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.

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.

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).

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.

Toxicity of polyethylene (PE) and polyvinyl chloride (PVC) microplastics (MPs), either virgin or spiked with chemicals, was evaluated in two short-lived fish using a freshwater species, zebrafish, and a marine species, marine medaka. Exposures were performed through diet using environmentally relevant concentrations of MPs over 4 months. No modification of classical biomarkers, lipid peroxidation, genotoxicity or F0 behaviour was observed. A significant decrease in growth was reported after at least two months of exposure. This decrease was similar between species, independent from the type of MPs polymer and the presence or not of spiked chemicals, but was much stronger in females. The reproduction was evaluated and it revealed a significant decrease in the reproductive output for both species and in far more serious numbers in medaka. PVC appeared more reprotoxic than PE as were MPs spiked with PFOS and benzophenone-3 compared to MPs spiked with benzo[a]pyrene. Further, PVC-benzophenone-3 produced behavioural disruption in offspring larvae. These results obtained with two species representing different aquatic environments suggest that microplastics exert toxic effects, slightly different according to polymers and the presence or not of sorbed chemicals, which may lead in all cases to serious ecological disruptions.

Healthy soils provide valuable ecosystem services (ES), but soil contamination can inhibit essential soil functions (SF) and pose risks to human health and the environment. A key advantage of using gentle remediation options (GRO) is the potential for multifunctionality: to both manage risks and improve soil functionality. In this study, an accessible, scientific method for soil health assessment directed towards practitioners and decision-makers in contaminated land management was developed and demonstrated for a field experiment at a DDX-contaminated tree nursery site in Sweden to evaluate the relative effects of GRO on soil health (i.e., the ‘current capacity’ to provide ES). For the set of relevant soil quality indicators (SQI) selected using a simplified logical sieve, GRO treatment was observed to have highly significant effects on many SQI according to statistical analysis due to the strong influence of biochar amendment on the sandy soil and positive effects of nitrogen-fixing leguminous plants. The SQI were grouped within five SF and the relative effects on soil health were evaluated compared to a reference state (experimental control) by calculating quantitative treated-SF indices. Multiple GRO treatments are shown to have statistically significant positive effects on many SF, including pollutant attenuation and degradation, water cycling and storage, nutrient cycling and provisioning, and soil structure and maintenance. The SF were in turn linked to soil-based ES to calculate treated-ES indices and an overall soil health index (SHI), which can provide simplified yet valuable information to decision-makers regarding the effectiveness of GRO. The experimental GRO treatment of the legume mix with biochar amendment and grass mix with biochar amendment are shown to result in statistically significant improvements to soil health, with overall SHI values of 141 % and 128 %, respectively, compared to the reference state of the grass mix without biochar (set to 100 %).

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.

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.

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.

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.

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 vertical bar C vertical bar 182,780 (vertical bar C vertical bar) and the aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor a-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 a-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.

Contaminated soils are a worldwide problem. Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in soil at former industrial areas, especially at old gasworks sites, gas stations and former wood impregnation facilities. Risk assessments of PAHs in contaminated soils are usually based on chemical analysis of a small number of individual PAHs, which only constitute a small part of the complex cocktail of hundreds of PAHs and other related polycyclic aromatic compounds (PACs) in the soils. Generally, the mixture composition of PAH-contaminated soils is rarely known and the mechanisms of toxicity and interactions between the pollutants are far from fully understood.

The main objective of this thesis was to characterize remediated PAHcontaminated soils by use of a chemical and bioanalytical approach. Bioassay specific relative potency (REP) values for 38 PAHs and related PACs were developed in the sensitive H4IIE-luc bioassay and used in massbalance analysis of remediated PAH contaminated soils, to assess the contribution of chemically quantified compounds to the overall aryl hydrocarbon receptor (AhR)-mediated activity observed in the H4IIE-luc bioassay. Mixtures studies showed additive AhR-mediated effects of PACs, including PAHs, oxy PAHs, methylated PAHs and azaarenes, in the bioassay, which supports the use of REP values in risk assessment. The results from the chemical and bioassay analysis showed that PAH-contaminated soils contained a large fraction of AhR activating compounds whose effect could not be explained by chemical analysis of the 16 priority PAHs. Further chemical identification and biological studies are necessary to determine whether these unknown substances pose a risk to human health or the environment. Results presented in this thesis are an important step in the development of AhR-based bioassay analysis and risk assessment of complex PAH-contaminated samples.

