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We investigated the lethal effects of a long fibrous microplastic (the range of lengths, widths, and thicknesses were 1.5-3.6 mm, 10-16 µm, and 7-8.5 µm, respectively) made of polyethylene terephthalate textile, on the marine copepod Acartia erythraea. In laboratory, starved copepods were observed to take in a piece of fibrous microplastic sedimented on the bottom. While no individual ingested the entire fibre, the tip of the ingested fibre reached deep into the gut of the copepods. This suggests that ingestion was not accidental but purposeful behaviour to take in non-living organic matter as a supplementary food source. All copepods that had the fibre in their mouths eventually died within 24-h because the fibre penetrated deep into the gut, preventing feeding and potentially causing stress. Our finding implies that a single piece of microplastic fibre remaining at the bottom of coastal zones could continue to kill copepods owing to its non-degradability.

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Microplastics in aquatic ecosystems harbor numerous microorganisms, including pathogenic species. The ingestion of these microplastics by commercial fish poses a threat to the ecosystem and human livelihood. Coastal lagoons are highly vulnerable to microplastic and microbiological pollution, yet limited understanding of the risks complicates management. Here, we present the main bacterial groups, including potentially pathogenic species, identified on microplastics in waters, sediments, and commercial fish from Ciénaga Grande de Santa Marta (CGSM), the largest coastal lagoon in Colombia. DNA metabarcoding allowed identifying 1760 bacterial genera on microplastics, with Aeromonas and Acinetobacter as the most frequent and present in all three matrices. The greatest bacterial richness and diversity were recorded on microplastics from sediments, followed by waters and fish. Biochemical analyses yielded 19 species of potentially pathogenic culturable bacteria on microplastics. Aeromonas caviae was the most frequent and, along with Pantoea sp., was found on microplastics in all three matrices. Enterobacter roggenkampii and Pseudomonas fluorescens were also found on microplastics from waters and fish. We propose management strategies for an Early Warning System against microbiological and microplastic pollution risks in coastal lagoons, illustrated by CGSM. This includes forming inter-institutional alliances for research and monitoring, accompanied by strengthening governance and health infrastructures.

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Microplastics at 10 sites along a 77 km transect of the river Thames estuary (UK) and 5 sites along 29 km of the Medway estuary were separated from sediment and analysed by ATR-FTIR spectroscopy. Microplastics were observed at all sites. Highest Thames concentrations were in urban London between Chelsea and West Thurrock (average 170.80 particles kg-1 ± 46.64, 3.36 mg kg-1 ± 1.79 by mass), mid-outer estuary sites were two to three times lower. Microplastics were slightly dominated by particles (54 %) over fibres (45 %), including polymer types ranked: polyethylene > PET > polypropylene > polyamide. Medway microplastics decreased seaward, with one urban-municipal site impacted by a combined-sewer-overflow containing a high proportion of fibres (Rochester, 484 particles kg-1, 7.39 mg kg-1 by mass). Microplastic abundance was correlated to organic carbon (TOC %) (R2 of 0.71 Thames and 0.96 Medway), but not sediment particle size. Sedimentary microplastics accumulation in the Thames was controlled by urbanisation-distance, and site hydrodynamics.

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As an important emerging pollutant, the fate of microplastics (MPs) in ecosystems is of growing global concern. In addition to hydrodynamics and animals, algae can also affect the transport of MPs in aquatic environments, which could potentially remove MPs from the water column. Although researchers have conducted many studies on the sink of MPs regulated by algae in both marine and freshwater environments, there is still a lack of comprehensive understanding coupled with the increasingly scattered study contents and findings. This review aims to provide a systematic discussion of the processes, mechanisms, and influencing factors, which are coupled with the sink of MPs changes by algae. The main processes identified include retention, flocculation, deposition, and degradation. The retention of MPs is achieved by adhesion of MPs to algae or embedment/encrustation of MPs within the epibiont matrix of algae, thereby preventing MPs from migrating with water currents. The extracellular polymeric substances (EPS) and enzymes produced by algal metabolic activities can lead not only to the formation of aggregates containing MPs but also to the biodegradation of MPs. The processes that algae alter the fate of MPs in aquatic environments are very complex and can be influenced by various factors such as algal attributes, microplastic characteristics and environmental conditions. This review provides insights into recent advances in the fate of aquatic MPs and highlights the need for further research on MPs-algae interactions, potentially shortening the knowledge gap in the sink of MPs in aquatic ecosystems.

