Microplastics in Limnic Ecosystems - Investigation of Biological Fate and Effects of Microplastic Particles and Associated Contaminants in Zebrafish (Danio rerio)-

Given the continually increasing global polymer production, environmental pollution from plastic debris has been widely perceived as an ecological threat with potentially adverse (eco-)toxicological impacts on various species and ecosystems. Especially small fragments of plastic debris measuring 1 ≤ 1000 µm in size − so-called microplastics (MPs) − can pose a risk to biota by physical or chemical hazards due to ingestion, sorption and transfer of anthropogenic environmental pollutants (e.g., pesticides, polycyclic aromatic hydrocarbons, pharmaceuticals). In recent years, there has been extensive research on the impact of streams and rivers as main entry routes for MPs into the marine environment. However, the evidence of adverse effects caused by the intake of MPs and the transfer of contaminants sorbed to MPs into freshwater ecosystems is still not conclusively documented. Therefore, this thesis addressed the impact of microplastic particles in limnic ecosystems by investigating the biological fate and effects of MPs and associated anthropogenic pollutants on different life stages of zebrafish (Danio rerio). To this end, effects of two common environmental pollutants (benzo(k)fluoranthene, chlorpyrifos) and two synthetic polymers (polystyrene, polymethyl methacrylate) were investigated with regard to alterations of complementary biomarkers during acute and chronic exposure, as well as the trophic transfer of MPs and MP-sorbed contaminants from invertebrate organisms to zebrafish and acute toxic effects of MPs pre-exposed in a natural aquatic ecosystem. Overall, the sorption of both anthropogenic pollutants to MPs could be confirmed under various exposure scenarios. In contrast to recent study results, no adverse effects were observed when zebrafish were exposed to clean, pristine polystyrene and polymethyl methacrylate. The ingestion of MPs was documented in the gastrointestinal tract of zebrafish and did not induce any physiological impairment. The uptake of both pollutants was verified for all investigated species by GC-MS and fluorescence measurements. In the acute exposure scenario using the fish embryo toxicity test (FET), zebrafish embryos displayed sublethal effects and morphological deformities related to the underlying mode of action of benzo(k)fluoranthene and chlorpyrifos. Exposure to MP-associated contaminants significantly reduced malformations and attenuated biomarker responses of acetylcholine esterase activity and CYP450 induction and hence indicated a reduced bioavailability of both pollutants for zebrafish embryos. Chronic exposure of adult zebrafish to both types of MPs and pollutants was designed to assess the potential impact of long-term exposure and possible bioaccumulation of these contaminants of emerging concern. However, exposure to MP-sorbed pollutants failed to alter biomarker responses over the prolonged experimental period. In addition, a novel approach was applied to monitor the uptake of benzo(k)fluoranthene in the intestinal epithelium using confocal laser scanning microscopy. However, no significant bioaccumulation or biotransformation of benzo(k)fluoranthene and chlorpyrifos in brain and liver tissue could be demonstrated using ultra performance liquid chromatography coupled with fluorescence detection and with a high-resolution mass spectrometer (UPLC-FLD/HRMS). In order to address the possible vector effects of MPs, the transfer of benzo(k)fluoranthene and polymethyl methacrylate particles in a simplified limnic food web were investigated, consisting of zooplankton (Daphnia magna), sediment-dwelling invertebrates (Chironomus riparius larvae) and zebrafish as highlevel predator. However, the trophic transfer of benzo(k)fluoranthene via MPs could not be confirmed by a combination of highly sensitive fluorescence tracking using CLSM, hepatic CYP450 induction, and advanced chemical-analytical methods. To account for the demand for environmentally relevant exposure scenarios and to improve risk assessment of MPs, a mixture of the most commonly used synthetic polymers (polyethylene, polypropylene, polystyrene, polyvinyl chloride) was exposed in a well-monitored surface water body. To evaluate the potential transfer of hazardous pollutants under natural conditions, the toxicity of the MP mixture was assessed in terms of acute toxic effects (FET), potential neurotoxic effects (AChE activity, larval visual motor response test) and effects of dioxin-like substances (EROD assay) using zebrafish embryos. In conclusion, the exposed MP mixture did not elicit significantly different effects than the natural particles from sediment and suspended matter samples. Eventually, it could only be confirmed that MPs could act as carriers for environmental contaminants following ingestion by various organisms. However, increased deleterious effects of MP-sorbed contaminants on various sensitive life stages of zebrafish could not be corroborated by acute or prolonged exposure, trophic transfer, or natural exposure. The findings suggest that the sorption of pollutants to MPs is more likely to reduce the bioavailability as a result of slow desorption within the organism. Although, MPs offer an alternative exposure route for aquatic organisms via ingestion. Even chronic exposure at environmentally relevant concentrations did not induce bioaccumulation of both highly lipophilic substances in zebrafish tissues. Lastly, the sorption behavior of MPs under realistic environmental exposure conditions was more likely to approximate the behavior of natural particles. Consequently, MPs might pose only a limited risk to limnic communities, especially with regard to the comparatively small fraction of plastic particles in freshwater ecosystems compared to the multitude of naturally occurring (a)biotic particles

