To Örebro University

Effective early warning systems for aquatic contamination require monitoring strategies capable of detecting subtle, long-term shifts in mixture-driven biological activity. Suspended particulate matter (SPM) serves as a carrier and reservoir for complex contaminant mixtures, facilitating their transport and persistence in aquatic systems, yet systematic toxicological time series for archived SPM remain scarce. Regulatory monitoring predominantly targets Priority Substances and River Basin Specific Pollutants, leaving the temporal trends of particle-associated mixture toxicity largely unresolved. Leveraging 18 years (2005–2022) of cryogenically archived annual SPM composites from the Rhine River, we conducted a spatiotemporal effect-based assessment integrating receptor-mediated effects, oxidative stress analysis and untargeted Cell Painting phenomics. This integrated toolbox enabled evaluation of pathway-specific responses and multi-compartment cellular perturbations associated with particle-bound contaminant mixtures. Polar SPM-associated chemicals elicited oxidative stress response and caused endocrine disruption through estrogen receptor α (ERα) activation and androgen receptor inhibition (anti-AR). Trend analysis showed spatiotemporal variation along the river, with statistically increasing trends of oxidative stress and anti-AR activity over time at Koblenz, driven by polar chemicals. Both polar and non-polar SPM extracts activated the aryl hydrocarbon receptor (AhR), indicating presence of compounds capable of triggering xenobiotic response pathways. Several subcellular compartments were affected, with mitochondrial features being among the most affected. These findings demonstrate that SPM-associated chemicals elicit diverse toxicological effects by acting on several receptors and impacting diverse cellular structures. Combining targeted and phenomics-based effect approaches provided comprehensive mechanistic insights and valuable information to support the early warning systems for chemical contamination in aquatic environments.

Humans are chronically exposed to mixtures of environmental contaminants. Exposure to endocrine-disrupting chemicals (EDCs) contributes to increased health impairment observed globally. This study aimed to evaluate the endocrine-disruptive and oxidative stress potential of a human-relevant, complex chemical mixture in vitro. By testing chemical class subgroup mixtures, the identity of toxicological drivers and mixture additivity could be investigated. A 50-component mixture was compiled based on Swedish human blood concentrations (xHBC), consisting of six subgroup mixtures: polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (PCB mixture), brominated flame retardants (BFR mixture), per- and polyfluoroalkyl substances (PFAS mixture), pesticide mixture, synthetic phenolic contaminants (phenol mixture), and phthalate mixture. These were tested in four chemically activated luciferase gene expression (CALUX) assays: dioxin responsive (DR-), estrogen receptor α (ERα-), androgen receptor. (AR-), and nuclear factor erythroid 2-related factor 2 (Nrf2)-CALUX, along with an adipocyte cell assay. The total mixture caused significant agonistic activity in DR- and ER-, and antagonistic activity in AR-CALUX at 0.1-15 xHBC, depending on the assay. Mixture additivity was assessed in ERα-, DR-, and anti-AR-CALUX using subgroup mixtures and the concentration addition (CA) model. The total mixture followed the CA model in ERα-, anti-AR- and DR-CALUX. The toxicological drivers of these activities were mainly the PCB and phenol mixture. A significant increase in differentiated adipocytes was observed at 100 xHBC. These results raise concerns regarding potential health effects on the endocrine system. The additive effects at human-relevant concentrations observed in this study motivate considering mixtures in regulatory contexts to protect the well-being of future generations.

The continuous release of synthetic chemicals into aquatic systems underscores the need for long-term assessments of contaminants of emerging concern (CECs). Archived suspended particulate matter (SPM) samples from the German Environmental Specimen Bank (ESB) (2005–2022) at two Rhine sites (Weil am Rhein and Koblenz) were analyzed using liquid chromatography–high-resolution mass spectrometry (LC-HRMS) suspect/non-target screening (SS/NTS) to evaluate temporal trends. Using retention-time indices and orthogonal MS/MS evidence (mzCloud, FISh, CFM-ID), 332 compounds were identified at varying Schymanski confidence levels (2.1: 3.0%; 2.2: 5.7%; 3.1: 53%; 3.2: 38%). Temporal analysis of LC-HRMS peak areas revealed that 25% of contaminants increased over time, with a higher proportion at Koblenz (29%) than Weil (18%). Conversely, several compounds exhibited statistically significant decreasing trends at both Koblenz (14%) and Weil sites (13%), consistent with regulatory measures, improved wastewater treatment, and shifts in industrial practices. Aquatic toxicity prediction (ECOSAR) indicated that 47% (154 of 332) of annotated structures were highly acutely toxic (LC₅₀/EC₅₀ ≤ 1 mg/L) to at least one test group (fish, Daphnia, or green algae). This study provides the first 18-year, site-specific non-target time series from a national archive and integrating orthogonal identification with hazard prediction to support chemical prioritization. Archived SPM enables retrospective and comparable assessment of particle-associated contaminants, complementing dissolved-phase monitoring, and supporting the identification of unmonitored emerging CECs. Crucially, long-term NTS of these SPM samples provides screening-level early-warning signals and a watch list for targeted confirmation and risk management.