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Microplastic (MP) pollution can exert significant pressure on soil ecosystems, however, the interactive effects of MPs on soil bacterial, fungal and protist communities remains poorly understood. Soil macrofauna, such as earthworms, can be directly affected by MPs, potentially leading to a range of feedbacks on the soil microbial community. To address this, we conducted a microcosm experiment to examine the effects of conventional (i.e., polyethylene, polystyrene) and biodegradable MPs (i.e. PBAT, polylactic acid) on the structure of the soil bacterial, fungal, and protist communities in the presence or absence of earthworms. We found that MP contamination negatively affected the diversity and composition of soil microbial and protist communities, with smaller-sized conventional MPs having the most pronounced effects. For example, compared with the unamended control, small-sized polyethylene MPs both significantly reduced the Shannon diversity of soil bacteria, fungi, and protist by 4.3 %, 37.0 %, and 9.1 %, respectively. Biodegradable MPs increased negative correlations among bacteria, fungi, and protists. However, earthworms mitigated these effects, enhancing the diversity and altering the composition of these communities. They also increased the niche width and stability of the soil microbial food web network. Our study indicated that earthworms help attenuate the response of soil microorganisms to MPs stress by influencing the diversity and composition of soil microorganisms and soil physicochemical properties and underscores the importance of considering macrofauna in MPs research.

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Microplastic pollution is one of the most pressing global environmental problems due to its harmful effects on living organisms and ecosystems. To address this issue, researchers have explored several techniques to successfully eliminate microplastics from water sources. Chemical coagulation, electrocoagulation, magnetic extraction, adsorption, photocatalytic degradation, and biodegradation are some of the recognized techniques used for the removal of microplastics from water. In addition, membrane-based techniques encompass processes propelled by pressure or potential, along with sophisticated membrane technologies like the dynamic membrane and the membrane bioreactor. Recently, researchers have been developing advanced membranes composed of metal-organic frameworks, MXene, zeolites, carbon nanomaterials, metals, and metal oxides to remove microplastics. This paper aims to analyze the effectiveness, advantages, and drawbacks of each method to provide insights into their application for reducing microplastic pollution.

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The wide microplastics (MPs) occurrence affects soil physicochemical and biological properties, thereby influencing its carbon cycling and storage. However, the regulation effect of MPs on soil organic carbon (SOC) formation and stabilization remains unclear, hindering the accurate prediction of carbon sequestration in future global changes under continuous MP pollution. Phospholipid fatty acids, amino sugars and lignin phenols were used in this study as biomarkers for microbial community composition, microbial necromass and plant lignin components, respectively, and their responses to conventional (polyethylene; PE) and biodegradable (polylactic acid; PLA) MPs were explored. Results showed PLA MPs had positive effects on soil microbial biomass, while the positive and negative effects of PE MPs on microbial biomass varied with MP concentration. PE and PLA MPs increased microbial necromass contents and their contribution to SOC, mainly due to the increase in fungal necromass. On the contrary, PE and PLA MPs reduced lignin phenols and their contribution to SOC, mainly owing to the reduction in vanillyl-type phenols. The response of microbial necromass to PLA MPs was higher than that to PE MPs, whereas the response of lignin phenols was the opposite. MPs increased SOC level, with 83%-200% and 50%-75% of additional SOC in PE and PLA treatments, respectively, originating from microbial necromass carbon. This finding indicates that the increase in SOC pool in the presence of MPs can be attributed to soil microbial necromass carbon, and MPs increased capacity and efficacy of microbial carbon pump by increasing microbial turnover and reducing microbial N limitation. Moreover, the increase in amino sugars to lignin phenols ratio in PE treatment was higher than that in PLA treatment, and the increase in SOC content in PLA treatment was higher than that in PE treatment, indicating a high possibility of SOC storage owing to PLA MPs.