How microplastics and adsorbed pollutants affect zebrafish development?

A presença de microplásticos no ecossistema aquático é, nos dias de hoje, uma realidade extremamente preocupante que acarreta sérios riscos para o meio ambiente e para a saúde pública. A capacidade dos microplásticos de adsorverem poluentes orgânicos, como é o caso do benzo[α]pireno (BaP), levanta preocupações adicionais, pois cria uma nova rota de entrada de compostos tóxicos na cadeia alimentar. No entanto, o conhecimento atual sobre o impacto dos microplásticos, inalterados e/ou contaminados, nos organismos aquáticos é ainda insuficiente e requer estudos adicionais. O trabalho desenvolvido no âmbito desta tese pretende assim fornecer novos dados sobre os efeitos biológicos dos microplásticos utilizando como modelo o peixe-zebra (Danio rerio). Pequenos teleósteos, como é o caso do peixe-zebra, oferecem vantagens significativas relativamente aos modelos animais clássicos e são atualmente utilizados como organismos de primeira linha para avaliar os riscos ambientais associados aos compostos tóxicos presentes nos meios aquáticos. Estudos toxicológicos requerem o uso de materiais inertes e condições controladas, todavia, nenhum dos sistemas atualmente comercializados é adequado para avaliar o efeito tóxico dos microplásticos. Estes sistemas contêm componentes feitos de polímeros plásticos que podem libertar partículas plásticas micrométricas, lixiviar os constituintes químicos ou adsorver os compostos químicos em estudo. O sistema de aquários autônomo ZEB316 foi desenvolvido no âmbito deste trabalho com o objetivo de suprimir esta necessidade e de facultar uma solução económica e fácil de implementar que permita a realização de estudos toxicológicos de última geração. Este sistema é construído com materiais inertes e resistentes à corrosão e proporciona boas condições de alojamento através de um sistema eficiente de recirculação e filtragem da água. A avaliação dos parâmetros da água e do desempenho do crescimento dos peixes mostrou que o sistema ZEB316 oferece condições de alojamento comparáveis à dos sistemas comercialmente disponíveis. A maioria dos resultados obtidos no âmbito desta dissertação provêm da análise de imagens de microscopia de campo claro e/ou fluorescência. Embora o uso de imagens de microscopia seja comum na maioria dos laboratórios e permita obter uma quantidade considerável de informação, a análise destas imagens é geralmente um processo moroso, em parte devido à falta de ferramentas automáticas/semiautomáticas dedicadas que permitam a sua análise. Nesse sentido, desenvolvemos diversas macros para o software ImageJ, com o objetivo de reduzir o erro associado à análise pelo usuário, aumentando assim a reprodutibilidade dos dados e a padronização das metodologias experimentais. Uma vez que o foco deste trabalho está centrado no estudo da osteotoxicidade, é de salientar o desenvolvimento de um conjunto de macros específicas para esse fim e denominadas ZFBONE. Este conjunto de ferramentas permite aos usuários avaliar, a partir de imagens 2D, parâmetros morfométricos de várias estruturas ósseas (por exemplo, opérculo, raios e escamas da barbatana caudal), mas também a extensão e a intensidade das colorações específicas do osso. Além disso, outras macros foram desenvolvidas para outros fins, por exemplo, para analisar os vários parâmetros morfométricos relativos aos embriões de peixe ou para avaliar cortes histológicos. Uma vez desenvolvidos os meios e técnicas necessários para a execução dos ensaios toxicológicos, e subsequente análise dos resultados, procedeu-se à realização das várias experiências que visam compreender efeito biológico dos microplásticos e contaminates no peixe-zebra. Assim, larvas de peixe-zebra foram produzidas e mantidas no sistema previamente descrito, ZEB316, e expostas cronicamente, durante o seu desenvolvimento, a partículas de polietileno de 20- 27 μm, inalteradas (MP) ou contaminadas com benzo[α]pireno (MP-BaP). Estas partículas foram adicionadas à dieta dos peixes através de uma suplementação a 1% m/m. Apesar de não se ter registado qualquer alteração ao nível dos parâmetros morfológicos aos 30 dias pós-fertilização (dpf), a presença de MP e MP-BaP acabou por ter um efeito negativo no crescimento dos espécimes aos 90 e 360 dpf. A fecundidade relativa, a morfologia do ovo/embrião e a área do vitelo também sofreram um impacto negativo em peixes-zebra alimentados com MP-BaP. No que respeita ao estado geral do esqueleto, os peixes expostos a dietas experimentais contendo MP e MP-BaP sofreram um aumento significativo na incidência de deformações esqueléticas aos 30 dpf quando comparados com peixes alimentados com a dieta controlo, bem como um desenvolvimento anómalo da barbatana caudal e escamas, e uma diminuição da qualidade do osso aos 90 dpf. Um comprometimento da formação óssea intergeracional foi também observado na prole de espécimes expostos a MP ou MP-BaP, que se refletiu numa redução do osso opercular dos descendentes aos 6 dpf. Além de um claro efeito no desenvolvimento ósseo, a análise histológica do intestino revelou ainda um número reduzido de células caliciformes em peixes alimentados com dieta MP-BaP, um claro sinal de inflamação intestinal. Finalmente, a exposição das larvas a MP-BaP levou a um aumento da expressão de genes associados à via de resposta