To Örebro University

Environmental pollutants often induce morphological alterations in developing organisms, yet assessments are commonly subjective, limiting reproducibility and sensitivity. We developed and validated a semi-automated brightfield high-content imaging (HCI) pipeline to quantitatively detect morphological changes in zebrafish embryos. Using FishInspector software, we adapted image analysis for microscopy systems without automated embryo positioning, extending applicability across standard laboratory setups.To validate the approach, zebrafish embryos were exposed for 96 hours to two previously characterized pollutant mixtures (PFOS + PCB126; PFOS + B[a]P + arsenate) known to cause developmental effects. The pipeline sensitively quantified phenotypes including reduced swim bladder and shortened body length. These endpoints reflect developmental delay, highlighting the method's ability to capture mechanistically relevant effects. Such changes may reduce physiological performance and behavior, ultimately impacting fish populations.While earlier subjective scoring identified some similar alterations, our findings underscore the advantages of quantitative, semi-automated morphology assessment. The method improves reproducibility, enables standardized comparisons across studies, and increases sensitivity to detecting subtle morphological effects. By integrating brightfield imaging with semi-automated analysis, this approach broadens the toxicological toolbox for developmental hazard assessment and mixture toxicity research.

Background: Poly- and perfluoroalkyl substances (PFAS) are persistent pollutants affecting wildlife and biodiversity. Perfluorooctane sulfonic acid (PFOS) and one of its short-chain substitutes, perfluorobutane sulfonic acid (PFBS), are widely found in environmental components, especially in water. PFOS has been highlighted as causing deleterious effects on various organisms while PFBS adversity is suspected but requires further investigation. In this study, zebrafish embryos were exposed from 2 h post-fertilization to 28 days post-fertilization to two different concentrations (0.2 mu g/L and 2 mu g/L) of PFOS or PFBS. We then investigated the impacts of these early exposures later in life on adult fish fitness, growth, morphology, behaviour, and liver lipidomic profiles.

Results: PFOS exposure significantly reduced egg production, and both PFOS and PFBS altered growth patterns, organ development, and anxiety-like behaviour. Lipidomic analyses revealed persistent shifts in liver lipid composition that correspond to these phenotypic changes.

Conclusions: Taken together, our findings indicate that early-life exposure to low levels of PFOS and PFBS leads to long-term, sex-specific impairments in zebrafish physiology and behaviour, with disruptions in lipid metabolism emerging as a potential underlying mechanism.

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.

The rapid development of nanoparticles (NPs) has raised significant concerns regarding their potential toxic effects on both human health and the environment. Zebrafish (Danio rerio) have emerged as a valuable in vivo model for nanotoxicity studies due to their physiological and genetic similarities to humans, high fecundity, transparent embryos, and rapid development. This chapter provides a comprehensive review of zebrafish as a model organism for investigating NP toxicity. It highlights key advantages, including their suitability for high-throughput screening and real-time visualization of NP biodistribution. The chapter discusses various NP uptake pathways, such as the gills, gastrointestinal tract, and blood-brain barrier, and explores the biological barriers that influence NP accumulation. Furthermore, it summarizes toxicological findings on teratogenic, immunotoxic, neurotoxic, and hepatotoxic effects of NPs across different zebrafish life stages. The use of zebrafish allows for the investigation of both acute and chronic NP exposure, offering insights into developmental and reproductive toxicity, oxidative stress, and genotoxicity. By bridging the gap between simple in vitro tests and more complex mammalian models, zebrafish serve as an essential model for assessing the potential risks of nanomaterials for human and environmental health.

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.