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Plastic pollution poses a significant threat to marine ecosystems. Microfibres from fabrics have become the most prevalent shape of microplastic found in the marine environment. The northern krill (Meganyctiphanes norvegica) is the most abundant euphausiid species in the northern hemisphere, playing a crucial role in various pelagic ecosystems. Anthropogenic microparticles in northern krill was assessed for the first time in samples collected in the Azores on two occasions - April 2019 (n = 480) and April 2023 (n = 480). Analysis of all individuals revealed 533 anthropogenic particles, with an average abundance of 0.56 ± 0.14 items per individual and, no significant differences between years. Microfibres were the most common shape (94.8 %), with the remaining items being fragments (5.2 %), and blue and black were the predominant colours. MicroFourier transform infrared spectroscopy analysis (µFTIR) of 22.1 % of the total number of particles, showed that they were mainly cellulosic (65.3 %) - either natural or semisynthetic - followed by polyester (7.6 %). Our finding of microplastics in the northern krill raises important questions due to its crucial role in marine food webs. The intake of anthropogenic particles, particularly those that are 100 % synthetic, suggests that the northern krill may act as a transfer vector of these pollutants to higher trophic levels.

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Marine organisms commonly encounter co-stress resulting from the coexistence of microplastics (MPs) and heavy metals pollution in marine environments. Nevertheless, the combined effects and toxicity mechanisms of MPs and heavy metals on marine organisms remain unclear. This study integrated growth, physiological, morphological, and biochemical markers to assess the individual and combined toxicity of polyvinyl chloride MPs (PVC MPs, 1 × 104 particles/L) and copper (Cu, 200 µg/L) on marine jacopever (Sebastes schlegelii). The results revealed that co-exposure to MPs and Cu had a more detrimental impact on jacopever compared to the single-exposure groups, as evidenced by the enhanced growth inhibition, respiratory stress, and hepatotoxicity. This phenomenon may be attributed to PVC MPs accelerating the accumulation of Cu in jacopever liver. Therefore, peroxidation damage occurred in the co-exposed liver and may result in liver dysfunction. These findings contribute valuable insights into the risks associated with the coexistence of MPs and heavy metal pollution in marine ecosystems.

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Microplastic (MP) pollution in urban environments is a pervasive and complex problem with significant environmental and human health implications. Although studies have been conducted on MP pollution in urban environments, there are still research gaps in understanding the exact sources, regulation, and impact of urban MP on the environment and public health. Therefore, the goal of this study is to provide a comprehensive overview of the complex pathways, harmful effects, and regulatory efforts of urban MP pollution. It discusses the research challenges and suggests future directions for addressing MPs related to environmental issues in urban settings. In this study, original research papers published from 2010 to 2024 across ten database categories, including PubMed, Google Scholar, Scopus, and Web of Science, were selected and reviewed to improve our understanding of urban MP pollution. The analysis revealed multifaceted sources of MPs, including surface runoff, wastewater discharge, atmospheric deposition, and biological interactions, which contribute to the contamination of aquatic and terrestrial ecosystems. MPs pose a threat to marine and terrestrial life, freshwater organisms, soil health, plant communities, and human health through ingestion, inhalation, and dermal exposure. Current regulatory measures for MP pollution include improved waste management, upgraded wastewater treatment, stormwater management, product innovation, public awareness campaigns, and community engagement. Despite these regulatory measures, several challenges such as; the absence of standardized MPs testing methods, MPs enter into the environment through a multitude of sources and pathways, countries struggle in balancing trade interests with environmental concerns have hindered effective policy implementation and enforcement. Addressing MP pollution in urban environments is essential for preserving ecosystems, safeguarding public health, and advancing sustainable development. Interdisciplinary collaboration, innovative research, stringent regulations, and public participation are vital for mitigating this critical issue and ensuring a cleaner and healthier future for urban environments and the planet.

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Low-cost and scalable technologies that allow people to measure microplastics in their local environment could facilitate a greater understanding of the global problem of marine microplastic pollution. A typical way to measure marine microplastic pollution involves imaging filtered seawater samples stained with a fluorescent dye to aid in the detection of microplastics. Although traditional fluorescence microscopy allows these particles to be manually counted and detected, this is a resource- and labour-intensive task. Here, we describe a novel, low-cost microscope for automated scanning and detection of microplastics in filtered seawater samples-the EnderScope. This microscope is based on the mechanics of a low-cost 3D printer (Creality Ender 3). The hotend of the printer is replaced with an optics module, allowing for the reliable and calibrated motion system of the 3D printer to be used for automated scanning over a large area (>20 × 20 cm). The EnderScope is capable of both reflected light and fluorescence imaging. In both configurations, we aimed to make the design as simple and cost-effective as possible, for example, by using low-cost LEDs for illumination and lighting gels as emission filters. We believe this tool is a cost-effective solution for microplastic measurement. This article is part of the Theo Murphy meeting issue 'Open, reproducible hardware for microscopy'.