Risk assessments of polycyclic aromatic hydrocarbons (PAHs) are complicated because these compounds exist in the environment as complex mixtures of hundreds of individual PAHs and other related polycyclic aromatic compounds (PACs). In this study, the hypothesis that concentration addition (CA) can be used to predict the aryl hydrocarbon receptor (AhR)-mediated activity of PACs in mixtures containing various combinations of PACs was tested. AhR-mediated activities of 18 mixtures composed of two to 23 PACs, which included PAHs, azaarenes and oxygenated PAHs, were examined by the use of the AhR-based H4IIE-luc bioassay. Since greater AhR-mediated activities have been observed in soils contaminated by PAHs, investigations were done to test whether soil extract matrix or the presence of non-effect PACs might affect responses of the H4IIE-luc bioassay. Our results showed that AhR-mediated activities of mixtures of PACs could be predicted by the use of concentration addition. Additive activities of PACs in multi component mixtures along with the insignificant effect of the soil matrix support the use of concentration addition in mass balance calculations and AhR-based bioassays in risk assessment of environmental samples. However, independent action (IA) could not be used to predict the activity of mixtures of PACs.

Risk assessments of polycyclic aromatic hydrocarbons (PAHs) are complicated because these compounds exist in the environment as complex mixtures of hundreds of individual PAHs and other related polycyclic aromatic compounds (PACs). In this study, the hypothesis that concentration addition (CA) can be used to predict the aryl hydrocarbon receptor (AhR) mediated potency of PACs in mixtures containing various combinations of PACs. Effects of 18 mixtures composed of two to 23 PACs, which included PAHs, azaarenes and oxygenated PAHs, were examined by use of the AhR based H4IIE-luc bioassay. Since greater AhR-mediated activities have been observed in soils contaminated by PAH, investigations were done to test whether soil extract matrix or presence of noneffect PACs might affect responses of the H4IIE-luc bioassay. AhR-mediated activity of the mixture of PACs could be predicted by use of concentration addition. Additive behavior of PACs in multi component mixtures supported the hypothesis that use of concentration addition could be used in risk assessment of PAC- mixtures. However, independent action (IA) could not be used to predict the activity of mixtures of PACs. 

The H4IIE-luc transactivation bioassay for aryl hydrocarbon receptor (AhR) agonists was used to investigate the relative potency factors (REPs) of 22 individual polycyclic aromatic hydrocarbons (PAHs) and their oxygenated-, methylated-, and N-containing derivatives (azaarenes), which are often present in PAH-contaminated soils. Naphthacene and dibenz[ah]acridine exhibited greater AhR-mediated potency, whereas lesser molecular-weight azaarenes were less potent AhR agonists. Six oxygenated PAHs had calculable REPs, but their potencies were less than their parent PAHs. Unlike the parent, unsubstituted PAHs, oxidation of methylated PAHs seemed to increase the AhR-mediated potency of the compounds, with 2-methylanthracene-9,10-dione being almost 2 times more potent than 2-methylanthracene. Both bioassay and gas chromatography-mass spectrometry analysis were used to examine the exposure time-dependent effects on the REPs at 24 h, 48 h, and 72 h of exposure in the H4IIE-luc transactivation bioassay. Changes in concentrations of 5 compounds including the model reference 2,3,7,8-tetrachlorodibenzo-p-dioxin in the cell culture wells were measured, and the amounts in the cell medium, in the cells, and adsorbed to the wells was determined and the influence on the REPs was studied. Declining REP values with increased duration of exposure were shown for all compounds, which we concluded were a consequence of the metabolism of PAHs and PAH derivatives in H4IIe-luc cells. The present study provides new knowledge regarding the degradation and distribution of compounds in the wells during exposure. Environ Toxicol Chem 2014;33:943-953. (c) 2014 SETAC