do BaP, ao mesmo tempo que impactou negativamente na expressão de genes envolvidos no estresse oxidativo. Os resultados obtidos indicaram um maior comprometimento do desenvolvimento ósseo no peixe-zebra quando sujeito a uma dieta contendo microplásticos contaminados com BaP (MP-BaP), por comparação com a dieta contendo microplásticos não contaminados (MP). Outros autores demostraram também que a presença de BaP afeta a formação das vértebras e o desenvolvimento generalizado do esqueleto, no entanto os mecanismos envolvidos nestes fenómenos permanecem pouco estudados. Desta forma, realizámos ensaios adicionais in vivo com o objetivo de avaliar os efeitos osteotóxicos do BaP durante o desenvolvimento e regeneração óssea em peixe-zebra. A exposição aguda de larvas de peixe-zebra entre os 3 e os 6 dpf ao BaP levou a uma redução do tamanho do osso opercular e uma diminuição quantitativa de células positivas para osteocalcina, indicando um efeito composto na maturação dos osteoblastos. Por sua vez, quando se trata de uma exposição crônica das larvas de peixe-zebra ao BaP, entre os 3 e os 30 dpf, verificou-se que o desenvolvimento do esqueleto axial é afetado, aumentando a incidência e gravidade das deformações esqueléticas. Em peixes jovens adultos, observou-se ainda que a exposição ao BaP afetou não só a mineralização dos raios da barbatana caudal e escamas recém-formados, como prejudicou também o seu padrão morfológico, fenómenos que têm por base uma remodelação óssea desequilibrada. Relativamente às análises de expressão genética de vários marcadores verificou-se que o BaP induziu a ativação das vias xenobióticas e metabólicas e impactou negativamente na formação e organização da matriz extracelular. Curiosamente, a exposição ao BaP regulou positivamente os marcadores de inflamação nas larvas e aumentou o recrutamento de neutrófilos. Uma interação direta entre neutrófilos e a matriz extracelular óssea, ou células responsáveis pela formação do osso, foi observada in vivo, o que sugere um papel dos neutrófilos nos mecanismos subjacentes à osteotoxicidade do BaP. Globalmente, os resultados obtidos no âmbito deste trabalho sugerem que a exposição crónica a microplásticos inalterados e/ou contaminados não prejudica apenas o crescimento dos peixes, mas também o seu desempenho a nível reprodutivo, saúde geral do seu esqueleto, e compromete a descendência por meio de efeitos intergeracionais. Além disso, este trabalho fornece novos dados sobre os principais mecanismos celulares e moleculares envolvidos na osteotoxicidade do BaP e aborda o possível papel dos neutrófilos na redução da resposta óssea inflamatória.The presence of microplastics in the aquatic ecosystem represents a major issue for the environment and human health. The capacity of organic pollutants such as benzo[α]pyrene (BaP) to adsorb onto microplastic particles raises additional concerns, as it creates a new route for toxic compounds to enter the food web. Current knowledge on the impact of pristine and/or contaminated microplastics on aquatic organisms remains insufficient, and this work aims at providing new insights by evaluating their biological effects in zebrafish (Danio rerio). The ZEB316, a standalone housing system built with inert materials, and a comprehensive set of ImageJ semi-automatic tools were first developed and optimized to perform state-of-the-art toxicological studies and obtain meaningful data from morphometric analysis of brightfield/ fluorescence images. Zebrafish larvae were exposed throughout their development to polyethylene microplastics, pristine or spiked with BaP, supplemented in the fish diet. While exposure up to 30 days post-fertilization only increased the incidence of skeletal deformities, long-term exposure to pristine/contaminated microplastics not only jeopardized fish growth, reproduction performance, and skeletal health, but it also caused an intergenerational effect. To further study the mechanisms underlying BaP osteotoxicity, several bone-related in vivo assays were used to evaluate the effects of waterborne exposure to BaP during bone development and regeneration. BaP inhibited osteoblast maturation and ECM mineralization and stimulated osteoclast activity, thus affecting bone remodeling. Transgenic and transcriptomic approaches suggested that besides the activation of xenobiotic and metabolic pathways, which may negatively impact extracellular matrix formation and organization, BaP activates inflammatory mechanisms that recruit neutrophils, which affect both osteoblast and osteoclast activity, possibly through a direct interaction of the neutrophils with the bone matrix. This work provides novel data on the effects of microplastics exposure during zebrafish development, in particular its osteotoxic effects, and gives new insights into the cellular and molecular players involved in BaP osteotoxicity.Marco Tarasco was supported by the Portuguese Foundation for Science and Technology (FCT) through the PhD grant SFRH/BD/128634/2017 and COVID/BD/151848/2021 and by NEUBIAS-COST STSM program as part of action CA15124. This work was funded by FCT through projects UID/Multi/04326/2019, UID/00350/2020, UIDB/04326/2020, UID/MAR/00350/2013 and from the operational programs MAR2020 and COMPETE 2020 through projects OSTEOMAR MAR-02.01.01-FEAMP-0057 and EMBRC.PT ALG-01-0145-FEDER-022121