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Microplastic (MP) pollution constitutes an emerging type of pollution threatening both aquatic and terrestrial ecosystems. The impact on aquatic ecosystems has been extensively studied, but the effect on terrestrial ecosystems and their inhabitants is mostly underexplored. In this study, we explored the effect of MP pollution on gut bacterial microbiome of endogeic (Aporrectodea caliginosa) and anecic (Lumbricus terrestris) earthworms. The experiments were performed in sandy soil with 0.2% of low-density polyethylene MPs (LDPE MPs). We observed that the endogeic earthworms had 100% survival, while anecic earthworms survived 25 days in the control (i.e. in absence of MPs) and 21 days in the treatment with LDPE MPs. The main driver of shifts in the diversity and composition of the bacterial communities in the gut of tested earthworms was the lifestyle of the worms, followed by the presence of MPs. The bacterial microbiome diversity was significantly different among the two types of earthworms, and the highest bacterial diversity was found in the gut of the endogeic earthworms. The effect of MPs on gut bacterial microbiome was clearly observed in the changes in the relative abundance of several phyla and families of the bacterial communities in both types of earthworms, although it was most evident in the anecic earthworms. The Actinobacteriota, Proteobacteria, and Firmicutes were the main groups enhanced in the MP treatments, suggesting enrichment of the bacterial communities with potential plastic degraders.

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Increasing microplastic (MP) pollution, primarily from anthropogenic sources such as plastic film mulching, waste degradation, and agricultural practices, has emerged as a pressing global environmental concern. This review examines the direct and indirect effects of MPs on crops, both in isolation and in conjunction with other contaminants, to elucidate their combined toxicological impacts. Organic fertilizers predominantly contain 78.6% blue, 9.5% black, and 8.3% red MPs, while irrigation water in agroecosystems contains 66.2% white, 15.4% blue, and 8.1% black MPs, ranging from 0-1 mm to 4-5 mm in size. We elucidate five pivotal insights: Firstly, soil MPs exhibit affinity towards crop roots, seeds, and vascular systems, impeding water and nutrient uptake. Secondly, MPs induce oxidative stress in crops, disrupting vital metabolic processes. Thirdly, leachates from MPs elicit cytotoxic and genotoxic responses in crops. Fourthly, MPs disrupt soil biotic and abiotic dynamics, influencing water and nutrient availability for crops. Lastly, the cumulative effects of MPs and co-existing contaminants in agricultural soils detrimentally affect crop yield. Thus, we advocate agronomic interventions as practical remedies. These include biochar input, application of growth regulators, substitution of plastic mulch with crop residues, promotion of biological degradation, and encouragement of crop diversification. However, the efficacy of these measures varies based on MP type and dosage. As MP volumes increase, exploring alternative mitigation strategies such as bio-based plastics and environmentally friendly biotechnological solutions is imperative. Recognizing the persistence of plastics, policymakers should enact legislation favoring the mitigation and substitution of non-degradable materials with bio-derived or compostable alternatives. This review demonstrates the urgent need for collective efforts to alleviate MP pollution and emphasizes sustainable interventions for agricultural ecosystems.

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Standardisation and validation of methods for microplastics research is essential. A major methodological challenge is the removal of planktonic organisms from marine water samples allowing for the identification of microplastics associated to planktonic communities. To improve the reproducibility and accuracy of digestion methods for the removal of planktonic biomass, we compared and modified existing chemical digestion methods. These digestion methods included an acidic digestion using nitric acid, alkaline digestions with potassium hydroxide (alkaline 1 digestion) and sodium hydroxide from drain cleaner (alkaline 2 digestion), an oxidative digestion using sodium dodecyl sulfate with hydrogen peroxide, and an enzymatic digestion using enzyme drain clean pellets. Chemical digestion of three densities of zooplankton communities (high, medium, and low) in the presence of five commonly found environmental microplastic pollutants (polyamide, polyethylene, polyethylene terephthalate, polypropylene, and polystyrene) were performed for each treatment. The chemical treatments were assessed for (i) their digestion efficiency of zooplankton communities by different biomass densities, and (ii) their impact on microplastic particles through the comparison of both chemical (Raman spectroscopy) and physical (length, width, and visual) changes, between the pre-treatment and post-treatment microplastic particles. The alkaline 1, alkaline 2 and oxidative methods demonstrated significantly better digestion efficiency (p < 0.05) than the modified enzymatic and acidic treatments. The acidic, alkaline 1, and alkaline 2, treatments caused the most damages to the microplastic particles. We suggest future studies to implement the oxidative digestion method with sodium dodecyl sulfate and hydrogen peroxide because of its high digestion efficiency, and low damage to microplastic particles. This method is similar to the wet peroxide oxidation digestion method used throughout the literature but can be implemented at a lower cost.