The H4IIE-luc transactivation bioassay for aryl hydrocarbon receptor (AhR) agonists was used to investigate relative potency factors (REPs) of 22 individual polycyclic aromatic hydrocarbons (PAHs) and their oxygenated-, methylated- and N-containing derivatives (azaarenes), which are often present in PAH-contaminated soils. Naphthacene and dibenz[ah]acridine exhibited greater AhRmediated potency, whereas lesser-molecular azaarenes were less potent AhR agonists. Six oxy-PAHs had calculable Relative potencies (REPs), but their potencies were less than their parent PAHs. Unlike the parent, unsubstituted PAHs, oxidation of methylated PAHs seemed to increase the AhR-mediated potency of the compounds, with methylanthracene-9,10-dione being almost two times more potent than methylanthracene. Both bioassay and gas chromatography–mass spectrometry (GC/MS) analysis were used to examine the exposure time dependent effects on the REPs at 24, 48 and 72 h of exposure in the H4IIE-luc transactivation bioassay. Changes in concentrations of five compounds including the model reference 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the cell culture wells were measured, and the amounts in the cell medium, the cells and adsorbed to the wells determined and the influence on the REPs was studied. Declining REP values with increased duration of exposure were shown for all compounds and proved to be a consequence of the metabolism of PAHs and PAH derivatives in H4IIe-luc cells. The present study provides new knowledge regarding the degradation and distribution of compounds in the wells during exposure.

Abstract Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in soil at former industrial areas; and in Sweden, some of the most contaminated sites are being remediated. Generic guideline values for soil use after so-called successful remediation actions of PAHcontaminated soil are based on the 16 EPA priority pollutants, which only constitute a small part of the complex cocktail of toxicants in many contaminated soils. The aim of the study was to elucidate if the actual toxicological risks of soil samples from successful remediation projects could be reflected by chemical determination of these PAHs. We compared chemical analysis (GC-MS) and bioassay analysis (H4IIE-luc) of a number of remediated PAH-contaminated soils. The H4IIE-luc bioassay is an aryl hydrocarbon (Ah) receptor-based assay that detects compounds that activate the Ah receptor, one important mechanism for PAH toxicity. Comparison of the results showed that the bioassay-determined toxicity in the remediated soil samples could only be explained to a minor extent by the concentrations of the 16 priority PAHs. The current risk assessment method for PAH-contaminated soil in use in Sweden along with other countries, based on chemical analysis of selected PAHs, is missing toxicologically relevant PAHs and other similar substances. It is therefore reasonable to include bioassays in risk assessment and in the classification of remediated PAH-contaminated soils. This could minimise environmental and human health risks and enable greater safety in subsequent reuse of remediated soils. 

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in soil at former industrial areas; and in Sweden, some of the most contaminated sites are being remediated. Generic guideline values for soil use after so-called successful remediation actions of PAH-contaminated soil are based on the 16 EPA priority pollutants, which only constitute a small part of the complex cocktail of toxicants in many contaminated soils. The aim of the study was to elucidate if the actual toxicological risks of soil samples from successful remediation projects could be reflected by chemical determination of these PAHs. We compared chemical analysis (GC-MS) and bioassay analysis (H4IIE-luc) of a number of remediated PAH-contaminated soils. The H4IIE-luc bioassay is an aryl hydrocarbon (Ah) receptor-based assay that detects compounds that activate the Ah receptor, one important mechanism for PAH toxicity. Comparison of the results showed that the bioassay-determined toxicity in the remediated soil samples could only be explained to a minor extent by the concentrations of the 16 priority PAHs. The current risk assessment method for PAH-contaminated soil in use in Sweden along with other countries, based on chemical analysis of selected PAHs, is missing toxicologically relevant PAHs and other similar substances. It is therefore reasonable to include bioassays in risk assessment and in the classification of remediated PAH-contaminated soils. This could minimise environmental and human health risks and enable greater safety in subsequent reuse of remediated soils.

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.

In the present study, relative potency factors (REPs) of 16 individual polycyclic aromatic hydrocarbons (PAHs) were investigated using the H4IIE-luc bioassay. Exposure time-dependent effects on the REPs were examined using 24, 48, and 72?h of exposure. Seven different mixtures of PAHs were tested for additivity at an exposure time of 24?h. Three of the PAH mixtures were also studied at 48 and 72?h of exposure. The mixture toxicities were predicted using the REP concept and the concentration addition (CA) model. Relative potency factor values investigated in the present study were similar to those reported in earlier studies. Declining REPs with an increasing exposure time were shown for all PAHs, indicating that this bioassay approach could be developed to assess the persistency of aryl hydrocarbon receptor (AhR) agonistic PAHs and in the risk assessment of complex PAH mixtures. The results from the mixture studies indicated that additive interactions of PAHs are time dependent. Generally, 48- and 72-h exposures resulted in biological effects that were similar to the CA and REP model predictions, while these models tended to underestimate the effect, to some extent, in the 24-h exposure, at least for the mixtures containing two to four PAHs. Thus, it cannot be ruled out that in the 24-h exposures, the tested PAH mixtures had slight synergistic effects. Further research is needed to identify and test additional AhR activating PAHs and investigate whether the effects in the H4IIE-luc bioassay are additive for more complex samples containing both PAHs and other AhR-activating contaminants. Also, the observed superinduction of luciferase by PAH-mixes warrants studies of whether this also can occur for relevant AhR-mediated endpoints in vivo. 