Understanding How Microplastics Affect Marine Biota on the Cellular Level Is Important for Assessing Ecosystem Function: A Review

Plastic has become indispensable for human life. When plastic debris is discarded into waterways, these items can interact with organisms. Of particular concern are microscopic plastic particles (microplastics) which are subject to ingestion by several taxa. This review summarizes the results of cutting-edge research about the interactions between a range of aquatic species and microplastics, including effects on biota physiology and secondary ingestion. Uptake pathways via digestive or ventilatory systems are discussed, including (1) the physical penetration of microplastic particles into cellular structures, (2) leaching of chemical additives or adsorbed persistent organic pollutants (POPs), and (3) consequences of bacterial or viral microbiota contamination associated with microplastic ingestion. Following uptake, a number of individual-level effects have been observed, including reduction of feeding activities, reduced growth and reproduction through cellular modifications, and oxidative stress. Microplastic-associated effects on marine biota have become increasingly investigated with growing concerns regarding human health through trophic transfer. We argue that research on the cellular interactions with microplastics provide an understanding of their impact to the organisms’ fitness and, therefore, its ability to sustain their functional role in the ecosystem. The review summarizes information from 236 scientific publications. Of those, only 4.6% extrapolate their research of microplastic intake on individual species to the impact on ecosystem functioning. We emphasize the need for risk evaluation from organismal effects to an ecosystem level to effectively evaluate the effect of microplastic pollution on marine environments. Further studies are encouraged to investigate sublethal effects in the context of environmentally relevant microplastic pollution conditions

Resolving the effects of environmental micro- and nanoplastics exposure in biota: A knowledge gap analysis

The pervasive spread of microplastics (MPs) and nanoplastics (NPs) has raised significant concerns on their toxicity in both aquatic and terrestrial environments. These polymer-based materials have implications for plants, wildlife and human health, threatening food chain integrity and ultimate ecosystem resilience. An extensive – and growing – body of literature is available on MP- and NP-associated effects, including in a number of aquatic biota, with as yet limited reports in terrestrial environments. Effects range from no detectable, or very low level, biological effects to more severe outcomes such as (but not limited to) increased mortality rates, altered immune and inflammatory responses, oxidative stress, genetic damage and dysmetabolic changes. A well-established exposure route to MPs and NPs involves ingestion with subsequent incorporation into tissues. MP and NP exposures have also been found to lead to genetic damage, including effects related to mitotic anomalies, or to transmissible damage from sperm cells to their offspring, especially in echinoderms. Effects on the proteome, transcriptome and metabolome warrant ad hoc investigations as these integrated “omics” workflows could provide greater insight into molecular pathways of effect. Given their different physical structures, chemical identity and presumably different modes of action, exposure to different types of MPs and NPs may result in different biological effects in biota, thus comparative investigations of different MPs and NPs are required to ascertain the respective effects. Furthermore, research on MP and NP should also consider their ability to act as vectors for other toxicants, and possible outcomes of exposure may even include effects at the community level, thus requiring investigations in mesocosm models