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Our comparative assessment is the first study to investigate microplastic body burden in native (Cambarus appalachiensis) and non-native (Faxonius cristavarius) crayfish along a semi-rural and urban stream across different seasons. Crayfish, sediment, and surface water were collected, processed, and characterized using µRaman spectroscopy to compare microplastic polymer types and shapes across compartments. Average surface water concentrations were significantly higher in our urban stream compared to our semi-rural stream (17.3 ± 2.4 particles/L and 9.9 ± 1.3 particles/L, respectively; P = 0.015). Average sediment concentrations were similar between urban and semi-rural streams (140 ± 14.5 particles/kg and 139 ± 22.5 particles/kg, respectively; P = 0.957). Our findings showed a significant interactive effect of season, site, and nativity (i.e., species) regarding microplastic body burden in crayfish (P = 0.004). The smaller, non-native crayfish amassed more microplastic particles than the native crayfish (0.4-2.0 particles/g versus 0.4-0.8 particles/g, respectively). Fibers and fragments were the most common polymer shapes across compartments, with white and black being the dominant particle colors. Our study identified 13 plastic polymer types in crayfish and three in surface water and sediment; polypropylene was the most common polymer across compartments. This study provides evidence that crayfish body burden of microplastics can differ across species, seasons, and locations, highlighting the need for future studies to consider that sublethal impacts associated with microplastic body burden may vary by region and species.

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Microplastic ingestion poses a significant concern for a plethora of marine organisms due to its widespread presence in marine ecosystems. Despite growing scientific interest, the effects on marine biota are not yet well understood. This study investigates the ingestion of microplastics (MPs) by mussels from various marine environments and assesses the associated effects that can be induced by MPs and associated toxic chemicals. Biomarkers of oxidative stress (catalase, lipid peroxidation), biotransformation (glutathione S-transferase), genotoxicity (micronuclei frequency) and neurotoxicity (acetylcholinesterase) were employed. Mussels, considered reliable bioindicators of MPs pollution, were sampled by hand from diverse locations under varied anthropogenic pressures, including a highly touristic Marine Protected Area (MPA) in the Ionian Sea, a mussel farm and a fish farm in the Aegean Sea. The results revealed the highest MP ingestion in mussels from the fish farm [0.21 ± 0.04 (SE) MPs/g or 0.63 ± 0.12 (SE) MPs/Ind.], likely due to plastic aquaculture equipment use. Stereoscopic observation revealed fibers, as the predominant shape of ingested MPs across all sites, and µFTIR polymer identification revealed the presence of various types, with polyethylene (PE) and polyamide (PA) being the most abundant. Significant physiological alterations in mussels related to MP ingestion levels were observed through biomarkers indicative of oxidative stress and biotransformation, as well as the Integrated Biomarker Response (IBR index). However, laboratory experiments with mussels exposed to controlled increasing PE concentrations for four weeks, did not show significant effects triggered by the PE ingestion, possibly indicating other environmental factors, such as contaminants from aquaculture environments, may influence biomarker levels in the field. Despite the observed effects, MP ingestion rates in mussels from the field were relatively low compared to other studies. Future research should continue to investigate the interactions between MPs and marine organisms in diverse environments to better understand and mitigate their impacts.