Tire wear particles (TWP) stand out as a major contributor to microplastic pollution, yet their environmental impact remains inadequately understood. This study delves into the cocktail effects of TWP leachates, employing molecular, cellular, and organismal assessments on diverse biological models. Extracted in artificial seawater and analyzed for metals and organic compounds, TWP leachates revealed the presence of polyaromatic hydrocarbons and 4-tert-octylphenol. Exposure to TWP leachates (1.5 to 1000 mg peq L-1) inhibited algae growth and induced zebrafish embryotoxicity, pigment alterations, and behavioral changes. Cell painting uncovered pro-apoptotic changes, while mechanism-specific gene-reporter assays highlighted endocrine-disrupting potential, particularly antiandrogenic effects. Although heavy metals like zinc have been suggested as major players in TWP leachate toxicity, this study emphasizes water-leachable organic compounds as the primary causative agents of observed acute toxicity. The findings underscore the need to reduce TWP pollution in aquatic systems and enhance regulations governing highly toxic tire additives.

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) and nitrogen heterocyclic polycyclic aromatic compounds (N-PACs) are toxic, highly leachable and often abundant at sites that are also contaminated with PAHs. However, due to lack of regulations and standardized methods for their analysis, they are seldom included in monitoring and risk-assessment programs. This intercomparison study constitutes an important step in the harmonization of the analytical methods currently used, and may also be considered a first step towards the certification of reference materials for these compounds. The results showed that the participants were able to determine oxy-PAHs with accuracy similar to PAHs, with average determined mass fractions agreeing well with the known levels in a spiked soil and acceptable inter- and intra-laboratory precisions for all soils analyzed. For the N-PACs, the results were less satisfactory, and have to be improved by using analytical methods more specifically optimized for these compounds.

In order to contribute to a comprehensive understanding of the regulating mechanisms of the aryl-hydrocarbon-receptor (AHR) in zebrafish embryos, we aimed to elucidate the interaction of proteins taking part in this signaling pathway during early development of the zebrafish (Danio rerio) after chemical exposure. We managed to illustrate initial transcription processes of the implemented proteins after exposure to two environmentally relevant chemicals: polychlorinated biphenyl 126 (PCB126) and β-Naphthoflavone (BNF). Using qPCR, we quantified mRNA every 4 h until 118 h post fertilization and found the expression of biotransformation enzymes (cyp1 family) and the repressor of the AHR (ahr-r) to be dependent on the duration of chemical exposure and the biodegradability of the compounds. PCB126 induced persistently increased amounts of transcripts as it is not metabolized, whereas activation by BNF was limited to the initial period of exposure. We did not find a clear relation between the amount of transcripts and activity of the induced CYP-proteins, so posttranscriptional mechanisms are likely to regulate biotransformation of BNF. With regard to zebrafish embryos and their application in risk assessment of hazardous chemicals, our examination of the AHR pathway especially supports the relevance of the time point or period of exposure that is used for bioanalytical investigations and consideration of chemical properties determining biodegradability.

Biotests like the fish embryo toxicity test have become increasingly popular in risk assessment and evaluation of chemicals found in the environment. The large range of possible endpoints is a big advantage when researching on the mode of action of a certain substance. Here, we utilized the frequently used model organism zebrafish (Danio rerio) to examine regulative mechanisms in the pathway of the aryl-hydrocarbon receptor (AHR) in early development. We exposed embryos to representatives of two chemical classes known to elicit dioxin-like activity: benzo[a]pyrene for polycyclic aromatic hydrocarbons (PAHs) and 2,3-benzofuran for polar O-substituted heterocycles as a member of heterocyclic compounds in general (N-, S-, O-heterocycles; NSO-hets). We measured gene transcription of the induced P450 cytochromes (cyp1), their formation of protein and biotransformation activity throughout the whole embryonic development until 5 days after fertilization. The results show a very specific time course of transcription depending on the chemical properties (e.g. halogenation, planarity, K_ow), the physical decay and the biodegradability of the tested compound. However, although this temporal pattern was not precisely transferable onto the protein level, significant regulation in enzymatic activity over time could be detected. We conclude, that a careful choice of time and end point as well as consideration of the chemical properties of a substance are fairly important when planning, conducting and especially evaluating biotests.