Unraveling the ecotoxicological effects of micro and nano-plastics on aquatic organisms and human health

Plastic pollution ranks among the most severe environmental disasters caused by humans, generating millions of tonnes of waste annually. The extensive and unregulated use of plastics has led to ecotoxicity and environmental imbalance. Microplastics (MPs) are prevalent in aquatic environments, and these MPs further degrade into even smaller particles known as nano-plastics (NPs). Both MPs and NPs impact the environment by readily absorbing organic pollutants and pathogens from their surroundings, owing to their bigger surface area to volume ratio. This review focuses on the source of origin, bioaccumulation, and potential impact of MPs and NPs on aquatic organisms and human health. Additionally, the review explores various methods employed for identification and quantification of these particles in aquatic ecosystems. Sufficient information is available on their characteristics, distributions, and effects on marine ecosystems compared with freshwater ecosystems. For plastic particles <10 μm, more toxicological effects were observed compared with larger size particles, in aquatic life. Understanding the mechanism of action and ecotoxicological effects of micro/nano-plastics on the health of aquatic life across various trophic levels, as well as human health, is of utmost importance. We address knowledge gaps and provide insights into future research approaches for a better understanding of the interactive mechanisms between binary pollutants.O

From nanoplastics to chemical pollutants: Exploring mixture toxicity in zebrafish

Doctoral thesis (PhD) – Nord University, 2024publishedVersio

Efeito de microplásticos e nanoplásticos no desenvolvimento embrionário : uma revisão integrativa

Na última década a quantidade de estudos detectando a presença de microplásticos (MP), e pouco tempo depois, nanoplásticos (NP), cresceu progressivamente. Essas partículas, advindas da fragmentação de plásticos maiores ou fabricadas propositalmente, foram encontradas em praticamente todos os ambientes, desde as águas marinhas, até alimentos, poeira doméstica, e mais recentemente, no corpo de animais marinhos, do ser humano e até no leite materno. Embora muito presentes na atualidade, seu impacto no ambiente e principalmente nos seres humanos ainda não é completamente compreendido. As condições ambientais influenciam a qualidade de vida dos seres vivos, e condições adversas podem resultar em alterações celulares, fisiológicas, embrionárias e em mudanças epigenéticas. Considerando a sensibilidade do período embrionário e fetal e a onipresença dos MPs e NPs, entender o efeito destas durante o desenvolvimento dos embriões é de suma importância. Na ausência de estudos experimentais em seres humanos, a análise desses efeitos baseia-se na translação de estudos em modelos animais. Sendo assim, este trabalho propõe-se a revisar, de maneira integrativa e seguindo a metodologia PRISMA, os estudos científicos que avaliam os efeitos ocasionados pela exposição à micro e nanoplásticos em modelos animais. Foram utilizadas como plataforma de pesquisa as bases de dados Biblioteca Virtual em Saúde (BVS), o Pubmed e o buscador Google Acadêmico. Os artigos analisados foram publicados entre 2010 a 2023, na língua inglesa. Após a análise dos artigos retornados e selecionados, os resultados observados foram organizados de acordo com os modelos animais avaliados (aves, bivalves, camundongos, copepódes, dáfnias, ouriços-do-mar, peixes-zebra, rãs, sépias e tunicados), divididos nos quatro filos correspondentes, e com as alterações encontradas (celulares, moleculares, fisiológicas e embrionárias). De forma geral, as MPs e NPs de todos os materiais plásticos acarretam inúmeros efeitos biológicos nos embriões, como alterações enzimáticas, metabólicas e fisiológicas e, em alguns casos, malformações embrionárias e redução da sobrevivência da prole. A partir da síntese dos resultados, foi preparado um material de divulgação científica voltado para gestantes, contendo indicações de como minimizar a exposição a MPs e NPs no período gestacional.In the last decade, the number of studies detecting the presence of microplastics (MP), and shortly afterwards, nanoplastics (NP), has grown progressively. These particles, which come from the fragmentation of larger plastics or are manufactured on purpose, have been found in practically every environment, from marine waters to food, household dust and, more recently, in the bodies of marine animals, humans and even breast milk. Despite being present everywhere, their impact on the environment and especially on humans is still not fully understood. Environmental conditions influence the quality of life of living beings and adverse conditions can generate cellular, physiological, embryonic and epigenetic changes. Considering the sensitivity of the embryonic and fetal period and the omnipresence of MPs and NPs, understanding their effect on embryo development is of great importance. In the absence of experimental studies in humans, the analysis of their impact is based on the translation of studies in animal models. Therefore, this work aims to review following the PRISMA methodology, scientific studies that evaluate the effects caused by exposure to MP and NP in animal models. The Biblioteca Virtual em Saúde (BVS), Pubmed and Google Scholar databases were used as search platforms. The articles analyzed were published between 2010 and 2023, in English. After analyzing the articles returned and selected, the results observed were organized according to the animal models evaluated (birds, bivalves, mice, copepods, daphnia, sea urchins, zebra fish, frogs, sepias and tunicates), divided into the four corresponding phyla, and the alterations found (cellular, molecular, physiological and embryonic). In general, MPs and NPs from all plastic materials have numerous biological effects on embryos, such as enzymatic, metabolic and physiological alterations and, in some cases, embryonic malformations and reduced survival of the offspring. Based on the synthesis of the results, materials for Science dissemination were prepared, aimed to pregnant people and containing indications on how to minimize exposure to MPs and NPs during pregnancy

