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The textile industry, while of major importance in the world economy, is a toxic industry utilizing and emitting thousands of chemical substances into the aquatic environment. The aim of this project was to study the potentially harmful effects associated with the leaching of chemical residues from three different types of textiles: sportswear, children’s bath towels, and denim using different fish models (cell lines, fish larvae and juvenile fish). A combination of in vitro and in vivo test systems was used. Numerous biomarkers, ranging from gene expression, cytotoxicity and biochemical analysis to behavior, were measured to detect effects of leached chemicals. Principle findings indicate that leachates from all three types of textiles induced cytotoxicity on fish cell lines (RTgill-W1). Leachates from sportswear and towels induced mortality in zebrafish embryos, and chemical residues from sportswear reduced locomotion responses in developing larval fish. Sportswear leachate increased Cyp1a mRNA expression and EROD activity in liver of exposed brown trout. Leachates from towels induced EROD activity and VTG in rainbow trout, and these effects were mitigated by the temperature of the extraction process. All indicators of toxicity tested showed that exposure to textile leachate can cause adverse reactions in fish. These findings suggested that chemical leaching from textiles from domestic households could pose an ecotoxicological threat to the health of the aquatic environment.

The role of microplastics as chemical vectors delivering environmental contaminants into biota has been proposed, but their environmental relevance remains an issue of a debate. In this paper we compared the propensity and relative importance of synthetic polymer microparticles [glassy polystyrene (PS) and rubbery polyethylene (PE)] and silica glass particles (SG) to act as vectors for hydrophobic organic chemicals (HOCs) into fish after ingestion. Particles were spiked with three HOCs [17α-ethinylestradiol, chlorpyrifos and benzo(α)pyrene], which differ in hydrophobicity and induce well-known biomarker responses. Three-spined stickleback were exposed to 8 different diets: control diets (1), diets with non-spiked particles (2–4), diets containing a mixture of particles spiked with 3 model contaminants (5–7) and, finally, diets loaded with only the chemical mixture (8), for 14 days. Chemical sorption onto the particles was quantified and chemical transfer into the fish was investigated via biomarkers (CYP1a, ERα, VTG, and AChE) in fish intestine, liver and brain and quantification of HOCs in fish muscle. Results demonstrated particle-mediated chemical transfer of moderately hydrophobic contaminants into fish. While PS and PE particles mediated higher chemical transfer and tissue accumulation of 17α-ethinylestradiol and chlorpyrifos than SG, the overall chemical transfer was found to be very low. The present work suggested that chemical sorption, desorption and subsequent transfer of chemicals in vivo depends on multiple interconnected factors, including physicochemical properties of particles and contaminants, as well as toxicokinetic and toxicodynamic interactions. The biomarker approach was, however, suboptimal for assessing chemical transfer when addressing particle-associated chemical mixtures.

Ingestion has been proposed as a prominent exposure route for plastic debris in aquatic organisms, including fish. While the consequences of ingestion of large plastic litter are mostly understood, the impacts resulting from ingestion of microplastics (MPs) are largely unknown. We designed a study that aimed to assess impacts of MPs on fish intestinal physiology and examined integrity of extrinsic, physical and immunological barriers. Rainbow trout were exposed to polystyrene (PS) MPs (100-400 μm) via feed for a period of 4 weeks. Fish were fed four types of diets: control, diets containing virgin PS particles, or particles exposed to two different environmental matrices (sewage or harbor effluent). Extrinsic barrier disturbance in intestinal tissue was evaluated via histology. The paracellular permeability toward ions and molecules was examined using Ussing chambers and mRNA expression analysis of tight junction proteins. Active transport was monitored as transepithelial potential difference, short-circuits current and uptake rate of amino acid