Existing regulatory frameworks often prove inadequate in identifying contaminants of emerging concern (CECs) and determining their impacts on biological systems at an early stage. The establishment of Early Warning Systems (EWSs) for CECs is becoming increasingly relevant for policy-making, aiming to proactively detect chemical hazards and implement effective mitigation measures. Effect-based methodologies, including bioassays and effect-directed analysis (EDA), offer valuable input to EWSs with a view to pinpointing the relevant toxicity drivers and prioritizing the associated risks. This review evaluates the analytical techniques currently available to assess biological effects, and provides a structured plan for their systematic integration into an EWS for hazardous chemicals in the environment. Key scientific advancements in effect-based approaches and EDA are discussed, underscoring their potential for early detection and management of chemical hazards. Additionally, critical challenges such as data integration and regulatory alignment are addressed, emphasizing the need for continuous improvement of the EWS and the incorporation of analytical advancements to safeguard environmental and public health from emerging chemical threats.

Polycyclic aromatic compounds (PACs) are prevalent in the environment, typically found in complex mixtures and high concentrations. Our understanding of the effects of PACs, excluding the 16 priority polycyclic aromatic hydrocarbons (16 PAHs), remains limited. Zebrafish embryos and in vitro bioassays were utilized to investigate the embryotoxic, behavioral, and molecular effects of a soil sample from a former gasworks site in Sweden. Additionally, targeted chemical analysis was conducted to analyze 87 PACs in the soil, fish, water, and plate material. CALUX® assays were used to assess the activation of aryl hydrocarbon and estrogen receptors, as well as the inhibition of the androgen receptor. Larval behavior was measured by analyzing activity during light and darkness and in response to mechanical stimulation. Furthermore, qPCR analyses were performed on a subset of 36 genes associated with specific adverse outcomes, and the total lipid content in the larvae was measured. Exposure to the sample resulted in embryotoxic effects (LC50 = 0.480 mg dry matter soil/mL water). The mixture also induced hyperactivity in darkness and hypoactivity in light and in response to the mechanical stimulus. qPCR analysis revealed differential regulation of 15 genes, including downregulation of opn1sw1 (eye pigmentation) and upregulation of fpgs (heart failure). The sample caused significant responses in three bioassays (ERα-, DR-, and PAH-CALUX), and the exposed larvae exhibited elevated lipid levels. Chemical analysis identified benzo[a]pyrene as the predominant compound in the soil and approximately half of the total PAC concentration was attributed to the 16 PAHs. This study highlights the value of combining in vitro and in vivo methods with chemical analysis to assess toxic mechanisms at specific targets and to elucidate the possible interactions between various pathways in an organism. It also enhances our understanding of the risks associated with environmental mixtures of PACs and their distribution during toxicity testing.

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.

Soil contaminants may restrict soil functions. A promising soil remediation method is amendment with biochar, which has the potential to both adsorb contaminants and improve soil health. However, effects of biochar amendment on soil-plant nitrogen (N) dynamics and N cycling microbial guilds in contaminated soils are still poorly understood. Here, a metal- and polycyclic aromatic hydrocarbon (PAH) contaminated soil was amended with either biochar (0, 3, 6 % w/w) and/or peat (0, 1.5, 3 % w/w) in a full-factorial design and sown with perennial ryegrass in an outdoor field trial. After three months, N and the stable isotopic ratio δ15N was measured in soil, roots and leaves, along with microbial responses. Aboveground grass biomass decreased by 30 % and leaf N content by 20 % with biochar, while peat alone had no effect. Peat in particular, but also biochar, stimulated the abundance of microorganisms (measured as 16S rRNA gene copy number) and basal respiration. Microbial substrate utilization (MicroResp™) was altered differentially, as peat increased respiration of all carbon sources, while for biochar, respiration of carboxylic acids increased, sugars decreased, and was unaffected for amino acids. Biochar increased the abundance of ammonia oxidizing archaea, while peat stimulated ammonia oxidizing bacteria, Nitrobacter-type nitrite oxidizers and comB-type complete ammonia oxidizers. Biochar and peat also increased nitrous oxide reducing communities (nosZI and nosZII), while peat alone or combined with biochar also increased abundance of nirK-type denitrifiers. However, biochar and peat lowered leaf δ15N by 2-4 ‰, indicating that processes causing gaseous N losses, like denitrification and ammonia volatilization, were reduced compared to the untreated contaminated soil, probably an effect of biotic N immobilization. Overall, this study shows that in addition to contaminant stabilization, amendment with biochar and peat can increase N retention while improving microbial capacity to perform important soil functions.