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As an emerging marine pollutant, microplastics represent a focal point in global monitoring and management efforts. With seawater accounts for 97 % of the total global water resources, scientific assessments of microplastics in seawater are crucial for pollution control and management of marine environments. This study focuses on investigating microplastics in near-shore seawater and proposes a rapid and accurate detection method using a constructed confocal Raman spectroscopy detection system. By optimizing the pretreatment process of seawater microplastic samples, the efficient removal of organic matter interference in microplastic detection is achieved. Employing fluorescent labeling addresses the issues of prolonged detection time and high false positive rates associated with traditional methods, enabling rapid differentiation between microplastics and other substances and significantly enhancing detection efficiency and accuracy. Additionally, the use of differential Raman spectroscopy effectively mitigates fluorescence signal interference, thus improving the signal-to-noise ratio of the spectra. By employing dual-wavelength laser excitation at 784 nm/785 nm, microplastics such as polyethylene (PE), polypropylene (PP), polystyrene (PS) ranging in size from 60 to 500 µm are successfully detected in seawater. The results demonstrate that the proposed pretreatment method for seawater microplastics and novel detection techniques enable rapid screening and comprehensive non-destructive detection of microplastics in seawater, thereby facilitating the characterization of marine microplastics and providing scientific support for enhancing the management of microplastic pollution and ecological risk control.

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Microscopic plastic (microplastic) pollutants threaten the earth's biodiversity and ecosystems. As a result of the progressive fragmentation of oversized plastic containers and products or manufacturing in small sizes, microplastics (particles of a diameter of 5 mm with no lower limit) are used in medicines, personal care products, and industry. The incidence of microplastics is found everywhere in the air, marine waters, land, and even food that humans and animals consume. One of the greatest concerns is the permanent damage that is created by plastic waste to our fragile ecosystem. The impossibility of the complete removal of all microplastic contamination from the oceans is one of the principal tasks of our governing body, research scientists, and individuals. Implementing the necessary measures to reduce the levels of plastic consumption is the only way to protect our environment. Cutting off the plastic flow is the key remedy to reducing waste and pollution, and such an approach could show immense significance. This review offers a comprehensive exploration of the various aspects of microplastics, encompassing their composition, types, properties, origins, health risks, and environmental impacts. Furthermore, it delves into strategies for comprehending the dynamics of microplastics within oceanic ecosystems, with a focus on averting their integration into every tier of the food chain.

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This study investigates the occurrence and characteristics of macroplastic and polymer microparticles in the Urias coastal lagoon's beach sediments, in northwest Mexico. Coastal lagoons, productive and vulnerable ecosystems, are impacted significantly by anthropogenic activities, leadings to their pollution by various contaminants, including plastics. Our research involved sampling sediments from four sites within the lagoon that were influenced by different human activities such as fishing, aquaculture, thermoelectric power plant operations, industrial operations, and domestic wastewater discharge. Our methodology included collecting macroplastics and beach sediment samples, followed by laboratory analyses to identify the plastic debris' size, shape, color, and chemical composition. The results indicated a notable presence of macroplastic items (144), predominantly bags, styrofoam, and caps made of polyethylene (PE), polypropylene (PP), and polyethylene terephthalate (PET). The polymer microparticles were mainly fibers, with cotton and polyester as the most common polymers, suggesting a significant contribution from clothing-related waste. The dominant colors of the microparticles were blue and transparent. High densities were observed in areas with slower water exchange. Our findings highlight the urgent need for better waste management practices to mitigate plastic pollution in coastal lagoons, preserving their ecological and economic functions.

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Concerns have been conveyed regarding the availability and hazards of microplastics (MPs) in aquatic biota due to their widespread presence in aquatic habitats. Zebrafish (Danio rerio) are widely used as a model organism to study the adverse impacts of MPs due to their several compelling advantages, such as their small size, ease of breeding, inexpensive maintenance, short life cycle, year-round spawning, high fecundity, fewer legal restrictions, and genetic resemblances to humans. Exposure of organisms to MPs produces physical and chemical toxic effects, including abnormal behavior, oxidative stress, neurotoxicity, genotoxicity, immune toxicity, reproductive imbalance, and histopathological effects. But the severity of the effects is size and concentration-dependent. It has been demonstrated that smaller particles could reach the gut and liver, while larger particles are only confined to the gill, the digestive tract of adult zebrafish. This thorough review encapsulates the current body of literature concerning research on MPs in zebrafish and demonstrates an overview of MPs size and concentration effects on the physiological, morphological, and behavioral characteristics of zebrafish. Finding gaps in the literature paves the way for further investigation.

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