In this study, we investigated biological effects associated with ingestion of polystyrene (PS) microplastic (MPs) in fish. We examined whether ingestion of contaminated PS MPs (100-400 μm) results in chemical stress in rainbow trout (Oncorhynchus mykiss) liver and we explored whether this exposure can affect the oxidative stability of the fillet during ice storage. Juvenile rainbow trout were fed for 4 weeks with four different experimental diets: control (1) and feeds containing virgin PS MPs (2) or PS MPs exposed to sewage (3) or harbor (4) effluent. A suite of ecotoxicological biomarkers for oxidative stress and xenobiotic-related pathways was investigated in the hepatic tissue, and included gene expression analyses and enzymatic measurements. The potential impact of MPs exposure on fillet quality was investigated in a storage trial where lipid hydroperoxides, loss of redness and development of rancid odor were assessed as indications of lipid peroxidation. Although, chemical analysis of PS MPs revealed that particles sorb environmental contaminants (e.g., PAHs, nonylphenol and alcohol ethoxylates and others), the ingestion of relatively high doses of these PS MPs did not induce adverse hepatic stress in fish liver. Apart from small effect on redness loss in fillets of fish exposed to PS MPs, the ingestion of these particles did not affect lipid peroxidation or rancid odor development, thus did not affect fillet's quality.

According to the United Nations, plastic pollution in the natural environment has been identified as one of the biggest environmental challenges of this century and has become a cause for an emerging international concern. It has been predicted that up to 12 million tons of plastic waste reach the aquatic environment annually. Therein, UV-radiation induced photo-oxidation, mechanical weathering and biological degradation contribute to the fragmentation of plastic litter to the micro- or even nanoscale. Microplastics (MPs) thus have become prominent pollutants in the aquatic environment, and their prevalence has been documented in every aquatic ecosystem studied. MPs enter aquatic food webs, also reaching humans, the top consumers in the food chain. The omnipresence of small microscopic plastic particles in the aquatic environment presents several ecotoxicological concerns. Firstly, MP fragments can interact with aquatic organisms and act as physical or mechanical stressors. Secondly, MPs can be toxic, as some polymers consist of potentially hazardous monomers. Synthetic, petroleum-derived polymers can also contain functional additives, impurities or chemical residuals, which are not chemically bound to the polymeric material and thus have the potential to leach out and cause diverse toxicological effects. Lastly, plastic polymers are known to absorb persistent hydrophobic organic pollutants from the environment. MPs have been suggested to act as vectors of environmental contaminants into organisms, promote bioaccumulation of toxic compounds, and cause biological effects in aquatic biota. It remains widely debated whether MPs are important vectors of chemicals for aquatic animals, including fish, and whether MP ingestion by edible fish species can impact human food quality and safety. This PhD project addressed some of these prevailing concerns, and investigated biological fate and impacts of MPs and associated chemicals in fish. It has been shown that exposure route can play an important role in particle-organism interactions and can determine the organismal uptake and localization of plastic particles in fish [Paper I]. Plastic nanoparticles interact with aquatic organisms: they can enter fish via contaminated prey (trophic transfer) and they can be directly ingested and/or adhere to organismal surfaces. Ingested nanoplastics can accumulate in the gastrointestinal tract and can then be internalized by the intestinal cells. Plastic ingestion is regarded as an environmentally relevant particle pathway in fish, and it facilitates their entrance into aquatic food chains. Studies included in this thesis also explored biological effects derived from the ingestion of larger, micro-sized plastic particles, at sizes commonly extracted from biological and environmental matrices, and which entail environmentally relevant chemical exposures [Papers II-III]. Direct impacts resulting from MP ingestion were found to be negligible, as no adverse effects were observed on fish intestinal physiology. Indirect, chemical exposure related effects resulting from ingestion of contaminated MPs were also minor. No indications of hepatic stress (oxidative stress, detoxification, endocrine disruption) were observed. It was concluded that MPs did not act as mechanical and chemical hazards upon ingestion, and are unlikely to cause adverse effects on organismal health. Although MPs showed capacity to associate with environmental contaminants [Papers II-IV], the transfer of pollutants from particles into fish via ingestion, as well as accumulation and biological impacts were suspected to be low [Papers II-IV]. The early findings presented in this thesis suggest that ingestion of MPs by commercial fish species does not significantly diminish the oxidative stability of commercial fish products, and MP-mediated chemical exposure does not pose an evident concern for human food quality and product shelf-life.

