contained the highest concentrations of PCBs, polycyclic aromatic hydrocarbons (PAHs), and polychlorinated dibenzodioxins/furans (PCDDs/Fs). The non-ortho-chlorinated PCBs in the extracts were probably major contributors to the EROD induction noted. The EROD-inducing potency of sediment extract from the lake downstream Lake Jarnsjon was higher than that of extract from the upstream lake. This indicates that EROD-inducing substances were transported from Lake Jarnsjon sediment to the lake downstream. The extracts from Lake Jarnsjon sediment and SPM were separated into three fractions containing aliphatic/monoaromatic, diaromatic, and polyaromatic compounds, respectively, which were tested in the in vitro chicken embryo Liver bioassay. In all extracts, the aliphatic/monoaromatic fractions were low in EROD-inducing potency, while the polyaromatic fractions from SPM were more potent than their corresponding diaromatic fractions, indicating that they contained high concentrations of aromatic hydrocarbon (Ah) receptor ligands. Only a small part of the EROD induction caused by the polyaromatic fractions could be attributed to the 15 analyzed PAHs in the samples. The EROD-inducing potencies of the diaromatic fractions correlated quite well with their concentrations of PCDDs/Fs and PCBs. The in vitro chicken embryo liver bioassay detected low concentrations of EROD inducers in the extracts and therefore proved useful for estimating the contents of Ah receptor ligands in environmental samples.

Lipophilic extracts of sediment, settling particulate matter (SPM) and blue mussel (Mytilus edulis) samples, collected at coastal locations on the east and west coast of Sweden, were HPLC-separated into three fractions containing 1. monoaromatic/aliphatic, 2. diaromatic (e,g, polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins and polychlorinated dibenzofuraus (PCDDs/Fs)), and 3, polyaromatic compounds (e,g, polycyclic aromatic hydrocarbons (PAHs)), The fractions were tested for dioxin-like effects using a sensitive bioassay, based on EROD-induction in cultured chicken embryo livers, The concentrations of PCDDs/Fs, PCBs and 15 PAHs in the samples were also determined, The polyaromatic fractions of the sediment samples were more potent as EROD-inducers than the diaromatic fractions, Only a small part of the EROD-induction caused by the polyaromatic fractions could be explained by the analysed PAHs in the samples, indicating presence of non-quantified polyaromatic EROD-inducing compounds in the samples, A greater pollution by EROD-inducing diaromatic and polyaromatic compounds on the east coast site than on the west coast site was seen, The filtration activities and faeces production of the mussels on the east coast experimental site increased the sedimentation of PCDDs/Fs, PCBs and PAHs, The described bioassay proved very useful in the assessment of dioxin-like compounds in both abiotic and biotic samples from the marine environment,

The 7-ethoxyresorufin-O-deethylase (EROD)-inducing potencies of a coplanar polychlorinated biphenyl (PCB) (3,3',4,3',5-pentachlorobiphenyl, IUPAC No. 126), a mixture of five polycyclic aromatic hydrocarbons (benzo(k)fluoranthene, benz(a)anthracene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene and chrysene), and lipophilic compounds extracted from the sediments in a PCB-contaminated lake and from sediments in lakes up- and downstream, were studied in rainbow trout sac-fry in a 43-day study. The compounds/extracts were injected into the yolk sacs of newly hatched sac-fry and hepatic EROD activities and mortality rates were measured at various times after the injections. Five livers from each group were also examined by transmission electron microscopy. All the compounds/extracts induced hepatic EROD activities in the sac-fry. Ten days after injection the EROD activity caused by PCB No. 126 (1.3 ng per embryo) was 40-fold compared to the control activity. This was the highest induction rate observed in the experiment. For the sediment extracts, the highest induction rates were observed at the first sampling occasion, which for these groups was on day 24. The extract from the Lake Jarnsjon sediment was more potent as an EROD inducer than the extracts of sediments from the lakes up- and downstream from Lake Jarnsjon. None of the sediment extracts caused any significant mortality. In sac-fry injected with the PAH mixture, EROD was only slightly induced. The highest dose of PAHs (10 mu g per embryo) caused about 90% mortality by 24 days after injection. When the livers were examined by transmission electron microscopy, morphological alterations (e.g. hepatocyte degeneration and hypertrophy) were seen in the groups injected with Lake Jarnsjon sediment extract, PCB No. 126 and the highest dose of the PAH mixture (10 mu g per embryo).