Prevalence, Fate and Effects of Plastic in Freshwater Environments

Plastic (and microplastic) pollution has been described as one of the greatest environmental challenges of our time, and a hallmark of the human-driven epoch known as the Anthropocene. It has gained the attention of the general public, governments, and environmental scientists worldwide. To date, the main focus has been on plastics in the marine environment, but interest in the presence and effects of plastics in freshwaters has increased in the recent years. The occurrence of plastics within inland lakes and rivers, as well as their biota, has been demonstrated. Experiments with freshwater organisms have started to explore the direct and indirect effects resulting from plastic exposure. There is a clear need for further research, and a dedicated space for its dissemination. This book is devoted to highlighting current research from around the world on the prevalence, fate, and effects of plastic in freshwater environments

Fluorescent Microplastic Uptake by Immune Cells of Atlantic Salmon (Salmo salar L.)

The ubiquitous presence of microplastics and their marine ecotoxicity are major public concerns. Microplastics are ingested accidentally by the marine fauna or are taken up indirectly through the food chain. These particles can accumulate in cells and tissues and affect the normal biological functions of organisms, including their defense mechanisms. There is limited information available about the response of immune cells to microplastics; the degree of uptake by the cells, the response of different organs or the impact of environmental concentrations of microplastic are matters that remain unclear. Moreover, very little is known about the toxicity of different polymer types. This study aimed to shed light on the physical impact of small microplastics (1–5 μm) on cells from Atlantic salmon. Immune cells from intestine, blood, and head kidney were exposed to green fluorescent polyethylene microplastic (PE-MP), yellow fluorescent polystyrene microplastic (PS-MP) and both. High (50 mg/L), medium (5 mg/L), and low (0.05 mg/L) concentrations were tested for 1, 24, 48, and 72 h to study cell mortality and microplastic uptake. Quantitative data of microplastic uptake by fish immune cells were obtained for the first time by imaging flow cytometry. Salmon immune cells showed a relatively low ability to phagocytose microplastics. Less than 6% of the cells ingested the particles after 48 h of exposure to high concentrations. Cells also phagocytosed microplastics at low concentrations although at low rates (<0.1%). PE-MPs was phagocytosed by higher percentage of cells compared to PS-MPs and the former bioaccumulated in time while the latter decreased over time. However, each cell generally phagocytosed more PS-MPs particles than PE-MPs. Cells from different tissues showed different responses to the microplastic polymers. In conclusion, this study shows that immune cells of Atlantic salmon can phagocytose microplastics, and the impact is dependent on the microplastic type. PE-MPs, the most abundant polymer in the oceans and a widely used plastic in salmon aquaculture, was more easily taken up than PS-MPs. Furthermore, the study demonstrates how imaging flow cytometry can be applied in microplastics research.publishedVersionUnit Licence Agreemen