Crumb rubber, derived from recycled tyres, has been used as a sustainable, low-cost infill material. However, growing evidence suggests that tyre crumb rubber leachates can be highly toxic, underscoring the need for comprehensive risk assessments of different types of rubber and the development of safer alternatives. This study evaluated the acute toxicity of leachates from various infill materials, including black recycled tyre rubbers, coloured thermoplastic rubbers, and a mineral-based material ("Bioflex"), on key models of marine plankton. Specifically, we determined the effects of micronised crumb rubber leachates on the microalga Rhodomonas salina, embryos and nauplii of the copepod Acartia tonsa, and early developmental stages of the sea urchin Arbacia lixula. We also evaluated the toxicity of leachates extracted using an organic solvent compared to those obtained using filtered seawater, using R. salina as a model organism. Toxicity varied significantly among rubber types, with EC50 values ranging from 22.1mgL-1 to >1505mgL-1, depending on the organism, and biological endpoint. One of the tested tyre rubber crumbs consistently exhibited high toxicity across all test species, with EC50 values as low as 50mgL-1 for specific life stages. Among thermoplastic rubbers, green crumb rubber also showed high toxicity towards R. salina, with an EC50 of 25.7mgL-1. In contrast, the mineral-based material was only toxic to the early stages of A. lixula, exhibiting relatively low toxicity (EC50 = 2305mgL-1). In most cases, the toxicity of seawater-based leachates was comparable to that of solvent-based extracts, indicating that most toxic compounds affecting R. salina are water-soluble. Metal analysis revealed that the mineral-based material contained lower concentrations of toxic metals, such as zinc, and lower levels of polycyclic aromatic hydrocarbons than the other rubbers. This may partially explain its reduced toxicity to plankton. Our findings demonstrate that leachates from recycled crumb rubber and thermoplastic rubbers are toxic to marine plankton, although the effects are species-specific and dependent on the material composition. Our results also suggest that mineral-based infill materials (e.g., "Bioflex") are ecologically safer alternatives to rubbers. Overall, this study indicates that leachate pollution from recycled tyre rubber and thermoplastic rubber particles poses a potential risk to the pelagic food web, highlighting the importance of restricting or banning their use in areas near sensitive aquatic environments.

This study applied a virtual effect-directed analysis (vEDA) approach, integrating effect-based analysis and chemical screening, to identify bioactive compounds in rubber infill from artificial turf. Bioreporter assays targeting diverse toxicological endpoints were selected to detect a wide range of potential endocrine-disrupting and genotoxic compounds. Of 21 samples, all except one showed aryl-hydrocarbon receptor (AhR) activity (14-31,400 ng benzo[a]pyrene equivalents/g), four induced p53 activity (0.04-0.86 µg actinomycin D equivalents/g) and two showed estrogen receptor α (ERα) activity (530 and 1020 pg estradiol equivalents/g). Chemical analysis quantified up to 87 polycyclic aromatic compounds (PAC) and gas chromatography high-resolution mass spectrometry-based suspect screening yielded 281 tentative identifications. Annotation with bioassay activity data from databases and predictive models revealed 29 AhR-, 32 ERα- and 18 p53-active compounds. Univariate analysis was used to prioritize compounds for further chemical and toxicological confirmation. Eighteen AhR agonists were confirmed, contributing 0-98% to the observed AhR activity in the samples. Phenylamine additives, detected at high concentrations, exhibited low AhR activating potency and contributed < 1%. In contrast, methylated chrysene isomers elicited relatively high potencies and contributed substantially (≤65%) to the observed AhR activity. N-Isopropyl-N'-phenyl-p-phenylenediamine (IPPD) was confirmed as p53 active and explained ∼50% of the observed activity in the most p53-active sample. Styrene-butadiene rubber (SBR) showed higher AhR- and p53 activities and concentrations of quantified compounds than the alternative materials. The study highlights differences in chemical hazards among rubber infill materials and demonstrates the utility of vEDA as an early-warning tool for identifying compounds of concern.