The 7-ethoxyresorufin O-deethylase (EROD)- and aryl hydrocarbon hydroxylase (AHH)-inducing potencies and lethalities of a technical preparation of polychlorinated naphthalenes (PCNs) (Halowax 1014, approx imate congener ratio: 20% tetrachloronaphthalenes, 40% pentachloronaphthalenes, 40% hexachloronaphthalenes), a mixture of 50% 1,2,3,5,6,7-hexachloronaphthalene and 50% 1,2,3,4,6,7-hexachloronaphthalene (HxCN-mix), and 1,2,3,4,5,6,7-heptachloronaphthalene (HpCN) were studied in chicken (Gallus domesticus) and elder duck (Somateria mollissima) embryos. Mortality and hepatic EROD activity were determined on day 10 of incubation in chicken embryos exposed to various doses of the PCNs via the air-sacs of the eggs on day 7. The HxCN-mix and Halowax 1014 proved to have both embryolethal and EROD-inducing properties, while the HpCN had low EROD-inducing potency and embryolethality. ED(50) values for EROD induction by the HxCN-mix and Halowax 1014 were estimated to be 0.06 mg/kg egg and 0.2 mg/kg egg, respectively. Fifty percent of the chicken embryos died (6/12) when given 3.0 mg/kg of the HxCN-mix while a similar dose of Halowax 1014 caused mortality in 4 out of 12 chicken embryos. The dose-response curve for EROD induction by Halowax 1014 exhibited a decline after the maximal level was reached. When Halowax 1014 (1.0 mg/kg, egg) was coinjected with 3,3',4,4',5-pentachlorobiphenyl (PCB IUPAC #126) (0.1 mu g/kg egg) no additive effects on EROD activity were found, but when the same dose of Halowax 1014 was coinjected with a dose of PCB #126, known to cause maximal induction (1.0 mu g/kg egg), the resulting EROD activity was lower than that caused solely by 1.0 mu g PCB #126/kg egg. These findings indicate that Halowax 1014 has both EROD-inducing and EROD-inhibiting properties. Mortality and EROD and AHH activities were determined on day 18 (chicken) or day 24 (elder) of incubation in embryos exposed to 1.0 mg/kg egg via the yolk-sac on day 4 (chicken) or day 5 (elder). The HxCN-mix and Halowax 1014 induced AHH and EROD in both chicken and elder, but the induction rates were higher in the elder embryos. The HxCN-mix and Halowax 1014 caused degenerative hepatic lesions and pericardial oedema in the chicken embryos but not in the elder embryos. The most toxic PCNs tested (the HxCN-mix and Halowax 1014) were approximately of the same EROD-inducing potency as previously found for the most toxic mono-ortho-chlorinated biphenyls (Brunstrom 1990), and 1000 times less toxic and potent as EROD inducers compared with PCB #126 (Brunstrom and Andersson 1988). HpCN was considerably less toxic and exhibited a low EROD-inducing potency. The chicken embryos were more sensitive to the hepatotoxic effects produced by Halowax 1014 and the HxCN-mix than the elder duck embryos, while the elder embryos were more responsive in terms of EROD and AHH induction. The two HxCNs studied usually make up approximately 1% of the total quantity of PCNs present in Halowax 1014 [when determined with gas chromatography (flame ionization detection)]. Therefore, the relatively high toxic potency of Halowax 1014 cannot be explained by its content of the two HxCNs.

A bioassay for the detection of dioxin-like compounds was used to estimate levels in sewage sludge from Swedish sewage treatment plants (STPs). The sludge extracts were HPLC-separated into three fractions containing a) monoaromatic/aliphatic, b) diaromatic (e.g. polychlorinated biphenyls [PCBs], polychlorinated dibenzodioxins and polychlorinated dibenzofurans [PCDDs/Fs]), and c) polyaromatic compounds (e.g. polycyclic aromatic hydrocarbons [PAHs]). The bioassay, which is based on EROD (7-ethoxyresorufin O-deethylase) induction in cultured chicken embryo livers detected dioxin-like activity in all unfractionated extracts and in the di- and polyaromatic fractions of all sludge extracts, but not in the monoatomatic/aliphatic fractions. The levels ranged between 6 and 109 pg bio-TEQ/g sludge (d.w.). In sediment samples from rural lakes in Sweden, levels of about 5 pg bio-TEQ/g (d.w.) have been found. The polyaromatic fractions of the sludge samples were potent in the bioassay, probably due to various PAHs and other polyaromatics in the sludge. The levels of six PAHs that are screened for in the sludge at Swedish STPs accounted for only 3-10% of the observed EROD-induction by the polyaromatic fractions. Consequently, many other polyaromatic EROD-inducing compounds were present in the sludge. Inclusion of a biological test like the chicken embryo liver bioassay in the screening of sludge would improve the ability to detect the presence of bioactive dioxin-like compounds. A theoretical estimation of bio-TEQ concentrations in farm-soil following long-term application of sludge with bio-TEQ concentrations similar to those observed in this investigation indicated that the bio-TEQ levels in soil would increase very slowly over time. The chicken embryo liver bioassay proved useful in assessing levels of dioxin-like compounds in sewage sludge and it gives valuable complementary information to chemical analysis data.