The Arctic is undergoing rapid changes, and biota are exposed to multiple stressors, including pollution and climate change. Still, little is known about their joint impact. Here, we investigated the cumulative impact of crude oil, warming, and freshening on the copepod species Calanus glacialis and Calanus finmarchicus. Adult females were exposed to ambient conditions (control; 0 °C + 33 psu) and combined warming and freshening: 5 °C + 27 psu (Scenario 1), 5 °C + 20 psu (Scenario 2) for 6 days. All three conditions were tested with and without dispersed crude oil. In Scenario 1, fecal pellet production (FPP) significantly increased by 40-78% and 42-122% for C. glacialis and C. finmarchicus, respectively. In Scenario 2, FPP decreased by 6-57% for C. glacialis, while it fluctuated for C. finmarchicus. For both species, oil had the strongest effect on FPP, leading to a 68-83% reduction. This overshadowed the differences between climatic scenarios. All variables (temperature, salinity, and oil) had significant single effects and several joint effects on FPP. Our results demonstrate that Arctic copepods are sensitive to environmentally realistic concentrations of crude oil and climate change. Strong reductions in feeding can reduce the copepods' energy content with potential large-scale impacts on the Arctic marine food web.

To facilitate the study of potential harmful compounds sorbed to microplastics, an effect-directed analysis using the DR CALUX^® assay as screening tool for Aryl hydrocarbon receptor (AhR)-active compounds in extracts of marine deployed microplastics and chemical analysis of hydrophobic organic compounds (HOCs) was conducted. Pellets of three plastic polymers [low-density polyethylene (LDPE), high-density polyethylene (HDPE) and high-impact polystyrene (HIPS)] were deployed at Heron Island in the Great Barrier Reef, Australia, for up to 8 months. Detected AhR-mediated potencies (bio-TEQs) of extracted plastic pellets ranged from 15 to 100 pg/g. Contributions of target HOCs to the overall bioactivities were negligible. To identify the major contributors, remaining plastic pellets were used for fractionation with a gas chromatography (GC) fractionation platform featuring parallel mass spectrometric (MS) detection. The bioassay analysis showed two bioactive fractions of each polymer with bio-TEQs ranging from 5.7 to 14 pg/g. High resolution MS was used in order to identify bioactive compounds in the fractions. No AhR agonists could be identified in fractions of HDPE or LDPE. Via a multivariate statistical approach the polystyrene (PS) trimer 1e- Phenyl-4e-(1- phenylethyl)-tetralin was identified in fractions of HIPS and in fractions of the blank polymer of HIPS.

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).

Several plasticizing chemicals induce endocrine disrupting effects in humans, and the indoor environment is suggested to be a source of exposure. As children are particularly vulnerable to the effects from exposure to endocrine disrupting chemicals (EDCs), it is essential to monitor exposure to EDCs such as phthalates and non-phthalate plasticizers in indoor environments intended for use by children. The aim of this study was to assess everyday plasticizer exposure among preschool-aged children in Sweden by measuring urinary plasticizer metabolite concentrations. In addition, it was investigated whether the concentrations would be altered as a result of the children spending part of the day at preschool, in comparison with weekend exposure, when they may spend more time in home environments or engage in various weekend and leisure activities. For this purpose, fourteen metabolites from eight phthalates (di-ethylhexyl phthalate, DEHP; di-n-butyl phthalate, DnBP; di-isobutyl phthalate, DiBP; butyl-benzyl phthalate, BBzP; di-iso-nonyl phthalate, DiNP; di-propylheptyl phthalate, DPHP; di-iso-decyl phthalate, DiDP; and di-ethyl phthalate, DEP) and one non-phthalate plasticizer (di-isononyl cyclohexane 1,2-dicarboxylate, DiNCH) were measured in 206 urine samples collected at four occasions, i.e. twice during the winter and twice during the spring from 54 children (mean 5.1 years, SD 0.94) enrolled at eight preschools in Sweden. A detection frequency (DF) of 99.9% for the 14 metabolites indicates a widespread exposure to plasticizers among children in Sweden. Compared to previous Swedish and international studies performed during approximately the same time period, high urinary concentrations of monobenzyl phthalate (MBzP), a metabolite from the strictly regulated BBzP, were measured in this study (median 17 ng/mL). Overall, high urinary phthalate metabolite concentrations were observed in this study compared to the US CDC-NHANES from the same time period and similar age-group. Compared to European studies, however, similar concentrations were observed for most metabolites and the urinary concentrations from few participating children exceeded the human biomonitoring guidance values (HBM-GV) for children. After days with preschool attendance, lower urinary concentrations of metabolites originating from DEP and phthalates that are strictly regulated within the EU REACH legislation (DEHP, DnBP, and DiBP) and higher concentrations of metabolites originating from DiNP, DPHP, and DiDP, i.e. less or non-regulated phthalates were found compared the urinary concentrations of these metabolites in weekends. This may indicate that factors in the indoor environment itself are important for the extent of the plasticizer exposure. All the analyzed metabolites were measured in lower concentrations in urine collected from children attending preschools built or renovated after the year 2000, while no seasonal differences were observed in this study.