The effectiveness of biochar amendment for remediation purposes depends on many factors related to the biochar and the contaminated site. Therefore, each application must be evaluated site-specifically. To facilitate full-scale implementation, more information from field studies on biochar-amended contaminated sites, as well as cost-effective approaches to evaluate the remediation efficacy of specific biochar materials are needed. We studied the effects of biochar and peat on metal solubility and bioavailability in a contaminated soil in a fully factorial field trial. The biochar was produced from wood via gasification in a floating fixed-bed reactor at 750 degrees C. Soil solutions from field-installed lysimeters, grass (Lolium perenne L), and earthworms (Eisenia fetida) were analyzed. In addition, a standardized batch leaching test (ISO 21268-2:2019) was performed to evaluate its feasibility to mimic soil solution concentrations. The results showed that biochar generally reduced the solubility and uptake of cationic metals. In situ solubility of Cu and Hg was reduced more than 80%, and Zn up to 70%. Soil solution concentrations of Cr increased in biochar-amended soils, but this effect was reduced by peat. Peat had small effects on in situ solubility of other metals. For cations, the batch test showed the same trends as the soil solution, with biochar decreasing solubility. However, mobilization of colloids during shaking in the batch test induced artefacts, leading to an overestimation of the solubility of some metals, especially Pb and Hg, an effect that was enhanced by peat applications.

As the volume of plastic waste from electrical and electronic equipment (WEEE) continues to rise, a significant portion is disposed of in the environment, with only a small fraction being recycled. Both disposal and recycling pose unknown health risks that require immediate attention. Existing knowledge of WEEE plastic toxicity is limited and mostly relies on epidemiological data and association studies, with few insights into the underlying toxicity mechanisms. Therefore, this study aimed to perform comprehensive chemical screening and mechanistic toxicological assessment of WEEE plastic-associated chemicals. Chemical analysis, utilizing suspect screening based on high-resolution mass spectrometry, along with quantitative target chemical analysis, unveiled numerous hazardous compounds including polyaromatic compounds, organophosphate flame retardants, phthalates, benzotriazoles, etc. Toxicity endpoints included perturbation of morphological phenotypes using the Cell Painting approach, inflammatory response, oxidative stress, and endocrine disruption. Results demonstrated that WEEE plastic chemicals altered the phenotypes of the cytoskeleton, endoplasmic reticulum, and mitochondria in a dose-dependent manner. In addition, WEEE chemicals induced inflammatory responses in resting macrophages and altered inflammatory responses in lipopolysaccharide-primed macrophages. Furthermore, WEEE chemicals activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, indicating oxidative stress, and the aryl hydrocarbon receptor (AhR). Endocrine disruption was also observed through the activation of estrogenic receptor-α (ER-α) and the induction of anti-androgenic activity. The findings show that WEEE plastic-associated chemicals exert effects in multiple subcellular sites, via different receptors and mechanisms. Thus, an integrated approach employing both chemical and toxicological methods is essential for comprehensive assessment of the toxicity mechanisms and cumulative chemical burden of WEEE plastic-associated chemicals.

Environmental context: The widespread use of the insecticide DDT has left a legacy of pollution that still threatens ecosystems today. This study presents a method to accurately measure the bioavailability of DDT and its breakdown products in contaminated soils. This will improve risk assessments and guide sustainable land management practices, helping to protect both the environment and human health.

Rationale: The insecticide dichlorodiphenyltrichloroethane (DDT) and its degradation products (collectively DDX) are persistent organic pollutants that pose significant environmental risks due to their persistence and bioaccumulation in ecosystems. Accurate quantification of DDX bioavailability in soil systems is crucial for effective land management and risk assessment.

Methodology: This study utilised equilibrium passive sampling with polyoxymethylene (POM) to determine the bioavailability of DDX in soil. The sorption dynamics of 10 DDX compounds were investigated (p,p′-DDT, o,p′-DDT, p,p′-dichlorodiphenyldichloroethane (p,p′-DDD), o,p′-DDD, p,p′-dichlorodiphenyldichloroethene (p,p′-DDE), o,p′-DDE, p,p′-dichlorodiphenylmethane (p,p′-DDM), p,p′-dichlorobenzophenone (p,p′-DBP), 1-chloro-2,2-bis(4-chlorophenyl)ethylene (p,p′-DDMU) and dicofol) and their POM–water partition coefficients (KPOM) were determined. The study involved interlaboratory comparisons, using soils from nine historically contaminated sites and ecotoxicology assessments (mortality, reproduction and bioaccumulation in earthworms, Eisenia fetida) to validate the POM method.