A bioassay for the detection of dioxin-like compounds was used to estimate uptake of dioxin-like compounds in carrots, oil seed rape seeds, zucchinis and cucumbers grown in soil amended with sewage sludge from Swedish sewage treatment plants (STP), This sensitive bioassay is based on 7-ethoxyresorufin O-deethylase (EROD)-induction in cultured chicken embryo livers and reflects the combined biological effect of all dioxin-like compounds in a sample, including ones that seldom are analyzed. The bioassay detected low concentrations of dioxin-like compounds in all carrot, zucchini and cucumber samples, but did not detect any dioxin-like compounds in the rape seeds. In carrots the concentrations were increased up to seven times when grown in soil amended with high applications of some of the sludge samples, while others did not increase the concentrations compared to control. More realistic sludge applications only increased the concentrations slightly. The sludge-fertilized carrots contained the highest concentrations of the investigated plants (up to 14 pg bioassay-derived TCDD equivalents (bio-TEQs)/g d.w.). In the carrots, differences in uptake of dioxin-like compounds depended on the sludge origin, which may be due to more easily bioaccumulated dioxin-like compounds in some sludge samples, or other components that facilitated uptake into the carrots. In the cucumbers, a more than two-fold increase (from 0.2 to 0.5 pg bio-TEQs/g d.w.) was observed in specimens grown in sludge-amended soil when compared to controls, suggesting a small uptake from the roots to the shoots. No sludge-dependent increase in uptake was seen in the zucchini fruits. The bio-TEQ levels were generally low in the consumable above ground plant parts of the investigated species. However, the question if repeated sludge application results in a soil accumulation of dioxin-like compounds, thereby increasing the risk of plant uptake, remains to be investigated.

Polycykliska aromatiska kolväten (PAHer) är relativt vanliga i förorenadeområden, särskilt på gamla gasverktomter, bensinstationer och tidigare impregneringsanläggningar.På grund av deras toxicitet så är sanering av PAH-förorenadeområden av hög prioritet. För att minska riskerna med PAH-förorenade jordar,både före och efter sanering, är det viktigt att åstadkomma en heltäckande riskbedömningoch säker klassning av dessa jordmassor. De generella riktvärden förPAH-förorenad mark som används idag är i regel baserade på kemisk analys av 16standard PAHer (PAH16), trots att det ofta förekommer 100-tals PAHer och PAHmetaboliteri jordarna.I detta projekt har vi genom att jämföra kemisk och biologisk analys (H4IIEluc)av ett flertal sanerade PAH-förorenade jordprover studerat om toxicitetenverkligen minskar i proportion till minskningen av PAHer i jordarna. H4IIE-luc ären mekanismspecifik bioanalys som detekterar alla ämnen som aktiverar Ahreceptorn,en av de två viktigaste mekanismerna bakom PAHers toxicitet. Jämförelsenav resultaten visade att den totala toxiciteten i de sanerade jordprovernainte gick att förklara med kemisk analys av PAH16 och att man därmed med dagensanalysmetodik riskerar att missa toxikologiskt relevanta PAHer och andra liknandeämnen. Vidare kemiska identifieringsstudier samt bioanalytiska studier krävs föratt ta reda på om dessa okända ämnen utgör en risk för människa eller miljö.Våra resultat visar på svagheten med kemisk analys av ett mindre antal ämnensom grundval för klassning av renade massor. Det är därför är rimligt att inkluderamekanismspecifika tester i riskbedömning och vid klassning av renade PAHförorenadejordar. Dels för att minimera riskerna som dessa jordar kan utgöra förmänniskor och miljö, dels för att man med en större säkerhet och i större utsträckningskall kunna återanvända sanerade jordmassor.

A sensitive bioassay, based on EROD induction in cultured chicken embryo livers, was used together with chemical analysis to determine levels of dioxin-like contaminants in particulate matter in Eman. Extracts of sediment and settling particulate matter (SPM) collected in the river system before, during, and after dredging of a PCB contaminated lake, Lake Jarnsjon, were separated into three fractions containing a) monoaromatic/aliphatic; b) diaromatic (e.g., PCBs and polychlorinated dibenzodioxins and dibenzofurans); and c) polyaromatic compounds (e.g., polycyclic aromatic hydrocarbons). The samples from Lake Jarnsjon contained the highest PCB concentrations and the diaromatic extracts from Lake Jarnsjon samples showed the highest activities of EROD-inducing diaromatic compounds. The dioxin-like activity of the diaromatic fraction in sediment collected after the dredging of Jarnsjon had only around 1% of the activity of the pre-dredging sediment, showing that the remediation was successful in terms of removal of the PCB-contaminated sediment from the lake. In SPM collected immediately downstream from Jarnsjon, levels of diaromatic dioxin-like compounds were elevated during the dredging, and decreased after that. The post-dredging concentrations were however higher than in SPM from lakes upstream of Jarnsjon, showing that elevated levels of dioxin-like diaromatic compounds were still present in the water System downstream of Jarnsjon a couple of years after the dredging.