Locomotion-based behavioural endpoints have been suggested as suitable sublethal endpoints for human and environmental hazard assessment, as well as for biomonitoring applications. Larval stages of the sand goby (Pomatoschistus minutus) possess a number of attractive qualities for experimental testing that make it a promising species in behavioural ecotoxicology. Here, we present a study aimed at developing a toolkit for using the sand goby as novel species for ecotoxicological studies and using locomotion as an alternative endpoint in toxicity testing. Exposure to three contaminants (copper (Cu), di-butyl phthalate (DBP) and perfluorooctanoic acid (PFOA) was tested in the early life stages of the sand goby and the locomotion patterns of the larvae were quantified using an automatic tracking system. In a photo-motor test, sand goby larvae displayed substantially higher activity in light than in dark cycles. Furthermore, all tested compounds exerted behavioural alterations, such as hypo- and hyperactivity. Our experimental results show that sand goby larvae produce robust and quantifiable locomotive responses, which could be used within an ecotoxicological context for assessing the behavioural toxicity of environmental pollutants, with particular relevance in the Nordic region. This study thus suggests that sand goby larvae have potential as an environmentally relevant species for behavioural ecotoxicology, and as such offer an alternative to standard model species. This article belongs to the Special Issue Aquatic Organisms for Environmental Monitoring

A major challenge in nanoecotoxicology is finding suitable methods to determine the uptake and localisation of nanoparticles on a whole-organism level. Some uptake methods have been associated with artefacts induced by sample preparation, including staining for electron microscopy. This study used light sheet microscopy (LSM) to define the uptake and localisation of fluorescently labelled nanoparticles in living organisms with minimal sample preparation. Zebrafish (Danio rerio) were exposed to fluorescent gold nanoparticles (Au NPs) and fluorescent polystyrene NPs via aqueous or dietary exposure. The in vivo uptake and localisation of NPs were investigated using LSM at different time points (1, 3 and 7 days). A time-dependent increase in fluorescence was observed in the gut after dietary exposure to both Au NPs and polystyrene NPs. No fluorescence was observed within gut epithelia regardless of the NP exposure route indicating no or limited uptake via intestinal villi. Fish exposed to polystyrene NPs through the aqueous phase emitted fluorescence signals from the gills and intestine. Fluorescence was also detected in the head region of the fish after aqueous exposure to polystyrene NPs. This was not observed for Au NPs. Aqueous exposure to Au NPs resulted in increased relative swimming distance, while no effect was observed for other exposures. This study supports that the route of exposure is essential for the uptake and subsequent localisation of nanoparticles in zebrafish. Furthermore, it demonstrates that the localisation of NPs in whole living organisms can be visualised in real-time, using LSM.

Zebrafish (Danio rerio) is not only a widely used species in the Fish Embryo Toxicity (FET) test but also an emerging model in behavioural ecotoxicology. By using automatic behaviour tracking technology, locomotion of developing zebrafish (ZF) larvae can be accurately recorded and potentially used in an ecotoxicological context to detect toxicant-induced behavioural alterations. In this study, we explored if and how quantitative locomotion data can be used for sub-lethal toxicity testing within the FET framework. We exposed ZF embryos to silver ions and nanoparticles, which previously have been reported to cause neurodevelopmental toxicity and behavioural retardation in early-life stages of ZF. Exposure to a broad range of silver (Ag(+) and AgNPs) concentrations was conducted, and developmental toxicity was assessed using FET criteria. For behavioural toxicity assessment, locomotion of exposed ZF eleutheroembryos (120hpf) was quantified according to a customised behavioural assay in an automatic video tracking system. A set of repeated episodes of dark/light stimulation were used to artificially stress ZF and evoke photo-motor responses, which were consequently utilized for locomotion profiling. Our locomotion-based behaviour profiling approach consisted of (1) dose-response ranking for multiple and single locomotion variables; (2) quantitative assessment of locomotion structure; and (3) analysis of ZF responsiveness to darkness stimulation. We documented that both silver forms caused adverse effects on development and inhibited hatchability and, most importantly, altered locomotion. High Ag(+) and AgNPs exposures significantly suppressed locomotion and a clear shift in locomotion towards inactivity was reported. Additionally, we noted that low, environmentally relevant Ag(+) concentrations may cause subordinate locomotive changes (hyperactivity) in developing fish. Overall, it was concluded that our locomotion-based behaviour-testing scheme can be used jointly with FET and can provide endpoints for sub-lethal toxicity. When combined with multivariate data analysis, this approach facilitated new insights for handling and analysis of data generated by automatized behavioural tracking systems.