Results: KPOM values for 9 of the 10 DDX compounds were successfully determined, allowing for accurate quantification of freely dissolved pore water concentrations of DDX in historically contaminated soils. The interlaboratory study highlighted important considerations in extraction and gas chromatography–mass spectrometry analysis, and the ecotoxicology study demonstrated the potential of POM passive sampling as a reliable tool for assessing DDX bioavailability (bioaccumulation in Eisenia fetida).

Discussion: The POM method proved to be a robust and reliable approach for quantifying freely dissolved DDX, with implications for improving the accuracy of risk assessments and guiding sustainable land management practices. The study also highlighted the need for careful consideration of analytical challenges, such as the potential degradation of DDX compounds during gas chromatography analysis, to ensure accurate quantification.

Indoor dust contains a complex mixture of chemicals, including endocrine-disrupting chemicals (EDCs), which may pose risks to children's health. As children spend most of their time indoors and have frequent dust contact, their exposure is heightened. This study quantified the endocrine disrupting potential of dust from children's indoor environments in Sweden, and assessed associations with flame retardants and plasticizers in dust, handwipes, and urine.

Fifty dust samples from 18 homes and 11 preschool units were analyzed for estrogen, anti-androgen, and thyroid receptor activities using human osteosarcoma cell-based luciferase reporter assays. Associations were evaluated with 21 legacy and 18 emerging halogenated flame retardants (HFRs) and 11 organophosphate esters (OPEs) in dust and handwipes, as well as nine plasticizers (eight phthalates and di-isononyl cyclohexane 1,2-dicarboxylate (DiNCH)) in dust, and 14 plasticizer metabolites in urine. Samples for biological and chemical analyses were collected from the same designated areas within a limited time frame.

Most dust samples exhibited estrogen receptor agonist (ER) and androgen receptor antagonistic (anti-AR) activity, while thyroid receptor (TR) induction was low. Preschool dust showed significantly higher estrogenic activity than home dust. No seasonal variation was observed. Associations were observed between dust hormonal activities and urinary plasticizer metabolites, as well as HFR and OPE concentrations in dust and handwipes. Relative potency (REP) analyses of 36 HFRs and OPEs revealed notable anti-AR activity for 2,2´,4,4´-tetrabromodiphenyl ether (BDE-47) (REP values 0.85±0.10 (EC25) and 0.93±0.07 (EC50)) and 2,2´,4,4´,6-pentabromo diphenyl ether (BDE-100) (REP values 2.74±0.29 (EC25) and 3.23±0.42 (EC50)). Additionally, BDE-100 showed low ER induction.

Climate change is driving extreme weather patterns, leading to prolonged droughts and more frequent intense precipitation events. These environmental changes will impact aquatic systems by altering essential water quality parameters such as temperature, redox potential, pH, suspended solids and organic matter, which influence pollutant solubility and determine ecosystem health as well as drinking water production. In this context, sediments play a crucial role as they represent both a sink and source of pollutants. Therefore, sediment toxicity testing is essential for accurate environmental risk assessments. However, there remains a gap regarding comprehensive sediment testing ap-proaches that integrate multiple biomarker responses.

The SEASON project uses an interdisciplinary approach combining strategies of environmental toxicology, analytical chemistry, andhydro geochemistry. The aim is to develop conceptual models for evaluating, understanding and predicting the impact of climate change effects on the fate, bioavailability, and toxicity of pollutants in the aquatic environment. This project focuses on risks associated with sediments contaminated by organic and inorganic pollutants, specifically metals and PFAS (per- and polyfluoroalkyl substances). By studying factors such as temperature, pH, microbial communities, and sediment-water interactions, the project seeks to understand how different climate change aspects affect pollutant behavior in aquatic ecosystems.

The project consists of four sub-projects. Three investigate different chemical groups and mixtures under varying water conditions, using zebrafish (Danio rerio) as the main model organism. These studies will include in vitro and in vivo assays, microcosm experiments, microbiome studies and chemical analyses. The fourth subproject will integrate the results to develop a predictive model for sediment risk assessment.

Sediment contact assays will be performed to evaluate the effects of contaminated samples on zebrafish embryos by measuring teratogenicity, developmental toxicity, behavioral changes, and gene expression. In microcosm studies, we will vary pH and mimic increased precipitation events to assess pollutant toxicity in adult zebrafish including sex-related toxicity differences, reproduction, and behavior. Effect-directed analysis (EDA) will be used to identify key toxicants in the samples. Microbiome analysis using metagenomic sequencing will focus on how contaminated sediments alter bacterial communities, which in turn can affect pollutant distribution and bioavailability. Chemical analyses will quantify PFAS, metals, and their speciation throughout the study. Ultimately, the project will integrate these data to develop models that increase our understanding of the impact of climate change on sediment contamination and aquatic ecosystem health.