As plastic debris breaks down in environments, it forms micro(nano) plastics that threaten organisms across ecosystems. These tiny particles are potentially harmful due to industrial additives ingested by organisms. Given the growing concern related to micro(nano) plastics, this study compared the toxicity of leachate from tire wear particles (TWPs) and a bioplastic alternative (Mater‑Bi ®) using two model organisms: zebrafish (Danio rerio) and nematodes (Caenorhabditis elegans). We investigated embryotoxicity and behavioral changes (photomotror response and tapping test) in zebrafish, alongside survival rates and swimming activity in nematodes. Interestingly, in zebrafish embryos, TWPs caused significant depigmentation (0.6 and 1.2 μg peq/mL, p<0.05 reducing visibility and decreasing activity under dark conditions in the larval photomotor response. Notably, activity in the tapping test increased across all tested concentrations of TWPs in zebrafish (p<0.05). In nematodes, the leachate was inducing hyperactivity (1.5 to 5 μg/mL), approximately a 1.5-fold increase in activity at 1.5 μg/mL (p<0.0001) without significantly impacting their survival. Although seemingly harmless to zebrafish a tall tested concentrations, bioplastics significantly reduced nematode activity (approximately 1.2 fold, p<0.008) at higher concentrations (>5 μg/mL). These findings emphasize the need for multi-species studies to understand the complex and variable impacts of micro(nano) plastics on various ecosystems.

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.

Marine environments receive plastic waste, where it suffers a transformation process into smaller particles. Among them, microplastics (MPs; <5 mm) are ingested by aquatic organisms leading to negative effects on animal welfare. The interactions between MPs, contaminants and organisms are poorly understood. To clarify this issue, European seabass (Dicentrarchus labrax L.) were fed with diets supplemented with 0 (control), polyethylene (PE) MPs (100 mg/kg diet), perfluorooctanesulfonic acid (PFOS, 4.83 μg/kg diet) or PFOS adsorbed to MPs (MPs-PFOS; final concentrations of 4.83 μg and 100 mg of PFOS and MP per kg of feed, respectively). Samples of skin mucus, serum, head-kidney (HK), liver, muscle, brain and intestine were obtained. PFOS levels were high in the liver of fish fed with the PFOS-diet, and markedly reduced when adsorbed to MPs. Compared to the control groups, liver EROD activity did not show any significant changes, whereas brain and muscle cholinesterase activities were decreased in all the groups. The histological and morphometrical study on liver and intestine showed significant alterations in fish fed with the experimental diets. At functional level, all the experimental diets affected the humoral (peroxidase, IgM, protease and bactericidal activities) as well as cellular (phagocytosis, respiratory burst and peroxidase) activities of HK leukocytes, being more marked those effects caused by the PFOS diet. Besides, treatments produced inflammation and oxidative stress as evidenced at gene level. Principal component analysis demonstrated that seabass fed with MPs-PFOS showed more similar effects to MPs alone than to PFOS. Overall, seabass fed with MPs-PFOS diet showed similar or lower toxicological alterations than those fed with MPs or PFOS alone demonstrating the lack of additive effects or even protection against PFOS toxicity.

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.

Microplastics (MPs) are globally present in the marine environment, but the biological effects on marine organisms at the molecular and cellular levels remain scarce. Due to their lipophilic nature, MPs can adsorb other contaminants present in the marine environment, which may increase their detrimental effects once ingested by organisms. This study investigates the effects of low-density polyethylene (PE) MPs with and without adsorbed benzo[a]pyrene in the gills proteome of the peppery furrow shell clam, Scrobicularia plana. Clams were exposed to PE MPs (11-13 μm; 1 mg L^-1) for 14 days. BaP was analyzed in whole clams' soft tissues, and a proteomic approach was applied in the gills using SWATH/DIA analysis. Proteomic responses suggest that virgin MPs cause disturbance by altering cytoskeleton and cell structure, energy metabolism, conformational changes, oxidative stress fatty acid, DNA binding and, neurotransmission highlighting the potential risk of this type of MPs for the clam health. Conversely, when clam gills were exposed to MPs adsorbed with BaP a higher differentiation of protein expression was observed that besides changes in cytoskeleton and cell structure, oxidative stress, energy metabolism and DNA binding also induce changes in glucose metabolism, RNA binding and apoptosis. These results indicate that the presence of both stressors (MPs and BaP) have a higher toxicological risk to the health of S. plana.