The fate of 7-ethoxyresorufin O-deethylase (EROD)-inducing compounds in source-separated organic household waste subject to anaerobic degradation (i.e. mesophilic/thermophilic anaerobic treatment) was investigated using organ cultures of embryonic chicken livers from fertilised hen eggs. This bioassay reflects the combined effect of all EROD-inducing, possibly dioxin-like compounds in a sample, including chemicals that are seldom or never analysed. All samples tested induced EROD in the bioassay, indicating the presence of dioxin-like compounds. In the anaerobic processes, the amounts of acid-resistant EROD-inducing compounds coming out of the reactors were considerably higher than the incoming amounts, especially for the low-temperature (mesophilic) process. This apparent production of EROD-inducing compounds may be due to de novo synthesis or to an increase in the EROD-inducing potency of the compounds in the material.

Polycyclic aromatic hydrocarbons (PAHs) are widespread fused-ring contaminants formed during incomplete combustion of almost all kind of organic materials from both natural and anthropogenic sources. Some PAHs have been shown to be carcinogenic to humans, and a wide range of PAHs are found in wildlife all around the globe including avian species. The purpose of this project was to assess the effects of a standard mixture of 16 PAHs (United States Environmental Protection Agency) on the hepatic fatty acid beta-oxidation in chicken embryos (Gallus gallus domesticus) exposed in ovo. The hepatic beta-oxidation was measured using a tritium release assay with [9,10-H-3]-palmitic acid (16:0) as substrate. Treated groups were divided into groups of 0.05, 0.1, 0.3, 0.5, and 0.8 mg PAHs/kg egg weight. The hepatic beta-oxidation was reduced after exposure in ovo to the 16 PAHs mixture compared to control. The mechanisms causing reduced fatty acid oxidation in the present study are unclear, however may be due to deficient membrane structure, the functionality of enzymes controlling the rate of fatty acid entering into the mitochondria, or complex pathways connected to endocrine disruption. To the best of our knowledge, this is the first time a PAH-caused reduction of hepatic beta-oxidation of fatty acids in avian embryos has been observed. The implication of this finding on risk assessment of PAH exposure in avian wildlife remains to be determined.

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.

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.

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 β-oxidation is suppressed while the peroxisomal fatty acid β -oxidation is increased. At higher doses, however, both β -oxidation pathways are upregulated.

An industrial sludge containing a complex mixture of nitroaromatic compounds was treated in industrial large-scale aerobic and anaerobic biodegradation processes, performed in compost sacs. The goal was to study changes in genotoxicity during the two different oxygen regimes using the umuC genotoxicity assay. The composting sac was actively aerated during 3 months and allowed to mature for another 3 months. The anaerobic sac was not aerated for 5 months and aerated during the last month in order to enhance degradation of remaining organic carbon. The sludge was obtained from the wastewater treatment plant at an industrial area in Karlskoga, Sweden. The biodegradation study was performed at a commercial waste treatment plant in Stockholm, according to the company routine procedure when treating household waste in sealed sacs.

The material from the non-aerated system showed increased genotoxicity in the acetone-soluble fraction after treatment, as did the water-soluble fraction. The subsequent aeration period did not decrease the toxicity below the genotoxicity limit. The increase in the water-soluble genotoxic compounds may pose an environmental problem during secondary storage or use of sludge treated this way, since leakage of water-dissolved genotoxic compounds may occur.

The composting process also generated genotoxicity, but this was restricted to acetone-soluble compounds, while the water-soluble compounds remained low in genotoxicity. The aerated process therefore seems more favorable in term of risk reduction of this industrial sludge, although it is necessary to optimize the aerated process in order to achieve non-toxic levels of potential genotoxic compounds extractable by organic solvents.