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 assed 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.

Complex chemical mixtures found in soils at contaminated sites typically includes polycyclic aromatic compounds (PACs), thus posing potential environmental and human health risks. Pressurized liquid extraction (PLE) followed by silica clean-up is one of the most often used extraction methods for PACs in soil. While silica clean-up provide satisfactory recovery of oxygenated polycyclic aromatic hydrocarbons (OPAHs), this technique provides limited recovery of azaarenes. In this work, we used PLE and in-cell clean up with basic silica to increase the recovery of OPAHs and azaarenes. The optimized selective pressurized liquid extraction (SPLE) method used 4 g basic silica, dichloromethane, 100% flush volume, 100 and 120 °C extraction temperatures, with two static cycles for each temperature, no rinse in between the two extractions, and 20 and 120 s purge for the first and second extraction temperature, respectively. The method was validated for a wide range of PAC groups, including OPAHs, azaarenes, alkylated PAHs, and sulfur heterocycles (SPACs), in total 87 PACs, using certified reference material and in comparison to the results from previous inter-laboratory data. Our SPLE method yielded results that are in agreement with certified values and inter-laboratory data from prior analysis. The SPLE method also yielded lower variation than the results from the inter-laboratory data for analysis of OPAH and azaarenes, suggesting better precision than previous methods. More importantly, the SPLE method increases sample analysis throughput as extra clean-up step is not necessary anymore. The SPLE method was then successfully applied to rapidly screen PACs in three soil samples.

Soil samples from a contaminated site in Sweden were analyzed to identify the presence of 78 polycyclic aromatic compounds (PACs) using gas chromatography coupled with mass spectrometry (GC-MS). The target analysis revealed large contributions not only from polycyclic aromatic hydrocarbons (PAHs), but also from alkylated- and oxygenated-PAHs (alkyl- and oxy-PAHs, respectively), and N-heterocyclics (NPACs). PAC profiles indicated primarily pyrogenic sources, although contribution of petrogenic sources was also observed in one sample as indicated by a high ratio of alkylated naphthalene compared to naphthalene. The aryl hydrocarbon receptor (AhR)-activity of the soil extracts was assessed using the H4IIe-pGudluc 1.1 cells bioassay. When compared with the calculated total AhR-activity of the PACs in the target list, 35–97% of the observed bioassay activity could be explained by 62 PACs with relative potency factors (REPs). The samples were further screened using GC coupled with Orbitrap™ high resolution MS (GC-HRMS) to investigate the presence of other PACs that could potentially contribute to the AhR-activity of the extracts. 114 unique candidate compounds were tentatively identified and divided into four groups based on their AhR-activity and environmental occurrence. Twelve substances satisfied all the criteria, and these compounds are suggested to be included in regular screening in future studies, although their identities were not confirmed by standards in this study. High unexplained bio-TEQ fractions in three of the samples may be explained by tentatively identified compounds (n = 35) with high potential of being toxic. This study demonstrates the benefit of combining targeted and non-targeted chemical analysis with bioassay analysis to assess the diversity and effects of PACs at contaminated sites. The applied prioritization strategy revealed a number of tentatively identified compounds, which likely contributed to the overall bioactivity of the soil extracts.