Since the mid-1970s, Sweden has been depositing 1 million ton d.w sludge/year, produced at waste water treatment plants. Due to recent legislation this practice is no longer a viable method of waste management. It is necessary to improve existing and develop new sludge management techniques and one promising alternative is the dewatering and treatment of sludge in constructed wetlands. The aim of this study was to follow reduction of organic carbon, BOD and nutrients in an industrial sludge containing nitro-aromatic compounds passing through constructed small-scale wetlands, and to investigate any toxic effect such as growth inhibition of the common reed Phragmites australis. The result showed high reduction of all tested parameters in all the outgoing water samples, which shows that constructed wetlands are suitable for carbon and nutrient removal. The results also showed that P. australis is tolerant to xenobiotics and did not appear to be affected by the toxic compounds in the sludge. The sludge residual on the top of the beds contained low levels of organic carbon and is considered non-organic and could therefore be landfilled. Using this type of secondary treatment method, the amount of sludge could be reduced by 50-70%, mainly by dewatering and biodegradation of organic compounds.

Goal, Scope and Background:  Sweden has prohibited the deposition of organic waste since January, 2005. Since 1 million tons of sludge is produced every year in Sweden and the capacity for incineration does not fill the demands, other methods of sludge management have to be introduced to a larger degree. One common method in the USA and parts of Europe is the use of wetlands to treat wastewater and sewage sludge. The capacity of reed beds to affect the toxicity of a complex mixture of nitroaromatics in sludge, however, is not fully elucidated. In this study, an industrial sludge containing explosives and pharmaceutical residues was therefore treated in artificial reed beds and the change in toxicity was studied. Nitroaromatic compounds, which are the main ingredients of many pharmaceuticals and explosives, are well known to cause cytotoxicity and genotoxicity. Recently performed studies have also showed that embryos of zebrafish (Danio rerio) are sensitive to nitroaromatic compounds. Therefore, we tested the sludge passing through constructed wetlands in order to detect any changes in levels of embryotoxicity, genotoxicity and dioxin-like activity (AhR-agonists). We also compared unplanted and planted systems in order to examine the impact of the root system on the fate of the toxicants. Methods:  An industrial sludge containing a complex mixture of nitroaromatics was added daily to small-scale constructed wetlands (vertical flow), both unplanted and planted with Phragmites australis. Sludge with an average dry weight of 1.25%, was added with an average hydraulic loading rate of 1.2 L/day. Outgoing water was collected daily and stored at −20°C. The artificial wetland sediment was Soxhlet extracted, followed by clean-up with multi-layer silica, or extracted by ultrasonic treatment, yielding one organic extract and one water extract of the same sample. Genotoxicity of the extracts was measured according to the ISO protocol for the umu-C genotoxicity assay (ISO/TC 147/SC 5/WG9 N8), using Salmonella typhimurium TA1535/pSK1002 as test organism. Embryotoxicity and teratogenicity were studied using the fish egg assay with zebrafish (Danio rerio) and the dioxin-like activity was measured using the DR-CALUX assay. Chemical analyses of nitroaromatic compounds were performed using Solid Phase Micro Extraction (SPME) and GC-MS. Results:  Organic extracts of the bed material showed toxic potential in all three toxicity tests after two years of sludge loading. There was a difference between the planted and the unplanted beds, where the toxicity of organic extracts overall was higher in the bed material from the planted beds. The higher toxicity of the planted beds could have been caused by the higher levels of total carbon in the planted beds, which binds organic toxicants, and by enrichment caused by lower volumes of outgoing water from the planted beds. Discussion:  Developmental disorders were observed in zebrafish exposed directly in contact to bed material from unplanted beds, but not in fish exposed to bed material from planted beds. Hatching rates were slightly lower in zebrafish exposed to outgoing water from unplanted beds than in embryos exposed to outgoing water from planted beds. Genotoxicity in the outgoing water was below detection limit for both planted and unplanted beds. Most of the added toxicants via the sludge were unaccounted for in the outgoing water, suggesting that the beds had toxicant removal potential, although the mechanisms behind this remain unknown. Conclusions:  During the experimental period, the beds received a sludge volume (dry weight) of around three times their own volume. In spite of this, the toxicity in the bed material was lower than in the sludge. Thus, the beds were probably able to actually decrease the toxicity of the added, sludge-associated toxicants. When testing the acetone extracts of the bed material, the planted bed showed a higher toxicity than the unplanted beds in all three toxicity tests. The toxicity of water extracts from the unplanted beds, detected by the fish egg assay, were higher than the water extracts from the planted beds. No genotoxicity was detected in outgoing water from either planted or unplanted beds. All this together indicates that the planted reed beds retained semi-lipophilic acetone-soluble toxic compounds from the sludge better than the unplanted beds, which tended to leak out more of the water soluble toxic compounds in the outgoing water. The compounds identified by SPME/GC in the outgoing water were not in sufficient concentrations to have caused induction in the genotoxicity test. Recommendations and Perspectives:  This study has pointed out the benefits of using constructed wetlands receiving an industrial sludge containing a complex mixture of nitroaromatics to reduce toxicity in the outgoing water. The water from planted, constructed wetlands could therefore be directed to a recipient without further cleaning. The bed material should be investigated over a longer period of time in order to evaluate potential accumulation and leakage prior to proper usage or storage. The plants should be investigated in order to examine uptake and possible release when the plant biomass is degraded.

GOAL, SCOPE AND BACKGROUND:

Sweden is meeting prohibition for deposition of organic waste from 2005. Since 1 million tons of sludge is produced every year in Sweden and the capacity for incineration does not fill the demands, other methods of sludge management have to be introduced to a higher degree. Two biological treatment alternatives are anaerobic digestion and composting. Different oxygen concentrations result in different microbial degradation pathways and, consequently, in a different quality of the digestion or composting residue, It is therefore necessary to study sludge treatment during different oxygen regimes in order to follow both degradation of compounds and change in toxicity. In this study, an industrial sludge containing explosives and pharmaceutical residues was treated with anaerobic digestion or composting, and the change in toxicity was studied. Nitroaromatic compounds, which are the main ingredients of both pharmaceutical and explosives, are well known to cause cytotoxicity and genotoxicity. However, little data are available concerning sludge with nitroaromatics and any associated dioxin-like activity. Therefore, we studied the sludge before and after the treatments in order to detect any changes in levels of Ah receptor (AhR) agonists using two bioassays for dioxin-like compounds.

METHODS:

An industrial sludge was treated with anaerobic digestion or composting in small reactors in a semi-continuous manner. The same volume as the feeding volume was taken out daily and stored at -20 degrees C. Sample preparation for the bioassays was done by extraction using organic solvents, followed by clean up with silica gel or sulphuric acid, yielding two fractions. The fractions were dissolved in DMSO and tested in the bioassays. The dioxin-like activity was measured using the DR-CALUX assay with transfected H4IIE rat hepatoma pGudluc cells and an EROD induction assay with RTL-W1 rainbow trout liver cells.

RESULTS AND DISCUSSION:

The bioassays showed that the sludge contained AhR agonists at levels of TCDD equivalents (TEQs) higher than other sludge types in Sweden. In addition, the TEQ values for the acid resistant fractions increased considerably after anaerobic digestion, resulting in an apparent formation of acid resistant TEQs in the anaerobic reactors. Similar results have been reported from studies of fermented household waste. There was a large difference in effects between the two bioassays, with higher TEQ levels in the RTL-W1 EROD assay than in the DR-CALUX assay. This is possibly due to a more rapid metabolism in rat hepatocytes than in trout hepatocytes or to differences in sensitivities for the AhR agonists in the sludge. It was also demonstrated by GC/FID analysis that the sludge contained high concentrations of nitroaromatics. It is suggested that nitroaromatic metabolites, such as aromatic amines and nitroanilines, are possible candidates for the observed bioassay effects. It was also found that the AhR agonists in the sludge samples were volatile.

CONCLUSIONS:

The sludge contained fairly high concentrations of volatile AhR agonists. The increase of acid resistant AhR agonist after anaerobic digestion warrants further investigations of the chemical and toxic properties of these compounds and of the mechanisms behind this observation.

RECOMMENDATION AND OUTLOOK:

This study has pointed out the benefits of using different types of mechanism-specific bioassays when evaluating the change in toxicity by sludge treatment, in which measurement of dioxin-like activity can be a valuable tool. In order to study the recalcitrant properties of the compounds in the sludge using the DR-CALUX assay, the exposure time can be varied between 6 and 24 hours. The properties of the acid-resistant AhR agonists formed in the anaerobic treatment have to be investigated in order to choose the most appropriate method for sludge management.

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 andVeringkanal were mutagenic in the Ames tester strain TA98 with metabolic activation (S9mix). 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/s11356-014-31850, 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.

A substantial formation of PBDF congeners was observed during photolytic decomposition of decaBDE in toluene. The decaBDE degradation was monitored by chemical and toxicological analysis and in all, twenty-seven mono- to hexasubstituted polybrominated dibenzofurans (PBDFs) were detected in toluene solutions of decabromodiphenyl ether (decaBDE) after irradiation with UV-A, UV-AB and UV-ABC. The concentration levels of PBDFs formed after 16 h of UV exposure increased with wider spectra and were determined to be 3.5, 4.2 and 14 microg/ml after UV-A, UV-AB and UV-ABC irradiation, respectively. In accordance, bioassay derived TEQs (bio-TEQs), determined with the DR-CALUX assay, increased with a similar pattern. The PBDFs formed after the three UV exposures accounted for 0.31%, 0.35% and 1.2% of the initial amount of decaBDE (molar basis). The PBDF congener patterns were consistent in all three UV experiments which imply that no alterations were induced in the PBDF formation or degradation processes due to differences in UV irradiation. However, these processes tended to increase with wider spectra and increasing radiation energy most likely due to the strong absorbance of for example decaBDE at shorter wavelengths. After total decaBDE decomposition the PBDF formation increased significantly in the UV-ABC experiment. The tetra to hexasubstituted BDFs constituted the majority of detected compounds in all experiments. In all samples, the estimated chemical TEQ indicate that the bio-TEQs observed are largely explained by the presence of non-2,3,7,8-substituted PBDFs with relatively low toxicological potencies.