Polycyclic aromatic compounds (PACs) encompass a diverse group of compounds, often found in historically contaminated sites. Different eperimental techniques have been used to remediate PAC-contaminated soils. This brief review surveyed more than 270 studies concerning remediation of PAC-contaminated soils and found that, while these studies often measured the concentration of 16 parent polycyclic aromatic hydrocarbons (PAHs) pre- and post-remediation, only a fraction of the studies included the measurement of PAC transformation products (PAC-TPs) and other PACs (n = 33). Only a few studies also incorporated genotoxicity/toxicity/mutagenicity analysis pre- and post-remediation (n = 5). Another aspect that these studies often neglected to include was bioavailability, as none of the studies that included measurement of PAH-TPs and PACs included bioavailability investigation. On the basis of the literature analysis, future remediation studies need to consider chemical analysis of PAH-TPs and PACs, genotoxicity/toxicity/mutagenicity, and bioavailability analyses pre- and post-remediation. These assessments will help address numerous concerns, including, among others, the presence, properties, and toxicity of PACs and PAH-TPs, risk assessment of soil post-remediation, and the bioavailability of PAH-TPs. Other supplementary techniques that aid in these analyses and recommendations for future analyses are also discussed.

Contaminants in the soil may threaten soil functions (SFs) and, in turn, hinder the delivery of ecosystem services (ES). A framework for ecological risk assessments (ERAs) within the APPLICERA - APPLICable site-specific Environmental Risk Assessment research project promotes assessments that consider other soil quality parameters than only contaminant concentrations. The developed framework is: (i) able to differentiate the effects of contamination on SFs from the effects of other soil qualities essential for soil biota; and (ii) provides a robust basis for improved soil quality management in remediation projects. This study evaluates the socio-economic consequences of remediation alternatives stemming from a Tier 1 ERA that focusses on total contaminant concentrations and soil quality standards and a detailed, site-specific Tier 3 Triad approach that is based on the APPLICERA framework. The present study demonstrates how Tier 1 and Tier 3 ERAs differ in terms of the socio-economic consequences of their remediation actions, as well as presents a novel method for the semi-quantitative assessment of on-site ES. Although the presented Tier 3 ERA is more expensive and time-consuming than the more traditional Tier 1 ERA approach, it has the potential to lower the costs of remediation actions, decrease greenhouse gas emissions, reduce other environmental impacts, and minimise socio-economic losses. Furthermore, the remediation actions stemming from the Tier 3 ERA were predicted to exert far less negative ES effects than the actions proposed based on the results of the Tier 1 ERA.

Current risk assessment of PAH-contaminated soils is usually based on chemical analysis of a small number of PAHs. The use of effect-based methods for risk assessment would yield results covering more of the effect of all the chemicals in the soil. To put such effect based data into context we tested a relative approach in which effects of contaminated soil were compared to clean rural and urban soils. This concept of relative risk assessment was tested by studying the mutagenic and AhR-agonistic potency of contaminated soil and urban soil compared to farm soil from selected ecological farms. A set of 21 soil samples was collected: 11 PAH-contaminated samples (collected in collaboration with three Swedish remediation companies), 5 urban samples (collected in Swedish cities) and 5 soil samples from ecological farms. The urban and rural samples were collected at the surface (0-10 cm deep), the contaminated samples were collected from piles during remediation (100-200 cm deep). To evaluate the toxicants in the soil sample, lipophilic sample extracts were tested in two different assays; (i) the Ames Fluctuation Assay (AFA) mutant strains TA98 and TA100 of Salmonella typhimurium with and without a metabolic activation system (rat-liver homogenate S9) to determine the mutagenic potential of the soil samples and (ii) the cell mechanism-specific H4IIE-luc assay to determine the Ahreceptor (AhR) activating potency of the soil extracts. The results showed clear mutagenicity, both direct and indirect, in one of the PAH-contaminated samples and three other PAH samples also demonstrated some mutagenic activity. The extracts from urban city soil showed mutagenicity in three of the 5 samples, while none of the ecological farm samples had mutagenic extracts. The bio-TEQ values were very high for all remediated samples and elevated in one urban sample. Bio-TEQ values were low in the ecological farm extracts. These findings demonstrate that the present investigation scheme using two different bioassays to determine the mutagenic potential and the Ah receptor activating potency of soil extracts is a suitable method for testing toxic properties of soil extracts. The concept of relative risk assessment using background samples from rural and urban areas and effect based testing shows promise for further development.