Uptake of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) was studied in earthworms collected from a sawmill site in Sweden with severe PCDD/Fs contamination (the hot spot concentration was 690,000 ng TEQ_WHO2005/kg d.w.) in order to investigate the transfer of PCDD/Fs from the site to the biota. PCDD/Fs concentrations in the collected earthworms were compared to PCDD/Fs concentrations in laboratory exposed earthworms (Eisenia fetida), which were exposed to contaminated soils from the sawmill site for 34 days. All analyses were performed by high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). PCDD/Fs concentrations in the earthworms ranged from 290 to 520,000 pg/g (f.w.). The main congeners found in both soils and earthworms were OCDF, 1234678-HpCDF, OCDD and 1234678-HpCDD. The study showed that the PCDD/Fs in the soil were biovailable to the earthworms and the PCDD/Fs concentrations in the soils correlated with the concentrations in the earthworms. Earthworm samples from soil with lower concentration had higher bioaccumulation factors than samples from soils with high concentration of contamination. Thus, a less contaminated soil could yield higher concentrations in earthworms compared to a higher contaminated soil. Assuming that when assessing risks with PCDD/F contaminated soil, a combination of chemical analysis of soil PCDD/Fs concentrations and bioavailability should be employed for a more comprehensive risk assessment.

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

To evaluate the sediment quality of selected sites in the catchment area of the River Neckar, an integrative assessment approach was used to assess the ecological hazard potential of dioxin-like sediment compounds. The approach is based on 7-ethoxyresorufin-O-deethylase (EROD) induction in embryonic chicken liver culture and comprehensive chemical analyses of polycyclic aromatic hydrocarbons (priority PAHs according to the US Environmental Protection Agency). The majority of the sediment extracts exhibited high potencies as EROD-inducers. In one sediment sample, which was influenced by a sewage treatment plant, a very high concentration of 930 ng bioassay 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (bio-TEQs)/g organic carbon could be determined. However, in none of the samples, more than 6% of the EROD-inducing potency could be explained by the PAHs analyzed chemically. Thus, non-analyzed compounds with EROD-inducing potency were present in the extracts. A fractionation of sediment samples according to pH allowed to localize the major part of EROD-inducing compounds in the neutral fractions. However, a significant portion of the EROD induction could also be explained by the acidic fractions. Following the concept of the Sediment Quality Triad according to Chapman, in situ alterations of macrozoobenthos were examined. A comparison of the results predicted by the EROD assay and chemical analyses with alterations in situ, as measured by means of the saprobic index and the ecotoxicological index according to Carmargo, revealed a high ecological relevance of the results of bioassays and chemical analyses for major sites.

Background and aim Despite intensive and continuous stocking and improvement of water quality since the 1970s, fish populations, especially those of the grayling (Thymallus thymallus), have declined over the last two decades in the upper Danube River (Germany). In order to assess 1) possible links between molecular/biochemical responses and ecologically relevant effects, and 2) if ecotoxicological effects might be related to the decline in fish catches in the upper Danube river, sediment samples and fish were collected at different locations and analyzed using a weight-of-evidence (WOE) approach with several lines of evidence. The objective of the presentation is to introduce the conceptual framework and to review results of the ongoing study. As previously addressed by Chapman and Hollert (2006) a variety of lines of evidence can be used in WOE studies. Briefly, 1) a comprehensive battery of acute and mechanism-specific bioassays was used to characterize the ecotoxicological hazard potential. 2) Histopathological investigations and the micronucleus assay with erythrocytes were applied, analyzing in situ parameters. 3) Diversity and abundance of benthic macroinvertebrates and fish as well as 4) persistent organic pollutants, endocrine disrupting substances, limnochemical parameters and the concentration of heavy metals were recorded. To identify organic contaminants a spotential causes of sediment toxicity assays, 5) effect directed analysis was applied. © 2009 Springer-Verlag.

Six different lipid matrices (the intact lipid (IL), four lipid fractions with different polarity, and the free fatty acids (FFAs) obtained by hydrolysis of the triacylglycerol (TAG) containing fraction) were obtained from salmon (Salmo salar) and eel (Anguilla anguilla), each collected at a contaminated and a comparatively uncontaminated catch site along the coast of Scandinavia. The lipid matrices were studied in toxicological test systems representing various biological functions of different organ systems from several species and trophic levels. The results were evaluated with particular respect to the concentrations of extractable organically bound chlorine (EOCl) in the matrices tested. In some test systems, the specimens with a higher EOCl concentration appeared to be more toxic. For example, the TAG containing fraction (F2) from Idefjord eel, having a higher EOCl content than F2 from Oslofjord eel, reduced the number and hatchability of eggs laid by zebrafish. Both IL and F2 of Idefjord eel increased mortality and reduced the oxygen/nitrogen-ratio in blue mussels. Non-polar compounds (F1) from Bothnian Sea salmon induced 7-ethoxyresurofin O-deethylase (EROD) activity in rainbow trout hepatocytes, whereas F1 from Senja salmon did not. F1 from Bothnian Sea salmon also reduced the number of T-cells in foetal mouse thymus anlagen in vitro compared with the cell number in anlagen exposed to F1 from Senja salmon. A positive correlation between EOCl concentration and test response was found for EROD activity in rainbow trout hepatocytes and for ATP-leakage in Erlich ascites tumour cells when testing the phospolipid containing fraction (F4). However, in most test systems the fish oils, irrespective of EOCl content, were of low toxicity, and the observed effects need to be verified in future studies.

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.

Perfluorooctane sulfonate (PFOS; C8F17SO3–) is a fully fluorinated organic compound which has been manufactured for decades and was used widely in industrial and commercial products. The recent toxicological knowledge of PFOS mainly concerns mono-substance exposures of PFOS to biological systems, leaving the potential interactive effects of PFOS with other compounds as an area where understanding is significantly lacking. However, a recent study, reported the potential of PFOS to enhance the toxicity of two compounds by increasing cell membrane permeability. This is of particular concern since PFOS has been reported to be widely distributed in the environment where contaminants are known to occur in complex mixtures. In this study, PFOS was evaluated alone and in combination with cyclophosphamide (CPP) to investigate whether a presence of PFOS leads to an increased genotoxic potential of CPP towards hamster lung V79 cells. Genotoxicity was investigated using the micronucleus(MN) assay according to the recent draft ISO/DIS 21427-2 method. PFOS alone demonstrated no genotoxicity up to a concentration of 12.5 μg/ml. However, PFOS combined with two different concentrations of CPP, with metabolic activation, caused a significant increase in the number of micronucleated cells compared to treatments with CPP alone. These results provide a first indication that PFOS has the potential to enhance the genotoxic action of CPP towards V79 cells, suggesting, together with the alterations in cell membrane properties shown previously, that genotoxicity of complex mixtures may be increased significantly by changes in chemical uptake. Together with an earlier study performed by the own working group, it can be concluded that PFOS alone is not genotoxic in this bioassay using V79 cells up to 12.5 μg/ml, but that further investigations are needed to assess the potential interaction between PFOS and other substances, in particular regarding the impact of membrane alterations on the uptake of toxic substances.

Objectives: Personnel working with electronic dismantling are exposed to polybrominated diphenyl ethers (PBDEs), which in animal studies have been shown to alter thyroid homeostasis. The aim of this longitudinal study was to measure plasma level of PBDEs in workers at an electronic recycling facility and to relate these to the workers’ thyroid status. Methods: PBDEs and three thyroid hormones: triiodothyronine (T3), thyroxin (T4) and thyroid stimulating hormone (TSH) were repeatedly analysed in plasma from 11 workers during a period of 1.5 years.Results: Plasma levels of PBDEs at start of employment were <0.5–9.1 pmol/g lipid weight (l.w.). The most common congener was PBDE #47 (median 2.8 pmol/g l.w.), followed by PBDE #153 (median 1.7 pmol/g l.w.), and PBDE #183 had a median value of <0.19 pmol/g l.w. After dismantling the corresponding median concentrations were: 3.7, 1.7 and 1.2 pmol/g l.w., respectively. These differences in PBDE levels were not statistically significant. PBDE #28 showed a statistically significantly higher concentration after dismantling than at start of employment (P=0.016), although at low concentrations (start 0.11 pmol/g l.w. and dismantling 0.26 pmol/g l.w.). All measured levels of thyroid hormones (T3, T4 and TSH) were within the normal physiological range. Statistically significant positive correlations were found between T3 and #183 in a worker, between T4 and both #28 and #100 in another worker and also between TSH and #99 and #154 in two workers. Conclusions: The workers’ plasma levels of PBDEs fluctuated during the study period. Due to small changes in thyroid hormone levels it was concluded that no relevant changes were present in relation to PBDE exposure within the workers participating in this study.